Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes

Abstract

Although radiotherapy (RT) is widely used to control tumor growth across many cancer types, there is a relatively high incidence of RT failure exhibited by tumor recurrence, therefore a clear need exists to achieve improved effectiveness of RT. The RT-elicited immune response largely impacts the efficacy of RT and includes immune cells that kill tumor cells, but also immunosuppressive cells, which dampen anti-tumor immunity. Using murine models in which syngeneic tumor cell lines (Colon38, Glioma261, Line1) are grown intramuscularly and treated with 15 Gy local RT, we assessed the effects of RT on both the systemic and intratumoral immune response. Here we demonstrate that RT stimulates increased production of two chemokines, CCL2 and CCL5, at the tumor site. Further, that this leads to increased CCR2+ CCR5+ monocytes in circulation and subsequently alters the intratumoral immune infiltrate favoring the largely immunosuppressive CCR2+ CCR5+ monocytes. Importantly, a CCR2/CCR5 antagonist administered daily (15 mg/kg subcutaneously) starting two days prior to RT reduces both circulating and intratumoral monocytes resulting in increased efficacy of RT in radioresponsive tumors. Overall, these data have important implications for the mechanism of RT and present a means to improve RT efficacy across many cancer types.

Keywords: chemokines; immune response; immunotherapy; myeloid cells; radiotherapy.

Figure 1. Radiotherapy increases peripheral blood IM

1x10⁵ Colon38 tumor cells were injected i.m. in C57BL/6 mice and left untreated or treated with 15 Gy radiation on day 7 of tumor growth (equivalent to day 0 on Figure 1 x-axis). A. Representative flow cytometry plots illustrating peripheral blood CCR2+ IM (arrow) from unirradiated and day 3-post RT treated mice. Plots are gated on CD45+, CD11b+ cells, and IM were shown to be Ly6G negative. B. The percentage of IM of CD45+ cells in the peripheral blood at various timepoints post-RT was determined by flow cytometry. In C. peripheral blood IM from naïve non-tumor-bearing mice + local RT (15 Gy) to leg, tumor-bearing, and tumor bearing + local RT (15 Gy) were plotted from the day 3 post-RT (day 10 of tumor growth) timepoint. # (p < 0.05) represents significance relative to naïve group and * represents significance to unirradiated tumor group as determined by ANOVA followed by a Tukey post-hoc test. n=4-10 for all groups at each time point.

Figure 2. Intratumoral IM are increased following RT

Colon38, Glioma261, and Line1 tumor cells were injected and irradiated as described in Figure 1 and the materials and methods. A. Representative dot plots of intratumoral IM (arrowed black box/circle) assessed by flow cytometry from day 4 post-RT (day 11 of tumor growth) unirradiated and irradiated tumors. Percentage of IM out of CD45+ cells are provided on plots. IM from Colon38 tumors were quantified by %IM of total CD45+ cells (B- top) and these data were normalized based on tumor size and shown as #IM/mg tumor (B- bottom). C. mRNA was isolated from Colon38 tumor homogenate and CCR2 expression was determined by RTPCR at various timepoints post-RT. D. Immunohistochemistry was performed on day 11 unirradiated and irradiated (4 days post-IR) Colon38 tumors as described in materials and methods. * (p < 0.05) represents significance as determined by t-test. n=4-8 for all groups at each time point.

Figure 3. Radiotherapy modulates various intratumoral cytokines and chemokines

Tumors were injected and irradiated as outlined in Figure 1, and mRNA from day 4 post-RT (day 11 of tumor growth) tumor homogenate was used to examine the expression of various cytokine and chemokines using an BioRad RTPCR plate array. A. A volcano plot illustrating changes in gene expression between irradiated and unirradiated tumors where red defines upregulated genes (2-fold induction), black defines no change, and green defines downregulated genes. Genes that exhibited a significant increase in expression in irradiated tumors (p<0.05) fall above the blue line and are listed in B. Significance determined by t-test. n=3 for each group.

Figure 4. Intratumoral CCL2 and CCL5 are induced by RT

Tumors were injected and irradiated as described in Figure 1. mRNA was isolated from tumor homogenate and CCL2 A. and CCL5 B. expression was determined by RTPCR at various timepoints post-RT. CCL2 C. and CCL5 D. protein expression was quantified by ELISA from day 4 post-RT (day 11 of tumor growth) tumor homogenate. * (p < 0.05) represents significance by t-test. n=4-5 for all groups at each time point.

Figure 5. A CCR2/CCR5 small molecule antagonist (CVC) reverses the radiation-induced increase of IM in peripheral blood and tumor

Tumors were injected and irradiated as described in Figure 1. Starting two days prior to irradiation, mice were treated daily with 15 mg/kg drug or vehicle control s.c. for 15 days. A. Peripheral blood was isolated from the different groups of mice on day 10 (day 3 post-RT) to assess levels of IM by flow cytometry. Day 11 (day 4 post-RT) tumors were dissociated and intratumoral IM B. or TAMs C. quantified by flow cytometry by percentage of total CD45+ immune cells (top panels) and by number of cell/mg tumor tissue (bottom panels). * (p < 0.05) represents significance as determined by t-test. n=4-6 for all groups.

Figure 6. Dual blockade of CCR2/CCR5 (CVC) improves RT efficacy in radioresponsive tumors

Tumors were injected and irradiated as described in Figure 1. Starting two days prior to irradiation, mice were treated daily with 15 mg/kg CVC (CCR2/CCR5 antagonist) or vehicle control s.c. for 15 days. Tumor growth was monitored in unirradiated along with poorly radioresponsive A. and strongly radioresponsive B. tumors. The same experiment was performed in conjunction with CD8+ T cell depletion (200 ug anti-CD8 given every 3 days starting on day 4). Untreated and anti-CD8 data from (A) is duplicated in (B) for reference. Individual growth curves (poor and strong responders combined) from both irradiated vehicle (black) and CVC (red) treated mice are presented in C, D. Percent mice with no evidence of disease (NED) was calculated when tumor size was reduced to non-tumor leg measurements between the irradiated +/- drug groups. * (p < 0.05) represents significance as determined by ANOVA followed by a Tukey post-hoc test. n=8 for both unirradiated groups; 3-4 for poor responders; 8-9 for strong responders. n=6 for anti-CD8 studies. n=12 for NED plot.