Sublethal exposure to alpha radiation (223Ra dichloride) enhances various carcinomas' sensitivity to lysis by antigen-specific cytotoxic T lymphocytes through calreticulin-mediated immunogenic modulation

Abstract

Radium-223 dichloride (Xofigo®; 223Ra) is an alpha-emitting radiopharmaceutical FDA-approved for the treatment of bone metastases in patients with advanced castration-resistant prostate cancer. It is also being examined clinically in patients with breast and lung carcinoma and patients with multiple myeloma. As with other forms of radiation, the aim of 223Ra is to reduce tumor burden by directly killing tumor cells. External beam (photon) and proton radiation have been shown to augment tumor sensitivity to antigen-specific CD8+ cytotoxic T lymphocytes (CTLs). However, little is known about whether treatment with 223Ra can also induce such immunogenic modulation in tumor cells that survive irradiation. We examined these effects in vitro by exposing human prostate, breast, and lung carcinoma cells to sublethal doses of 223Ra. 223Ra significantly enhanced T cell-mediated lysis of each tumor type by CD8+ CTLs specific for MUC-1, brachyury, and CEA tumor antigens. Immunofluorescence analysis revealed that the increase in CTL killing was accompanied by augmented protein expression of MHC-I and calreticulin in each tumor type, molecules that are essential for efficient antigen presentation. Enhanced tumor-cell lysis was facilitated by calreticulin surface translocation following 223Ra exposure. The phenotypic changes observed after treatment appear to be mediated by induction of the endoplasmic reticulum stress response pathway. By rendering tumor cells more susceptible to T cell-mediated lysis, 223Ra may potentially be effective in combination with various immunotherapies, particularly cancer vaccines that are designed to generate and expand patients' endogenous antigen-specific T-cell populations against specific tumor antigens.

Keywords: CTL-mediated lysis; alpha radiation; calreticulin; immunogenic modulation; radium-223.

Figure 1. ²²³Ra alpha radiation inhibits tumor proliferation, with minimal effects on cell viability

Human prostate, breast, and lung carcinoma cells were mock-irradiated (0 Gy, open bars) or treated with either 10 Gy of photon radiation (EBRT, closed bars) or 2–40 Gy of ²²³Ra (red circles). EBRT-treated cells were irradiated and then cultured for an additional 96 h. Alpha-irradiated cells were cultured with an appropriate amount of ²²³Ra so that the total cumulative dose was 2, 4, 10, or 40 Gy after 96 h. Cells were harvested, counted, and stained with AO/PI viability dye. Cell viabilities for each treatment, normalized to that of the mock-irradiated samples, are italicized. Data are representative of 2–3 independent experiments.

Figure 2. Sublethal exposure to ²²³Ra increases CTL lysis of breast, prostate, and lung carcinoma cells in vitro

(A) Tumor cells were either left untreated (0 Gy, white bars) or exposed to 4 Gy (grey bars) or 10 Gy (black bars) of ²²³Ra over a 96-h incubation period, then used as targets in an overnight CTL lysis assay. CEA-, MUC-1-, and brachyury-specific CD8⁺ T cells were used as effectors at an E:T ratio of 30:1. (B) CTL HLA restriction was verified by incubating tumor cells with anti-HLA-A2 blocking mAb (top) or by performing the killing assay with CEA⁺HLA-A2⁻ AsPC-1 cells as target controls (bottom). Experiments were repeated 1–3 times with similar results. *denotes statistical significance relative to untreated cells (P < 0.05).

Figure 3. Sublethal doses of ²²³Ra significantly increase HLA expression in various tumor types

Human prostate, breast, and lung carcinoma cells were mock-irradiated (0 Gy) or exposed to a cumulative dose of ²²³Ra totaling 4 or 10 Gy over a 96-h incubation period. Changes in HLA-ABC expression were determined by immunofluorescence imaging post-irradiation. (A) HLA-ABC expression was quantified using ImageJ software. The data presented are mean fluorescence intensity (MFI) values normalized to their respective mock-irradiated controls. (B) HLA-ABC immunofluorescence stains (green, 20× magnification). Upper right panels: 4′6-diamidino-2-phenylindole nuclear stain (blue). Lower right panels: isotype control. Data are representative of 2–3 independent experiments.

Figure 4. Tumor cells exposed to ²²³Ra have increased expression of calreticulin

Immunofluorescence imaging was used to analyze calreticulin upregulation in human prostate, breast, and lung cancer cells following exposure to ²²³Ra. (A) Calreticulin expression was quantified using ImageJ software. The data presented are MFI values normalized to their respective mock-irradiated controls. (B) Calreticulin immunofluorescence stains following a cumulative dose of either 4 or 10 Gy radiation over a 96-h treatment period (green, 20× magnification). Upper right panels: 4′6-diamidino-2-phenylindole nuclear stain (blue). Lower right panels: isotype control. Data are representative of 2–3 independent experiments.

Figure 5. ²²³Ra activates the ER stress response in LNCaP cells

(A) LNCaP cells stably transduced with a firefly luciferase ER stress reporter element were either mock-irradiated (0 Gy) or exposed to a total dose of 10 Gy ²²³Ra over a 96-h incubation period. At the indicated time points, firefly and Renilla luciferase activities were determined using a Dual Luciferase Reporter Assay System. The results are shown as the ratio of firefly luciferase activity to Renilla luciferase control. (B) Functional role of cell-surface calreticulin on CTL-mediated lysis. LNCaP cells were either mock-irradiated or exposed to 10 Gy ²²³Ra, then co-cultured with CEA-specific CD8⁺ T cells in the presence of calreticulin blocking peptide (CBP, right) or lymphocytic chriomeningitis virus control peptide (LCMV, left). (C) Schematic illustrating the immunomodulatory effects of ²²³Ra on tumor cells. *denotes statistical significance relative to untreated cells (P < 0.05).