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

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.

First is to gate on single cells only using FSC-H vs FSC-A, then SSC-H vs SSC-A, then FSC-H vs FSC-W.
Next is to gate on the live CD45+ cells for total immune infiltrates.Then the CD45+ cells are sub-gated into CD11b+ and CD11c+ cells.CD11b+ cells are then sub-gated into Ly6G+ (granulocytes), Ly6C+ (monocytes), and Ly6G-Ly6C-cells (mostly macrophages and DCs, here referred to as TAMs).The CD11c+ cells are subdivided into CD8+ DCs, CD11b+ DCs and other DCs.Then the activation status of each cell population is examined by expression of CD11b, CD86 and MHC class II on their cell surface.CD86 and MHC II are used as surrogate for increased antigen presentation while CD11b is used as an activation marker.

Granulocytes
Fig. S1: RT, VTP and Cryoablation enhances anti-tumor responses in 4T1 breast cancer.Balbc mice were implanted with 4T1 tumors and treated according to the schema in Figure 1A and as described in methods.Shown are individual tumor growth curves (n=10 mice per group) for 8 cohorts are shown.Table: tumor free numbers were assessed at day 98 post implantation.
Fig. S3: Summary of myeloid cell kinetics and activation status in the spleen, draining lymph node (TDLN) and tumor.Shown are heatmaps of the log 10 fold change (Log FC) of the cell populations and their activation markers for each treatment group normalized to the control.The gating strategy to identify each cell population is shown in Figure S2.
Fig.S4: TAMs and APCs correlated positively with tumor burden.Plots of frequencies of myeloid cells (as a % of CD45+ cells) vs. tumor burden (weight (g)) from experiments outlined in Figure1A.Each plot represents pooled data from all treatment groups and all time points examined (n=120).Pearson correlation was used to determine the r and p values.The green boxes indicate the correlations that were statistically significant.

Figure S5 :
Figure S5: Activation of Myeloid cells in the spleen and draining LN of animals treated with localized therapies.10 5 4T1 mouse breast cancer cells were injected subcutaneously in the right hind limb of 6-8 week female Balbc mice (n=5 mice per group).Ten days later when the tumors reached 50-60mm 2 in size, mice were treated with 15 Gy radiation (RT), VTP or Cryo therapy according to the doses listed in Fig 1A and methods.(A,B ) Left: The frequencies +/-SEM of CD11b+ myeloid cells and CD11c+MHC II+ APCs (as a % of CD45+ cells) in the spleen (A) and draining LN (B) from each treatment group at the indicated time points.Right: Heatmaps representing the fold change (Log FC) of CD86, MHC II and CD11b expression based on mean fluorescence intensity (MFI) on immune cells in the spleen (A) and draining LN (B).*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.

Fig. S6 :Figure S7 .
Fig. S6: Gating strategy to identify each T cell population and their activation/differentiation status.Shown is the gating strategy of a representative tumor sample to identify each T cell population and their activation markers.First is to gate on single cells only using FSC-H vs FSC-A, then SSC-H vs SSC-A, then FSC-H vs FSC-W.Next is to gate on the live CD45+ cells for total immune infiltrates.Then the CD45+ cells are subgated into CD4+ and CD8+ T cells.The CD4+ cells are further divided into Tregs and Teffs based on Foxp3 expression.All three T cell populations (CD8+ T cells, CD4+ Teffs and CD4+ Tregs) are then examined for their differentiation status by CD62L and CD44 expression.Naïve T cells are CD62L+CD44-, central memory T cells (T CM ) are CD62L+CD44+ and effector memory T cells (T EM ) are CD62L-CD44+.The T cell populations are further examined for the proliferation (Ki67) and activation status (Granzyme B, CD25).

Figure S8 .Figure S9 .Figure S10 :
Figure S8.Heatmap summary of T cell frequencies and their activation status in the spleen.Heatmaps from the experiments outline in Figure 1 schema, shown are the log 10 fold change (Log FC) of T cell populations and their activation markers for each treatment group normalized to the control.The gating strategy to identify each cell population is shown in Figure S6.Red arrows highlight significant changes observed in Granzyme B and Ki67.

Figure S11 :
Figure S11: CD11b+ myeloid cells are confined to the stroma or necrotic areas of 4T1 tumors.4T1 mouse breast cancer cells were injected and treated with RT, VTP or Cryoablation therapy according to the doses and schedule in Figure 1A.6 days after treatment, tumors and spleens were harvested and processed for immunohistochemistry (IHC).Shown are representative images of serial sections for H&E, CD8 and CD11b for each treatment group.Bar = 200µm.

Figure S12 :
Figure S12: IHC analysis show a decrease in CD11b+ myeloid cells in the spleens of animals treated with VTP and Cryoablation.10 5 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, spleens were harvested and processed for immunohistochemistry (IHC).Shown are representative images of serial sections for H&E, CD8 and CD11b for each treatment group.Bar = 1mm.