Bariatric surgery is a sustainable weight loss approach, including vertical sleeve gastrectomy (VSG). Obesity exacerbates tumor growth, while diet-induced weight loss impairs progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters response to therapy. Using a pre-clinical model of obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy were investigated. Weight loss by VSG or weight-matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as diet in reducing tumor burden despite achieving similar weight and adiposity loss. Leptin did not associate with changes in tumor burden; however, circulating IL-6 was elevated in VSG mice. Uniquely, VSG tumors displayed elevated inflammation and immune checkpoint ligand PD-L1+ myeloid and non-immune cells. VSG tumors also had reduced T lymphocytes and markers of cytolysis, suggesting an ineffective anti-tumor microenvironment which prompted investigation of immune checkpoint blockade. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden. Finally, we compared transcriptomic changes in adipose tissue after bariatric surgery from patients and mouse models. A conserved bariatric surgery-associated weight loss signature (BSAS) was identified which significantly associated with decreased tumor volume. Findings demonstrate conserved impacts of obesity and bariatric surgery-induced weight loss pathways associated with breast cancer progression.

There is increasing evidence suggesting the role of microbiome alterations in relation to pancreatic adenocarcinoma and tumor immune functionality. However, molecular mechanisms of the interplay between microbiome signatures and/or their metabolites in pancreatic tumor immunosurveillance are not well understood. We have identified that a probiotic strain (Lactobacillus casei) derived siderophore (ferrichrome) efficiently reprograms tumor-associated macrophages (TAMs) and increases CD8 + T cell infiltration into tumors that paralleled a marked reduction in tumor burden in a syngeneic mouse model of pancreatic cancer. Interestingly, this altered immune response improved anti-PD-L1 therapy that suggests promise of a novel combination (ferrichrome and immune checkpoint inhibitors) therapy for pancreatic cancer treatment. Mechanistically, ferrichrome induced TAMs polarization via activation of the TLR4 pathway that represses the expression of iron export protein ferroportin (FPN1) in macrophages. This study describes a novel probiotic based molecular mechanism that can effectively induce anti-tumor immunosurveillance and improve immune checkpoint inhibitors therapy response in pancreatic cancer.

Myeloid-derived suppressor cells (MDSCs) are an immature innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. Conversely, efficient cytotoxic T cell priming is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to CD8(+) T cells, a process that requires a specific subtype of dendritic cells (DCs) called con-ventional DC1 (cDC1) which are often dysfunctional in cancer. One way to activate cDC1 is ligation of CD40 which is abundantly expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when com-bined with agonistic CD40, may result in long-lasting protective immunity. In this study, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation, and recruitment to the tumor microenvironment. Our findings demonstrate that PKC agonists decreased MDSC expansion from hematopoietic progenitors and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers via activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmacytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive activity and enhanced MDSC cross-priming capacity both in vitro and in vivo. Finally, com-bination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth with a significant increase in intratumoral activated CD8(+) T cells and tissue-resident memory CD8(+) T cells in a syn-geneic breast cancer mouse model. In sum, this work proposes a novel promising strategy to simultaneously target MDSCs and promote APC function that may have highly impactful clinical relevance in cancer patients.

Immunotherapy has revolutionized cancer treatment showing unprecedented long-term antitumor responses. However, most patients do not respond to immunotherapies due at least partly to immune suppression. Immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses both innate and adaptive immunity. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC breast cancer patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to immunotherapy. On the other hand, cross-presenting dendritic cells (DCs) are essential to generate an antitumor immune response. Breast cancer patients who harbor higher numbers of these DCs have a better prognosis than patients with lower DC numbers. Several strategies aiming at achieving an effective combination with immunotherapy are under active investigation. The central dogma of these strategies consists of inducing T cells into “immunologically cold tumors” which are defined by having low neoantigen burden and a paucity of T cells and DCs. Hence, strategies that enhance cross-presenting DCs and T cell antitumor potential while altering MDSC’s suppressive function are likely to be effectively combined with immunotherapy for a maximum therapeutic benefit. Protein Kinase C (PKC) is a family of enzymes that play a critical role in cell signaling controlling the balance between survival and cell death. With the discovery in 1980s that PKC is a receptor for the tumor-promoting phorbol esters, the dogma that PKC is an oncoprotein was fueled. This led to more than three decades of failed clinical trials trying to inhibit PKC in cancer. Recent evidence suggests that PKC isozymes are generally inactivated in cancer and that most mutations affecting PKC isozymes are in fact loss of function mutations. This suggests that PKC is a tumor suppressor rather than an oncoprotein and that strategies in cancer treatment should focus on restoring PKC, rather than inhibiting it. To date, the role of PKC isozymes in antitumor immunity is unknown. Herein, our novel data suggest that PKC agonism using established agonists reduced MDSC generation from bone marrow (BM) progenitors specifically via activation of the PKC delta (PKCδ) isoform. PKC agonism induced MDSC differentiation to cross-presenting CD103+ DCs both ex-vivo and in vivo. Additionally, PKC agonist-treated purified MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. In contrast, PKC agonism significantly increased the generation of cross-presenting DCs (cDC1) from BM progenitors. Treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumor and spleen. These findings propose PKC as a common pathway in myeloid cells to tip the balance from immune suppression to effective antitumor immunity. Citation Format: Mehdi Chaib, Jeremiah Holt, Laura Sipe, Ajeeth Pingili, Deidre Daria, Liza Makowski. Pkc agonism restricts immune suppression and promotes antigen cross-presentation in triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-46.

