Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death

Abstract

Established tumors are typified by an immunosuppresive microenvironment. Countering this naturally occurring phenomenon, emerging evidence suggests that radiation promotes a proimmunogenic milieu within the tumor capable of stimulating host cancer-specific immune responses. Three cryptic immunogenic components of cytotoxic-agent induced cell death-namely, calreticulin cell surface exposure, the release of high mobility group box 1 (HMGB1) protein, and the liberation of ATP-have been previously shown to be critical for dendritic cell (DC) activation and effector T-cell priming. Thus, these immune-mobilizing components commonly presage tumor rejection in response to treatment. We initially set out to address the hypothesis that radiation-induced immunogenic cell death (ICD) is dose-dependent. Next, we hypothesized that radiation would enhance chemotherapy-induced ICD when given concomitantly, as suggested by the favorable clinical outcomes observed in response to analogous concurrent chemoradiation regimens. Thus, we designed an in vitro assay to examine the 3 hallmark features of ICD at clinically relevant doses of radiation. We then tested the immunogenic-death inducing effects of radiation combined with carboplatin or paclitaxel, focusing on these combinations to mimic chemoradiation regimens actually used in clinical trials of early stage triple negative [NCT0128953/NYU-10-01969] and locally advanced [NYU-06209] breast cancer patients, respectively. Despite the obvious limitations of an in vitro model, radiotherapy produced both a dose-dependent induction and chemotherapeutic enhancement of ICD. These findings provide preliminary evidence that ICD stimulated by either high-dose radiotherapy alone, or concurrent chemoradiation regimens, may contribute to the establishment of a peritumoral proimmunogenic milieu.

Keywords: Immunogenic cell death; carboplatin; ionizing radiation; oxaliplatin; paclitaxel.

Figure 1. Ionizing radiation induces ATP release into the ECM. (A and B) TSA cells were stably transfected with a pGEN2.1 plasmid encoding a firefly luciferase reporter sequence flanked by a folate receptor (FR) leader sequence and glycophosphatidylinositol (GPI) anchor sequence (A, top panel). ATP remains intracellular when tumor cells are in their native condition, correlating with minimal luciferase activity. ATP release upon immunogenic cell death increases pericellular ATP that, in the presence of oxygen, magnesium, and D-luciferin, reacts with the external membrane-bound luciferase and produces photons that can be detected by luminometery (A, bottom panel). (B) The amount of luminescence detected from 2 x 10⁴ cells per well (96-well plate) pGEN2.1-pMe-Luc transfected TSA cells after 24 h of exposure to increasing doses of ionizing radiation (IR) ranging from 0–20 Gray (Gy) reported as fold-change in relative luminescent units (RLU) in comparison to the luminescent signal detected from non-irradiated cells, normalized to 1. Shown are the mean RLU (n = 8 wells/group) ± SD.

Figure 2. Radiotherapy promotes calreticulin surface translocation. (A–C) TSA cells were stably transfected with a pEZ-M02 plasmid encoding a calreticulin (CRT) fusion protein with the modular HaloTag^® reporter,and endoplasmic reticulum targeting KDEL sequences (A, top panel) corresponding to the translation of a fusion CRT-HaloTag-KDEL protein (A, middle panel). CRT remains in the ER in non-stressed cells, whereas upon immunogenic cell death (ICD) CRT translocates to the cell surface (A, bottom panel). Externally localized CRT-HaloTag-KDEL is irreversible bound by membrane impermeable HaloTag^® Alexa Fluor 488 ligand activating its fluorescent properties that can then be detected via fluorescence microscopy or flow cytometry (A, middle and bottom panels). (B and C) pEZ-M02-CRT-HaloTag-KDEL transfected TSA cells were treated for 24 h with the indicated dosage of ionizing radiation (IR, delivered at time 0 h) were exposed to the impermeable HaloTag^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via cytoflourimetric analysis. (B) Representative histograms at each dose of IR. (C) Mean fluorescent intensity (MFI) detected from cells irradiated with the indicated IR dosage vs. non-irradiated cells normalized to 1. Shown are the MFI (n = 10 000 cells/group) ± SD.

Figure 3. Radiation therapy promotes HMGB1 release. (A–C) TSA cells were stably transfected with a pCMV6-AN-RFP plasmid (A, top panel) comprising the high mobility group box 1 (HMGB1) protein coding open-reading frame sequence fused with a C-terminal red fluorescent protein (RFP) tag (A, middle panel). Under native conditions, HMGB1 remains in the nucleus, whereas HMGB1 is released from cells undergoing ICD, detectable in the conditioned medium of cultured HMGB1-RFP TSA cells via fluorimetry (A, bottom panel). (B) Chimeric HMGB1-RFP protein can be detected in the nucleus of untreated cells via confocal fluorescence microscopy. (C) Transfected cells were exposed to ionizing radiation (IR) ranging from 0–20 Gray (Gy), as indicated. Released HMGB1-RFP was detected in the conditioned medium 72 h after treatment via cytofluorimetric analysis and reported as fold change in relative fluorescent units (RFU) in comparison to untreated control cells, normalized to 1. Shown are the MFI (n = 6 wells/group) ± SD.

