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Case Reports
. 2020 Oct;8(2):e000537.
doi: 10.1136/jitc-2020-000537.

Low-dose radiation treatment enhances systemic antitumor immune responses by overcoming the inhibitory stroma

Hampartsoum B Barsoumian  1 , Rishab Ramapriyan  1 , Ahmed I Younes  1 , Mauricio S Caetano  1 , Hari Menon  1 , Nathan I Comeaux  1 , Taylor R Cushman  1 , Jonathan E Schoenhals  1 , Alexandra P Cadena  1 , Timothy P Reilly  2 , Dawei Chen  1 , Fatemeh Masrorpour  1 , Ailin Li  1 , David S Hong  3 , Adi Diab  4 , Quynh-Nhu Nguyen  1 , Isabella Glitza  4 , Renata Ferrarotto  5 , Stephen G Chun  1 , Maria Angelica Cortez  1 , James Welsh  6
Affiliations
Case Reports

Low-dose radiation treatment enhances systemic antitumor immune responses by overcoming the inhibitory stroma

Hampartsoum B Barsoumian et al. J Immunother Cancer. 2020 Oct.

Abstract

Background: Despite some successes with checkpoint inhibitors for treating cancer, most patients remain refractory to treatment, possibly due to the inhibitory nature of the tumor stroma that impedes the function and entry of effector cells. We devised a new technique of combining immunotherapy with radiotherapy (XRT), more specifically low-dose XRT, to overcome the stroma and maximize systemic outcomes.

Methods: We bilaterally established 344SQ lung adenocarcinoma tumors in 129Sv/Ev mice. Primary and secondary tumors were irradiated with either high-dose or low-dose of XRT with systemic anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte associated protein 4 administration. Survival and tumor growth were monitored for the various groups, and secondary tumors were phenotyped by flow cytometry for immune populations. Tumor growth factor-beta (TGF-β) cytokine levels were assessed locally after low-dose XRT, and specific immune-cell depletion experiments were conducted to identify the major contributors to the observed systemic antitumor effect.

Results: Through our preclinical and clinical studies, we observed that when tumor burden was high, there was a necessity of combining high-dose XRT to 'prime' T cells at the primary tumor site, with low-dose XRT directed to secondary (metastatic) tumors to 'modulate the stroma'. Low-dose XRT improved the antitumor outcomes of checkpoint inhibitors by favoring M1 macrophage polarization, enhancing natural killer (NK) cell infiltration, and reducing TGF-β levels. Depletion of CD4+ T cells and NK cells abrogated the observed antitumor effect.

Conclusion: Our data extend the benefits of low-dose XRT to reprogram the tumor environment and improve the infiltration and function of effector immune cells into secondary tumors.

Keywords: immunotherapy; lung neoplasms; radioimmunotherapy; radiotherapy; tumor microenvironment.

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Conflict of interest statement

Competing interests: JW serves on the science advisory boards for Alpine Immune Sciences, RefleXion Medical, Mavu Pharma, MolecularMatch and Checkmate Pharmaceuticals, and he is the founder of Healios Oncology, MolecularMatch, and OncoResponse companies. MD Anderson Cancer Center has a trademark for RadScopal (TM). JW has research support collaborations with Bristol Myers Squibb, Checkmate Pharmaceuticals, Mavu pharma, Merck, and Nanobiotix. AD is an advisory board member for Nektar Therapeutics. IG is a consultant for Bristol Myers Squibb and Array Biopharma. DSH has consulting/advisory role in Alpha Insights, Axiom, Adaptimmune, Baxter, Bayer, Genentech, GLG, Group H, Guidepoint Global, Infinity, Janssen, Merrimack, Medscape, Numab, Pfizer, Seattle Genetics, Takeda, Trieza Therapeutics. DSH has research/grant funding from AbbVie, Adaptimmune, Amgen, AstraZeneca, Bayer, Bristol Myers Squibb, Daiichi-Sankyo, Eisai, Fate Therapeutics, Genentech, Genmab, Ignyta, Infinity, Kite, Kyowa, Lilly, LOXO, Merck, MedImmune, Mirati, MiRNA, Molecular Templates, Mologen, NCI-CTEP, Novartis, Pfizer, Seattle Genetics, Takeda. DSH's ownership interests include MolecularMatch (Advisor), OncoResponse (founder), Presagia Inc (Advisor).

