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Abstract
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).
Conclusions Vaccination with Lm-LLO-ISG15 significantly controlled RCC tumor burden in both subcutaneous and orthotopic models, as compared to that of controlled Lm. Interestingly, treatment with Lm-LLO-ISG15 resulted in distinct anti-tumor responses in subcutaneous versus orthotopic RCC tumors.
Listeria-based vaccines is efficacious against RCC tumorsListeria-based vaccines is efficacious against both subcutaneous and orthotopic tumors. (A) Experimental schema for subcutaneous studies. Briefly, 1×106 Renca cells in 100 µl PBS were implanted subcutaneously into the right hind flank and mice were subsequently vaccinated i.p. with Lm-based vaccines. (B) Tumor kinetic curve throughout the course of the experiment and (C) final tumor mass at the end of the experiment of both Lm-based vaccines. (D) Experimental schema for orthotopic studies. Briefly, 5×104 Renca cells expressing luciferase (Renca-luc) were implanted directly into the left kidney and mice were subsequently vaccinated i.p. with Lm-based vaccines. D-luciferin (150mg/kg, Perkin Elmer) was injected i.p. to the mice and bioluminescence signals was detected by IVIS Imaging system at 10 min post-injection. (E) The tumor kinetic curve was plotted by using total flux (photon/sec) of the region of interest (ROI). (F) Final tumor mass at the end of the experiment of both Lm-based vaccines. All statistical analysis was done with Prism 8 GraphPad software version 8.3.0., using unpaired student t-test. *p<0.05, **p<0.01. i.p. : intraperitoneal, Lm-LLO-OVA: Listeria-based vaccine targeting non-specific antigen, i.e., chicken ovalbumin. Lm-LLO-ISG15: Listeria-based vaccine targeting interferon-stimulated gene 15 (ISG15)
Distinct T cell response between s.c. and orthotopic tumorsDistinct T cell response between subcutaneous and orthotopic tumors to Lm-LLO-ISG15. Data obtained from flow cytometry analysis for subcutaneous (A-D) and orthotopic studies (E-H). Briefly, tumor tissues were harvested, and minced, and single-cell suspensions prepared. Tumor cells were stimulated with Cell Activation Cocktail kit (423303, Biolegend) for 5-6 hours and processed as normal samples. (A, C). Distribution of multi-cytokine produced by live CD4+ in subcutaneous tumors. (B, D). Distribution of multi-cytokine produced by live CD8+ in subcutaneous tumors. (E, G). Distribution of multi-cytokine produced by live CD4+ in orthotopic tumors. (F, H). Distribution of multi-cytokine produced by live CD8+ in orthotopic tumors. All statistical analysis was done with Prism 8 GraphPad software version 8.3.0., using unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001. Lm-LLO-OVA: Listeria-based vaccine targeting non-specific antigen, i.e., chicken ovalbumin. Lm-LLO-ISG15: Listeria-based vaccine targeting interferon-stimulated gene 15 (ISG15), IFN-γ: interferon-gamma, IL-2: interleukin-2, TNF-α: tumor necrosis factor-alpha
Distinct MDSC response between s.c. and orthotopic tumorsDistinct myeloid-derived suppressor cells (MDSCs) response between subcutaneous and orthotopic tumors to Lm-LLO-ISG15. Data obtained from flow cytometry analysis for subcutaneous (A-C) and orthotopic studies (D-F). Briefly, tumor tissues were harvested to prepare single cell suspension, and stained with appropriate fluorochrome-conjugated anti-mouse monoclonal antibodies. Frequency of (A). total myeloid cells, (B). m-MDSCs, and (C). pmn-MDSCs from total viable cells in subcutaneous tumors. Frequency of (D). total myeloid cells, (E). m-MDSCs, and (F). pmn-MDSCs from total viable cells in orthotopic tumors. All statistical analysis was done with Prism 8 GraphPad software version 8.3.0., using unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001. OVA: Listeria-based vaccine targeting non-specific antigen, i.e., chicken ovalbumin. ISG15: Listeria-based vaccine targeting interferon-stimulated gene 15 (ISG15), m-MDSCs: monocytic myeloid-derived suppressor cells, pmn-MDSCs: polymorphonucleic myeloid-derived suppressor cells