Imaging Calreticulin for Early Detection of Immunogenic Cell Death During Anticancer Treatment

Expression of preapoptotic markers and CRT in CT26 cells after immunogenic treatment. CT26 cells were treated with oxaliplatin (500 μM), doxorubicin (25 μM), mitoxantrone (3 μM), or gemcitabine (15 μM) for 0, 1, 2, and 4 h. (A) Levels of preapoptotic proteins pPERK, pEIF2α, and Cas-8 were analyzed by Western blotting. (B) Expression of preapoptotic-related markers pPERK, pEIF2α, and Cas-8 in CT26 cells was quantified using densitometry analysis of Western blots after treatment for 0, 1, 2, and 4 h with oxaliplatin, doxorubicin, mitoxantrone, or gemcitabine. Relative expression was calculated to respective control using Student t test, and level of expression was expressed as mean of 3 independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001. (C) Western blot analysis of translocated CRT (ecto-CRT) in plasma membrane and total CRT in CT26 cell lysates with and without immunogenic treatment. CT26 cells were treated with or without immunogenic (oxaliplatin, doxorubicin, or mitoxantrone) and nonimmunogenic (gemcitabine) drugs for 0, 1, 2, or 4 h. DXR = doxorubicin; GEM = gemcitabine; MTX = mitoxantrone; OXP = oxaliplatin; PMP = plasma membrane protein; TP = total protein.

Flow cytometry analysis of CRTpep binding to ecto-CRT after immunogenic and nonimmunogenic drug treatment in CT26 cells. (A) Flow cytometry analysis of CRTpep binding to ecto-CRT after immunogenic and nonimmunogenic drug treatment in CT26 cells. Binding of FITC-CRTpep to ecto-CRT in CT26 cells after 2 and 4 h of anticancer drug (immunogenic and nonimmunogenic) treatment was determined by flow cytometry. Percentage cellular uptake was based on detected mean fluorescence levels of untreated control cells. After anticancer drug treatment, CT26 cells were preincubated with CRTpep (200 μM) for 1 h, followed by incubation with FITC-CRTpep (2 μM), and then were subjected to flow cytometry to detect uptake using fluorescence generated by ecto-CRT. (B) Quantitative assessment of binding of FITC-CRTpep to ecto-CRT in CT26 cells after 2 and 4 h of anticancer drug (immunogenic and nonimmunogenic) treatment that was determined by flow cytometry. Data are mean (±SD) fluorescence level. ***P < 0.001 (n = 3). DXR = doxorubicin; GEM = gemcitabine; MTX = mitoxantrone; ns = not significant; OXP = oxaliplatin.

Immunofluorescence staining and analysis of CRTpep binding to ecto-CRT after immunogenic and nonimmunogenic drug treatment in CT26 cells. Binding of FITC-CRTpep to ecto-CRT in CT26 cells after 2 and 4 h of anticancer drug (immunogenic and nonimmunogenic) treatment was determined by confocal laser scanning microscopy (×40 magnification) after immunofluorescence staining. Green = FITC-CRTpep; blue = 4′,6-diamidino-2-phenylindole–stained nuclei; red = cell membrane stained with wheat germ agglutinin 555. (Scale bar = 50 μm.) For blocking assay, anticancer drug–treated cells were further incubated with 200 μM unlabeled CRTpep followed by 2 μM FITC-CRTpep. (Scale bar = 50 μm.) DAPI = 4′,6-diamidino-2-phenylindole; DXR = doxorubicin; GEM = gemcitabine; MTX = mitoxantrone; OXP = oxaliplatin; WGA = wheat germ agglutinin.