Figures

Figure 1
Figure 1
L-XRT hampers tumor growth, activates T cells, increases NK cells and M1 macrophages, and downregulates the inhibitory cytokine TGF-β. (A), The one-tumor mouse model was established by injecting 344SQ-P cells (0.5 × 106) into the right hind legs of 129Sv/Ev mice (n=5/group), after which L-XRT (1 Gy × 2 fractions) was delivered when tumors reached around 7–8 mm in diameter. Anti-PD1 (200 µg/inj.) and anti-CTLA-4 (50 µg/inj.) were administered intraperitoneally on days 5, 8, 12, and 16 post 344SQ-P injection. Mice were euthanized when tumor reached 14 mm, and survival was plotted by the Kaplan-Meier method. Experiment was repeated twice, and similar patterns were detected. (B and C) dLNs from the L-XRT dose groups were harvested at 3 days after radiation and analyzed with flow cytometry. Both CD4 (No XRT vs 1 Gy × 2, p=0.0318) and CD8 cells (No XRT vs 1 Gy × 2, p=0.0001) were activated after two 1 Gy fractions, as depicted by the activation marker CD44. (D and E), spleens (spl), dLNs, and TILs were harvested at 48 hours after two 1 Gy fractions for phenotyping by flow cytometry. (D) Cells were gated on the CD45+ population and then on CD49b+ to identify NK cells (TILs no XRT vs TILs 1 Gy × 2, p=0.0328). (E) Cells were gated on CD45+ and then on Gr1intermediate and CD11b+ to identify macrophages, and further gated on F4/80+ CD38hi to identify M1 macrophages (No XRT vs 1 Gy × 2, p=0.0463). (F) NanoString molecular counts (nCounter immunology panel) showed significant reduction in local TGF-β expression 24 hours after two 1 Gy fractions of L-XRT (p=0.0007). Data for each group was represented as mean±SD and Student’s t-tests were used to compare groups. *p≤0.05 was considered to indicate statistical significance. dLNs, draining lymph nodes; L-XRT, low-dose radiation; PD1, programmed cell death protein 1; TGF-β, tumor growth factor beta; TILs, tumor-infiltrating leukocytes.
Figure 2
Figure 2
H-XRT to the primary tumor plus L-XRT to the secondary tumor (RadScopal technique) plus double-agent immunotherapy is required for optimal systemic antitumor outcomes and reduction of TGF-β at secondary sites. (A) Treatment timeline. Primary tumors were injected subcutaneously on day 0 and secondary tumors injected on day 4. H-XRT (three 12 Gy fractions) was delivered to primary tumors on days 7, 8, and 9; L-XRT (two 1 Gy fractions) was delivered to secondary tumors on days 12 and 13. Double-agent immunotherapy (anti-PD1 + anti-CTLA-4) was given intraperitoneally on days 5, 8, 12, and 16. (B) Survival curves for the indicated radioimmunotherapy treatment groups. ‘RadScopal’ refers to H-XRT to the primary and L-XRT to the secondary tumors. Experiment was repeated twice and data were pooled. Median survivals following the order of group labels were (23, 30, 34, 35.5, 31, N/A, 35, and 35 days post tumor inoculation, respectively). (C) Tumor growth curves for primary (1°) and secondary (2°) tumors after the indicated treatments. Mice were euthanized when either the primary or secondary tumors reached 14 mm in diameter. (D) Secondary tumors were harvested on day 14, 1 day after delivery of the second L-XRT fraction. TILs were enriched by Histopaque separation and RNA was isolated for RT-PCR with TGF-β primers and peptidylprolyl isomerase A (PPIA) controls. Delta–delta CT values were calculated and TGF-β expression was reported using 2−ΔΔCT values. (E) Systemic TGF-β levels were assessed by serum BioPlex assay on day 16. *p≤0.05 was considered statistically significant. H-XRT, high-dose radiation; L-XRT, low-dose radiation; PD1, programmed cell death protein 1; PPIA, peptidylprolyl isomerase A; TGF-β, tumor growth factor beta; TILs, tumor-infiltrating leukocytes.
Figure 3
Figure 3
H-XRT to the primary tumor plus L-XRT to the secondary tumor is effective in other solid tumor models, is immune-mediated, and depends on CD4 T cells and NK cells. (A) The two-tumor LLC model was established in C57BL/6 mice (n=5/group). Survival curves are shown for the indicated groups, with log-rank tests used to compare groups (RadScopal vs control, p=0.0047; RadScopal vs RadScopal + anti-PD1 + anti-CTLA-4, p=0.0198). (B) In a two-tumor nu/nu mouse model lacking functional T cells (n=4/group), neither H-XRT + anti-PD1 nor RadScopal + anti-PD1 showed any efficacy. (C) A two-tumor 344SQ-P model was established in 129Sv/Ev mice, which were then given anti-CD4, anti-CD8, or anti-NK antibodies to deplete the corresponding immune cells. Survival was plotted by the Kaplan-Meier method and log-rank tests used to compare groups. *p≤0.05 was considered statistically significant. H-XRT, high-dose radiation; LLC, Lewis lung carcinoma; L-XRT, low-dose radiation; NK, natural killer; PD1, programmed cell death protein 1.
