Cryoablation of primary breast cancer tumors induces a systemic abscopal effect altering TIME (Tumor Immune Microenvironment) in distant tumors

Abstract

Introduction: Despite recent advances, triple-negative breast cancer (TNBC) patients remain at high risk for recurrence and metastasis, which creates the need for innovative therapeutic approaches to improve patient outcomes. Cryoablation is a promising, less invasive alternative to surgical resection, capable of inducing tumor necrosis via freeze/thaw cycles. Necrotic cell death results in increased inflammatory signals and release of preserved tumor antigens, which have the potential to boost the local and systemic anti-tumor immune response. Thus, compared to surgery, cryoablation enhances the activation of T cells leading to an improved abscopal effect, defined as the occurrence of a systemic response after local treatment. We previously showed with a bilateral-tumor mouse model of TNBC that cryoablation of the primary tumor leads to increased infiltration of distant (abscopal) tumors by tumor infiltrating lymphocytes (TILs) and decreased rates of recurrence and metastasis. However, the early drivers of the cryoablation generated abscopal effect are still unknown and knowledge of the mechanism could provide insight into improving the anti-tumor immune response through pharmacologic immune modulation in addition to cryoablation.

Methods: One million 4T1-12B-luciferase expressing cells were transplanted into the mammary fat pad of BALB/c mice. Two weeks later, left (primary) tumors were either resected or cryoablated. A week after the procedure, right (abscopal) and left tumors, along with spleen, tumor-draining lymph node and blood were collected and processed for flow cytometry and/or RNA-sequencing and immunofluorescence.

Results: Here we show that cryoablation of mouse mammary carcinomas results in smaller abscopal tumors that harbor increased frequencies of anti-tumor cells [such as natural killer (NK) cells], accompanied by a systemic increase in the frequency of migratory conventional type 1 dendritic cells (cDC1; CD103⁺ XCR1⁺), compared to resection. The changes in cell frequencies are mirrored by the immune gene signature of the abscopal tumors, with cryoablation inducing genes involved with NK cell activation and leukocyte-mediated toxicity, including IL11ra1 and Pfr1.

Conclusions: These results better define the early mechanisms through which cryoablation improves tumor elimination, which is mediated by enhanced frequencies of anti-tumoral cells such as NK and cDC1s at the abscopal tumor and in the spleen of mice treated with cryoablation, respectively.

Keywords: RNA-seq analysis; abscopal effect; breast cancer; cryoablation; immune response.

Figure 1

Experimental approach. (A) Schematic showing the breast cancer cryoablation procedure and the abscopal effect. Through a small incision on the skin, the cryoprobe is inserted into the tumor; as liquid nitrogen is released, an ice ball forms around the tumor, causing it to freeze. The freeze/thaw cycles lead to tumor necrosis, which results in release of preserved tumor antigens, damage associated molecular patterns (DAMPs), and inflammatory cytokines, which will be recognized by antigen presenting cells. The higher availability of inflammatory signals and tumor antigens will boost the activation of anti-tumor T cells that can recognize both local and distant tumor cells. The generation of a systemic response after a local treatment is called “abscopal effect”. (B) Schematic of experimental approach using our murine model of breast cancer. 1x10⁶ 4T1-12B cells were bilaterally transplanted in the mammary fat pad of BALB/c mice. Two weeks later, mice were divided into two groups and received their according treatment: resection or cryoablation. One-week post treatment, animals were sacrificed and cryoablated tumors, abscopal tumors, TdLNs, spleens, and peripheral blood were collected and processed for analysis. Created in BioRender. Melkus, M. (2024) BioRender.com/w50o762.

Figure 2

Abscopal tumors from cryoablation present lower weight compared to those from resection. (A) Cryoablation of the tumors. Skin is retracted away from the tumors, and the probe is laid flat on top of it. Far right panel shows a completely frozen tumor. (B) Representative IVIS pictures of mice from the resection and cryoablation groups, imaged 24 hours pre- and post-procedures. (C) Pictures of organs post-sacrifice. Far left panel shows the abscopal versus the cryoablated tumor in the mouse. The right panels show pictures of the primary tumors, abscopal tumors, and spleen, from left to right; top pictures are from the resection group, while bottom pictures are from the cryoablation group. (D) Graphs showing individual mouse tumor and spleen weights. The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed to compare resected vs cryoablation, with p<0.05 (*) considered significant. There is a one-week difference when tumors were isolated when comparing the resected to the cryoablated tumors (primary tumors). R, resection group; C, cryoablation group; AR, abscopal tumors from resection; AC, abscopal tumors from cryoablation. n = 17.

