The outcome of peritoneal carcinomatosis, often occurring due to appendix or colorectal cancer, has dramatically improved with the combination of cytoreductive surgery (CRS) and hyperthermic (43 °C) intraperitoneal chemotherapy (HIPEC). Nevertheless, recurrence of the disease presumably due to remnant cancer cells limits survival of the patients suggesting further optimization in HIPEC treatment. Therefore, it is important to understand mechanisms operating behind HIPEC treatment. Since chemotherapy may induce immunogenic changes, we analyzed effects of MitomycinC/Doxorubicin (M/D), widely used chemotherapeutics in HIPEC settings, on the immunogenicity of colorectal cancer cells in-vitro.
Colorectal cell-lines were treated with M/D for 30 minutes with and without hyperthermia (430C). After the treatment, cells were further incubated at 370C for 72 hours. The expression of immunogenic cancer-testig antigens (CTA) was analyzed using qRT-PCR and western blot. To assess DC maturation, we set up a co-culture between differentially treated colorectal cells and monocyte-derived DC`s. We analyzed surface markers such as HLA-DR, CD 86 and CD 83 to assess DC maturation using flow cytometry. Further, the activation of cytotoxic T-cells was measured by intracellular IFN-y staining after co-culture with DC`s that were pre-incubated with treated and untreated colorectal cancer cells.
Initial qRT-PCR screening of CTA revealed that two namely, Cyclin A1 and SSX-4 were upregulated after HIPEC treatment. Compared to the control condition (no drug, 370C), M/D in HIPEC condition led to an increase of Cyclin A1 up to 53 folds and SSX-4 to 30 folds. The amount of Cyclin A1 protein was doubled compared to the control treatment (p = 0.0015, CI mean volume intensity 0.1989 – 0.4233). After co-culturing with HIPEC treated colorectal cancer cells, we noticed significant expression in CD 83, a DC activation marker. DCs that were activated upon incubation with HIPEC-treated cancer cells were able to prime cytotoxic T-cells leading to enhance IFN-y production.
HIPEC treatment can cause immunogenic changes in colorectal cancer cells. This could explain a part of the mechanism, how HIPEC treatment may work and inclusion of an immunotherapy may improve outcome of this treatment.
Kuno Lehmann.
Has not received any funding.
All authors have declared no conflicts of interest.