Chitin immunomodulation stimulates anti-PD-1 therapy in an intraductal model for triple-negative breast cancer

Chitin is one of the most abundant polysaccharides on earth, functioning as structural component in fungal and bacterial cell walls, and in the exoskeleton of nematodes, insects and crustaceans, but is not synthesized by mammals. Consequently, chitin is recognized by the mammalian immune system via chitinases and it is also blocking enzymatically-inactive and immunosuppressive chitinase-like proteins such as chitinase 3-like 1. The concomitant chitin-induced immune sensitization has been shown to prime potent anti-tumor immune responses in mice and humans. Here, we aimed to explore chitin-induced immunomodulation as potential therapeutic strategy for aggressive and difficult to treat triple-negative breast cancer (TNBC). To this end, we used an in-house characterized immunocompetent mouse model that relies on the intraductal inoculation of triple-negative 4T1 tumor cells in mouse mammary glands and recapitulates human TNBC. Mice were treated with 1 mg of chitin every 3 days for 2 weeks through intraperitoneal injections upon invasive tumor progression. Potential synergy of chitin with immune checkpoint blockade (ICB) was additionally checked through combination with anti-PD-1 therapy (200 µg/mouse every week). Our results show that chitin significantly reduces 4T1 primary tumor growth and alleviates ICB resistance, providing add-on tumor reduction in combination with anti-PD-1. Yet, only the chitin and anti-PD-1 combination treatment significantly reduces both lymph node and lung metastases, which is also reflected by reduced splenomegaly. The efficacy of chitin treatment is further demonstrated by significantly reduced levels of chitinase 3-like 1, both in the primary tumor and in serum of chitin-treated mice. In line with its function as anti-tumor immunomodulator, effective chitin treatment establishes a hot tumor microenvironment with reduced myeloid-derived suppressor cells, M2 macrophages, and regulatory T-cell production, as well as increased infiltration of CD8α+ T-cells with enhanced anti-tumor specifics (i.e. positivity for granzyme B, Ki67 and IFN-γ) that was also mimicked in the axillary lymph nodes. Combination with anti-PD-1 resulted in an add-on increase in the number of anti-tumorigenic CD8α+ T-cells, supporting additional disease reduction. Based on these novel insights, chitin treatment could provide a response to the clinical demand for better TNBC therapeutics that relieve immunosuppression and enhance immunotherapeutic success in patients

One cisplatin dose suffices to maintain anti-tumor immunity and enhances anti-PD-1 efficacy in an intraductal model for triple-negative breast cancer

Cytotoxic cisplatin chemotherapy has a direct killing effect on tumor cells and significantly modulates the host immune system. This cisplatin-mediated immunomodulation is attractive in combination with immune checkpoint blockers, as it may enhance their efficacy e.g. in immunotherapy-resistant triple-negative breast cancer (TNBC). To better understand the combination therapy-induced immune response, an immunocompetent 4T1-based intraductal model was used that recapitulates the complete human TNBC process. Treatments with cisplatin and programmed death (PD)-1 blocking antibody were started once primary tumors met the invasive carcinoma stage and continued until deadly metastases developed. The model showed resistance to single weekly anti-PD-1 therapy, but became responsive upon combination therapy with cisplatin. Interestingly, repetitive cisplatin dosing (every 5 days) provided similar disease reduction as one cisplatin dose at start of treatment, indicating that early cisplatin-mediated immunomodulation suffices to stimulate and maintain anti-tumor immunity in support of PD-1 blockade. In line with this observation, cytotoxic T-cells (CTLs) were increased in lymphoid tissues (i.e. spleen and lymph nodes) while immunosuppressive myeloid cells were reduced in both the primary tumor as well as lymphoid tissues at 2 days after the first cisplatin dose. Both significant cisplatin-mediated changes remained for 2 weeks and the combination with anti-PD-1 further increased the number of CTLs, providing an add-on disease reduction. Collectively, our results highlight that single-dosed cisplatin is a promising immunotherapeutic ally, holding potential for reducing toxicity without treatment efficacy loss in TNBC

Characterization of the immune landscape of canine and feline mammary tumors as a basis for candidate biomarker, drug and toxicity studies

