Heterologous prime-boost cellular vaccination induces potent antitumor immunity against triple negative breast cancer

Abstract

Introduction: Triple negative breast cancer (TNBC) is the most aggressive and hard-to-treat subtype of breast cancer, affecting 10-20% of all women diagnosed with breast cancer. Surgery, chemotherapy and hormone/Her2 targeted therapies are the cornerstones of treatment for breast cancer, but women with TNBC do not benefit from these treatments. Although the prognosis is dismal, immunotherapies hold significant promise in TNBC, even in wide spread disease because TNBC is infiltrated with more immune cells. This preclinical study is proposing to optimize an oncolytic virus-infected cell vaccine (ICV) based on a prime-boost vaccination strategy to address this unmet clinical need.

Methods: We used various classes of immunomodulators to improve the immunogenicity of whole tumor cells in the prime vaccine, followed by their infection with oncolytic Vesicular Stomatitis Virus (VSVd51) to deliver the boost vaccine. For in vivo studies, we compared the efficacy of a homologous prime-boost vaccination regimen to a heterologous strategy by treating 4T1 tumor bearing BALB/c mice and further by conducting re-challenge studies to evaluate immune memory responses in surviving mice. Due to the aggressive nature of 4T1 tumor spread (akin to stage IV TNBC in human patients), we also compared early surgical resection of primary tumors versus later surgical resection combined with vaccination.

Results: In vitro results demonstrated that immunogenic cell death (ICD) markers and pro-inflammatory cytokines were released at the highest levels following treatment of mouse 4T1 TNBC cells with oxaliplatin chemotherapy and influenza vaccine. These ICD inducers also contributed towards higher dendritic cell recruitment and activation. With the top ICD inducers at hand, we observed that treatment of TNBC-bearing mice with the influenza virus-modified prime vaccine followed by VSVd51 infected boost vaccine resulted in the best survival. Furthermore, higher frequencies of both effector and central memory T cells along with a complete absence of recurrent tumors were observed in re-challenged mice. Importantly, early surgical resection combined with prime-boost vaccination led to improved overall survival in mice.

Conclusion: Taken together, this novel cancer vaccination strategy following early surgical resection could be a promising therapeutic avenue for TNBC patients.

Keywords: Immunogenic cancer vaccine; immune effector cells; oncolytic virotherapy; triple negative breast cancer; tumor microenvironment.

Figure 1

Immunogenic cell death of mouse and human TNBC cells can be induced following in vitro treatment with the chemotherapeutic agent oxaliplatin and the season influenza vaccine. (A) Western blot analysis of HMGB1 from cell-free supernatants, (B) luminometry measurement of relative ATP from cell-free supernatants, and (C) measurement of cell surface calreticulin of TNBC cell lines treated with ICD inducers at their IC50 concentrations or infected with VSVd51 at 10 MOI for 24h. The results were compared to non-treated cells. All data are representative of at least three similar experiments where n=3 for technical replicates, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; (n.s., no significance).

Figure 2

ICD inducers improve the release of immunogenic mediators from TNBC cells. Cytokine and chemokine levels from (A) mouse 4T1 and human (B) BT-549 cell line culture supernatants were quantified by ELISA following treatment of with IC50 concentrations of ICD inducers or infected with 10 MOI of VSVd51 for 24h. The results were compared to non-treated cells. ELISA was performed using supernatant pooled from 3 independent experiments, where *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; (n.s., no significance).

Figure 3

Enhanced recruitment and activation of mouse dendritic cells following exposure to TNBC spheroids treated with oxaliplatin or influenza vaccine. Live cell images from (A) 4T1 spheroids co-cultured with deep red CellTracker-labelled BMDCs, 24h following their treatment with ICD inducers at IC50 concentrations. LPS (1μg/ml) treated spheroids were used as positive controls. (B) Quantification of microscopy images representing the number of infiltrated and aggregated immune cells around treated spheroids. Dashed lines delineate the edge of spheroids. The results are compared to non-treated cells. (C) Flow cytometry analysis of maturation markers of BMDCs following 48h of exposure to CM of 4T1 cells treated with indicated ICD inducer at IC50 concentration or 10 MOI of VSVd51. LPS treated (1μg/ml) BMDCs were used as a positive control. (D) Flow cytometry analysis of phagocytic BMDCs labelled with deep red CellTracker and co-cultured with Bodipy TMR labelled 4T1 cells. All data are representative of at least three similar experiments where n=3 for technical replicates, *P < 0.05; **P < 0.01; ****P < 0.0001; (n.s., no significance).

