Comparative study of immune response to local tumor destruction modalities in a murine breast cancer model

Abstract

Introduction: Immunotherapy is revolutionizing the management of multiple cancer types. However, only a subset of patients responds to immunotherapy. One mechanism of resistance is the absence of immune infiltrates within the tumor. In situ vaccine with local means of tumor destruction that can induce immunogenic cell death have been shown to enhance tumor T cell infiltration and increase efficacy of immune checkpoint blockade.

Methods: Here, we compare three different forms of localize tumor destruction therapies: radiation therapy (RT), vascular targeted photodynamic therapy (VTP) and cryoablation (Cryo), which are known to induce immunogenic cell death, with their ability to induce local and systemic immune responses in a mouse 4T1 breast cancer model. The effects of combining RT, VTP, Cryo with anti-PD1 was also assessed.

Results: We observed that RT, VTP and Cryo significantly delayed tumor growth and extended overall survival. In addition, they also induced regression of non-treated distant tumors in a bilateral model suggesting a systemic immune response. Flow cytometry showed that VTP and Cryo are associated with a reduction in CD11b+ myeloid cells (granulocytes, monocytes, and macrophages) in tumor and periphery. An increase in CD8+ T cell infiltration into tumors was observed only in the RT group. VTP and Cryo were associated with an increase in CD4+ and CD8+ cells in the periphery.

Conclusion: These data suggest that cell death induced by VTP and Cryo elicit similar immune responses that differ from local RT.

Keywords: breast cancer; cryoablation; immune checkpoint blockade; radiation therapy; vascular photodynamic therapy.

Figure 1

RT, VTP and Cryoablation elicit anti-tumor responses in 4T1 breast cancer. (A) Experiment schema of the comparative study with with RT, VTP and Cryo. 2 x 10⁵ 4T1 mouse breast cancer cells were injected subcutaneously in the hind limb of 6-8 week female Balbc mice (n=10 mice per group). 12 days later, mice were treated with RT, VTP or Cryo therapy. For combinations with anti-PD-1, mice were injected intraperitoneally with anti-PD-1 antibody every 3 days for a total of 3 doses. (B) Comparison average tumor growth and (C) overall survival of 4 cohorts relative to the control. (D) Comparison of the average tumor growth +/- SEM and overall survival for individual cohorts with and without anti-PD1 are shown. Statistics were calculated using student T-tests on day 30 for tumor growth and the Logrank method for survival: *p ≤ 0.05, ***p ≤ 0.005.

Figure 2

Activation of Myeloid cells in the tumor of animals treated with localized therapies. 2 x 10⁵ 4T1 mouse breast cancer cells were injected subcutaneously in the right hind limb of 6-8 week female Balbc mice (n=5 mice/group). 12 days later when the tumors reached 50-60mm2 in size, mice were treated with 15 Gy radiation (RT), VTP or Cryo therapy according to the doses listed in Figure 1A and methods. (A) Experiment schema of timeline of tumor challenge, treatment dates and tissue harvest for flow cytometry. (B) bivariate plot to show the gating strategy for total immune infiltrates in the tumors (live CD45⁺) and fold change in The frequencies of CD45⁺ cells of each treatment group represented at fold change over the control in the tumor at the indicated time points. (C) Heatmaps representing the log10 fold change (Log FC) of the immune cell populations in the tumor. (D) Pearson correlations of tumor burden (weight (g)) vs. frequencies of myeloid cells (as a % of CD45⁺ cells) from experiments outlined in (A). (E) Histogram plots showing relative expression levels of two activation markers associated with antigen presentation (CD86 and MHC II) on each myeloid cell in the tumor at day 3 post treatment. Bar graphs represent fold change in expression over the control group. Statistics were calculated using a student T-test: *p ≤ 0.05, ***p ≤ 0.005.

Figure 3

Activation of T cells in the tumor, spleen and draining LN of animals treated with localized therapies. 2 x 10⁵ 4T1 mouse breast cancer cells were injected subcutaneously in the right hind limb of 6-8 week female Balbc mice and treated with RT, VTP or Cryo therapy according to the doses and schedule in Figure 1A . 3, 6 and 9 days after the start of treatment, tumor, spleen and draining LNs were harvested and processed for flow cytometry. (A) Heatmaps represents the log10 fold change (Log FC) of the CD8⁺ T cells, CD4⁺ Teffs and CD4⁺ Tregs as well as the CD8:Treg and CD4:Treg ratios in the tumor, spleen and LN at 3, 6, and 9 days post treatment. (B) Heatmaps represents the log₁₀ fold change (Log FC) relative to the control group of T cell activation markers in the spleen with the indicated therapies. (C) Quantification of effector memory (Tem) and proliferating (Ki67+) CD4⁺ Teffs (CD4⁺Foxp3^-) in the spleen 9 days after treatment. *p ≤ 0.05.

Figure 4

IHC analysis show an increase in CD11b⁺ myeloid cells infiltrating the tumors of animals treated with RT, VTP and Cryoablation. 2 x 10⁵ 4T1 mouse breast cancer cells were injected subcutaneously in the right hind limb of 6-8 week female Balbc mice and treated with RT, VTP or Cryo therapy according to the doses and schedule in Figure 1A . 6 days after treatment, tumors and spleens were harvested and processed for immunohistochemistry (IHC). (A) Shown are representative images of serial sections for H&E, CD8 and CD11b for each treatment group. Bar = 100µm. (B, C) Relative densities of CD8 and CD11b expression in the tumor and spleens. Shown are % of tissue stained positive for each marker calculated and normalized per tissue area using the HALO Image Analysis Software. N=3 mice/group. Statistics were calculated using student’s T-test: *p ≤ 0.05, **p ≤ 0.01. n/a means statistics could not be calculated since the Cryo group had only one analyzable tumor.

Figure 5

RT, VTP and Cryoablation incudes systemic T cell activation and promotes regression of secondary tumors. (A, B) 2 x 10⁵ 4T1 cells were injected subcutaneously in the right hind limb of 6-8 week female Balbc mice (5 mice/group) and treated with RT, VTP or Cryo therapy according to the doses and schedule in Figure 1A . 6 days after treatment, tumors, LNs and spleens were harvested and single cells suspension were stimulated with PMA/Ionomycin in the presence of Golgi inhibitors for 6hr then processed for intracellular cytokine staining (ICS). (A) Representative plots of IFNγ vs. TNFα by CD4⁺ Teffs and CD8⁺ T cells from the spleen. (B) Frequencies of IFNγ⁺TNFα⁺ CD4⁺ Teffs and CD8⁺ T cells in the spleen. (C) 10⁵ 4T1 cells were injected subcutaneously in the right hind limb on day 0 and left flank on day 5 of female Balbc mice (10 mice/group). Twelve days after the initial tumor injection, the right hind limb tumors were treated with 15 Gy RT, VTP or Cryoablation as outlined in the treatment schedule. Tumor size was measured using calipers, shown are the average tumor size (mm²) +/- SEM in both primary treated and secondary non-treated tumors. Statistics were calculated using a student T-test. For tumor growth curves, statistics were calculated on day 29 post tumor inoculation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.