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... Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research ABT-510 Is an Effective Chemopreventive Agent in the Mouse 4-Nitroquinoline 1-Oxide Model of Oral Carcinogenesis Rifat Hasina,1 Leslie E. Martin,1 Kristen Kasza,2 Colleen L. Jones,1 Asif Jalil1 and Mark W. Lingen1 Abstract Despite numerous advances, the 5-year survival rate for head and neck squamous cell cancer (HNSCC) has remained largely unchanged. This poor outcome is due to several variables, including the development of multiple primary tumors. Therefore, it is essential to supplement early detection with preventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we sought to define an appropriate dose and duration of administration that would predict the histologic timeline of HNSCC progression. Additionally, we sought to determine the timing of the onset of the angiogenic phenotype. Finally, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can slow/delay the development of HNSCC. We determined that 8 weeks of 100 g/mL 4-NQO in the drinking water was the optimal dosage and duration to cause a sufficient incidence of hyperkeratoses, dysplasias, and HNSCC over a period of 32 weeks with minimal morbidity and mortality. Increased microvessel density and vascular endothelial growth factor expression in hyperkeratotic lesions provided evidence that the initiation of the angiogenic phenotype occurred before the development of dysplasia. Importantly, ABT-510 significantly decreased the overall incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). These findings suggest that our refinement of the 4-NQO model allows for the investigation of the histologic, molecular, and biological alterations that occur during the premalignant phase of HNSCC. In addition, these data support the hypothesis that inhibitors of angiogenesis may be promising chemopreventive agents. At current rates, approximately 400,000 cases of head and led Slaughter et al. (4) to propose the concept of field cancerization. This theory suggests that multiple individual primary tumors may develop independently in the upper aerodigestive tract as a result of years of chronic exposure to carcinogens. The occurrence of these new primary tumors can be particularly devastating for individuals whose initial lesions are small. Their 5-year survival rate for the first primary tumor is considerably better than patients with late-stage disease. However, second primary tumors are the most common cause of treatment failure and death among early-stage HNSCC patients (5). Therefore, it is insufficient treatment to address only the initial lesion. To improve the outcome of such patients, some form of chemopreventive treatment is essential. Chemoprevention can be defined as the systemic use of natural or synthetic agents to reverse or halt the progression of premalignant lesions. Chemopreventive agents are being tested for their efficacy in the preclinical and clinical settings for several malignancies, including HNSCC (6). However, the initial promising responses have not been consistently reproduced and toxicity was often a significant issue. Therefore, more effective and better tolerated therapy is needed for premalignant oral disease. Angiogenesis, the growth of new blood vessels from preexisting ones, is an essential phenotype in several physiologic and pathologic processes, including growth and development, wound healing, reproduction, arthritis, and tumor formation neck squamous cell cancer (HNSCC) will be diagnosed worldwide this year (1). Despite numerous advances in therapy, the long-term survival for these patients has remained largely unchanged. Several factors contribute to this poor outcome. First, oral cancer is often diagnosed in an advanced stage. The 5-year survival rate of early-stage oral cancer is approximately 80%, whereas the survival drops to 19% for late-stage disease (2). Second, the development of multiple primary tumors has a major effect on survival. The rate of second primary tumors in these patients has been reported to be 3% to 7% per year, higher than for any other malignancy (3). The observation of frequent second primary tumors in oral cancer Authors' Affiliations: Departments of 1Pathology, Medicine, and Radiation and Cellular Oncology and 2Health Studies, The University of Chicago, Chicago, Illinois Received 11/13/08; revised 2/7/09; accepted 3/4/09; published OnlineFirst 3/31/09. Grant support: Abbott Laboratories and NIH grant DE012322. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mark W. Lingen, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Chicago, IL 60637. Phone: 773-702-5548; Fax: 773-702-9903; E-mail: mark.lingen@uchospitals. edu. 2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0211 www.aacrjournals.org 385 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research (7). Whether active neovascularization occurs is dependent on the relative concentrations of inducers and inhibitors of angiogenesis present in a given tissue microenvironment. Therefore, the inhibition of tumor-associated angiogenesis, using natural or synthetic inhibitors of angiogenesis, is an attractive target for therapy that has been gaining traction in the field of oncology. Like all solid tumors, HNSCCs must develop multiple direct and indirect ways to induce angiogenesis. Importantly, the expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. In addition to their main biological/molecular effects, some of the drugs currently under investigation in the chemopreventive setting have potential antiangiogenic activity. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Animal models that faithfully recapitulate the human condition are critical to further our understanding of the molecular, biological, and clinical aspects of various diseases, including cancer. The hamster buccal pouch and the rat tongue models of oral carcinogenesis are well-established surrogate models for the human condition. However, although the hamster buccal pouch and rat tongue models have been extensively investigated, they have several limitations, resulting in the recent development of mouse oral cancer models that have several advantages (1218). In particular, mouse models enable the development and testing of new approaches to prevention and treatment, identification of early diagnostic markers, and an understanding of the biology and genetics of tumor initiation, promotion, and progression in an animal model whose genome is most similar to humans (1922). Although the 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis has gained increased attention as an alternative model, several parameters require further investigation. For example, although various treatment protocols have been described, the optimal administration, timing, and dosage of 4-NQO required to develop lesions that clinically mimic the human condition (singular or synchronous neoplasms) have not been fully established. In addition, a detailed and systematic investigation of the histologic changes during progression in the 4-NQO mouse model before the development of HNSCC has not been done. Finally, the timing and mechanisms of the development of various tumor-related phenotypes have not been determined in this model. Each of these issues is of critical importance and must be addressed to determine how closely this model system mimics the human condition such that it can be used effectively for future preclinical studies. Therefore, the purpose of this work was 4-fold. First, we sought to establish a dosage and treatment schedule that resulted in the development of singular or occasionally synchronous HNSCC rather than innumerable lesions throughout the oral cavity over a protracted period of time. Second, we established a predictable timeline for the histologic progression of mucosal lesions in mice treated with 4-NQO. Third, we sought to clarify when the expression of the angiogenic phenotype can be first observed in this model. Finally, ABT510, a mimetic peptide of thrombospondin-1 (Fig. 1; refs. 23, 24), was administered to 4-NQOtreated mice as a proof of principle to test the hypothesis that inhibitors of angiogenesis can be successfully used as chemopreventive agents for HNSCC. Materials and Methods Administration of 4-NQO Two hundred thirty male CBA mice, 6 to 8 wk of age, were purchased from The Jackson Laboratory and housed in the Animal Resource Facility under controlled conditions and fed normal diet and autoclaved water. All animal procedures were carried out in accordance with Institutional Animal Care and Use Committee approved protocols. Mice were administered 4-NQO in their drinking water on a continuous basis at the required dose for the required duration. 4-NQO powder (Sigma) was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at 20C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol (Sigma) and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of either 50 or 100 g/mL. A fresh batch of water was prepared every week for each of the 8 or 16 wk of carcinogenic treatment. Normal autoclaved drinking water was resumed at the end of this period. Control mice not receiving 4-NQO were given water containing vehicle only. Treatment with ABT-510 ABT-510, a synthetic peptide that mimics the antiangiogenic activity of the naturally occurring protein thrombospondin-1, was provided by Abbott Laboratories. The peptide was dissolved in sterile Fig. 1. Chemical structure of ABT-510. ABT-510 is a nonapeptide derived from the antiangiogenic fragment of the second type 1 repeat of thrombospondin-1. The key structures in the synthesis and the chemical structure from which this figure was derived can be found in the original publication by Haviv et al. (23). Cancer Prev Res 2009;2(4) April 2009 386 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 2. Histopathology of 4-NQOinduced oral lesions in the mouse tongue. Photomicrographs show the histopathologic progression in this model system: histologically normal (control; A), hyperkeratosis (B), epithelial dysplasia (C), and squamous cell carcinoma (D). 5% dextrose, immediately filter sterilized, and stored at 4C. Mice receiving ABT-510 treatment were given a daily i.p. injection of 50 mg/kg body weight for the required duration of 4, 8, 12, 16, 20, or 24 wk. The route of administration and dosage given was determined based on previously published studies (2429). polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+, DAKO). CD31 antigen retrieval was done using the Dako Target Retrieval System (pH 9.0) in a decloaking chamber. The primary antibody PECAM (Santa Cruz Biotechnology) was applied at 1:200 dilution in PBS for 1 h at room temperature. Antibody binding was visualized using the LSAB kit (DAKO). For determination of cell proliferation, sections were treated in ET buffer using decloaking chamber, incubating at 1:300 dilution using a Ki-67 antibody (NeoMarkers) for 1 h at room temperature. This was followed by anti-rabbit polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+). All three immunohistochemistry stains were developed with 3,3-diaminobenzidine chromogen and counterstained with hematoxylin. Corresponding negative control experiments were done by omitting the incubation step with the primary antibody. Histologic examination Mice were sacrificed in accordance with Institutional Animal Care and Use Committee recommendations. Specifically, cervical dislocation was done after anesthesia by i.p. injection of xylazine and ketamine. Immediately following death, the tongues were excised, longitudinally bisected, and processed in 10% buffered formalin and embedded in paraffin. Fifty 5-m sections from each specimen were then cut and the 1st, 10th, 20th, 30th, 40th, and 50th slides were stained with H&E for histopathologic analysis. Histologic diagnoses were rendered using established criteria. Hyperkeratoses were characterized by a thickened keratinized layer, with or without a thickened spinous layer (acanthosis), and an absence of nuclear or cellular atypia. Dysplasias were characterized as lesions that showed various histopathologic alterations, including enlarged nuclei and cells, large and/or prominent nucleoli, increased nuclear to cytoplasmic ratio, hyperchromatic nuclei, dyskeratosis, increased and/or abnormal mitotic figures, bulbous or teardrop-shaped rete ridges, loss of polarity, and loss of typical epithelial cell cohesiveness. Because of the subjective nature of grading of epithelial dysplasia and its limited ability to predict biological progression (30, 31), we chose to not assign descriptive adjectives of severity to the dysplastic lesions. Rather, we grouped all lesions showing cytologic atypia but lacking evidence of invasion into the single category of dysplasia. HNSCCs were characterized by lesions that showed frank invasion into the underlying connective tissue stroma. Scoring of immunohistochemistry A combined scoring method that accounts for intensity of staining as well as percentage of cells stained was used for the evaluation of VEGF as previously described (32). Strong, moderate, weak, and negative staining intensities were scored as 3, 2, 1, and 0, respectively. For each of the intensity scores, the percentage of cells that stained at that level was estimated visually. The resulting combined score consisted of the sum of the percentage of stained cells multiplied by the intensity scores. For example, a case with 10% weak staining, 10% moderate staining, and 80% strong staining would be assigned a score of 270 (10 1 + 10 2 + 80 3 = 270) out of a possible score of 300. The determination of microvessel density (MVD) using CD31 as a marker was done as previously described (33). Briefly, using low-power magnification, the region containing the most intense area of tumor neovascularization was chosen for counting in each of the tumors. For the normal control tissue, MVD was determined by finding the most intense area of neovascularization directly below the overlying mucosa. Individual microvessels were counted using a 100 field (10 objective lens and 10 ocular lens). Any brown staining endothelial cells that were clearly separate in appearance were counted as individual vessels. Ten