Introduction: Emerging evidence suggests that tumor extracellular matrix (ECM) may play a role in tumor-immune interactions. Breast tumors with high immune infiltrates have a distinct ECM profile, and T cell exclusion has been linked to specific ECM signatures. Despite this evidence suggesting a link between immune infiltrates and tumor matrix, it remains unclear whether ECM can directly affect the ultimate step in tumor clearance by the immune system, T cell mediated cytotoxicity.
Methods: We compared clearance of 4T1 mammary gland carcinoma cells (MCC) seeded on ECM arrays by T cells isolated from spleens of MHC mismatched strain of mice. Briefly, 4T1 were seeded at 10k/ml for 1 hour, cultured for 24 hours then cocultured with T cells for 2 hours before fixing and staining. For RNA sequencing, ECM proteins (Collagen 1 -Col1, Collagen 4- Col4, Fibronectin -Fn or Vitronectin- Vtn) were coated onto plates at 250ug/ml, then 4t1 were added for 24 hours, then T cells were added for 24 hours followed by RNA isolation and sequencing.
Results: We compared number of cells per spot with and without T cells across all ECM combinations and found that co-culture with T cells reduced the average number of MCCs, but this difference did not reach statistical significance. Only in the following conditions did MCC number significantly decrease: Col1 alone, Col6 alone, Fn alone, Vtn alone and Col6+ Eln (Fig 2B). In Col4 containing conditions, MCC cell number increased in the presence of T cells. Intensity of CD274 (PD-L1) and the MHC class 1 protein H2-Kd varied with substrate (p<10−19, p<10−22 respectively) with significantly higher expression of PD-L1 in Col1 and Vtn conditions vs. Col4 or Laminin, and higher H2-Kd in Vtn conditions. These findings demonstrate a defect in T cell mediated MCC clearance in some ECM conditions that is distinct from the PD-L1 checkpoint. Comparing transcriptomes across, we observed that all MCC+ T cell conditions separated from MCC alone conditions (Fig. 3B), largely due to expression of known T cell related genes (such as Ptprc, Trbc2, Sell, Itk, and Il7r). Differentially regulated gene counts between MCC+ T cells and MCC alone conditions were lowest in the Col4 condition (Fig. 3BC-E), and significance and number of genes from T cell associated ontologies were lowest in the Col4 conditions (Fig. 3E). We observed that MCC on Col4 upregulated cytokines including Ccl2, Cxcl3, Cxcl10, and Tgfβ2, compared to both Fn and Vtn conditions, suggesting that this condition could suppress immune activation through altered cytokine expression.
This work was funded by LDRD 19-SI-003 under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. LLNL-ABS-8235222
Citation Format: Claire Robertson, Aimy Sebastian, Aubree Hinckley, Naiomy Rios-Arce, William Hynes, Wei He, Nicholas Hum, Elizabeth Wheeler, Gabriela Loots, Matthew Coleman, Monica Moya. Extracellular matrix modulates T cell clearance of malignant cells in vitro [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P043.