Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell-Mediated Killing - PubMed (original) (raw)

. 2016 May 1;95(1):120-130.

doi: 10.1016/j.ijrobp.2016.02.022. Epub 2016 Feb 13.

Anthony S Malamas 1, Michael B Bernstein 2, Kwong Y Tsang 1, April Vassantachart 2, Narayan Sahoo 2, Ramesh Tailor 2, Rajesh Pidikiti 2, Chandan P Guha 3, Stephen M Hahn 2, Sunil Krishnan 2, James W Hodge 4

Affiliations

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell-Mediated Killing

Sofia R Gameiro et al. Int J Radiat Oncol Biol Phys. 2016.

Abstract

Purpose: To provide the foundation for combining immunotherapy to induce tumor antigen-specific T cells with proton radiation therapy to exploit the activity of those T cells.

Methods and materials: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences.

Results: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibility leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells.

Conclusions: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.

Published by Elsevier Inc.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors have no potential conflicts of interest.

Figures

Figure 1

Figure 1. Exposure of distinct human tumor types to sublethal doses of photon or proton radiation significantly increases expression of histocompatibility leukocyte antigens

Human prostate (LNCaP), breast (MDA-MB-231), lung (H1703), and chordoma (JHC7) cells were mock irradiated (0 Gy) or exposed to a single dose of 8 Gy (A) photon or (B) proton radiation. After 96 h, HLA-ABC (green) expression was examined by immunofluorescence (10× magnification). Upper inset: HLA-ABC mean fluorescence intensity (MFI) normalized to controls. Middle inset: DAPI nuclear stain (blue). Lower inset: isotype control. Data are representative of 2 independent experiments.

Figure 2

Figure 2. Exposure of human carcinoma cells to sublethal doses of photon or proton radiation significantly increases sensitivity to antigen-specific CTL lysis

Human prostate (LNCaP), breast (MDA-MB-231), lung (H1703), and chordoma (JHC7) tumor cells were mock-irradiated (0 Gy; open bars) or exposed to a single dose of 8 Gy (closed bars) (A) photon or (B) proton radiation. After 96 h, cells were used as targets in a CTL-lysis assay using CEA-, MUC-1-, brachyury-, or PSA-specific CD8+ T cells as effectors. To verify that effector T cells were HLA-restricted, CTLs were incubated with CEA+, HLA-A2/A24− AsPC-1 carcinoma cells (MDA-MB-231 inset, panel A). To verify antigen specificity, PSA-specific CTLs were incubated with PSA− AsPC-1 cells exposed to 0 or 8 Gy (panel A, lower right). Results are presented as mean ± S.E.M. from 3–6 replicate wells. This experiment was repeated 1–3 times with similar results. *, statistical significance relative to controls.

Figure 3

Figure 3. Carcinoma cells recovering from exposure to photon or proton radiation have increased surface and intracellular expression of calreticulin

Calreticulin (green) expression was examined by immunofluorescence (10× magnification) 96 h after a single 8-Gy dose of (A) photon or (B) proton radiation of human prostate (LNCaP), breast (MDA-MB-231), lung (H1703), or chordoma (JHC7) cells. Upper inset: MFI normalized to that of mock-irradiated controls. Middle inset: DAPI nuclear stain (blue). Lower inset: isotype control. Data are representative of 2 independent experiments. This experiment was repeated 2 times with similar results.

Figure 4

Figure 4. Carcinoma cells recovering from exposure to photon or proton radiation show increased calreticulin expression on the cell surface, resulting in heightened sensitivity to CTL-mediated killing

Cell-surface expression of calreticulin (green) was examined by confocal immunofluorescence (10× magnification) in human prostate (LNCaP) and breast (MDA-MB-231) cells 96 h after a single dose of 8 Gy (A) photon or (B) proton radiation. Blue denotes DAPI nuclear stain. (C–D) Functional role of cell-surface calreticulin on CTL-mediated lysis. MDA-MB-231 cells were mock irradiated (0 Gy) or exposed to a single dose of 8 Gy (C) photon or (D) proton radiation. After 96 h, cells were used as targets in a CEA-specific CTL-lysis assay in the presence of calreticulin-blocking peptide (CBP, open bars) or LCMV control peptide (closed bars). Results are presented as mean ± S.E.M. from 6 replicate wells. *, statistical significance relative to controls.

Figure 5

Figure 5. Residential CSCs exposed to proton radiation harbor increased calreticulin expression on the cell surface

(A) Flow cytometry was utilized to identify the stem-like (CD133hi CD44hi, designated CSC) and non-stem-like (CD133lo CD44lo, designated non-CSC) cells in LNCaP populations 96 h after mock (0 Gy) or proton radiation (8 Gy). Insets: percentage and viability of each population. (B) Calreticulin cell-surface expression in non-CSC and CSC populations after mock (closed histograms) or proton (open histograms) radiation. (C) MDA-MB-231 stem-like (CD44+CD24−) and non-stem-like (CD44+CD24+) cell populations. Insets: percentage and viability of each population. (D) Cell-surface expression of calreticulin in non-CSC and CSC populations after mock (closed histograms) or proton (open histograms) radiation. Viability was examined using LIVE/DEAD Fixable Violet Dead Stain. Cell surface expression of markers was evaluated on live cells gated by FSC/SSC and LIVE/DEAD staining.

Comment in

References

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