random fields within this hotspot area were viewed and counted at 100. Results were expressed as the total number of microvessels observed in the hotspot region of each individual tumor. For Ki-67, the labeling indices were determined by randomly analyzing at least 500 nuclei in 10 high-powered fields (400 magnification) for Immunohistochemistry Antigen retrieval was achieved on deparaffinized sections and endogenous peroxidase activity was quenched in 3% hydrogen peroxide and blocked in milk peroxidase. For vascular endothelial growth factor (VEGF) detection, slides were treated in ET buffer in a decloaking chamber and mouse primary antibody (Santa Cruz Biotechnology) was applied at 1:50 dilution in PBS for 1 h at room temperature. Antibody binding was visualized with anti-mouse www.aacrjournals.org 387 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research each tissue section. Labeling indices were expressed as a percentage of the total number of cells. quent deaths were observed in study A. At week 4 (n = 4), 75% of the tongues showed hyperkeratotic lesions, whereas 25% contained dysplasia. In the week 8 mice (n = 4), 25% had hyperkeratosis, 25% had dysplasia, and 50% had carcinoma. At 12 weeks (n = 4), 75% contained dysplasia and 25% had squamous cell carcinoma. Finally, by 24 weeks (n = 4), 25% showed dysplasia and 75% contained squamous cell carcinoma. Pilot study B (50 g/mL for 16 weeks) was terminated early because of an excessive number of deaths. Of the initial mice, 40% (8 of 20) died either during carcinogen treatment or within 6 weeks after the completion of the carcinogen treatment. In pilot studies C and D, mice were treated with 100 g/mL 4-NQO for either 8 weeks (pilot study C) or 16 weeks (pilot study D). In pilot study C (100 g/mL for 8 weeks), there were no deaths during the study. At week 4 (n = 5), 100% of animals showed hyperkeratotic lesions (Fig. 2B). At week 8 (n = 5), 60% showed hyperkeratosis and 40% contained epithelial dysplasia (Fig. 2C). By 12 weeks (n = 5), 100% of animals had dysplasia. Finally, at week 24 (n = 5), 40% had dysplasia and 60% showed squamous cell carcinoma (Fig. 2D). Similar to pilot study B, unacceptable mortality rates were observed in pilot study D after animals were treated with 100 g/mL for 16 weeks, resulting in the premature termination of this arm of the study as well. Although this was a relatively small sample size, these results showed that a sequential histologic progression could be observed using this type of carcinogenic induction protocol. It further suggested that the majority of the dysplastic and cancerous lesion were likely to be found starting at 12 weeks after carcinogen treatment. Therefore, to expand on the pilot studies, a cohort of 55 additional mice was treated with 100 g/mL 4-NQO for 8 weeks and tongues were subsequently harvested at weeks 16, 20, and 24. At week 16 (n = 20), 15% contained hyperkeratosis, 55% had dysplasia, and 30% showed squamous cell carcinoma. At 20 weeks (n = 15), 13% had hyperkeratosis, 60% contained dysplasia, and 23% showed squamous cell carcinoma. By 24 weeks after carcinogen treatment (n = 20), 25% of specimens contained dysplasia and 75% of the tissues showed squamous cell carcinoma. Collectively, the data for the 100 g/mL 4-NQO administered for 8 weeks show a reproducible timeline of histologic progression. Specifically, the data show that hyperkeratotic Data analysis For comparison of immunohistochemical scores across groups, ANOVA was done. A transformation of the data (square root or natural log) was used, as needed, to stabilize the variance across groups. If a significant overall difference was found by ANOVA, then pairwise comparisons were done with a Bonferroni adjustment for multiple comparisons. A test for trend was also done using ANOVA with the appropriate linear contrasts, and these results were confirmed using a nonparametric trend test as described by Cuzick (34). For comparison of ABT-510 treatment groups, Fisher's exact test was done by collapsing data across the six sacrifice times. All analyses were done using Stata version 10 (StataCorp). Results Histopathologic progression of 4-NQOtreated mice Articles describing the mouse 4-NQO model have typically reported the incidence of dysplasia and/or cancer at the end of the prescribed treatment protocols. However, these articles have not systematically characterized the sequential timing of histologic atypia development after carcinogen treatment over a protracted period of time. This is an important aspect of the model because a more thorough understanding of the histologic and molecular progression in this system is required if it is to be used to model oral premalignancy as well as preclinical chemoprevention studies. To carefully characterize the development of the histopathologic changes in this model, a series of pilot studies were done to identify the optimal carcinogen dosage and duration of exposure required to develop a predictable time line of progression. The animals were sacrificed at planned intervals after completion of carcinogen treatment, and their tongues were excised and examined histologically. No histopathologic changes were noted in the tongue mucosa from the control mice (Fig. 2A). Mice were treated with 50 g/mL 4-NQO for 8 weeks (pilot study A) and 16 weeks (pilot study B). They were subsequently randomly assigned to groups and sacrificed at 4, 8, 12, and 24 weeks after 4-NQO treatment. From the initial 20 animals in pilot study A (50 g/mL for 8 weeks), 4 mice died during treatment from undetermined causes. However, no subse- Fig. 3. Incidence of each histologic diagnosis of control and ABT-510 treatment groups at each sacrifice time. The number located at the top of each bar indicates the total sample size of each group. ABT-510 significantly decreased the incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 388 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 4. Immunohistochemical staining of Ki-67 in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for Ki-67 and labeling indices were quantified (B and C). Ki-67 labeling indices in the specimens containing hyperkeratosis, dysplasia, and HNSCC were all significantly greater when compared with the normal mucosa (P < 0.001). to the mouse tongue epithelium from untreated control mice (n = 5) contained only occasional capillaries that were relatively evenly dispersed throughout the connective tissue stroma (Fig. 5A). However, the number and distribution of microvessels at the mucosal/connective tissue interface increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 5A). With increasing histologic atypia, the vessels were more densely packed directly adjacent to the basal cell layer. Quantification of MVD during histologic progression revealed a statistically significant difference (P < 0.001 by ANOVA) in CD31 scores when normal epithelium (mean SD: 23.2 4.3) was compared with hyperplasia (90.2 6.7), dysplasia (191.6 10.6), and squamous cell carcinoma (309.3 17.9). Pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Furthermore, there was a significant linear trend in CD31 levels across the four naturally ordered groups (i.e., increasing CD31 levels with increasing disease severity; P for trend < 0.001; Fig. 5B and C). lesions predominate at weeks 4 and 8, dysplasias are the most common diagnosis at weeks 12, 16, and 20, and carcinoma is the predominant histologic finding at week 24 (Fig. 3). Ki-67 Expression during histologic progression in 4NQOtreated mice Expression of Ki-67, a nuclear proliferation-associated antigen that is specific for cells in the active phases of the cell cycle, was determined via immunohistochemistry to quantify the relative proliferative rates of normal, hyperkeratotic, dysplastic, and malignant mouse tongue mucosa. Cells positive for Ki-67 expression showed distinct nuclear staining. In normal mucosa, Ki-67 expression was limited to basilar and occasional parabasilar cells (Fig. 4A), whereas greater suprabasilar labeling was observed with increasing histologic atypia (Fig. 4A). There was a statistically significant difference (P < 0.001 by ANOVA) in labeling indices when normal epithelium (mean SD: 16.4 3.0) was compared with hyperplasia (23.5 2.6), dysplasia (32.3 3.5), and squamous cell carcinoma (47.8 3.6). Subsequent pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Additionally, there was evidence for an increasing linear trend in Ki-67 levels across the four ordered groups (P for trend < 0.001; Fig. 4B and C). Expression of VEGF during the histologic progression in 4-NQOtreated mice Like all solid tumors, HNSCC must develop direct and indirect mechanisms to induce the production of new blood vessels. Several dozen candidate angiogenic molecules are produced by oral keratinocytes, and VEGF is an important angiogenic factor in both physiologic and pathologic settings, including HNSCC (35). Therefore, we did immunohistochemistry for VEGF to quantify its expression at various stages of histologic progression in the mouse 4-NQO model. Expression of VEGF by tongue keratinocytes from untreated control mice (n = 5) was rare and limited to the basilar and parabasilar cells (Fig. 6A). In addition, occasional stromal cells as well as endothelial lined vascular channels stained positively. The intensity of VEGF expression as well as the overall expression Increased MVD occurs before histologic atypia in 4NQOtreated mice The induction of blood vessel growth is an early phenotypic change in both human HNSCC as well as in the hamster buccal pouch and rat tongue models of oral carcinogenesis (811). To determine the timing of the expression of the angiogenic phenotype in the 4-NQO mouse model, we did immunohistochemistry for CD31 to quantify MVD as a surrogate for in vivo angiogenesis activity. The connective tissue stroma adjacent www.aacrjournals.org 389 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research of the cytokine by various keratinocytes present in the different layers of the epithelium increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 6A). Quantification of VEGF expression among histologic stages revealed a statistically significant difference (P < 0.001 by ANOVA) when normal epithelium (mean SD: 17.0 5.7) was compared with hyperplasia (70.6 14.0), dysplasia (144.9 35.4), and squamous cell carcinoma (237.6 41.4). Each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Most importantly, there also was evidence for an increasing linear trend in VEGF levels with disease progression (P for trend < 0.001; Fig. 6B and C). normal, 40% contained hyperkeratosis, and 10% had dysplasia. At week 8 (n = 10), 10% were normal, 70% contained hyperkeratosis, and 20% showed dysplasia. By week 12 (n = 10), 10% were normal, 30% contained hyperkeratosis, and 60% had dysplasia. At week 16 (n = 12), 58% had hyperkeratosis, 25% had dysplasia, and 17% showed carcinoma. Progressing to week 20 (n = 16), 25% had hyperkeratosis, 25% had dysplasia, and 50% showed carcinoma. Finally, at week 24 (n = 21), 33% had hyperkeratosis, 38% had dysplasia, and 29% showed carcinoma. With respect to overall incidence of cancer, a 46% reduction in HNSCC was observed, with the 4-NQO group having an incidence rate of 37.3% and the ABT-510 treatment group having an incidence of 20.3% (P = 0.021). In addition, the combined incidence of dysplasia and HNSCC was 82.7% in the 4-NQO control group and 50.6% in the ABT-510 treatment group. This difference was highly statistically significant (P < 0.001). Effects of ABT-510 administration in 4-NQOtreated mice The expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Therefore, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can be used as chemopreventive agents in the realm of HNSCC. Based on our findings described above, mice were administered 4-NQO (100 g/mL) in their drinking water for 8 weeks. At the completion of this initiation phase, the mice were returned to normal water and given daily i.p. injections of ABT-510 (50 mg/kg/d) and sacrificed at regular intervals over the next 24 weeks. During the 24-week chemopreventive period, no significant differences in food or fluid consumption among the groups were observed. Similarly, there was no difference in body weight or activity between the control and the treatment group mice (data not shown). Data for the incidence of tongue lesions are shown in Fig. 3. At week 4 (n = 10), 50% of the tongues were histologically Discussion Animal models of HNSCC have traditionally used either 7,12-dimethylbenz(a)anthracene or 4-NQO as the carcinogenic agent. The induction of HNSCC using 4-NQO has been achieved in several different rodent species, including mice, hamsters, and rats, and has been generally found to be preferable for several reasons. First, in contrast to the topical application of 7,12-dimethylbenz(a)anthracene, 4-NQO can be delivered via the drinking water, thereby making the outcomes more predictable. Second, the molecular alterations induced in mouse mucosa by 4-NQO closely mimic the human disease. For example, similar to the human condition, epidermal growth factor receptors are overexpressed in this model (36). Similar to humans, altered expression of p53 as well as mutation of p53 have been shown in 4-NQO models (37). In addition, 4-NQO induces activating point mutations in the H-ras oncogenes (38). Although H-ras mutations are infrequent events in U.S. HNSCC (39), they are common in the Fig. 5. Expression of the angiogenic phenotype in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for CD31 and MVD was quantified (B and C). MVD in the specimens containing hyperkeratosis, dysplasia, and HNSCC was all significantly greater when compared with the normal mucosa (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 390 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 6. Expression of VEGF in the 4-NQO mouse model of HNSCC. A, tissue sections containing representative areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for VEGF and expression levels were quantified (B and C). Expression of VEGF in the specimens containing hyperkeratosis, dysplasia, and HNSCC was significantly greater when compared with normal mucosa (P < 0.001). opment of singular or