Figure 4

Increased migration and functionality of human DC and effector immune cells following co-culture with oxaliplatin and influenza vaccine treated human TNBC spheroids. Live cell images from (A) BT-549 and MD-MBA-231 spheroids co-cultured with deep red CellTracker-labelled human DCs, 24h following their treatment with ICD inducers at IC50 concentrations. LPS (1μg/ml) treated spheroids were used as positive controls. (B) Quantification of microscopy images representing the number of infiltrated and aggregated immune cells around the treated spheroids. Dashed lines delineate the edge of spheroids. The results are compared to non-treated cells. Flow cytometry analysis of maturation markers on human DCs following 48h of exposure to CM of (C) BT-549 or (D) MDA-MB-231 cells treated with indicated ICD inducers at IC50 concentration or 10 MOI of VSVd51. LPS treated (1μg/ml) human DCs are used as a positive control. (E) Flow cytometry quantification of migrated purified human CD3⁺/CD8⁺ T cells and CD3^-/CD56⁺ NK cells towards CM from BT-549 and MDA-MB-231 cells incubated with indicated treatments; MCP-1 (50ng/ml) was used as a positive control for immune cell migration. Flow cytometry analysis of purified human CD3⁺/CD8⁺ T cells and CD3^-/CD56⁺ NK cells in tri-cultures with human DCs previously exposed to CM from (F) BT-549 or (G) MDA-MB-231 treated spheroids with indicated treatments. The results are compared to non-treated cells. All data are representative of at least three similar experiments where n=3 for technical replicates, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; (n.s., no significance).

Figure 5

Heterologous prime-boost vaccination improves survival in the BALB/c-4T1 model of TNBC. (A) Timeline of in vivo BALB/c-4T1 experiment. BALB/c mice were orthotopically implanted with 1x10⁵ 4T1 cells followed by a complete primary tumor resection on indicated days. Two days postoperatively, mice received 1 dose of the prime vaccine in the cleared mammary fat pad (FLU-4T1, Oxa-4T1, irradiated 4T1, ICV). Nine days postoperatively, mice received their 4T1-ICV boost vaccine. Immune functional analyses, re-challenge and monitoring were performed as indicated. (B) Immune cell suspensions from the peripheral blood of mice following indicated treatments were stained with T cell markers (CD3⁺, CD8⁺, IFNγ⁺, CD107a⁺) and analyzed by flow cytometry. (C) Kaplan-Meier survival analysis of mice receiving prime-boost ICV. n=10-12 mice/group. *P < 0.05; **P < 0.01; (n.s., no significance), log-rank test. (D) Tumor growth measurements comparing re-challenged 4T1 tumors with their corresponding primary tumor from 4T1-ICV and FLU-4T1-ICV treatment cohorts. (E) Flow cytometry analysis of central memory T cells in the axillary lymph node and effector memory T cells in the blood of FLU-4T1+ICV treated cohort before and after re-challenging with 4T1 tumors. (F) Representative lung pictures from FLU-4T1+ICV re-challenged and 4T1-ICV treated cohorts. (G) Kaplan-Meier survival analysis of mice receiving early vs. late surgery and prime boost ICV. n=10-12 mice/group. *P < 0.05; **P < 0.01; (n.s., no significance), log-rank test. All flow cytometry data are representative of three similar experiments where n=4 mice/treatment, *P < 0.05; **P < 0.01; (n.s., no significance).

Figure 6

Model of heterologous prime-boost vaccination for TNBC. Adjuvant vaccination with a prime cellular vaccine results in the release of immunogenic cell death biomarkers (DAMPs, cytokines, chemokines) that recruit and activate antigen presenting cells to cross-present tumor associated antigens to tumor-targeted T cells. This is followed by the administration of a boost oncolytic virus-infected cellular vaccine to further focus the immune response on tumor antigens. An enhanced secondary immune response is instigated by this heterologous prime-boost vaccination to reduce metastatic and recurrent disease of TNBC.