Characterization of the tumor microenvironment in the mouse oral cancer (MOC1) model after orthotopic implantation in the buccal mucosa

Abstract

Background: Preclinical models are invaluable for studies of head and neck cancer. There is growing interest in the use of orthotopic syngeneic models, wherein cell lines are injected into the oral cavity of immunocompetent mice. In this brief report, we describe injection of mouse oral cancer 1 (MOC1) cells into the buccal mucosa and illustrate the tumor growth pattern, lymph node response, and changes in the tumor immune microenvironment over time.

Methods: MOC1 cells were injected into the buccal mucosa of C57BL6 mice. Animals were sacrificed at 7, 14, 21, or 27 days. Tumors and lymph nodes were analyzed by flow cytometry.

Results: All mice developed tumors by day 7 and required euthanasia for tumor burden and/or weight loss by day 27. Lymph node mapping showed that these tumors reliably drain to a submandibular lymph node. The proportion of intratumoral CD8+ T cells decreased over time, while neutrophilic myeloid cells increased dramatically. Growth of orthotopic MOC2 and MOC22 also showed similar growth patterns versus published data in flank tumors.

Conclusions: When used orthotopically in the buccal mucosa, the MOC1 model induces a robust lymph node response and distinct pattern of immune cell infiltration, with peak immune infiltration by day 14.

Keywords: head and neck cancer; mouse model; oral cancer; orthotopic model; syngeneic mouse model.

Figure 1:

Photograph of buccal MOC1 tumors. A, photograph of injection into the left buccal mucosa. The anesthetized mouse is gently restrained by an assistant. The bevel of the needle is just barely inserted at the skin/mucosa junction near the oral commissure. B, A right buccal tumor has been injected with methylene blue followed by dissection of the tumor draining lymph node (TDLN), which is adjacent to the submandibular salivary gland.

Figure 2:

Natural history of the tumor, animal, and tumor draining lymph node (TDLN) after buccal inoculation of MOC1 cells. A, tumor weight in milligrams. B, animal weight in grams. C, total cells collected after mechanical digestion of the TDLN. D, Proportion of CD3+CD4+ and CD3+CD8+ T lymphocytes in the TDLN. E and F, total CD8+ T cells sorted from non-draining lymph nodes (NDLN) versus TLDN 7–10 days after MOC1 implantation in the buccal mucosa of wildtype (E) or OT-1 mice (F). Data represent mean ± standard error of the mean. In A and B, **p<0.01, ***p<0.001, ****p<0.0001 versus day 28. In E and F, *p<0.05, ***p<0.001 versus NDLN.

Figure 3:

Tumor kinetics for orthotopic MOC1, MOC2, and MOC22 models. Growth curves for each model were stopped when the first animal met endpoint criteria (tumor size or weight loss). Data are mean ± SEM, n = 5 (MOC2) – 9 (MOC1/22) animals per group.

Figure 4:

Lymphocytes patterns in the tumor evolved over time. A, Tumor infiltrating lymphocytes (TIL) including CD3+CD8+ T cells, CD3+CD4+ T cells, and CD45+NK1.1+ natural killer (NK) cells. B, surface expression of CD107a on CD8+ TIL. C, surface expression of PD-1 on CD8+ TIL. D, surface PD-L1 expression on CD45-EpCAM+ tumor cells. MFI, mean fluorescence intensity. Data represent mean ± standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus day 7.

Figure 5:

Some myeloid cell subsets also shifted over time. A, intratumoral neutrophilic (Ly6G^hiLy6C^int) and monocytic (Ly6G^neg Ly6C^hi) myeloid cells. B, ratio (log) of intratumoral CD8+ T cells to neutrophilic myeloid cells. C, intratumoral CD11b+F4/80+ macrophages, subcategorized as M1-like (CD206^lo) and M2-like (CD206^hi). D, intratumoral CD11c+F4/80- dendritic cells. Data represent mean ± standard error of the mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus day 28 or as indicated. For C, the differences in total and M1 macrophages from day 14 to day 21 were the only significant findings (p<0.05).