occasionally synchronous oral lesions. This aspect was critical because we wanted to refine the model to ensure that the mice developed lesions in a fashion that was most similar to the human condition rather than forming innumerable ones. In addition, the prescribed dosage and timing of treatment resulted in a predictable histologic progression over the 24-week period of time. Specifically, the data show that after the completion of carcinogen treatment, the predominant histopathologic diagnoses are hyperkeratosis at weeks 4 and 8, dysplasia at weeks 12, 14, and 16, and HNSCC at week 24 (Fig. 3). Importantly, the establishment of the progression timeline in this model will allow for future studies aimed at investigating the molecular and biological alterations that occur during the premalignant phase of HNSCC as well as for the validation of potential diagnostic biomarkers. The induction of new blood vessel growth is a critical tumor phenotype in all malignancies, including HNSCC. There is considerable interest in determining how cells, progressing from normal to tumorigenic, make this switch. In some animal models, a distinct switch to the angiogenic phenotype is seen (44). In other cases, the cells developing into tumors sequentially become more angiogenic in a stepwise fashion (8, 45). Although the phenotype has been studied using animal models as well as human cells and tissue, the mechanisms about how this occurs in HNSCC are unknown. However, although the phenotypic changes in these models are similar to the human condition, the specific mechanisms involved in the induction of new blood vessel growth can be quite different. For example, although the major angiogenic factor in the hamster buccal pouch model is transforming growth factor (8), this growth factor has not been found to be a significant participant in the induction of angiogenesis in human HNSCC. Rather, a different subset of growth factors, including interleukin-8 and VEGF, seems to play predominant roles (35). As there were no data about the angiogenic phenotype in the 4NQO mouse model, we sought to determine the timing of the rest of the world (40). Therefore, because the majority of the 400,000 cases of HNSCC are outside of the United States/Europe and may therefore harbor H-ras mutations (in conjunction with epidermal growth factor receptor and p53 alterations), we believe that this is an excellent model from an experimental, histologic, and molecular perspective. Finally, 4-NQOinduced lesions develop in the absence of nonspecific inflammatory changes. This is a critical point because substances such as 7,12-dimethylbenz(a)anthracene can be significant irritants, resulting in chronic inflammation, necrosis, sloughing of tissue, and the formation of organizing granulation tissue (41). The etiology, type of inflammatory cell infiltrate, and therefore perhaps mechanisms in this type of injurious situation are likely to be different from what one sees in the human condition. Furthermore, these factors make it difficult to study premalignant lesions because inflammation itself can cause cytologic and/or morphologic changes that can be confused with dysplasia. Because 4-NQO treatment does not result in this type of injurious nonspecific inflammation, it is more likely to reflect the events that occur during human HNSCC. Although 4-NQO has been used as a carcinogenic agent to induce tumors in animal models since the 1950s, and in the oral cavity since the 1970s (42, 43), a thorough review of the literature failed to reveal studies that were specifically designed to characterize the histopathologic changes that develop over time in the 4-NQO mouse model. Furthermore, the route of administration, the amount of carcinogen used, and the duration of treatment schedules have been highly variable. Therefore, we sought to characterize the optimal dosage and timing of 4-NQO treatment and to establish a timeline for the reproducible development of lesions showing hyperkeratosis, dysplasia, and HNSCC. Using two different doses and two different durations of treatment, we determined that 100 g/mL in the drinking water for 8 weeks provided the most preferable results. This decision was based on the fact that this treatment protocol resulted in the devel- www.aacrjournals.org 391 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research cumulative decrease of 39% in the incidence of both dysplasia and HNSCC between the control and ABT-510 treatment groups (82.7% versus 50.6%; P < 0.001), the rates being higher in the control groups at each time point except for week 4 where there was only one case of dysplasia. Overall, these cumulative data strongly support the contention that ABT-510 was effective at decreasing the incidence of dysplasia and HNSCC in the mouse 4-NQO model of oral carcinogenesis. The integration of inhibitors of angiogenesis into the treatment regimens of various diseases is increasing in frequency (47, 48). However, although there has been considerable discussion about the potential role of antiangiogenic agents in the area of chemoprevention, there are limited data to support the role of this class of drugs in this clinical setting (49, 50). ABT-510 is a mimetic peptide of thrombospondin-1 and acts as an inhibitor of angiogenesis by modulating the ability of endothelial cells to respond to various angiogenic factors. Specifically, ABT-510 binds to CD36, thereby inducing caspase-8 mediated endothelial cell apoptosis. This mechanism has been shown to block angiogenesis in vitro and in vivo as well as slow tumor growth in preclinical studies (2429). Further, it has been tested clinically for the treatment of inflammatory bowel disease and in cancer therapy as a single agent or in combination with chemotherapeutic agents (5155). Because it directly abrogates the ability of endothelial cells to respond to angiogenic factors, rather than altering the expression of these factors by tumor and/or stromal cells, one does not typically observe the direct modulation of various angiogenic factors in tumor cells. In keeping with these previous observations, we did not observe an alteration in the expression of VEGF by the oral keratinocytes in the treatment group animals (data not shown). However, the nearly 2-fold decrease in the incidence of HNSCC in the ABT-510 group at 24 weeks shows that the drug has a potent effect in vivo in this animal model. One of the long-term goals of chemoprevention must be the development of treatments that can be easily taken by at-risk individuals for prolonged periods of time with minimal side affects to achieve widespread acceptance and long-term compliance. This would be particularly important in the case of high-risk patients who have not yet developed their first malignancy. As such, because ABT-510 is not an orally administered agent, it is unlikely that it would be found acceptable in its current form. Nonetheless, our proof-of-principle findings support the hypothesis that inhibitors of angiogenesis may have activity as chemopreventive agents. Furthermore, many of the chemopreventive agents currently under investigation, such as epidermal growth factor receptor tyrosine kinase inhibitors and cyclooxygenase-2 inhibitors, have multiple activities, including the inhibition of angiogenesis. However, the toxicities observed at the current prescribed dosages may preclude them from being used widely as chemopreventive agents. Therefore, the combination of one or both of these agents at lower doses in concert with other antiangiogenic agents may reduce toxicities and improve efficacy. The data presented here show proof of principle that the induction of new blood vessel growth by premalignant cells may be one such phenotype that could be targeted in a chemoprevention cocktail. appearance of this phenotype as well as identify the angiogenic factors that might be involved. The determination of MVD in tissue sections, using endothelial cell markers such as factor VIII, CD31, and/or CD34, is an accepted surrogate marker for in vivo angiogenesis. In the 4-NQOtreated animals, we observed a statistically significant increase in MVD as early as the hyperkeratotic stage, thereby showing that, much like other models as well as the human condition, the expression of the angiogenic phenotype is an early phenotypic change (Fig. 6). Furthermore, we observed sequentially increasing levels of VEGF expression at the stages of hyperkeratosis, dysplasia, and HNSCC (Fig. 6B and C). The mechanisms for this sequential increase are not known at this time. However, one could hypothesize that the progressive increase in VEGF expression might in part be the result of potential tumor-stroma paracrine interactions (46). The design of the study did not allow us to functionally validate whether VEGF was the key angiogenic factor produced in this animal model. However, the concordant increased expression of both MVD and VEGF supports the hypothesis that VEGF plays a central role in the expression of the angiogenic phenotype in the mouse 4-NQO model. We have shown that daily treatment with ABT-510 for 24 weeks resulted in limited toxicity and significantly decreased the incidence of dysplasias and carcinomas in the mouse 4-NQO model of HNSCC (Fig. 3). The rationale for the 24-week treatment schedule was based on the observed cancer incidence and histologic diagnosis in our preliminary studies. As a result of these findings, animals were subsequently sacrificed at the same regular intervals over the 24-week time course to synchronize the treatment arm results with the initial histologic observations. Specifically, we observed most HNSCC (72%) at week 24, whereas dysplasia was the predominant histologic diagnosis at weeks 16 (55%) and 20 (60%). Because we were testing the hypothesis that ABT-510 would reduce the incidence of HNSCC, we believe it was most appropriate to carry out the prevention study to a time point where the predominant histologic diagnosis in the control group would be expected to be HNSCC. Overall, we observed a 46% cumulative reduction in the incidence of HNSCC between the control and treatment groups (37.3% versus 20.3%; P = 0.021). We did observe a small increase of HNSCC in the treatment group at week 20 when compared with the 20-week control group. However, this difference was not statistically significant (P = 0.273). The reasons for this observation are unknown but may be the result of uneven cohort sizes between the control and treatment groups within and at different sacrifice points. For example, the week 20 treatment group contained fewer animals (n = 16) than some of the other time points, such as week 24 (n = 21). The differences in cohort sizes at the given time points were a reflection of the fact that our initial experiments determined that week 24 should be the major cancer end point of this study. As such, although we were interested in synchronizing the treatment arm results with the histologic studies, we also felt it was most important to have the largest n at the major end point of the study. Further, it should be noted that the 16- and 24-week time points showed significant differences in the incidence of HNSCC between the control and treatment groups. 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... Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Cancer Research Tumor and Stem Cell Biology Genetic Identication of SEMA3F as an Antilymphangiogenic Metastasis Suppressor Gene in Head and Neck Squamous Carcinoma Colleen L. Doci1, Constantinos M. Mikelis1, Michail S. Lionakis2, Alfredo A. Molinolo1, and J. Silvio Gutkind1 Abstract Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCCs are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its coreceptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LEC) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis. Cancer Res; 75(14); 293748.
2015 AACR. Introduction demonstrate intratumoral lymphangiogenesis that is associated with invasion, metastasis, and decreased survival (68). However, the relative contribution of angiogenesis and lymphangiogenesis to cancer progression and metastasis is still not fully understood. HNSCC is one of the ten most common cancers globally and less than half of patients diagnosed with nonlocalized disease will survive beyond 5 years (9, 10). This is partially attributed to the propensity of HNSCC to metastasize to locoregional lymph nodes, which is the single most signicant prognostic indicator and decreases the overall survival rate by more than 50% (1113). Furthermore, approximately 40% of the lymph nodes in the human body are located within the head and neck region (14), and HNSCC demonstrates signicant intratumoral lymphangiogenesis compared with other solid cancers, suggesting that lymphangiogenesis may play a pivotal role in HNSCC metastasis and survival. Therefore, HNSCC may represent a clinically relevant condition to begin to dissect the specic role of lymphangiogenesis in metastasis. The emergence of deep-sequencing approaches for human disease has led to the identication of a multitude of aberrant molecules in cancer that may contribute to its pathogenesis. While conducting analyses on altered molecules in the HNSCC genome, we observed that SEMA3F is among the top 1% underexpressed genes (15). SEMA3F is a member of the class 3 semaphorin family originally characterized in axonal guidance (16). In addition, semaphorins have been shown to play multiple roles in normal and pathologic angiogenesis by acting on their receptors, plexins and neuropilins (1720). Interestingly, SEMA3F can bind to One of the dening hallmarks of cancer is ability to form new vasculature to facilitate the growth and metastatic spread of cancer cells (1). Metastasis is the leading cause of morbidity in patients with a variety of solid tumors, where cancer cells often disseminate through blood and lymphatic vessels (1). Thus, the presence of intra- and peritumoral vasculature is a critical diagnostic and prognostic biomarker (2). Angiogenesis is required for tumors to grow beyond a critical limit and tumor-associated blood vessels have been suggested to participate in metastasis (3). Signicantly less is known about the role of lymphangiogenesis in cancer, although lymphatic invasion is one of the most relevant diagnostic parameters for solid tumors (2, 4, 5). Specically, melanoma and head and neck squamous cell carcinomas (HNSCC) 1 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland. 2Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: J. Silvio Gutkind, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, Bldg 30, Rm 320, Bethesda, MD 20892. Phone: 301-496-6259; Fax: 301-4020823; E-mail: sg39v@nih.gov doi: 10.1158/0008-5472.CAN-14-3121
