Background In the past few decades, subcutaneous tumors have been used extensively in vivo studies to test the therapeutic efficacy of several forms of anti-cancer agents including immunotherapy. However, the recent growing demand for preclinical models that reflect human diseases more accurately, has led to the attention of orthotopic tumor models. There is a growing body of evidence demonstrating the difference in response to immunotherapy between orthotopic and subcutaneous mouse models. Our work illustrated the distinct anti-tumor response to immunotherapy between subcutaneous versus orthotopic syngeneic mouse models of renal cell carcinomas (RCC).
Methods For subcutaneous studies, 1 × 106 Renca cells were implanted subcutaneously (s.c.) into the right hind flank and mice were subsequently vaccinated i.p. on day 5, 12, and 19 with Lm-based vaccines. For orthotopic studies, 5 × 104 Renca cells expressing luciferase (Renca-luc) were implanted directly into the right kidney as previously described and mice were subsequently vaccinated i.p. on day 5, 12, and 17 with Lm-based vaccines. All mouse experiments were performed in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) at the TTUHSC. For flow cytometry analysis, tumor tissues were harvested to prepare single-cell suspensions and stained with appropriate fluorochrome-conjugated anti-mouse monoclonal antibodies. Data were acquired on BD Fortessa and analyzed with FlowJo software version 10.7.0. All statistical analysis was done with Prism 8 GraphPad software version 8.3.0., using unpaired student t-test.
Results Listeria-based vaccines targeting interferon-stimulated gene 15, Lm-LLO-ISG15, significantly controlled tumor burden in both subcutaneous and orthotopic models as compared to that of controlled Lm (figure 1). Interestingly, while anti-tumor efficacy of Lm-LLO-ISG15 in the subcutaneous model was associated with activation of both CD8+ and CD4+ T cells, anti-tumor response in orthotopic models was mainly engaged by the activation of CD4+ T cells. The multi-cytokines producing T cells were higher in subcutaneous tumors as compared to that of orthotopic models (figure 2). In addition, treatment with Lm-LLO-ISG15 increased a higher influx of total myeloid cells as well as both monocytic- and polymorphonuclear-monocytic-derived suppressor cells (MDSCs) to the tumor microenvironment (TME) compared to Lm-LLO-OVA in subcutaneous tumors. In contrast, total myeloid cells, m-MDSCs, and pmn-MDSCs were significantly lower in Lm-LLO-ISG15 group compared to controlled Lm in orthotopic models (figure 3).
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