Immune checkpoint blockade (ICB) has improved outcomes in some cancers. A major limitation of ICB is that most patients fail to respond, which is partly attributable to immunosuppression. Obesity appears to improve immune checkpoint therapies in some cancers, but impacts on breast cancer (BC) remain unknown. In lean and obese mice, tumor progression and immune reprogramming were quantified in BC tumors treated with anti-programmed death-1 (PD-1) or control. Obesity augments tumor incidence and progression. Anti-PD-1 induces regression in lean mice and potently abrogates progression in obese mice. BC primes systemic immunity to be highly responsive to obesity, leading to greater immunosuppression, which may explain greater anti-PD-1 efficacy. Anti-PD-1 significantly reinvigorates antitumor immunity despite persistent obesity. Laminin subunit beta-2 (Lamb2), downregulated by anti-PD-1, significantly predicts patient survival. Lastly, a microbial signature associated with anti-PD-1 efficacy is identified. Thus, anti-PD-1 is highly efficacious in obese mice by reinvigorating durable antitumor immunity.

Background Immunotherapies that reinvigorate T cell responses have transformed the treatment of many cancers showing unprecedented durable antitumor responses. However, most patients do not respond to immunotherapy due in part to immunosuppression. Immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an innate cell population that expands in pathological conditions such as cancer and suppresses T cells via production of immunosuppressive factors. In contrast, immunotherapy success is dependent on the ability of antigen-presenting cells (APCs) to cross-present tumor antigens to cytotoxic T cells. Immunogenic cross-presentation by APCs requires a specific subtype of dendritic cells (DCs) called conventional DC1 (cDC1) which are dysfunctional in cancer. Novel ways to increase cDC1 function are promising and under active investigation. One of these ways is ligation of CD40 which is primarily expressed by myeloid cells and its agonism leads to myeloid cell activation. Thus, targeting MDSCs while simultaneously expanding cross-presenting DCs represents a promising strategy that, when combined with agonistic CD40, will likely result in long-lasting protective immunity. Methods Using in vitro, ex vivo, in vivo and adoptive transfer systems, we investigated the effect of PKC agonists PEP005 and prostratin on MDSC expansion, differentiation to APC-like cells and recruitment to the TME. MDSC suppressive capacity was investigated using functional coculture assays with CD8+ T cells. Furthermore, we assessed the effect of PKC agonists on MDSC cross-priming capacity using in vitro coculture assay with OT-I CD8+ T cells as well as adoptive transfer experiments. We also investigated the effect of PKC agonists on cDC1 expansion from the BM in vitro and in vivo. Finally, we tested the efficacy of PKC agonism in combination with agonistic CD40 using the E0771 murine breast cancer orthotopic mouse model. Results Herein, we show that PKC agonists decreased MDSC expansion from hematopoetic progenitors in the BM and induced M-MDSC differentiation to an APC-like phenotype that expresses cDC1-related markers and the transcription factor Irf8. Simultaneously, PKC agonists favored cDC1 expansion at the expense of cDC2 and plasmocytoid DCs (pDC). Functionally, PKC agonists blunted MDSC suppressive function of T cells and promoted MDSC cross-priming capacity. Finally, combination of PKC agonism with agonistic CD40 mAb resulted in a marked reduction in tumor growth while synergistically increased intratumoral activated CD8+ T cells and tissue-resident memory CD8+ T cells. Conclusions In sum, we propose a novel promising strategy to simultaneously target MDSCs and promote APC function that may have potential clinical relevance in cancer patients.

Abstract Immunotherapy has revolutionized cancer treatment showing unprecedented long-term antitumor responses. However, most patients do not respond to immunotherapy. Increasing preclinical and clinical evidence suggests that high tumor mutational burden does not always correlate with immunotherapy efficacy. Interestingly, cancers that have been associated with viruses imply that the pre-existing presentation of viral antigens may confer an increased response rate to immunotherapy. This suggests an important role of antigen presentation, particularly by the myeloid compartment in predicting immunotherapy efficacy. On the other hand, immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses T cells. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to immunotherapy. Hence, strategies that enhance antigen cross-presentation while altering MDSC's suppressive function represent a promising therapeutic approach to overcome immunotherapy resistance. Protein Kinase C (PKC) is a family of kinases composed of 11 isoforms that play a critical role in cell signaling. PKC delta (PKCδ) is the most abundant isoform in myeloid cells and plays an important role in dendritic cell (DC) function. To date, the role of PKCδ in myeloid cells in cancer is unknown. Using varied informatic approaches in patient databases, we found that BC patients with both high expression of PRKCD (PKCδ gene) and either high expression of CD8+ T cell or low expression of MDSC gene signatures in tumors had a significantly greater overall survival compared to other groups, suggesting support for activation of PKCδ. Novel preliminary data suggests that PKC agonism using FDA-approved PEP005 and prostratin reduced MDSC generation from BM progenitors specifically via activation of PKCδ isoform. PKC agonism induced MDSC differentiation to CD103+ DC-like cells both ex-vivo and in adoptive transfer experiment. Additionally, PEP005-treated MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. Importantly, PEP005 enhanced MDSC cross-priming capacity in both in vitro and in adoptive transfer experiments. Finally, treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumors. These findings propose PKCδ as a novel target in myeloid cells to tip the balance from immune suppression to effective antitumor immunity. Citation Format: Mehdi Chaib, Jeremiah Holt, Laura Sipe, Ajeeth Pingili, T.j. Hollingsworth, Deidre Daria, Neil Hayes, Liza Makowski. Myeloid PKCd activation inhibits innate immune suppression and promotes antigen cross-presentation in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 118.