Figure 4. Cytotoxicity induced by platinum and ionizing radiation combinatorial treatment in TSA cells. (A and B) The differential cytotoxic effects of platinum compounds ± ionizing radiation (IR) at a dosage of 2 Gray (Gy) was evaluated via colony forming assay. TSA cells (200 cells/well in a 6-well plate; n = 6) were exposed to increasing doses of the indicated platinum agent [0–5 μM] for 48 h ± IR [2 Gy], delivered at time 0 h. After incubating the cells for 10 d, the colonies formed were fixed, stained, and subsequently counted. (A) The percent colonies formed are displayed, normalized to untreated cells (100% colony formation). (B) Corresponding images of crystal violet stained colonies. Shown are the mean colony formation ± SD. Statistical analyses were performed using a paired Student’s t test; P values < 0.05 were considered statistically significant.

Figure 5. Pericellular ATP in platinum and ionizing radiation treated TSA cells. (A and B) TSA cells transfected with pGEN2.1 vector encoding plasma membrane localized luciferase (pMe-Luc) were used to assay the effects of platinum ± ionizing radiation (IR) on pericellular ATP concentrations. The amount of luminescence detected from 2 x 10⁴ pGEN2.1-pMe-Luc transfected TSA cells/well (96-well plate) in the presence of D-luciferin. (A) Luminescence detected after 24 h of exposure to increasing doses of oxaliplatin (0–10 μM). (B) Luminescence detected after 24 h of exposure to increasing doses of the indicated platinum agent [0–5 μM] ± increasing doses of IR, ranging from 0–20 Gray (Gy) delivered at time 0 h. Values are reported as fold-change in relative luminescent units (RLU) in comparison to the luminescent signal detected from non-irradiated cells, normalized to 1. Shown are the means (n = 8 wells/group) ± SD. Statistical analyses were performed by paired Student’s t test; P values < 0.05 were considered statistically significant.

Figure 6. Calreticulin translocation to the cell surface in platinum and ionizing radiation treated TSA cells. (A and B) Externalization of calreticulin (CRT) was monitored using pEZ-M02-CRT-Halotag-KDEL stably transfected TSA breast cancer cells treated for 24 h with platinum ± the indicated dosage of ionizing radiation (IR, delivered at time 0 h) exposed to the impermeable HaloTag^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via fluorescence cytometry. (A) Representative histograms from the indicated treatment. (B) Mean fluorescent intensity (MFI) detected from cells irradiated with the indicated platinum agent ± IR vs. non-irradiated cells normalized to 1. Shown are the MFI (n = 10 000 cells/group) ± SD. Statistical analysis was performed by paired Student’s t test; P values < 0.05 were considered statistically significant.

Figure 7. HMGB1 release from platinum and ionizing radiation treated TSA cells. (A and B) Red-fluorescence protein (RFP) tagged high mobility group box 1 (HMGB1) expressing TSA cells were used to analyze the combined effect of platinum and ionizing radiation (IR) on the release of HMGB1 from TSA mammary carcinoma cells. (A) HMGB1-RFP stably transfected TSA cells were exposed to oxaliplatin (0–100 μM) or IR (0–20 Gray) delivered at time 0 h. Released HMGB1-RFP was detected in the conditioned medium 24–72 h (as indicated) after treatment via fluorimetry and reported as a fold change in relative fluorescent units (RFUs) in comparison to untreated cells (24 h). (B) HMGB1-RFP was detected in the conditioned medium of transfected cells exposed for 72 h with increasing doses of platinum (0–20 μM) ± radiation therapy (RT) at a dosage of 2 Gray (Gy) delivered at time 0 h. The RFU is plotted relative to untreated control cells, normalized to 1. Shown are the mean (n = 6/group) ± SD. Statistical analyses were performed by paired Student’s t test: P values < 0.05 were considered statistically significant.

Figure 8. Immunogenic cell death is enhanced in paclitaxel and ionizing radiation treated TSA cells. (A–D) The cytotoxic effects of paclitaxel (PTX) ± ionizing radiation (IR) were evaluated via colony forming assay and molecular markers of immunogenic cell death. (A) TSA cells were plated at 200 cells/well in a 6-well plate. After adherence, cells were exposed to 50 nM paclitaxel ± radiation therapy (RT) at a dosage of 2 Gray (Gy) delivered at time 0 h and evaluated via colony forming assay. After incubating the cells for 10 d, the colonies formed were fixed, stained, and counted. The percent colonies formed are displayed are displayed, normalized to untreated cells (100% colony formation). (B) To assay release of ATP, TSA cells stably transfected with a plasma membrane localized luciferase (pMe-Luc) plated at 2 x 10⁴ cells per well (96-well plate) were exposed to 1 μM paclitaxel ± 2 Gy IR, delivered at time 0 h. The relative luminescent units (RLUs) detected 24 h later are shown in comparison to untreated cells, normalized to 1. Shown are the mean RLU (n = 8 wells/group) ± SD (C) To assay calreticulin (CRT) externalization, CRT-Halotag-KDEL stably transfected TSA cells were treated for 24 h with 100 nM paclitaxel ± 10 Gy IR delivered at time 0 h were exposed to the impermeable HaloTag^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via fluorescence cytometry and reported as fold change in mean fluorescent intensity (MFI) vs. untreated cell levels, normalized to 1. Shown are the MFI (n = 10 000 cells/group) ± SD (D) The release of high mobility group box 1 (HMGB1) protein was assayed using red fluorescence protein (RFP)-tagged HMGB1. TSA cells stably transfected with HMGB1-RFP stably were exposed to 1 μM paclitaxel ± 2 Gy IR, delivered at time 0 h. Released HMGB1-RFP was detected in the conditioned medium 24–72 h after treatment via fluorimetry and reported as fold change in relative fluorescent units (RFU) vs. untreated cell levels, normalized to 1. Shown are the mean RLU (n = 6 wells/group) ± SD.