Tumor specific induction of surface calreticulin by doxorubicin in pancreatic ductal adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDAC) accounts for most pancreatic cancer cases. Despite advances in surgery and chemotherapy, the 5-year survival rate is only 13%. The aggressive nature of PDAC combined with delayed diagnosis highlights the need for new targeted diagnostic and treatment strategies. Calreticulin (CALR) is an endoplasmic reticular chaperone protein that can translocate to the cell surface under stress conditions such as chemotherapy. We hypothesized that systemic doxorubicin induces tumor specific surface translocation of CALR in PDAC. Methods Mouse 2838c3 PDAC cells were injected subcutaneously into mice. Once the tumors were palpable, the mice were treated with two intraperitoneal injections of doxorubicin (10 mg/kg) or saline, spaced 48 hours apart and then sacrificed. Tumors and major organs (heart, lung, kidney, pancreas, colon, small intestine, liver, and spleen) were harvested and prepared for either flow cytometry or immunofluorescence analyses. For flow cytometry, tumors and organs were dissociated into non-permeabilized, fixed single cell suspensions and stained with a fixable live/dead dye and an anti-CALR antibody. Immunofluorescence staining was performed under non-permeabilized conditions using an anti-CALR antibody and wheat germ agglutinin (WGA) membrane mask. Signal intensity was quantified in Fiji as integrated density/area. Unpaired t-test with Welch's correction was utilized for comparison. Results Doxorubicin treatment significantly increased surface CALR expression in PDAC tumors compared to saline-treated controls as measured by flow cytometry (3.0-fold increase; p < 0.05) and immunofluorescence (1.3-fold increase; 9623 AU ± 522.6 vs. 7341 AU ± 485.9, mean ± SEM, p = 0.0021). Increases in surface CALR were consistent across biological replicates. Importantly, there were no significant differences in CALR expression after doxorubicin induction in non-tumor tissues such as liver, spleen, heart, lung, kidney, pancreas, colon, or small intestine via flow cytometry or immunofluorescence analyses.