Figure 4
Figure 4
H-XRT to the primary tumor plus L-XRT to the secondary tumor plus anti-CTLA-4 and anti-PD1 checkpoint inhibitors increase CD4+ T-effector cells, enhance NK cell function, and increase M1 macrophage polarization in secondary tumors. On day 19, we isolated TILs from the secondary tumors of the two-tumor 129Sv/Ev mouse model to phenotype lymphoid and myeloid subpopulations by flow cytometry (n=5–6/group). (A) Percentages of CD4+ T cell lymphocytes. (B) Percentages of Treg lymphocytes. (C) Percentages of CD49+ NK cells producing granzyme B in the lymphocyte population. (D) Total numbers of CD49+ granzyme B+ NK cells per mg of tumor weight. (E) Percentages of DCs from leukocytes. (F) Total numbers of DCs per mg of tumor weight. (G) Percentages of F4/80+ CD38hi M1 TAMs in the Gr1intermediate CD11b+ population that was gated on the CD45+ parent population. (H) Total numbers of M1 TAMs per mg of tumor weight. (I) Percentages of F4/80+ CD206+ M2 TAMs in the Gr1intermediate CD11b+ population that was gated on the CD45+ parent population. (J) Total numbers of M2 TAMs per mg of tumor weight. Data for each group was represented as mean±SD. One-way ANOVA statistical analysis followed by Tukey’s multiple comparison tests were conducted to compare the multiple treatment groups. p≤0.05 was considered to indicate statistical significance. *p≤0.05, **p≤0.01, ***p≤0.001. ANOVA, analysis of variance; DCs, dendritic cells; H-XRT, high-dose radiation; L-XRT, low-dose radiation; NK, natural killer; PD1, programmed cell death protein 1; TAMs, tumor-associated macrophages; TILs, tumor-infiltrating leukocytes; Tregs, T regulatory cells.
Figure 5
Figure 5
Prospective treatment of secondary tumors with L-XRT shows significant therapeutic benefit in patients. (A) Scans from a 54-year-old male with metastatic human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma refractory to previous treatment with anti-PD1 therapy. A single lesion in the upper left lobe of the lung was treated with H-XRT (four 12.5 Gy fractions) and a large abdominal lesion was concurrently treated with L-XRT (four 1.5 Gy fractions) and ongoing pembrolizumab (anti-PD1). A PET scan obtained 6 months after L-XRT showed a decrease of more than 50% in the volume of the abdominal lesion with near-complete resolution of avidity. (B) Spider plot depicting tumor response curves of secondary lesions that received L-XRT in nine patients who received concurrent H-XRT to primary tumors and systemic anti-PD1 or anti-PD-L1 immunotherapy. H-XRT, high-dose radiation; L-XRT, low-dose radiation; PD, progressed disease; PD1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; PET, positron emission tomography; PR, partial response.
Figure 6
Figure 6
Illustration of the proposed effects from ‘RadScopal’ therapy. At the primary tumor site, high-dose stereotactic radiation (H-XRT) releases tumor-associated antigens and upregulates MHC-I molecules to initiate T cell priming. APCs then present those neoantigens to CD4+ and CD8+ T cells in the dLNs for further activation. At the secondary (abscopal) tumor site, L-XRT modulates the stroma to overcome its inhibitory barriers. Pronounced mechanisms involve M1 macrophage polarization, downregulation of TGF-β, activation and infiltration of CD4+ T cells, and increases in proportion and activity of NK cells. The addition of double-agent checkpoint-inhibitor immunotherapy (anti-PD1 plus anti-CTLA-4) further augments the systemic effects of the radiation by blocking Tregs and attenuating T cell exhaustion, thereby prolonging and enhancing the antitumor response. APCs, antigen-presenting cells; dLNs, draining lymph nodes; L-XRT, low-dose radiation; PD1, programmed cell death protein 1; TGF-β, tumor growth factor beta; Tregs, T-regulatory cells.
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