Characterization and Differentiation of the Tumor Microenvironment (TME) of Orthotopic and Subcutaneously Grown Head and Neck Squamous Cell Carcinoma (HNSCC) in Immunocompetent Mice.

For the development and evaluation of new head and neck squamous cell carcinoma (HNSCC) therapeutics, suitable, well-characterized animal models are needed. Thus, by analyzing orthotopic versus subcutaneous models of HNSCC in immunocompetent mice, we evaluated the existence of adenosine-related immunosuppressive B- and T lymphocyte populations within the tumor microenvironment (TME). Applying the SCC VII model for the induction of HNSCC in immunocompetent C3H/HeN mice, the cellular TME was characterized after tumor initiation over time by flow cytometry. The TME in orthotopic grown tumors revealed a larger population of tumor-infiltrating lymphocytes (TIL) with more B cells and CD4+ T cells than the subcutaneously grown tumors. Immune cell populations in the blood and bone marrow showed a rather distinct reaction toward tumor induction and tumor location compared to the spleen, lymph nodes, or thymus. In addition, large numbers of immunosuppressive B- and T cells were identified within the TME but also in secondary lymphoid organs, independently of the tumor initiation site. The altered immunogenic TME may influence the response to any treatment attempt. Moreover, when analyzing the TME and other lymphoid organs of tumor-bearing mice, we observed conditions reflecting largely those of patients suffering from HNSCC suggesting the C3H/HeN mouse model as a suitable tool for studies aiming to target immunosuppression to improve anti-cancer therapies.

Comprehensive Exome Analysis of Immunocompetent Metastatic Head and Neck Cancer Models Reveals Patient Relevant Landscapes.

Immunocompetent metastatic head and neck cancer (HNC) models, although scarce, can help understanding cancer progression and therapy responses in vivo. Their comprehensive genome characterizations are essential for translational research. We first exome-sequenced the two most widely used spontaneous metastatic immunocompetent models, namely AT-84 and SCC VII, followed by comprehensive genomic analyses with three prior-sequenced models (MOC2, MOC2-10, and 4MOSC2), together with patient tumors for utility assessment. AT-84 and SCC VII bear high HNC tumor resemblance regarding mutational signatures-Trp53, Fanconi anemia, and MAPK and PI3K pathway defects. Collectively, the five models harbor genetic aberrations across 10 cancer hallmarks and 14 signaling pathways and machineries (metabolic, epigenetic, immune evasion), to extents similar in patients. Immune defects in HLA-A (H2-Q10, H2-Q4, H2-Q7, and H2-K1), Pdcd1, Tgfb1, Il2ra, Il12a, Cd40, and Tnfrsf14 are identified. Invasion/metastatic genome analyses first highlight potential druggable ERBB4 and KRAS mutations, for advanced/metastatic oral cavity cancer, as well as known metastasis players (Muc5ac, Trem3, Trp53, and Ttn) frequently captured by all models. Notable immunotherapy and precision druggable targets (Pdcd1, Erbb4, Fgfr1, H/Kras, Jak1, and Map2k2) and three druggable hubs (RTK family, MAPK, and DNA repair pathways) are frequently represented by these models. Immunocompetent metastatic HNC models are worth developing to address therapy- and invasion/metastasis-related questions in host immunity contexts.

Recombinant canstatin inhibits VEGF-A-induced lymphangiogenesis and metastasis in an oral squamous cell carcinoma SCC-VII animal model.

We describe the inhibitory effects of recombinant canstatin on tumor growth and lymphangiogenesis induced by an oral squamous cell carcinoma (SCC) using an orthotropic oral SCC animal model. Recombinant canstatin treatment decreased final tumor volumes and weights, as well as densities of blood and lymphatic vessels. Lung metastasis of oral SCC was significantly reduced in recombinant canstatin-treated animals. Recombinant canstatin reduced vascular endothelial growth factor (VEGF)-A expression in SCC-VII cells treated with the hypoxia mimetic agent, CoCl2 . VEGF-A induced in vivo lymphatic vessel formation in a Matrigel plug, but this was remarkably reduced in a recombinant canstatin-treated Matrigel. Recombinant canstatin suppressed the expression of vascular endothelial growth factor receptors (VEGFR)-1 and -2 stimulated by VEGF-A. Based on immunohistochemical analysis, recombinant canstatin significantly reduced the expression of VEGF-A, VEGFR-1, and -2 in SCC-VII-induced tumors. Recombinant canstatin did not affect the expression of VEGF-C or VEGFR-3. In addition, recombinant canstatin suppressed the VEGF-A-induced phosphorylation of VEGFR-1 and -2. Our results indicate that recombinant canstatin exhibits antitumoral and antilymphangiogenic activities against oral SCC cells. Antilymphangiogenic signaling by recombinant canstatin is probably mediated by the suppression of the integrin αvß3/VEGFR-1 and/or -2 signaling induced by VEGF-A. Our results also suggest that recombinant canstatin has a high potential to inhibit oral SCC-induced tumors and lymphatic metastasis.

Development of a squamous cell carcinoma mouse model for immunotoxicity testing.

An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds. Thus, there is a need for alternative testing strategies. A novel 3-stage tumor model in syngeneic C3H/HeN mice was evaluated here to study the effects of immunosuppressive drugs on tumor promotion and progression in vivo. The model employed a skin squamous cell carcinoma cell line (SCC VII) due to the increased prevalence of squamous cell carcinoma (SCC) in humans associated with immunosuppression after transplants. Local invasion, colonization and tumor progression were evaluated. The validation set of immunosuppressive drugs included: Cyclosporin (CSA), cyclophosphamide (CTX), azathioprine, etanercept, abatacept and prednisone. Local invasion was evaluated by histological assessment as well as fluorescence trafficking from Qdot(®)-labeled tumor cells from the site of inoculation to the draining lymph node. Colonization was evaluated by lung colony counts following intravenous inoculation. Tumor progression was assessed by morphometric analysis of lesion area, angiogenesis and growth fraction of established metastatic neoplasia. Immunosuppressive drugs in the validation set yielded mixed results, including decreased progression. The methods and results described herein using an in vivo syngeneic mouse tumor model can provide insight about the assessment of immunosuppressive drugs in carcinogenicity risk assessment.