2015 American Association for Cancer Research. www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2937 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. neuropilin 2 (NRP2), and early studies indicated that SEMA3F expression prevents the growth of metastatic melanoma cells that express high levels of NRP2 (21). However, the relevance of SEMA3F expression in cancers lacking NRP2 has not been investigated. Furthermore, NRP2 is a coreceptor highly expressed on lymphatic endothelial cells (LEC). Therefore, these observations prompted us to explore whether SEMA3F loss may contribute to HNSCC lymphangiogenesis, and hence impact on cancer progression and metastasis. Materials and Methods The following represent a brief summary of the procedures. Please see the Supplementary Material for additional detailed methods. Cell culture 293T-17, HaCat, COS-7, UMSCC2, and UMSCC17B cells were cultured in DMEM 10% FBS. LECs and HMVECs were cultured in EGM2-MV and human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 (Lonza). All cells were cultured at 37 C in 5% CO2. UMSCC2 and UMSCC17B stable cell lines were achieved by selection with 1 mg/mL blasticidin. Transfection of plasmids and siRNAs can be found in the Supplementary Methods. All cell lines underwent DNA authentication (Genetica DNA Laboratories, Inc.) before the described experiments to ensure consistency in cell identity. The Cancer Genome Atlas analysis Data regarding the copy number of PIK3CA and SEMA3F in head and neck cancer was downloaded from the cBio Portal for Cancer Genomics (http://www.cbioportal.org/public-portal/ accessed February 5, 2014). Immunohistochemistry Tissue arrays containing normal and oral cancer tissues were purchased from US BioMax Inc. Histopathology of tongue sections was performed as previously described (22). Formalin-xed parafn-embedded (FFPE) slides were stained and for tissue arrays were classied on the basis of the intensity and the percentage of positive cells quantied as described (23). Correlations were determined using Pearson coefcient. SEMA3F purication Serum-free conditioned medium (CM) from 293T-17 cells expressing NTAP-SEMA3F construct was collected, dialyzed, then isolated using HisTALON cobalt beads (Clontech). DFLAG control was generated by incubating puried SEMA3F with anti-FLAG conjugated beads (Sigma-Aldrich) and collecting the unbound supernatant. Immunoblotting Cells were lysed in radioimmunoprecipitation assay buffer and concentration was determined using Bio-Rad DC protein assay. Twenty micrograms total protein was separated by SDS-PAGE and transferred to polyvinylidene diuoride membrane overnight at 4 C. Membranes were blocked for 1 hour at room temperature in 5% milk in TBST and then probed with primary antibodies overnight at 4 C. Membranes were washed four times in Trisbuffered saline 1% Tween-20, probed with horseradish peroxidaseconjugated secondary antibodies for 1 hour at room tem- 2938 Cancer Res; 75(14) July 15, 2015 perature in 5% milk, washed four times in TBST, and detected using chemiluminescent substrate (Millipore). Immunouorescence For LECs and normal oral keratinocytes (NOK), cells were xed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. LECs were stained with phalloidin-GFP (Invitrogen) and counterstained with Hoescht 33342 (Invitrogen). NOKs were stained with SEMA3F (Sigma-Aldrich) or 58K Golgi Protein (Abcam), imaged with anti-rabbit AlexaFluor 488 (Invitrogen) or anti-goat AlexaFluor 546 (Invitrogen), and counterstained with Hoescht 33342 (Invitrogen). For Matrigel and orthotopic tumor sections, FFPE slides were prepared and stained using the immunohistochemistry protocol described, and then counterstained Hoescht 33342 (Invitrogen). The images were taken using an Axio Imager Z1 microscope equipped with an ApoTome system. Cell adhesion and collapse assays For adhesion assays, LEC were treated and plated on collagencoated plates. Nonadherent cells were removed by washing and adherent cells xed and stained. For collapse, LECs were transfected with LifeAct GFP and treated, or treated, xed, and stained with uorescent phalloidin and nuclear counterstain. Cell area and perimeter were assessed using ImageJ. For heterologous assays, transfected COS-7 cells were treated as indicated. For all assays, quantication was performed using ImageJ from three independent experiments. Statistical signicance was determined using one-way ANOVA. For movies, cells were imaged on an Olympus IX-81 inverted confocal microscope with images obtained every 30 seconds for a total of 3 hours, and analyzed using ZEN software (Carl Zeiss). In vivo lymphangiogenesis assay and FACS Basement membrane extract (Trevigen) plugs with growth factors and inhibitors, as indicated, were injected subcutaneously into the ank of nude mice. Single-cell suspensions from plugs were prepared as described (24) and cell populations determined by FACS. Statistics were determined using ANOVA from three independent experiments. Cells were resuspended in PBS, stained with a LIVE/DEAD uorescent dye (L-23105; Invitrogen), and incubated with CD16/32 (2.4G2; BD Biosciences) to block Fc receptors. For staining, cells were incubated with Alexa Fluor 488-conjugated LYVE-1, PE-Cy7-conjugated CD31, allophycocyanin (APC)-conjugated TER-119, APC-Cy7conjugated CD45 and eFluor 450-conjugated CD102 (eBioscience or BD Biosciences) washed, and analyzed on a 5-laser LSRFortessa (BD Biosciences). Data were analyzed using FACS Diva (BD Biosciences) and FlowJo software (Treestar). Quantication of cell types was performed using PE-conjugated uorescent counting beads (Spherotech). Migration assay Cells were treated as indicated for overnight Boyden migration. Membranes were xed with methanol, counterstained with hematoxylin, and imaged on an Axiovert microscope. Calculations were based on 18 imaging elds each from three independent experiments. Statistical signicance was determined using ANOVA. Proliferation assay Cancer cell lines were plated to 60% conuence and transferred to serum-free media with doxycycline for 20 hours. Cells were Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor incubated an additional 4 hours with 1 mCi [methyl-3H]-thymidine (PerkinElmer). Proliferation was determined by liquid scintillation counting. Statistical signicance was determined using ANOVA. Orthotopic tumor xenografts in SCID/NOD mice All animal studies were carried out according to NIHapproved protocols (ASP# 10-569), in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Female SCID/NOD mice (NCI) were housed in appropriate sterile lter-capped cages, and fed and watered ad libitum. Each animal was injected with 105 cells in 50 mL of serum-free media in the tongue. Animals in the control group were fed regular chow, while animals in the prevention group were fed 6 g doxycycline/kg chow 1 week before injection and throughout the study. All animals underwent evaluation of the tongue for disease onset every 3 days, and the observed lesions were assessed for length and width, and tumor volume was determined as described previously (22). Animals were euthanized at the indicated time points and the cervical lymph nodes assessed for evidence of metastases by H&E staining. Results SEMA3F expression is lost during HNSCC progression While analyzing gene expression alterations in HNSCC in available datasets, we observed that SEMA3F is among the 1% most downregulated genes [2.09-fold decrease, P 3.6E20; ref. 15] and is localized to 3p21, one of the most commonly deleted loci in HNSCC (25). To investigate whether reduced SEMA3F expression is caused by genomic alterations, we interrogated the The Cancer Genome Atlas (TCGA) Head and Neck Cancer databases, using PIK3CA, one the most frequently amplied genes in HNSCC that is localized to the long arm of the same chromosome (26) as a control (Fig. 1A). Nearly 75% of HNSCC patients showed heterozygous loss of SEMA3F, suggesting that this semaphorin may represent a potential tumor suppressor (Fig. 1A). We assessed the KaplanMeier univariate survival of patients with heterozygous loss of SEMA3F (Fig. 1B) and the Cox proportional hazard multivariate survival, taking into consideration lymph node metastasis (Fig. 1C). In both analyses, SEMA3F was a strong negative indicator of survival and a statistically signicant independent biomarker [HR 2.1, P 0.01 and exp(b) 1.95, P 0.03, respectively]. The median survival of patients with heterozygous loss was nearly half of patients expressing normal levels of SEMA3F (Fig. 1D). In addition, patients with SEMA3F heterozygous loss showed signicantly increased percentage of metastatic lymph nodes and lymphovascular invasion (Fig. 1E and F). SEMA3F loss was reected at the protein level, as immunohistochemical evaluation of SEMA3F using a SEMA3F-specic antibody (Supplementary Fig. S1) revealed progressive loss of expression with advancing cancer severity. While normal oral epithelium demonstrated high level of SEMA3F expression, this was almost completely abolished in advanced cancers (Fig. 1G). Quantication of a large collection of HNSCC tissues revealed that nearly 70% of the proliferating basal cells in normal oral epithelium expressed high levels of SEMA3F, while over half of HNSCC samples express little to no SEMA3F (Fig. 1H). Interestingly, this correlated with enhanced tumor vascularity, with markers for blood and lymphatic endothelium www.aacrjournals.org signicantly increased in the absence of SEMA3F (Fig. 1I and Supplementary Fig. S2). These data support that decreased expression of SEMA3F correlates with poor clinical outcome, increased tumor vascularity, invasion, and metastasis, thus suggesting that SEMA3F may function as a tumor and metastasis suppressor in HNSCC. SEMA3F is a chemorepulsant for LECs As the SEMA3F coreceptor NRP2 is highly expressed on LECs (27), we asked whether SEMA3F has a functional impact on these endothelial cells. We engineered a SEMA3F construct that would preserve its extracellular secretion and posttranslational processing while allowing for coexpression of N-terminal tandem afnity purication (NTAP) tags (Fig. 2A). SEMA3F was expressed, fully processed, and the secreted form of the protein was capable of purication using histidine afnity resin (Fig. 2A). Class 3 semaphorins function through a mechanism that involves negative regulation of integrin adhesion to the extracellular matrix, thereby modulating the attachment of endothelial cells to extracellular matrices through cytoskeletal remodeling (28). Secreted SEMA3F was functionally active in HUVEC adhesion assays, and this effect is specic as FLAG-depleted SEMA3F CM but not IgGdepeleted CM was no longer able to inhibit attachment (Fig. 2B). Furthermore, SEMA3F caused a dose-dependent decrease of LEC attachment, which was abolished by FLAG-mediated depletion (Fig. 2C). SEMA3F also induced a nearly 80% collapse of the LEC actin cytoskeleton, while decreasing cell perimeter by less than 3% (Fig. 2D). Using either puried SEMA3F or FLAG-depleted control, we documented the cellular collapse using live-cell imaging in LECs expressing uorescent actin (Supplementary Movies S1 and S2). SEMA3F induced signicant retraction of the LEC cytoskeleton, while little change in actin was observed in the FLAGdepleted control (Fig. 2E). Together, this indicates that SEMA3F can be puried in a biologically active form that is capable of negatively regulating the function of LECs. SEMA3F inhibits LEC function in vivo We next asked whether SEMA3F can function as antilymphangiogenic factor in vivo. Matrigel combined with different growth factors were implanted in anks of nude mice and invaded cells were identied and quantied by FACS. As proangiogenic growth factors such as VEGF-A and -C use neuropilins as coreceptors (16), sphingosine-1-phosphate (S1P), and basic broblast growth factor (bFGF) were substituted to circumvent any potential competition with SEMA3F. A stepwise gating strategy was employed to identify specic types of vascular-associated cells and endothelial cells while excluding immune-reactive cells like macrophages (Supplementary Fig. S3). Using Matrigel alone as a negative control and S1P and bFGF as a positive control, we tested the ability of puried SEMA3F to block chemoattractant-mediated endothelial cell recruitment in a dose-dependent, quantitative in vivo setting (Fig. 3). Gross evaluation and H&E-staining Matrigel plugs demonstrated strong inltration of vasculature in the positive control that was attenuated by SEMA3F addition (Fig. 3A, top and middle). Immunouorescence against vascular (CD31) and lymphatic (LYVE-1) vessels demonstrate a SEMA3F dose-dependent decrease in S1P- and bFGF-induced vessel recruitment (Fig. 3A, bottom). SEMA3F caused approximately 70% decrease in the abundance of red blood cells, long-regarded as the standard for angiogenesis implantation assays (Fig. 3B; ref. 29). Similarly, Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2939 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 1. SEMA3F expression is lost during head and neck cancer progression. A, SEMA3F (3p21.13p21.2) and PIK3CA (3q26.22q26.3) are located on chromosome 3 and their genomic copy number in head and neck cancer patients according to TCGA was calculated. Clinical outcome for patients with normal (CN 2) or SEMA3F heterozygous loss (CN 1) were interrogated and KaplanMeier (B) and Cox proportional hazard survival curves (C), median survival (D), evidence of metastatic disease (E), and lymphovascular invasion (F) were determined. G, immunohistochemistry of SEMA3F in primary tumor sections of normal oral epithelium and cancer. H, quantication of SEMA3F staining in a tissue array including normal oral epithelium, stage I, II, III, and IV cancers cases. Tissues were evaluated for intensity and percentage of positive cells, with 0 being absent staining and 4 being intense staining in all cells. I, the same array was also stained for podoplanin (lymphatic vessels) and CD31 (blood vessels), and correlation to SEMA3F expression was calculated using Pearson coefcient. Statistical signicance compared to normal was determined by Student t test,  , P < 0.01;    , P < 0.001. 