Abstract

Background/Objectives: Preclinical studies of head and neck squamous cell carcinoma (HNSCC) commonly use subcutaneous heterotopic (flank) tumor models for simplicity; however, orthotopic models may better reflect the native tumor environment. Direct comparisons of the tumor immune microenvironments (TIME) and tumor-draining lymph nodes (tdLNs) between these models remain limited. Better understanding of site-specific immune differences could improve model selection and interpretation of translational HNSCC studies. Methods: ROC1 tumors were established in murine heterotopic and orthotopic sites, followed by assessment of tumor growth kinetics, survival, and the tumor microenvironment. Immune composition of tumors, blood, tdLNs, and spleen was evaluated at three tumor progression timepoints using multiparameter spectral flow cytometry. Results: Heterotopic and orthotopic tumor models showed similar growth kinetics and survival. Immune profiling revealed increased infiltration of CD3+ T-cells, natural killer (NK) cells, and myeloid populations in both models. Heterotopic tumors were enriched in dendritic cells (DCs), plasmacytoid DCs, and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas orthotopic tumors showed increased macrophages, granulocytic MDSCs, and M-MDSCs. Despite temporal variation, both TIMEs were dominated by macrophages, DCs, and CD3+ T-cells. Late-stage heterotopic tumors contained more CD4+ T-cells. Reduced T-cell cytotoxicity (PD-1, CD107a) and increased immune checkpoint expression across myeloid cells indicated an immunosuppressive TIME. Systemically, effector cells were preserved despite suppressive cell trafficking, and tdLNs in both models exhibited immunosuppressive PD-L1 expression. Conclusions: Heterotopic and orthotopic ROC1 tumors share key immune features, but site-specific differences in the TIME and tdLNs reveal tissue-dependent regulation. These local effects align with systemic changes, supporting global tumor-associated immunosuppression.

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