Development of a Novel Calreticulin Reporter for Determination of Immunogenic Cell Death

Abstract

Immunogenic cell death (ICD) initiates a series of damage associated molecular patterns (DAMPs) that are able to activate an immune response. Of these, translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the outer leaflet of the cell membrane is a hallmark event that leads to activation of antigen presenting cells (APC), thus priming an immune response. Induction of ICD in cancer cells may provide a manner of activating an immunological anti‐tumor response, which may be utilized in immunotherapy. Currently, a few chemotherapeutic drugs, such as doxorubicin, are known to induce ICD and have been proposed to be repurposed for this use. Thus, it is likely that other known drugs may exhibit ICD induction and may be utilized in immunotherapy. However, to evaluate such drugs, there is a need to develop a high throughput method of detecting ICD.Here, a novel ICD reporter based on translocation of CRT and the HiBit‐tag expression system is described. This system utilizes a recombinant HiBit‐tag that associates with cell impermeable LgBit, thereby forming a bioluminescent protein complex. HiBit‐tagged CRT will complex with LgBit when translocated to the outer leaflet of the cell membrane, which may be detected bioluminescently by addition of D‐luciferin. Thus, the intensity of bioluminescence may be correlated to translocation of CRT, a hallmark of ICD.To create the novel ICD reporter, the human CRT gene was amplified using polymerase chain reaction (PCR) from plasmid HsCD00322958 (Harvard Medical School, MA), and inserted onto the 3′ end of the HiBit tag (pBiT3.1‐N, Promega, WI) using restriction enzyme digestion (PspXI and SbfI) followed by ligation. The recombinant HiBiT‐CRT plasmid was transformed into competent DH5α E. coli cells using the heat‐shock method. The HiBit‐CRT cassette was validated by DNA Sanger sequencing (Genscript, Piscataway, NJ). In order to transfect human ovarian adenocarcinoma cells (SKOV‐3) with the ICD reporter, the optical concentration of geneticin was determined by performing a kill curve. In brief, SKOV‐3 cells were maintained in McCoy's 5A cell medium supplemented with 10 % fetal bovine serum (FBS) at 37°C and 5% CO2, and then treated with varying concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, 1 mg/mL, 1.25 mg/mL, 1.5 mg/mL, 1.75 mg/mL, 2 mg/mL). The cell viability was evaluated daily for 7 days using bright field microscopy. The optimal concentration was determined to be 1.25 mg/mL. Next, SKOV‐3 cells will be transfected with the ICD reporter, and used to screen the Approved Oncology Drug Set VI (kind gift from the National Cancer Institute) for compounds that induce ICD.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.