2940 Cancer Res; 75(14) July 15, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 2. SEMA3F is a chemorepulsant that negatively impacts the function of lymphatic endothelial cells. A, Coomassie staining and Western blot analysis of serum-free CM from cells transfected with NTAP-SEMA3F. B, attachment of HUVEC cells CM from NTAP-SEMA3F cell supernatants alone or after FLAG or IgG immunoprecipitation depletion. C, attachment of LECs in the presence of increasing amounts of puried SEMA3F or supernatant after FLAG depletion. D, immunouorescent staining of actin (green) and nuclei (blue) in LEC treated with 100 ng/mL SEMA3F for 6 hours. Quantication of cell area and perimeter were determined using ImageJ using 25 independent elds. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. E, still images captured during live-cell imaging of LEC transfected with LifeAct GFP and treated with 1 mg/mL SEMA3F or DFLAG. we saw 60% to 70% decreased abundance of total CD31 CD102 endothelial cells (Fig. 3C) and vascular endothelial cells expressing little to no LYVE-1 (LYVE-1low), consistent with an antiangiogenic function previously reported for SEMA3F (Fig. 3D; refs. 21, 30, 31). Remarkably, SEMA3F prevented the recruitment of LECs (dened as LYVE-1hi), with a 70% to 85% reduction in the abundance of LECs with respect to control plugs (Fig. 3E). Thus, SEMA3F has potent antiangiogenic activity and an even more robust antilymphangiogenic function. NRP and plexin A coreceptor complexes are sufcient for SEMA3F signaling NRP2 and plexin A family members are capable of binding to SEMA3F (32, 33), but the relative contribution of each member of this family to SEMA3F-mediated signaling is unknown. Primary HUVEC, human microvascular endothelial cells (HMVEC), and LEC express NRP1, NRP2, and plexin A family members to varying degrees, while COS-7 cells do not express any of these receptors endogenously, thus serving as a heterologous system to investigate SEMA3F signaling (Fig. 4A). Each of the NRP and plexin A family members were expressed in COS-7 cells to test their relative www.aacrjournals.org contribution to SEMA3F signaling alone and in coreceptor complexes (Fig. 4B). As a control, we also tested the effect of SEMA3A, as this semaphorin signals primarily through NRP1-Plexin A1 and NRP1-Plexin A3 (34). Neither NRPs nor plexins alone were sufcient for SEMA3F or SEMA3A signaling (Fig. 4C and D). SEMA3A was most effective in cells expressing NRP1 and plexin A1, as predicted. SEMA3F potently induced the collapse of COS-7 cells that expressed NRP2 and plexin A3, although cells expressing NRP2 and plexin A1 also collapsed when compared with controls. Interestingly, a modest collapse was observed in SEMA3F-treated cells that expressed NRP1 and plexin A3, and to a lesser degree in those that expressed NRP1 and plexin A1. Some broader effect of SEMA3A was also observed, as NRP2-Plexin A1 complexes and NRP1-Plexin A4 complexes showed collapse. Plexin D1, a receptor for SEMA3E, did not appear to play a role in SEMA3F function (Supplementary Fig. S4). On the basis of these results, we summarized the relative contribution of different receptor combinations to SEMA3Finduced cytoskeletal collapse (Fig. 4E). Finally, we performed live cell imaging of the collapse in COS-7 cells transfected with NRP2 control or NRP2 and plexin A3 treated with SEMA3F Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2941 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 3. SEMA3F inhibits in vivo lymphangiogenesis. A, Matrigel plugs containing the indicated factors were examined for gross histology (top row), H&E (middle row), and immunouorescence for CD31 (red) and LYVE1 (green; bottom row). FACS analysis of the number of total red blood cells (B), total CD102 CD31 endothelial cells low (C), LYVE-1 vascular endothelial cells (D), hi and LYVE-1 lymphatic endothelial cells (E) are shown. Values were expressed as a percentage of the stimulated vehicle control from three independent experiments. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. (Supplementary Movies S3S4). Control cells demonstrated no changes in the actin cytoskeleton, while expression of NRP2 and plexin A3 was sufcient to induce a rapid collapse phenotype with signicant alterations in the actin cytoskeleton (Fig. 4F). NRP2 is predominantly required for SEMA3F signaling To determine whether both endogenous NRP1 and NRP2 are necessary for SEMA3F signaling, we abolished expression of these receptors using siRNAs in LECs. Little to no NRP expression remained in siRNA-targeted cells, with no apparent cross-specicity between the knockdown sequences (Fig. 5A). Loss of NRP1 expression only modestly attenuated the collapse of the cells upon SEMA3F treatment; however, signicantly less collapse was observed in SEMA3F-treated LEC that do not express NRP2 (Fig. 5B). In a doseresponse collapse (Fig. 5C) and attachment (Fig. 5D) assay, loss of NRP1 slightly inhibited the effectiveness of low doses of SEMA3F. In both assays, however, NRP2 knockdown had a pronounced effect, suggesting that NRP2 is the dominant 2942 Cancer Res; 75(14) July 15, 2015 SEMA3F receptor in LECs. This was further documented by logdose response analysis (Fig. 5E). While loss of NRP1 increased the IC50 of SEMA3F slightly compared with control, NRP2-decient cells required nearly ten times more SEMA3F to achieve the same effect. Taken together, these ndings suggest that while NRP1 may play a role in transmitting SEMA3F signal in the absence of NRP2, NRP2 is predominately required for SEMA3F biologic responses in LECs. A subset of HNSCC cells express NRP2 and respond to the antitumor activity of SEMA3F NOKs and most HNSCC cancer cells do not express NRP1 or NRP2. However, a subset of HNSCC cell lines acquired expression of NRP2 (Fig. 6A). Aligned with this observation, increased NRP2 mRNA expression correlates with loss of SEMA3F (P 0.003), poor prognosis, and decreased survival (Supplementary Fig. S5). To test the effect of SEMA3F in HNSCC cells with or without NRP2, we chose two representative highly tumorigenic and Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 4. NRP and plexin A coreceptors coordinate SEMA3F function. A, Western blot analysis demonstrating expression of the NRP, plexin, and VEGFR family members in a panel of immortalized and primary epithelial and endothelial cells. B, Western blot analysis demonstrated specic expression of each NRP and plexin A family member in COS-7 cells. C, COS-7 cells stably expressing GFP were transfected and treated as indicated. Collapse was quantied using ImageJ relative to vehicle control based on 25 imaging elds, each from quadruplicate wells in three independent experiments. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. D, representative images of cells transfected with different receptor combinations and treated with vehicle, SEMA3F, or SEMA3A. E, table summarizing the relative contributions of the different receptor combinations tested. F, confocal images of COS-7 cells transfected with LifeAct-GFP, NRP2, and/or plexin A3 and treated with SEMA3F or DFLAG. Images were taken 1 and 3 hours after treatment with SEMA3F. metastatic HNSCC cell lines (22), UMSCC17B, which do not express NRPs, and UMSCC2, with high level expression of NRP2. As an experimental approach, we used a doxycyclineinducible system and conrmed that doxycycline addition induced SEMA3F expression without changing the expression of NRP1 or NRP2 in these cells (Fig. 6B). In both cell types, expression of the rtTA3 tetracycline-dependent transactivating complex alone had no effect on proliferation (Fig. 6C and D). SEMA3F expression inhibited proliferation of UMSCC2 cells signicantly; however, this effect was abrogated upon NRP2 knockdown (Fig. 6C). Conversely, in UMSCC17B cells induction of SEMA3F did not alter proliferation (Fig. 6D). As a gainof-function strategy, transient expression of NRP2 in UMSCC17B rendered them sensitive to the growth inhibitory activity of SEMA3F (Fig. 6D). Induction of SEMA3F in UMSCC2 cells also blocked endogenous and directed migration (Fig. 6E). Interestingly, siRNA-mediated loss of NRP2 decreased the endogenous migration of these cells compared with control siRNA, although there was no difference between vehicle- and doxycyline-treated groups (Fig. 6E). www.aacrjournals.org Ectopic expression of NRP2 in UMSCC17B cells was sufcient to enhance cell migration over vector control, and induction of SEMA3F did not alter endogenous UMSCC17B migration unless NRP2 was introduced to these cells (Fig. 6F). Taken together, this supports that HNSCC cells expressing NRP2 may exhibit increased migratory capacity, but that loss of SEMA3F may need to precede gain of NRP2 expression. SEMA3F functions as a potent HNSCC metastasis suppressor SEMA3F inhibited the biologic activity of LECs and intratumoral lymphangiogenesis is highly prevalent in HNSCC, suggesting that SEMA3F may suppress metastasis in vivo through paracrine mechanisms. To test this hypothesis in the context of NRP2 expression or absence, we injected UMSCC2-rtTA3-SEMA3F and UMSCC17B-rtTA3-SEMA3F cells into the tongues of mice and assessed tumor growth, metastasis, and intratumoral vascularity. Consistent with our observations in vitro, doxycycline induction of rtTA3 alone had no effect on tumor growth (Supplementary Fig. S6). SEMA3F induction inhibited tumor growth in the UMSCC2-injected animals (Fig. 7A) and this correlated with a Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2943 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 5. NRP2 is predominantly required for SEMA3F function in LEC. A, Western blot analysis of endogenous NRP1 and NRP2 in LEC in the presence of control (siCtrl), NRP1, or NRP2 siRNA. B, uorescent imaging of LEC transfected with the indicated siRNA and then treated with vehicle or 100 ng/mL SEMA3F. Parental and siCtrl-transfected LECs treated with increasing doses of SEMA3F and evaluated for collapse (C) or attachment (D). For both collapse and attachment assays, values are reported relative to vehicle control and quantication was done by ImageJ, based on 25 imaging elds, each from quadruplicate wells in three independent experiments. E, IC50 of SEMA3F was calculated from the dose curve generated in C and D. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. statistically signicant decrease in cervical lymph node metastasis (Fig. 7C). Interestingly, SEMA3F induction in UMSCC17B tumors, which do not express NRP2, had no effect on tumor size (Fig. 7B). However, a similar decrease in lymph node metastasis was observed (Fig. 7D). This suggested that the paracrine signaling of SEMA3F to the tumor stroma may be sufcient for suppression of metastasis, while exerting an additional autoinhibitory effect on tumor growth in HNSCC cells that acquired expression of NRP2. Tongue sections were evaluated by immunohistochemistry for CD31 and LYVE1 and the intratumoral microvessel density (MVD) was quantied. In both the UMSCC2 and UMSCC17B tumors, induction of SEMA3F signicantly decreased intratumoral lymphangiogenesis and modestly decreased CD31 vasculature, albeit not to a statistically signicant degree (Fig. 7E and F). As a control, MVD outside the tumor was quantied and was not affected for either CD31 or LYVE1 (Fig. 7E and F). Immunouorescence staining revealed a high rate of cancer cells inltrating into lymphatic vessels in control animals that were not observed in SEMA3F-induced cohorts (Fig. 7G and H, white arrows). These results demonstrate that SEMA3F exerts a potent antilymphangiogenic and metastasis suppressor function in HNSCC, and that 2944 Cancer Res; 75(14) July 15, 2015 SEMA3F may also inhibit growth of a subset of HNSCC expressing NRP2. Discussion The tumor microenvironment is regulated through a complex autocrine and paracrine signaling network that is often hijacked and exploited by cancer cells to facilitate their survival, growth, and dissemination. In HNSCC, the interplay of these factors is shifted to promote intratumoral lymphangiogenesis and metastasis, which contribute to poor prognosis and outcome (6, 8). Our ndings suggest that SEMA3F is a key antilymphangiogenic molecule that may function at the core of these cell regulatory networks during HNSCC progression. Indeed, loss of SEMA3F is a frequent event in HNSCC, which correlates with increased tumor vascularity and metastasis. We show that SEMA3F can directly affect LEC function in vitro and in vivo through specic receptor complexes. In COS-7 reconstitution assays, NRP2 and plexin A3 were sufcient to mediate SEMA3F biologic responses, while NRP2-Plexin A1, NRP1-Plexin A3, and NPR1-Plexin A1 complexes can also transduce SEMA3F signals, albeit with decreasing efciency. NRP2 is the major coreceptor for SEMA3F in LECs, Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 6. NRP2 expression varies in HNSCC and mediates tumor cell responsiveness to SEMA3F. A, Western blot analysis for the expression of receptors in normal oral keratinocytes (NOKSI) and panel of HNSCC cell lines. B, Western blot analysis showing expression of inducible SEMA3F and knockdown or reintroduction of NRP2 expression in UMSCC2 and UMSCC17B cells, respectively. Proliferation assay of UMSCC2-rtTA3-SEMA3F (C) or UMSCC17B-rtTA3-SEMA3F (D) after SEMA3F induction. Cells were treated with control siRNA or NRP2 siRNA (C) or empty vector or NRP2 (D). Migration assay of UMSCC2-rtTA3SEMA3F (E) or UMSCC17B-rtTA3SEMA3F (F) cells with no stimulation (vehicle) or towards 20% FBS. Cells were treated with control siRNA or NRP2 siRNA (E) or empty vector or NRP2 (F). Proliferation and migration were reported as a percentage of control. