Imaging calreticulin for early detection of immunogenic cell death during anticancer treatment

Abstract

Surface-exposed calreticulin (ecto-CRT) is a well-known "eat-me" signal exhibited by dying cells that contributes to their recognition and destruction by the immune system. We assessed the use of a CRT-specific binding peptide for imaging ecto-CRT during immunogenic cell death and its utility for early prediction of treatment response. Methods: A synthetic CRT-specific peptide, KLGFFKR (CRTpep), was labeled with fluorescein isothiocyanate or 18 F, and the characteristics of ecto-CRT were evaluated in a colon cancer cell line in vitro and in vivo. Results: In vitro flow cytometry, immunofluorescence staining, and in vivo small-animal PET imaging results showed that CRTpep detected preapoptotic cells treated with immunogenic drugs or radiation but not those treated with the nonimmunogenic drug or a nontherapeutic dose of immunogenic drug. Conclusion:The present results indicate that the CRT-specific peptide would enable the prediction of therapeutic response, thereby facilitating early decisions on continuation or discontinuation of immunogenic treatment.

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

“…The membranes were incubated with a CRT-specific primary antibody (1:1000 dilution; Abcam) overnight at 4 °C, followed by incubation with an HRP-conjugated secondary antibody (1:5000 dilution; Invitrogen/Thermo Fisher Scientific) for 30 min at room temperature. The ecto-CRT levels in drug-treated cells were measured and the relative levels were calculated by normalization to the levels in PBS-treated control cells using intensity ratio of each band via applying the densitometric quantifications [ 8 , 14 ].…”

“…The cultured cancer cells were grown on a glass coverslip for 48 h and treated with anticancer agents (15 µM GEM, 3 µM MTX, or 25 µM DOX) for 4 h [ 8 ]. The cells were fixed with cold acetone for 10 min and incubated with 1% bovine serum albumin (BSA) in PBS for 10 min.…”

“…CRT exposed on the cell surface of the plasma membrane (ecto-CRT) is a primitive pro-phagocytic signaling protein [ 6 ]. The cell surface expression of ecto-CRT is highly induced by chemo-, radio-, and ablative immunotherapies [ 7 , 8 ]. Expression of ecto-CRT serves as an “eat-me” signal on the cell surface, resulting in ICD.…”

“…Conversely, gemcitabine (GEM), a non-immunogenic drug, does not result in the translocation of CRT to the plasma membrane and fails to induce ICD [ 17 , 18 , 19 , 20 ]. Most importantly, ecto-CRT can also be used as a marker for the prediction of early therapeutic responses to anticancer agents [ 8 , 10 , 19 ], which has a significant impact on the development of cancer immunotherapies.…”

“…LPR binds to the NH 2 -terminal heparin-binding domain of CRT [ 43 ]. Previously, a CRT-targeting synthetic peptide, Int-α (KLGFFKR), was reported, and its dissociation constant (Kd) against CRT was 1.868 µM [ 8 , 44 , 45 ]. The CRT-binding peptide, Hep-I (GQPMYGQPMY), was also reported to target to CRT with an efficiency of 34% and 11% in the solid and soluble phases, respectively [ 43 , 46 , 47 ].…”