Objectives/Goals: Clinical relevance of preclinical animal models is commonly in question. Herein, we investigated locoregional tumor immune microenvironment (TIME) differences in tumor-bearing murine oral cancer models, unresponsive to traditional immunotherapy, and also developed an oral tumor resection model to ultimately enhance translational relevance. Methods/Study Population: Here, we utilized carcinogen-induced, HPV-negative preclinical oral cancer models. For TIME studies, ROC1 cells were maintained as published. ROC1 tumors were established in the murine flank and oral cavity of wildtype C57Bl/6 mice, and tumor growth kinetics were assessed at each site. At distinct stages of tumor growth, tumors were harvested, as well as their respective corresponding inguinal and cervical tumor-draining lymph nodes (tdLNs). Multiparameter 28-marker spectral flow cytometry was performed to analyze immune cell populations at each site. For tumor resection studies, MOC2 tumors were similarly maintained and established in the oral cavity. MOC2 tumors were accessed via midline transcervical incisions. Upon tumor excision, wounds were closed with multiple interrupted Vicryl sutures. Results/Anticipated Results: We anticipated no differences between heterotopic and orthotopic tumor sites. Both sites displayed an initial period of delayed ROC1 tumor growth followed by rapid progression. Comprehensive analyses revealed low T cell infiltration overall and increases in select myeloid cells (i.e., macrophages and dendritic cells) over time in both models. Other immune cell types, however, generally increased over time in the flank. Differences between corresponding tdLNs further indicate deviating changes in immunosuppressive phenotypes (i.e., regulatory T cells and macrophages) and immune checkpoint marker expression. Additionally, MOC2 oral tumors were successfully resected with no visible remaining tumor. No subsequent healing complications were observed, and tumor recurrence occurred within 1 week post-surgery. Discussion/Significance of Impact: Tissue-specific TIME and tdLN differences may impact antitumor treatment and response. Ability to resect orthotopic tumors allows for modeling of standard-of-care treatment for oral cancer. These studies can enable tailoring of therapeutic strategies and provide insight into model selection and data interpretation from translational studies.