Cancer Res Treat.  2023 Jan;55(1):167-178. 10.4143/crt.2022.094.

Molecular and Immune Profiling of Syngeneic Mouse Models Predict Response to Immune Checkpoint Inhibitors in Gastric Cancer

• Lee D
• Choi J
• Oh HJ
• Ham IH
• Lee SH
• Nomura S
• Han SU
• Hur H

• KMID: 2538002
• DOI: http://doi.org/10.4143/crt.2022.094

Abstract

Purpose
Appropriate preclinical mouse models are needed to evaluate the response to immunotherapeutic agents. Immunocompetent mouse models have rarely been reported for gastric cancer. Thus, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) in immunocompetent mouse models using various murine gastric cancer cell lines.
Materials and Methods
We constructed subcutaneous syngeneic tumors with murine gastric cancer cell lines, YTN3 and YTN16, in C57BL/6J mice. Mice were intraperitoneally treated with IgG isotype control or an anti–programmed death-ligand 1 (PD-L1) neutralizing antibody. We used immunohistochemistry to evaluate the tumor-infiltrating immune cells of formalin-fixed paraffin-embedded mouse tumor tissues. We compared the protein and RNA expression between YTN3 and YTN16 cell lines using a mouse cytokine array and RNA sequencing.
Results
The mouse tumors revealed distinct histological and molecular characteristics. YTN16 cells showed upregulation of genes and proteins related to immunosuppression, such as Ccl2 (CCL2) and Csf1 (M-CSF). Macrophages and exhausted T cells were more enriched in YTN16 tumors than in YTN3 tumors. Several YTN3 tumors were completely regressed by the PD-L1 inhibitor, whereas YTN16 tumors were unaffected. Although treatment with a PD-L1 inhibitor increased infiltration of T cells in both the tumors, the proportion of exhausted immune cells did not decrease in the non-responder group.
Conclusion
We confirmed the histological and molecular features of cancer cells with various responses to ICI. Our models can be used in preclinical research on ICI resistance mechanisms to enhance clinical efficacy.

Keyword

Immune checkpoint inhibitors; Stomach neoplasms; Macrophages; Syngeneic mouse; Tumor immunity

Figure

• 

  Fig. 1 Histological features of syngeneic mouse tumors. (A) 1×106 of YTN3 (n=10) and YTN16 (n=5) cells were subcutaneously injected into C57BL/6J mice. (B) Tumor volume and weight of YTN3 (top) and YTN16 (bottom) tumors. Values are presented as mean±standard error of the mean (SEM). (C) Representative images of H&E staining and immunohistochemistry for α-smooth muscle actin (α-SMA) (×200, scale bars=50 μm). (D) Representative images for immunohistochemistry; CD8α for cytotoxic T cell, F4/80 for macrophage, EOMES for exhaustion marker, and programmed death-ligand 1 (PD-L1) for immune checkpoint (×200, scale bars=50 μm). (E) Immunohistochemistry staining. Positive cells were counted per mm2. **p < 0.01, ****p < 0.0001.

• 

  Fig. 2 Molecular characteristics of YTN3 and YTN16 murine gastric cancer cell lines. (A) Schematic figure for RNA-sequencing and mouse cytokine array workflow. (B) Gene ontology (GO) biological process analysis for differentially expressed genes (|fold-change| ≥ 2, p < 0.05) using DAVID database. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Mouse cytokine array panels for conditioned media of YTN3 and YTN16 cells (left). Mean pixel density was quantified for each target protein (right). (D) The expression of genes coding cytokine array–targeted proteins from RNA sequencing. *p < 0.05, ***p < 0.001.

• 

  Fig. 3 Response to programmed death-ligand 1 (PD-L1) inhibitor in syngeneic mouse tumors. (A) The schematic figure for anti–PD-L1 antibody treatment in vivo. 1×106 of YTN3 (n=15) and YTN16 (n=16) cells were subcutaneously injected in C57BL/6J mice. IgG isotype control or anti–PD-L1 antibody (12.5 mg/kg) was administered once a week, intraperitoneally. (B) Subcutaneous tumors of YTN3 and YTN16 cell lines after anti–PD-L1 antibody treatment. (C) Tumor volume and weight of YTN3 (top) and YTN16 (bottom) tumors. Values are presented as mean±standard error of the mean (SEM). **p < 0.01; ns, not significant.

• 

  Fig. 4 Reprograming of tumor microenvironment after programmed death-ligand 1 (PD-L1) inhibitor treatment. (A) Representative images for immunohistochemistry; CD8α, CD4, and EOMES (×200, scale bars=50 μm). (B) Immunohistochemistry. Positive cells were counted per mm2. The percentage of exhausted immune cells is represented as a percentage of EOMES+ cells to CD8α+ or CD4+ cells. ***p < 0.001.

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