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;   , P < 0.01;    , P < 0.001. although NRP1 can transmit SEMA3F signals to a lesser extent. Overall, our in vitro and in vivo studies indicate that SEMA3F is potent antilymphangiogenic molecule. Thus, SEMA3F loss may represent an early event in HNSCC, enabling intratumoral lymphangiogenesis that may contribute to the high prevalence of HNSCC cases presenting with locoregional lymph node invasion, heralding a poor clinical outcome. NRP2 is a multiligand coreceptor that can both promote and inhibit the development of venous and lymphatic vasculature, in the stroma and on some tumor cells themselves (3537). In endothelial cells, NRP2 associates with plexin A family members and VEGFR2 and VEGFR3, and these complexes have been implicated in their proangiogenic and -lymphangiogenic signaling (38, 39). Certain malignant epithelial cells express NRP2 without concomitant expression of VEGF receptors (40). The observation that NRP2 but not VEGF receptors are expressed in a subset of HNSCC cells raises the possibility that in these cells NRP2 may act as a coreceptor for plexins or other partners, acting as a gain-of-function alteration during cancer development and www.aacrjournals.org progression. Indeed, exogenous NRP2 expression in HNSCC cells enhanced their endogenous migration while loss of upregulated NRP2 in HNSCC cells decreased their endogenous migration. This allows us to hypothesize that cancer cells may be selected for their NRP2 expression due to an increased growth or migratory potential, but that SEMA3F loss may need to precede NRP2 gain in HNSCC (Fig. 7I). Lymphatic metastasis is mainly attributed to increased expression and signaling of proangiogenic factors (2, 5, 4143). However, expression and secretion of these growth factors alone may not be sufcient for tumor-associated lymphangiogenesis and cancer dissemination. For example, in some cancers VEGF-C or VEGF-D expression levels do not correlate with lymph node metastasis (44, 45), suggesting that other factors may counteract the high level expression of prolymphangiogenic cytokines and attenuate their signaling capacity and prognostic value in vivo. On the basis of our current results, one possible explanation is that SEMA3F could repel LECs and thus dominate the activity of VEGFA and VEGF-C, rendering their expression alone insufcient for Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2945 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 7. SEMA3F functions as a tumor and metastasis suppressor in vivo. Tumor growth of UMSCC2-rtTA3-SEMA3F (A) and UMSCC17B-rtTA3-SEMA3F (B) after SEMA3F induction via doxycycline chow. Cervical lymph node metastasis was evaluated as the percentage of metastatic lymph nodes in control and doxycycline-fed animals for UMSCC2-rtTA3-SEMA3F (C) and UMSCC17BrtTA3-SEMA3F (D). MVD for lymphatic (LYVE-1) and blood (CD31) vessels was evaluated in the tumor and muscle of tongues for UMSCC2-rtTA3-SEMA3F (E) and UMSCC17B-rtTA3-SEMA3F (F). MVD was reported relative to the average density in vessels/mm. Statistical signicance was determined using Student t test,  , P < 0.05;   , P < 0.01;    , P < 0.001. Immunouorescent staining of tumors from control and doxycycline-fed animals for UMSCC2-rtTA3-SEMA3F (G) and UMSCC17B-rtTA3-SEMA3F (H) revealed a higher density and size of vessels in control animals. Lymphovascular invasion by cancer cells is indicated by white arrowheads. I, proposed mechanism for the role of SEMA3F loss in HNSCC. See text for details. 2946 Cancer Res; 75(14) July 15, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor lymphangiogenesis and metastasis. This hypothesis is supported by our ndings that induction of SEMA3F alone in orthotopic HNSCC cells signicantly inhibits intratumoral lymphangiogenesis and metastasis, even in HNSCC cells that do not express NRP2. Thus, loss of SEMA3F expression in HNSCC may simultaneously enhance VEGF-mediated signaling on endothelial cells and alleviate repressive semaphorin functions, resulting in increased lymphangiogenesis. Our observation may have a broad impact in multiple highly prevalent human malignancies. In an integrative multiplatform analysis, 3p deletion was identied as a key genomic signature shared by a squamous-like subtype of solid cancers (46). Furthermore, chromosomal loss at 3p21, which is expected to result in the genomic deletion of SEMA3F, has been observed for lung, breast, and kidney cancers in addition to HNSCC (4750). Immunohistochemistry on HNSCC tumors reveals a complete absence of SEMA3F expression in most advanced cases, suggesting that epigenetic regulation or transcriptional dowregulation may contribute to reduced SEMA3F expression of the remaining SEMA3F allele in cancers with SEMA3F heterozygous loss. We can conclude that SEMA3F is a potent chemorepellent molecule for lymphatic and vascular endothelial cells, which acts through NRP2-Plexin A and to a lesser extent NRP1-Plexin A signaling complexes. Loss of SEMA3F is an early event in HNSCC and likely many other highly prevalent human malignancies. This information can in turn be exploited for therapeutic purposes, as most HNSCC lesions may retain one intact SEMA3F allele. This suggests that reactivation of SEMA3F expression or ectopic SEMA3F delivery may offer the opportunity to halt intratumoral lymphangiogenesis and suppress metastasis regardless of the NRP2 expression status of the HNSCC lesion. We can conclude that SEMA3F-NRP2 represents a novel regulatory axis during HNSCC development, progression, and metastasis, thus providing new prognostic markers and therapeutic options in this highly aggressive disease. Disclosure of Potential Conicts of Interest No potential conicts of interest were disclosed. Authors' Contributions Conception and design: C.L. Doci, J.S. Gutkind Development of methodology: C.L. Doci, C.M. Mikelis, A.A. Molinolo, J.S. Gutkind Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.L. Doci, C.M. Mikelis, M.S. Lionakis, A.A. Molinolo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.L. Doci, M.S. Lionakis, A.A. Molinolo Writing, review, and/or revision of the manuscript: C.L. Doci, J.S. Gutkind Study supervision: J.S. Gutkind Acknowledgments The authors thank Dr. Roberto Weigert for providing the LifeAct plasmid. They also thank Dr. Gera Neufeld for providing plexin A1, A3, and A4 receptor plasmids and puried Sema3E. Grant Support This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, and in part by the National Institute of Allergy & Infectious Diseases, NIH (Z01DE00558-23 and Z01DE00551-23). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis. Proc Natl Acad Sci U S A 2012;109:158949. 44. Lahat G, Lazar A, Wang X, Wang WL, Zhu QS, Hunt KK, et al. Increased vascular endothelial growth factor-C expression is insufcient to induce lymphatic metastasis in human soft-tissue sarcomas. Clin Cancer Res 2009;15:263746. 45. Ishikawa M, Kitayama J, Kazama S, Nagawa H. Expression of vascular endothelial growth factor C and D (VEGF-C and -D) is an important risk factor for lymphatic metastasis in undifferentiated early gastric carcinoma. Jpn J Clin Oncol 2003;33:217. 46. Hoadley KA, Yau C, Wolf DM, Cherniack AD, Tamborero D, Ng S, et al. Multiplatform analysis of 12 cancer types reveals molecular classication within and across tissues of origin. Cell 2014;158: 92944. 47. Kok K, Naylor SL, Buys CH. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv Cancer Res 1997;71:2792. 48. Sekido Y, Ahmadian M, Wistuba II, Latif F, Bader S, Wei MH, et al. Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene. Oncogene 1998;16:31517. 49. Roche J, Boldog F, Robinson M, Robinson L, Varella-Garcia M, Swanton M, et al. Distinct 3p21.3 deletions in lung cancer and identication of a new human semaphorin. Oncogene 1996;12:128997. 50. Hesson LB, Cooper WN, Latif F. Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene 2007;26:7283301. Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Genetic Identification of SEMA3F as an Antilymphangiogenic Metastasis Suppressor Gene in Head and Neck Squamous Carcinoma Colleen L. Doi, Constantinos M. Mikelis, Michail S. Lionakis, et al. Cancer Res 2015;75:2937-2948. Published OnlineFirst May 7, 2015. 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... Oral Oncology 49 (2013) 93101 Contents lists available at SciVerse ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology DSG3 as a biomarker for the ultrasensitive detection of occult lymph node metastasis in oral cancer using nanostructured immunoarrays Vyomesh Patel a, Daniel Martin a, Ruchika Malhotra b, Christina A. Marsh a, Colleen L. Doi a, Timothy D. Veenstra f, Cherie-Ann O. Nathan c, Uttam K. Sinha d, Bhuvanesh Singh e, Alfredo A. Molinolo a, James F. Rusling b, J. Silvio Gutkind a, a Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4330, United States Departments of Chemistry and Cell Biology, University of Connecticut, Storrs, CT, United States Department of Otolaryngology, Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, United States d Department of Otolaryngology, Head and Neck Surgery, University of Southern California, Keck School of Medicine, Los Angeles, CA, United States e Laboratory of Epithelial Cancer Biology, Head and Neck Service, Memorial Sloan-Kettering Cancer Center, New York, NY, United States f Laboratory of Proteomics and Analytical Technologies, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, Frederick, MD, United States b c a r t i c l e i n f o Article history: Received 26 April 2012 Received in revised form 19 July 2012 Accepted 1 August 2012 Available online 23 September 2012 Keywords: DSG3 Head and neck cancer Desmosomes Biomarker Sentinel lymph nodes Nanosensors Immunoarray Lymph nodes metastasis Proteomics SCC s u m m a r y Objectives: The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC. Materials and methods: DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates. Results: We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microuidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes. Conclusion: We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients. Published by Elsevier Ltd. Introduction With more than 500,000 new cases annually, squamous cell carcinomas of the head and neck (HNSCC) represent one of the ten most common cancers globally,1 and result in more than 11,000 deaths each year in the US alone.2 The 5 year survival of newly diagnosed HNSCC patients is 50%, and despite new treatment approaches, it has improved only marginally over the past decades.3 HNSCC has a high propensity to metastasize to loco Corresponding author. Address: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, Building 30, Room 211, Bethesda, MD 20892-4330, United States. Tel.: +1 301 496 6259; fax: +1 301 402 0823. E-mail address: sg39v@nih.gov (J.S. Gutkind). regional lymph nodes due to the presence of a rich lymphatic network and the overall high number of lymph nodes in the neck region.38 Even in patients without clinical evidence of lymph node involvement (N0), there is a high incidence of occult lymph node metastasis, ranging from 10% to 50%.4,5,7 The diagnosis of cervical lymph node metastasis an essential requirement for clinical staging and treatment,9 and is now widely accepted as the most important factor in HNSCC prognosis.3,5,6,10 However, due to limitations in the accurate diagnosis of lymph node metastasis, patients with clinically negative nodes often undergo elective neck resection or radiation,11,12 with the consequent associated morbidity and adverse impact in the quality of life.12 Clinical assessments of lymph node metastases include physical examination, imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography, and 1368-8375/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.oraloncology.2012.08.001 Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 94 V. Patel et al. / Oral Oncology 49 (2013) 93101 [18F]-2-uorodeoxyglucose positron emission tomography scans (PET).13,14 However, poor spatial resolution, false positive detection of reactive lymph nodes, and limited sensitivity under 5 mm size,1518 can add to potential false negative results. Histopathological, immunohistochemical (IHC), and molecular approaches to evaluate sentinel lymph node biopsies have improved the detection rate of metastatic disease in some cancers,19,20 but histopathology-based methods can often miss micrometastases, while more sensitive techniques such as IHC and real-time PCR for validated cancer markers are time consuming and require stringent handling procedures and technical expertise. A recent proteomic analysis of parafn embedded normal oral mucosa and HNSCC lesions revealed a very high abundance of Desmoglein 3 (DSG3) in both non-neoplastic epithelium and cancer lesions.21 DSG3 is a transmembrane glycoprotein involved in cell-to-cell adhesion that is exclusively expressed in stratied epithelium.22 These observations prompted us to explore whether the assessment of DSG3 protein levels could be used to investigate the presence of malignant squamous epithelial cells in cervical lymph nodes, and hence serve as a predictive biomarker for metastasis. In this regard, high sensitivity electrochemical immunoassays have recently gained acceptance in biomedicine.23 For example, we have developed immunosensors based on nanostructured electrodes coupled to microuidics and multilabel strategies to achieve highly sensitive detection of protein cancer biomarkers in serum.24,25 We have combined these strategies into a simple microuidic immunoarray26,27 and here explore the suitability of this platform for the rapid and sensitive detection of DSG3 protein. We show that this system can be used to rapidly detect and quantify DSG3 in frozen human tissue sections, distinguishing between clinically positive and negative cervical lymph nodes. Overall, these studies may help develop point-of-care procedures aiding in the diagnosis of invaded lymph nodes in HNSCC patients, thereby facilitating educated decisions regarding appropriate therapeutic intervention modalities, and decreasing the morbidity often associated with HNSCC. Materials and methods Reagents, antibodies, and cell culture All chemicals and reagents were from SigmaAldrich (St. Louis, MO), unless indicated. The following antibodies: goat-anti-human DSG3 [AF1720]; mouse-anti-human DSG3 [MAP1720], biotin