Pulsed radiotherapy to mitigate high tumor burden and generate immune memory

Abstract

Radiation therapy (XRT) has a well-established role in cancer treatment. Given the encouraging results on immunostimulatory effects, radiation has been increasingly used with immune-check-point inhibitors in metastatic disease, especially when immunotherapy fails due to tumor immune evasion. We hypothesized that using high-dose stereotactic radiation in cycles (pulses) would increase T-cell priming and repertoire with each pulse and build immune memory in an incremental manner. To prove this hypothesis, we studied the combination of anti-CTLA-4 and Pulsed radiation therapy in our 344SQ non-small cell lung adenocarcinoma murine model. Primary and secondary tumors were bilaterally implanted in 129Sv/Ev mice. In the Pulsed XRT group, both primary and secondary tumors received 12Gyx2 radiation one week apart, and blood was collected seven days afterwards for TCR repertoire analysis. As for the delayed-Pulse group, primary tumors received 12Gyx2, and after a window of two weeks, the secondary tumors received 12Gyx2. Blood was collected seven days after the second cycle of radiation. The immunotherapy backbone for both groups was anti-CTLA-4 antibody to help with priming. Treatment with Pulsed XRT + anti-CTLA-4 led to significantly improved survival and resulted in a delayed tumor growth, where we observed enhanced antitumor efficacy at primary tumor sites beyond XRT + anti-CTLA-4 treatment group. More importantly, Pulsed XRT treatment led to increased CD4⁺ effector memory compared to single-cycle XRT. Pulsed XRT demonstrated superior efficacy to XRT in driving antitumor effects that were largely dependent on CD4⁺ T cells and partially dependent on CD8⁺ T cells. These results suggest that combinatorial strategies targeting multiple points of tumor immune evasion may lead to a robust and sustained antitumor response.

Keywords: immune memory; immunotherapy; lung cancer; pulsed radiation; radiotherapy.

Figure 1

(A) Experimental design for Pulsed XRT (12Gy given on days 9, 10, 15, 16) and Delayed Pulsed XRT (12Gy given on days 9, 10, 22, 23) in 344SQ-Parental murine model. (B) Experimental design for Pulsed XRT (12Gy given on days 7, 8, 14, 15) and Delayed Pulsed XRT (12Gy given on days 7, 8, 21, 22) in 344SQ-Resistant model.

Figure 2

The combination of Pulsed XRT and anti-CTLA-4 treatment prolonged survival in both 344SQ-Parental and 344SQ-Resistant murine models. Survival curves of various groups in the 344SQ-Parental (A) and 344SQ-Resistant (B) models respectivley. (C) Normalized lung metastasis counts in 344SQ-Parental. (D) Noramlized lung metastasis counts in 344SQ-Resistant. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Pulsed radiation with anti-CTLA-4 checkpoint inhibitor generated a reverse abscopal effecton primary tumors of both 344SQ-Parental and 344SQ-Resistant models. (A) Primary tumor growth curves in the 344SQ-Parental model. (B) Primary tumor growth curves in the 344SQ-Resistant model. (C) Secondary tumor growth curves in the 344SQ-Parental model. (D) Secondary tumor growth in the 344SQ-Resisatnt model. Growth lines were compared between groups using two-way ANOVA and significance was set at p < 0.05. *p < 0.05, **p < 0.01, ***p<0.001, ****p < 0.0001.

Figure 4

Flow cytometry analysis of lymphocytes for immune memory in the blood and spleen of treated mice on day 26. (A) CD4⁺ Central memory in blood as percentage and cell count. (B) CD4⁺ Effector memory in blood as percentage and cell count. (C) CD8⁺ central memory in spleen as percentage and cell count. (D) CD27⁺ CD19⁺ memory B cells in spleen as percentage and cell count. %, percentage; #, cell number; CM, Central memory; EM, Effector memory. Statistical signficance was determined between two groups using Student’s t-test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5

TCR repretoire analysis of different experimental groups. (A) Proportion of clonotypes with specific counts. The proportion of clonotypes with specific counts was higher in the pulsed XRT and anti-CTLA-4 group. DP, Delayed Pulsed XRT; P, Pulsed XRT. (B–D) Specific clones (CAVSRNNNNRIFF, p = 0.036; CAASGTGGYKVVF, p = 0.049; CATGGSNAKLTF, p = 0.053) in the T-cell receptor repertoire that were significantly high in the Pulsed XRT + α-CTLA-4 group.