N. M. Kunda1, J. Qin1, G. Qiao1, B. S. Prabhakar2, A. V. Maker1,2 1University Of Illinois At Chicago,Division Of Surgical Oncology, Department Of Surgery, College Of Medicine,Chicago, IL, USA 2University Of Illinois At Chicago,Department Of Microbiology & Immunology, College Of Medicine,Chicago, IL, USA
Introduction:
Anti-tumor immune responses have been shown to improve outcomes in patients with advanced stage colon cancer. Immunogenic cell death (ICD) is a specific mechanism of drug-induced apoptosis that can stimulate an anti-tumor immune response through activation of specific T-cell responses. Mitoxantrone (MTX) is an anthracenedione antineoplastic agent known to trigger ICD, however, it has not been evaluated as a potential immunotherapeutic potentiator in colon cancer or its metastases.
Methods:
Murine colon cancer CT-26 cells were treated with 1 μM MTX. For cell cycle studies, cells were fixed with 70% ethanol and DNA staining was performed using DAPI. DNA content was measured with FACS and cell cycle distribution was analyzed. Treated colon cancer cells were then injected subcutaneously into the right flank of Balb/c mice on days 0 and 7. On day 14, isolated colorectal liver metastases were induced using our well-established model of intrasplenic tumor cell injection of untreated CT-26 cells. All animals were sacrificed on day 28 after establishment of liver metastases.
Results:
At clinical concentrations of MTX, the fraction of cells in the G2-M phase increased from 15.6%-38.8%, and the fraction of cells in the G1 phase decreased correspondingly from 62.6% to 32.8%, consistent with cell cycle growth arrest. The fraction of cells in the sub-G1 phase increased from 0.8% to 11.2%, consistent with DNA degradation and apoptosis. Liver metastatic tumor burden and average liver weights were significantly decreased in the MTX-treated group compared to control vaccinated animals (4.6g vs 1.8g, p<0.05). The percentages of infiltrating NK cells and activated T-cells in metastatic liver tumors were significantly higher in the MTX-treated group compared to controls (p<0.05). Splenocytes from treated animals also trended towards increased populations of CD3 cells (7.9% vs 17.9%), NK cells (3.6% vs 6.3%), & CD8 cells (24.1% vs 28.6%) compared to control-treated animals.
Conclusion:
Treatment of colon cancer cells with MTX induced cell cycle arrest, and vaccination of animals with MTX-treated colon cancer cells induced significantly increased immune cell infiltration and decreased tumor burden in distant colorectal liver metastases. Evaluation of MTX as a stimulator of immunogenic cell death and as a strategy to induce anti-tumor immunity for the treatment of advanced stage colorectal cancer is warranted.