Figure 3

Cryoablation of primary tumor leads to enhanced local infiltration by naïve and inflammatory cells one-week after the procedure. (A) Lymphoid populations at the primary tumors. Subpopulations of T cells were analyzed as frequency of the parent (CD4⁺ or CD8⁺), while the parent immune populations were analyzed as frequency of the live immune cells (CD45⁺). (B) Myeloid populations at the primary tumors, analyzed as frequency of the live immune cells (CD45⁺). The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed comparing resected vs cryoablated tumors, with p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****) considered significant. There is a one-week difference when tumors were isolated when comparing the resected to the cryoablated tumors (primary tumors). R, resection group; C, cryoablation group; CM, central memory; E/EM, effector/effector memory. n = 17.

Figure 4

TdLNs from the cryoablated and resected tumors present similar frequency of immune populations. Graphs depict the frequency of different lymphocyte and DC populations at the lymph nodes draining treated tumors, analyzed as frequency of the parent (CD4⁺ or CD8⁺) and frequency of the live immune cells (CD45⁺), respectively. The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed comparing lymph nodes draining the resected vs cryoablated tumors. All lymph nodes were collected at 3 weeks. R, resection group; C, cryoablation group; CM, central memory; E/EM, effector/effector memory. n = 13.

Figure 5

Abscopal tumors from cryoablation present increased frequencies of anti-tumor cells. (A) Representative tSNE map from one experimental repeat, showing the cluster localization of different lymphoid populations at the abscopal tumors. (B) Representative tSNE heatmap from one experimental repeat for concatenated abscopal tumors from resection (AR) and abscopal tumors from cryoablation (AC) samples. (C) Frequency of lymphoid populations on abscopal tumors from resection and cryoablation. Parent immune populations were analyzed as frequency of the live immune cells (CD45⁺), while subpopulations of T cells were analyzed as frequency of the parent (CD4⁺ or CD8⁺). The frequencies of each immune population were normalized by the average of the resected tumor frequencies, matched by experiment. The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed to compare resection vs cryoablation, with p<0.05 (*) and p<0.01 (**) considered significant. AR, abscopal from resection; AC, abscopal from cryoablation; CM, central memory; E/EM, effector/effector memory. n = 17.

Figure 6

Cryoablation decreases immunosuppression in abscopal tumors. (A) Representative tSNE map from one experimental repeat, showing the cluster localization of different myeloid populations at the abscopal tumors. (B) Representative tSNE heatmap from one experimental repeat for concatenated abscopal tumors from resection (AR) and abscopal tumors from cryoablation (AC) samples. (C) Frequency of myeloid populations on abscopal tumors from resection and cryoablation. All immune populations were analyzed as frequency of the live immune cells (CD45⁺). The frequencies of each immune population were normalized by the average of the resected tumor frequencies, matched by experiment. The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed to compare resection vs cryoablation, with p<0.05 (*) considered significant. AR, abscopal from resection; AC, abscopal from cryoablation. n = 17.

Figure 7

Cryoablation boosts the generation of migratory cDC1s. Graphs show the frequency of immune populations at the spleen [(A), n = 17], abscopal tumor-draining lymph node [(B), n = 17], and peripheral blood [(C), n = 17]. All immune populations were analyzed as frequency of the live immune cells (CD45⁺), except for the CD4⁺ and CD8⁺ subpopulations (analyzed as frequency of the parent). The different dot colors indicate the experiment. Unpaired Student’s t-test for normally distributed data or Mann-Whitney test for non-normally distributed data was performed comparing resection vs cryoablation, with p<0.05 (*) considered significant. R, resection group; C, cryoablation group; AR, abscopal from resection; AC, abscopal from cryoablation; CM, central memory; E/EM, effector/effector memory.

Figure 8

Cryoablation of primary tumor modifies the immune signature of distant tumors. (A) Venn diagram for differentially expressed genes for each tumor treatment condition compared to baseline control tumors. (B) Differential gene expression analysis on RNA sequencing of abscopal tumors from cryoablation in comparison to abscopal tumors from resection. (C) STRING analysis of abscopal tumors from cryoablation versus abscopal tumors from resection. (D) Gene Ontology of enriched immune pathways and associated genes in abscopal tumors from cryoablation, compared to abscopal tumors from resection. (E) Gene set enrichment analysis (GSEA) of significantly enriched immune pathways in abscopal tumors from cryoablation, compared to abscopal tumors from resection. R, resected tumor; AR, abscopal tumor from resection; AC, abscopal tumor from cryoablation. n = 5.