Discussion
It is well established that increased infiltration and activation of lymphocytes into primary and metastatic colon cancer is associated with improved patient outcomes, and we have previously demonstrated that strategies to increase tumorinfiltrating lymphocytes in the tumor microenvironment can result in antitumor immune responses and tumor regression of CRLM.10,21,23-26 Currently available checkpoint blockade immunotherapies that have generated antitumor immune responses in many tumor histologies have had limited efficacy in most gastrointestinal tumors and specifically on CRLM.4,6 Therefore, new strategies to enhance lymphocyte proliferation and activation in this disease are needed. One approach to incite an antitumor immune response in colon cancer is through generation of ICD.15 We show herein that treatment of murine colorectal cancer cells with MTX generates cell cycle arrest accompanied by dynamic increases in cell surface expression of CRT, consistent with prior reports of ICD in this tumor histology. Moreover, vaccination of animals with MTX-treated colon cancer cells, as compared with vaccination with wild-type tumor cells, resulted in marked decrease in CRLM growth and significant increases in tumorinfiltrating lymphocytes. The cell cycle of CT26 cells was blocked at the G2/M phase, consistent with previous studies and most likely secondary MTX-induced DNA damage.27,28 Furthermore, previous reports have demonstrated that MTX treatment can induce multiple hallmarks of ICD including CRT translocation, ATP secretion and HMGB1 release,19,20,29 and we also detected increased expression of CRT at the cell surface after MTX treatment, confirming that the tumor cells treated with MTX have the potential of enhancing antitumor immunity.