labeled goat-anti-human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA). The mouse anti-human DSG3 antibody [32-6300] from Invitrogen (MA, USA), and rabbit-anti-cytokeratin Wide Spectrum Screening [N1512] from Dako (CA), were used for immunohistochemistry (IHC). The a-tubulin antibody [11H10] was from Cell Signaling Technology (MA, USA). Biotinylated peroxidase and streptavidin coated magnetic beads were from Invitrogen. Antirabbit and anti-mouse biotinylated secondary antibodies were from Vector, Burlingame, CA, US. HN12, HN13 and HN30 cells were described previously.28 Cal27 and Jurkat cells were from ATCC (VA); and primary human cells from Lonza (MD). See Supplemental information for additional information. Human clinical tissues and tissue microarrays (TMAs), immunohistochemistry and immunouorescence Formalin xed, parafn-embedded, and freshly frozen HNSCC and lymph node samples were obtained anonymized with Institutional Review Board approval. Five lm sections from all tissues underwent standard H&E staining for histopathological evaluation and immunostaining. Tissue microarrays used include TMA MC2081 US (Biomax, MD) with 208 representative cases of colorectal, breast, prostate and lung cancers, and normal tissue; TMA LC810 (Biomax, MD), consisting of 40 cases of different types of lung cancers with their matched metastatic lymph nodes (total 80 tissue cores); and the Head and Neck Tissue Microarray Initiative, including 317 HNSCC cases.29 Tissue processing and analysis are described in detail in Supplemental information. All slides were scanned at 400 magnication using an Aperio CS Scanscope (Aperio, CA) and quantied using the available Aperio algorithms. Immunodetection of DSG3 was quantied according to percent of tumor cells stained (125%, 2650%, 5175%, or 76100%).29 For immunouorescence, 10 lm cryosections were immunostained with goat-anti-human DSG3 (AF1720), mouse-anti-vimentin and DAPI containing. See Supplemental information for additional details. Western blot analysis of cell and tissue extract, and microuidic immunoarrays systems for DSG3 A detailed description of the procedures used for tissue lysate preparation, SDSPAGE gel analysis and Western blotting, and the fabrication of the microuidic immunoarrays made of gold nanoparticles layered with glutathione are described in detail in the Supplemental information. Briey, the immunoarrays consisting of eight sensor elements, made of gold nanoparticles layered with glutathione, were rst coated with the capture antibody and transferred to a microuidic chamber. In parallel, biotinylated horseradish peroxidase and a biotinylated secondary antibody were attached to streptavidin-coupled magnetic beads and collected with a magnet. Next, 5 ll of 5750 fg/mL of recombinant DSG3 protein standards or 4 ll tissue extract were diluted 1:6000 in RIPA buffer and added to the bioconjugates. The bioconjugates with captured proteins were then magnetically separated, washed, resuspended in a nal volume of 110 lL, and immediately injected into the microuidic channel housing the immunoarrays. At this step, the ow was stopped, incubated for 20 min, washed, and hydroquinone solution was passed through the channel. The amperometric signal was developed by injecting 50 lL of 100 lM H2O2. Tissue lysates used for Western blot analysis and microuidic immunoarrays were made from primary tumors (n = 4), lymph node ( ) (n = 3), and lymph node (+) (n = 3). Results In a previous proteome-wide analysis of HNSCC progression, we noted that DSG3 was highly expressed in normal oral mucosa and HNSCC lesions.21 To further investigate a possible use for DSG3 as a predictive biomarker, we rst assessed DSG3 expression by immunohistochemistry in an independent cohort of human normal and malignant oral squamous tissues. By H&E histological evaluation, normal squamous epithelium shows a dened basement membrane with layers of differentiating keratinocytes, whereas in the malignant counterpart, this organized pattern is lost (Fig. 1A). Normal tissues sections stained for DSG3 show that it is predominantly expressed in the basal and suprabasal layers of the normal squamous epithelium, while in SCC DSG3 expression is restricted to cancer cells. Stromal cells were negative. We next evaluated DSG3 expression in a HNSCC tissue microarray (TMA) containing 317 evaluable cores.29 DSG3 was readily detected in all HNSCC cores and localized to tumors cells (Fig. 1B). Within these cases, well-differentiated carcinomas (n = 120) had the highest percentage of DSG3-positive cells (90%). The moderate- (n = 119), and poorlydifferentiated (n = 66) cores showed slightly lower proportion of DSG-reactive cells (80% and 70%, respectively), the remaining 12 cores consisting of corresponding to non-squamous tissues were Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 95 V. Patel et al. / Oral Oncology 49 (2013) 93101 Normal HNSCC H&E A 100 100 100 DSG3 100 B DSG3 immunostaining WD % positive cancer cells C 100 100 n: 120 119 66 76-100 51-75 26-50 1-25 80 60 40 20 0 PD 30 10 1 3 0.3 Recombinant DSG3 (ng) 100 Tubulin PD DSG3 100 HN13 HN30 CAL27 HaCaT Total cell lysates JURKAT HMVEC LEC HUVEC HN12 MD D MD 0.1 WD Figure 1 Validation of DSG3 expression in normal and malignant HNSCC. Normal oral mucosa biopsies and HNSCC were evaluated for DSG3 by IHC. (A) DSG3 is expressed throughout the normal epithelium, but is stronger in the basal and parabasal layers. Diffuse expression was seen in the epithelial component of all HNSCC. (B) Representative well (WD), moderate (MD) and poorly (PD) differentiated HSCC cases are shown. (C) DSG3 was expressed in all tumors regardless of differentiation, with increased expression in WD cases. Numbers of cases analyzed is depicted. (D) Total cell extracts from non-squamous (Jurkat, HMVEC, LEC, HUVEC) and oral-squamous (HN12, HN13, HN30, Cal27), and epidermal-squamous (HaCaT) were processed for Western blot analysis. Native DSG3 and its glycosylated forms were readily detected in squamous cells extracts, while absent from the non-squamous counterparts. These levels were compared with human recombinant DSG3 that was processed in a background of Jurkat cell lysate. Tubulin staining indicates equivalent loading and protein integrity. negative for DSG3 (Fig. 1C). The data demonstrates that DSG3 is highly expressed in human oral squamous epithelium and HNSCC. We next sought to assess in vitro the specicity of the epithelial expression of DSG3 in a panel of squamous and non-squamous model cells. The latter included Jurkat cells (immortalized T lymphocyte cells), HMVEC (skin human microvascular endothelial cells), LEC (lymphatic endothelial cells), and HUVEC (human umbilical vein endothelial cells). HaCaT cells are squamous, nonoral immortalized epidermal keratinocytes, while the oral squamous cell carcinoma lines included HN12, HN13, HN30 and Cal27.28 DSG3 was readily detected in all squamous oral cancer cell lines and HaCaT cells, with lower levels in HN12 and higher in Cal27 (Fig. 1D). No expression for DSG3 was observed in any of four non-squamous lines, while levels of a-tubulin indicated equal loading as well as protein integrity. The data seem to indicate that DSG3 is exclusively expressed in squamous epithelial-derived cells. To further validate the specicity of DSG3 expression, we evaluated TMAs containing cores representing the four most common cancers (breast, lung, prostate, and colon cancer) for DSG3 levels, and scored cases based on the presence or absence of DSG3 expression. As seen in Fig. 2A), DSG3 is poorly expressed in breast and prostate cancers, as well in adenocarcinoma of the lung (ADC), which likely reects the glandular epithelial cell origin of these human malignancies. In colon carcinoma, the expression was variable, and in all cases the pattern of expression was diffused and not the characteristic membrane lace-like pattern. In contrast, Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 96 V. Patel et al. / Oral Oncology 49 (2013) 93101 A TMA major cancer types Lung ADC Breast n: 42 47 16 45 37 13 298 100 + - Lung 100 m Colon % Cases 80 100 m 60 40 20 100 m OSCC Lung SCC Lung ADC Prostate Breast Prostate OSCC Rectum 0 100 m Colon 100 m 100 m Lung cancer B TMA lung cancer 100 SCC Met 100 m 100 m SCLC ADC n=30 % positive tumors SCC 80 60 40 20 100 m n=42 100 m n=6 0 SCC ADC SCLC Figure 2 Immunoreactivity of DSG3 in common tumor types. (A) Multi-tumor TMAs (lung, breast, colon, prostate), and an oral specic TMA were assessed for DSG3 expression by IHC, and the staining scored for the presence of specic staining as (+) or ( ). Most squamous cell lung cancers stained positive for DSG3, but very few of the adenocarcinomas gave positive reaction. All cores from the OSCC TMA scored positive. (B) In lung cancer, DSG3 expression was positive in most squamous cell carcinomas (SCC) including lymph node metastasis (SCC Met), while few cases of adenocarcinomas (ADC) gave positive reaction, and all small cell lung carcinoma samples (SCLC), were negative. DSG3 is highly expressed in tumors derived from cells of squamous epithelial origin, such as lung squamous carcinoma (SCC) and additional oral squamous carcinoma (OSCC) that were included in these arrays, showing a membrane localized staining. All stainings were scored blindly and tabulated (Fig. 2A). All cancers of squamous origin (oral and lung SCC) were strongly positive for DSG3 expression. As lung cancers include SCC, ADC, and small cell lung carcinomas (SCLCs), we examined further the specicity of DSG3 expression in these distinct lung cancer lesions. All lung SCC show strong membrane localized staining in both the primary tumor and metastasis, while ADC and SCLC show marginal to no DSG3 expression, as reected by scoring their corresponding tissue cores (Fig. 2B). Collectively, our results indicate the high specicity of DSG3 expression in oral and lung SCC lesions. Based on the observation that DSG3 is highly expressed in HNSCC, we wanted to determine if the presence of this protein in cervical lymph nodes of the neck region could be used as a predictive biomarker of HNSCC invasion. To this end, we evaluated formalin-xed, parafn-embedded and anonymized tissue sections of non-metastatic (N0) or metastatic (N+) human cervical lymph node biopsies from patients diagnosed with HNSCC for expression of DSG3 and cytokeratin, a squamous-specic protein marker. Negative lymph nodes were negative (Fig. 3A) whereas clusters of tumor cells stained positive for membrane-localized DSG3 (inset, top right), can be seen throughout the invaded lymph node (N+), indicating the metastatic spread of squamous tumor cells of the primary tumor lesions from the oral cavity (Fig. 3A). Noteworthy, small clusters of 23 isolated tumor cells, constituting micrometastases were readily detected by the presence of DSG3 protein, and this size tumor island could potentially be missed by histopathological evaluation (Inset). Next, we screened a larger cohort of metastatic and non-metastatic human cervical lymph nodes for cytokeratin and DSG3 expression (n = 35). All metastatic (n = 30), but not non-metastatic cases (n = 5) were positive for DSG3. Serial sections stained for H&E and cytokeratin conrmed the epithelial nature of the malignant DSG3-positive cells. (Fig. 3B, and low magnication of a whole lymph node in Suppl. Fig. 1). This indicates that DSG3 expression can help identify small numbers of malignant squamous tumor cells in lymph nodes, and hence the metastatic nature of the primary lesion. The sensitivity and specicity of this detection suggests that DSG3 may hold promise for accurately detecting micrometastasis in cervical lymph nodes in newly diagnosed HNSCC patients. Our previous study adapted amperometric sandwich immunoassays to a microuidic system for the ultrasensitive, multiplexed detection of secreted biomarker proteins.26 Here, we used a similar strategy for detecting DSG3 in complex tissue extracts using the microuidic immunoassay system. As shown in Fig. 4A, DSG3 Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 97 V. Patel et al. / Oral Oncology 49 (2013) 93101 A N- 100m B N+ 100m N+ 100m DSG3 N+ 50m 100m H&E N+ 50m DSG3 50m 100m N- 100m DSG3 50m CK 50m DSG3 50m Figure 3 Specic detection of DSG3 in human cervical lymph nodes. (A) Formalin xed and parafn embedded tissue sections of non-metastatic (N ) and metastatic lymph nodes (N+) show DGS3 expression only in N+, with the staining localized to the malignant squamous cells (n = 30). All N cases were negative (n = 5). B. The epithelial specicity of DSG3 immunoreactivity was further conrmed using simultaneous cytokeratin (CK) staining. A representative example is shown, whereby the H&E stained tumor island is matched with CK and DSG3 expression, with no non-specic staining. An example of a N case stained for DSG3 is shown. capture antibodies are attached to up 8 sensor elements, and streptavidin-coated paramagnetic beads (MB) loaded with 400,000 biotin-HRPs and thousands of secondary biotin-labeled antibodies to DSG3 (Ab2) are used to capture the protein off-line. After washing and magnetic separation, the MBs that have bound DSG3 (DSG3MB) are injected into the microuidic channel. Incubation at stopped ow allows the sensor antibodies to capture DSG3-MBs, and amperometric signals are developed by injecting hydrogen peroxide to activate HRP and hydroquinone to mediate the amperometic oxidation, resulting in peak currents proportional to DSG3 concentration (Fig. 4B). Noteworthy, the entire assay from incubation of sample with Ab2-MB-HRP to measurement takes 50 min. Remarkably, using this approach we were able to accurately and reproducibly detect DSG3 protein at levels down to 5 fg mL 1 in complex tissue extracts, with minimal non-specic binding. We next used these protocols for capturing DSG3 from clinical samples of human HNSCC. Desmosomes are notoriously insoluble, and multiple buffers tested, RIPA buffer afforded excellent solubility and retaining antigenicity of DSG3 extracted directly from cryosections of HNSCC and lymph node tissue. Protein extracts were made from a single 10 lm cryosection from each sample and used as input. Picogram levels of DSG3 protein were detected in all tumor samples (T14), and this was conrmed by Western blot analysis, where high levels of the protein were also detected in total cell extracts that were used for these analyses (Fig. 4C and D). The specicity of DSG3 identication was further conrmed by simultaneous uorescence microscopy of DSG3 and vimentin, as a stromal marker, in frozen sections of a series of metastatic and non-metastatic lymph nodes. As seen in Fig. 5A, only vimentin (red) was identied in non-metastatic lymph nodes (in blue, nuclear DAPI staining), whereas all metastatic lymph nodes showed pockets of very strong staining for DSG3 (green). To further explore the sensitivity of the method, we decided to analyze metastatic lymph node tissues in which the number of malignant epithelial cells was known. For this, H&E stained slides of each case was scanned, analyzed histologically, the malignant areas identied, and the number of cells quantied using the Aperio nuclear algorithm (Aperio, Vista, CA). The number of cancer cells per lymph node section is indicated in Table 1. No tumor cells were be present in the negative lymph nodes, while all three metastatic lymph nodes (13) evaluated had differing number of tumor cells. Noteworthy, positive lymph node 1 had less than Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 98 V. Patel et al. / Oral Oncology 49 (2013) 93101 A B Ab1 DSG3 (fg mL-1) 40 750 500 250 30 DSG3 PDDA HRP Ab2 I, nA 100 AuNPs 50 20 25 5 10 10 0 0 1 m streptavidin coated magnetic bead -10 0 400 800 1200 1600 t, s Ab2 -MB-HRP 40 35 y = -7.4659 + 14.987log(x) R = 0.99315 30 I, nA 25 20 15 10 5 Voltage + H2O2 HQ amperometric signal 0 100 10 C 1000 [DSG3], pg mL-1 1000 [DSG3], fg mL-1 D Primary tumor T1 T2 T3 T4 100 10 DSG3 1 0.1 0.01 Tubulin Figure 4 Rapid and ultrasensitive detection of DSG3 in human HNSCC samples using nanosensors. (A) Scheme used for the ultrasensitive detection by the microuidic immunoarray showing a single sensor in the array with capture DSG3 antibodies attached. Proteins are captured off-line on Ab2-magnetic bead (MB)-HRP bioconjugates , and after magnetic separation and washes, the MBs are injected into the immunoarray containing 8 sensors. A single immunoarray sensor is depicted. Following incubation, amperometric signals are generated by applying 0.3 V versus Ag/AgCl to the sensors by injecting a mixture of HRP-activator H2O2 and mediator hydroquinone (HQ). (B) Varying recombinant DSG3 protein concentrations were used to generate a calibration plot. The sensitivity of DSG3 sensor using recombinant protein was 5646 nA mL [fg protein] 1 cm 2. (C) Protein extracts of primary human oral squamous carcinomas (T14) made with RIPA buffer were processed for detection of DSG3. High DSG3 levels were found to be present in all the samples, and this was conrmed by Western blot analysis of the same extracts for DSG3 (D). Tubulin was used as loading control. 1000 tumor cells, while the remaining had between 13,000 and 16,000 cells (Table 1). Using the nanosensors, we evaluated total protein extracts from lymph nodes for levels of DSG3. As seen in Fig. 5A, DSG3 was essentially not detected in the normal lymph nodes, with marginal values likely a reection of very limited residual non-specic binding of Ab2-MB-HRP complex, giving rise to minimal amperometric signal. In contrast, all of the positive, metastatic lymph nodes showed high levels of DSG3 protein expression. To validate these measurements, the same protein extracts were also analyzed by Western blotting (Fig. 5B). No DSG3 was detected in the negative lymph nodes, while in all the positive lymph nodes, bands for DSG3 and its multiple glycosylated forms were readily seen, and the intensity of the corresponding bands correlated with DSG3 levels quantied by the nanosensors. When total DSG3 was normalized by the number of tumor cells in each metastatic lesion, positive lymph nodes expressed approximately 150 fg DSG3 per tumor cell (Table 1), well above the threshold for DSG3 detection. Together, this indicates that the nanosensor-based detection of DSG3 could be a highly sensitive and specic method for the identication of squamous carcinoma metastases in clinical practice. This rapid method was capable of measuring DSG3 levels even from as little as a single cell, suggesting that this technique may represent a potent tool for the ultrasensitive detection of the presence or absence of lymph node invasion in human oral cancer patients. Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 99 V. Patel et al. / Oral Oncology 49 (2013) 93101 Lymphnodes(+) 1 2 2 H&E Lymphnodes(-) 1 50m 100m 100m 50m 100m 50m 100m 50m 100m 50m 100m 50m 100m 50m IF 100m 50m DAPI/Vimentin/DSG3 Lymph node (-) Lymph node (+) 1000 [DSG3], pg mL-1 1 2 3 1 2 3 100 10 DSG3 1 0.1 0.01 1 2 3 1 2 3 Tubulin Lymph node - Lymph node + Figure 5 Detection of DSG3 in metastatic human cervical lymph nodes. H&E stained cryosections of representative non-metastatic ( ) and metastatic (+) human cervical lymph nodes were scanned and the total number of tumor cells per section was quantied (Table 1). Serial sections of these lymph nodes were evaluated by immunouorescence for DSG3 and detected only in metastatic lymph nodes (green). Vimentin (red) was used to identify stromal tissue, and nuclei of all cells were stained blue with DAPI (Fig. 5A). Protein extracts made from single cryosections of lymph nodes were used for the detection of DSG3 by Western blot analysis and DSG3 quantication using nanosensors. DSG3 levels were similar to background for all non-metastatic samples, while DSG3 levels in all metastatic cases were proportional to the number of invading HNSCC cells (Fig. 5B). Table 1 DSG3 detection in metastastic lymph nodes. Cryosections of non-metastatic (N ) (n = 3) and metastatic (N+) (n = 3) human cervical lymph nodes were collected and analyzed by nanosensor detection. The total number of tumor cells per cryosection was evaluated, and used to estimate the total amount of DSG3 per tumor cell. Samples Detected DSG3 (pg/mL) Tumor cells per section Detected DSG3 (fg/tumor cell) N 1 N 2 N 3 N+1 N+2 N+3 0.01 0.03 0.02 427 6274 4512 697 13,843 16,576 202 150 90 Discussion The spread of primary HNSCC lesions to locoregional lymph nodes has often already occurred at the time of diagnosis, thus compromising the prognosis and long-term survival of HNSCC patients.3 Accurate diagnosis of lymph node metastases remains difcult, and many patients that do not present cancer dissemination to the lymph nodes (N0) may be subjected to unnecessary elective surgery. On the other hand, small lesions may be difcult to identify within the lymph nodes in cryosections when histophatologic evaluation is performed while the patient is in the operating room. Hence, some patients may miss a therapeutic opportunity due to false negative diagnosis of lymph node metastasis. Here, we demonstrate that DSG3 is expressed in normal oral squamous mucosa, and in all HNSCC lesions and their metastatic cervical lymph nodes. Indeed, the presence of DSG3 in lymph nodes can be exploited to detect Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 100 V. Patel et al. / Oral Oncology 49 (2013) 93101 micrometastatic lesions, which can serve as a sensitive marker of HNSCC progression. We also show the feasibility of using a rapid, low-cost nanostructured immunoarray device for the detection of DSG3 protein in metastatic lymph nodes in newly diagnosed HNSCC patients, which can improve diagnosis and guide the most effective therapeutic options. Most current technologies for cancer detection and diagnostics are not suitable for the differentiation of normal versus metastatic lymph nodes at early stages of cancer progression, and efforts to address this gap have been met with mixed results. Currently, the gold standard for identication of metastasis is the serial sectioning and histopathological analysis of tissue specimens by H&E staining and immunohistochemistry.30 This provides key information needed for TNM (tumor-node-metastasis criteria) classication of HNSCC patients. However, a risk remains that micrometastases may go undetected in otherwise negative lymph nodes. IHC performed on serial sections for cytokeratin may help in detecting metastases, but unfortunately this low-throughput method requires signicant investment of time and expense, and it is often performed after surgery. Considering the false-negative rate and the sampling error that are encountered by H&E examination alone, a reliable and rapid predictive test to determine lymph node metastases is needed.31 Application of new technologies such as real-time quantitative PCR (qPCR), to look at mRNA levels of molecules expressed by oral squamous tissues have shown encouraging results.32 While this improves upon some of the limitations of IHC detection of cytokeratins, independent studies have found some inconsistencies in the precise cytokeratin to be analyzed. For example, from a three-marker analysis, only cytokeratin 14 was reliably detected by qPCR in several oral cancer cell lines and tissues, and sensitive enough to detect down to a single cancer cell in a background of Jurkat cells, essentially representing a model of lymph node metastasis.33 In another study, cytokeratin 17 was demonstrated to be far superior at discriminating positive lymph nodes while cytokeratin 14 was less informative, although in parallel histological analysis, this was only achieved if metastasis had exceeded 450 lm, leaving a high probability of micrometastasis going undetected.34 While the sensitivity of qPCR for detecting cytokeratins is unquestionable, its ability to reliably and consistently detect these molecules in a single tumor cell embedded within normal lymphatic tissues still remains a challenge. The met-receptor is over-expressed in several metastatic carcinomas including HNSCC,35,36 and with minimal to none in lymphatic cells, its presence in lymph nodes may be exploited for predicting metastasis. Indeed, qPCR analysis detected met expression in 40% of invaded lymph nodes, and interestingly exceeding the sensitivity of cytokeratins, which were tested in the same sample cohort.37 Use of multiple markers may improve detection of metastatic lymph nodes, and in this regard, mRNA for DSG3 (referred to as pemphigus vulgaris antigen, PVA) and TACSTD1 (tumor-associated calcium signal transducer 1), have been previously reported to be highly expressed in HNSCC, and successfully integrated into a multiplex qRT-PCR assay for metastatic prediction, achieving a remarkable accuracy.38,39 DSG3 mRNA levels in lymph nodes have been also touted as potential predictors of HNSCC progression.38,40 Generally, the use of qPCR greatly improves the sensitivity of detection of target genes, but the need of high quality RNA extracted from tissues remains a signicant technical hurdle, such as the presence of contaminants and RNA degradation that can severely interfere with data interpretation. It is noteworthy that mRNA levels may not accurately reect protein expression, as many post translational regulatory processes may allow or prevent the accumulation of translated products, and for predictive biomarkers, the presence of the target protein may be better suited. In this regard, our proteomics analysis of HNSCC and normal oral epithelial tissues suggested that DSG3 is preferentially expressed in squamous tissues.21 By examining hundreds of cancer lesions representing some of the most prevalent human malignancies we now show that DSG3 is highly expressed in all tumors derived from cells of squamous epithelial origin, such as lung SCC and HNSCC, with more variable expression in adenocarcinomas of the colon, prostate, breast and lung, likely reecting their glandular epithelial cell of origin. For HNSCC, we noticed a lower expression of DSG3 in poorly differentiated lesions, aligned with prior reports.41 However, all HNSCC cases analyzed expressed DSG3, albeit in some lesions not all tumor cells expressed this marker. Thus, although the possibility exists that in some invaded lymph nodes the level of DSG3 may be below our detection limit, our collective ndings indicate that DSG3 is highly expressed in all human SCCs but not expressed in normal lymph nodes, and that DSG3 can be detected even in small clusters of malignant cells invading the lymph nodes, thus serving as a marker for the metastatic spread of the cancerous lesions. We now exploited this information, the availability of highly specic monoclonal antibodies detecting different epitopes in DSG3, and our recently established biomarker detection platform using microuidic immunoarray devices featuring nanostructured electrodes,26 to develop an assay system enabling the rapid and ultrasensitive detection of DSG3 protein in complex tissue extracts, with minimal non-specic binding. The method was sensitive enough to detect isolated tumor cells, and certainly small groups of cells in a single cryosection of a positive lymph node. Taken together, we can conclude that the ability to quantitate femtogram levels of DSG3 can be used for the intraoperational detection of the presence of even few invasive human squamous epithelial cells in cryosections of lymph nodes of HNSCC patients, hence aiding the pathologists and surgeons to make informed decisions about appropriate treatment options. We expect that similar approaches can be used to optimize the detection of additional cancer biomarkers in lymph node sections, thus increasing the basis for successful clinical prediction. Collectively, combined with a simple work-ow and a short assay time, these features described in this study, hold promise for the development of point-of-care clinical screening techniques to identify HNSCC patients with metastatic disease. Indeed, the encouraging results described in this proof of principle study may provide the rationale for future validation of this diagnostic strategy in larger multicenter studies. Conict of interest statement None declared. Acknowledgements This work was supported by the Intramural Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health, and through grant R01EB014586 from the National Institute of Biomedical Imaging and Bioengineering (JRF). Appendix A. 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