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Background Dishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of β-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression. Methods DVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05. Results DVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = − 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC. Conclusion Our study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

Introduction: Triple-negative and HER2+ breast cancers (BCs) are high-risk subtypes that spread easily and are hard to treat. Thus, newer therapeutic approaches that can prevent local recurrence and metastatic spread of disease are required. Cryoablation, a technique that kills tumor cells through rapid freeze/thaw cycles, preserving tumor-associated antigens, is approved to treat small low-risk breast tumors but has not been as successful for high-risk BCs. A promising area of BC cryoablation research is its combinational use with immune checkpoint inhibitors (ICIs) to enhance the anti-tumor immune response and to generate distant tumor cell targeting – the abscopal effect. Using a murine model of high-risk metastatic BC, we investigated cryoablation in conjunction with anti-CTLA4 and anti-PD-L1.Methods: BALB/c mice were bilaterally transplanted in the mammary fat pad with 4T1-12b-luciferase-expressing metastatic BC cells. Two weeks after transplant, all mice had their left tumors cryoablated; 24 hours pre- and post-cryoablation, mice received an intraperitoneal injection containing PBS (control, n=5) or 100 µg of either anti-CTLA4 (n=5) or anti-PD-L1 (n=5). Right tumors were not manipulated and represented distant metastatic tumors to examine the immune abscopal effect. Mice were sacrificed one-week post-cryoablation; cryoablated (left) and abscopal (right) tumors, peripheral blood and spleen were collected and processed to obtain isolated cells for flow cytometry analysis of immune cell populations.Results: In vivo fluorescence imaging and mouse necropsies revealed cryoablated tumors undergoing necrosis. A trend of reduced tumor weights was observed in abscopal tumors of ICI-treated groups. Flow cytometry analysis showed that the frequency of total T cells was similar across all groups in both the cryoablated and abscopal tumors, spleens, and blood. However, when examining different activation states of T cell subsets, we found anti-CTLA4 treated mice had increased T cell activity with higher percent of effector and effector memory CD4+ T cells in the abscopal tumors compared to the non-treated group, where similar trends were observed for the CD8+ T cells. Additionally, a higher percent of activated, effector, and effector memory CD4+ and CD8+ T cells were found in the blood of mice treated with anti-CTLA4, compared to the non-treated mice. Interestingly, we did not observe broad T cell activation with anti-PD-L1 treatment but found increased levels of naïve CD8+ T cells in abscopal tumors compared to the other groups.Conclusions: Cryoablation in combination with anti-CTLA4 increased T cell activation in abscopal tumors and blood after treatment, while the combination with anti-PD-L1 increased naïve CD8+ T cells. The observed differences are in accordance with the distinct mechanism of action for each drug. Further studies will investigate each strategy in long-term survival experiments. The goal is to identify and develop predictive biomarkers for efficacy of cryoablation in combination with ICIs that can be translated to the clinic. Citation Format: Flavia Sardela de Miranda, Rachel Babcock, Maribel Castro, Sonia Y Khan, Carsen Roach, Thomas Hintelmann, Kathryn Furr, Chang H Lee, Geetha P Boligala, Fahmida Rasha, Luis Brandi, Harvinder S Gill, Kevin Pruitt, Rakhshanda L Rahman. Breast cancer cryoablation in combination with anti-CTLA-4 increases T cell activation in a murine tumor model [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B18.