Introduction: Mammary tumors are common in dogs and, to a lesser extent, also in cats. Especially in the latter companion animal species, these tumors often present in aggressive forms with metastases in the sentinel lymph node (SLN). Due to their spontaneous nature, similar exposure to environmental risk factors and comparable genomics, both canine and feline mammary tumor patients are increasingly proposed as superior models for human breast cancer. However, current knowledge of the canine and feline mammary tumor microenvironment (TME) is limited and hampers translational studies focusing on for example biomarker detection, dose titration of candidate drugs, and PK/PD and toxicity of novel immuno- as well as repurposed therapies. Therefore, the most relevant immune cell types were defined side-by-side in the canine and feline mammary TME, providing the first comparative insights into TME immunosuppression. In addition, the analysis was extended to the SLN, evaluating the impact of metastatic progression on SLN immunosuppression. Methods: Retrospectively collected canine and feline mammary adenocarcinoma, including weakly (grade 1), moderately (grade 2) and highly invasive/metastatic (grade 3) FFPE samples, and their SLN were characterized by immunohistochemistry. Investigated immune cell markers included ionized calcium-binding adaptor molecule (IBA)-1 for macrophages, CD163 for immunosuppressive (i.e. M2-polarized) macrophages, major histocompatibility complex (MHC)II for antigen-presenting cells, CD20 for B-cells, CD3 for T-cells, and additionally granzyme B and programmed death (PD)-1 for activated canine T-cells. Results: IBA-1+ and CD163+ immunosuppressive macrophages were localized in the stroma surrounding the tumor mass in the canine and feline TME, and MHCII+ antigen-presenting cells were identified as most abundant immune cells in these stromal areas. For both species, the number of these immune cells was significantly higher in grade 3 compared to grade 1 and 2 mammary tumors, indicative for an enhanced immunosuppressive leukocyte reaction upon tumor progression. Moreover, IBA-1+ and CD163+ macrophages were also significantly more present in grade 3-associated SLNs, suggesting immunosuppression at this metastatic site upon advanced disease stages. Immunosuppression in the canine mammary TME was further characterized based on the location of granzyme B+ and PD-1+ activated T-cells. Remarkably, despite the prominent CD3+ T-cell influx, PD-1 and granzyme B stainings remained either scattered throughout or restricted to CD20+ B-cell-rich islands in the TME stroma, rendering tumor-infiltrated CD3+ T-cells inactive. Conclusions: Our results highlight the immunosuppressive nature of the canine and feline mammary TME, which is highly relevant for translatable studies that can ultimately benefit both companion animals themselves and human patients

Progression of canine mammary tumors is associated with an immunosuppressed microenvironment

Breast cancer is the most common and deadliest type of cancer in women. Although preclinical studies mostly rely on mice, scientists have also turned to dogs as valuable models given their spontaneous mammary tumor growth and similarity to the human genome. Moreover, dogs - in contrast to traditional rodent models - have equal exposure to environmental cancer-associated risk factors as humans. To this end, dogs may be of great interest for therapeutic testing, benefitting both humans and animals from a cross-health perspective. Yet, properly conducting such trials first requires characterization of the canine mammary tumor microenvironment (TME). Here, we investigated immune cell types in retrospectively collected samples, comprising both weakly invasive (grade 1) and highly invasive/metastatic (grade 3) canine mammary adenocarcinoma, using immunohistochemistry: CD45 as pan-leukocyte marker, CD163 as marker for immunosuppressive M2 macrophages, and CD3 as T-cell marker all showed a significantly increased staining in grade 3 vs grade 1 mammary tumors. M2 macrophages were primarily located in stromal areas surrounding the tumor mass, whereas T-cells were also located within the tumor core. Despite the increased T-cell influx, granzyme B and PD-1 staining identified reduced T-cell activation in grade 3 vs grade 1 mammary tumors, indicative of an immunosuppressed TME comparable to the human breast TME. As immunosuppression is the most important cause for immunotherapeutic failure in humans, these first data warrant further exploration of canine mammary tumors in preclinical studies as model for the human patients

Priming of the murine mammary gland with Staphylococcus chromogenes IM reduces bacterial growth of Streptococcus uberis : a proof-of-concept study

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth

One cisplatin dose provides durable stimulation of anti-tumor immunity and alleviates anti-PD-1 resistance in an intraductal model for triple-negative breast cancer

Aggressive triple-negative breast cancer (TNBC) is classically treated with chemotherapy. Besides direct tumor cell killing, some chemotherapeutics such as cisplatin provide additional disease reduction through stimulation of anti-tumor immunity. The cisplatin-induced immunomodulation in TNBC was here investigated in-depth using immunocompetent intraductal mouse models. Upon primary tumor transition to invasive carcinoma, cisplatin was injected systemically and significantly reduced tumor progression. Flow cytometric immunophenotyping was corroborated by immunohistochemical analyses and revealed both differential immune cell compositions and positivity for their programmed death (PD)-1 and PD-ligand (L)1 markers across body compartments, including the primary tumor, axillary lymph nodes and spleen. As key findings, a significant decrease in immunosuppressive and a concomitant increase in anti-tumor lymphocytic cell numbers were observed in the axillary lymph nodes and spleen, highlighting their importance in cisplatin-stimulated anti-tumor immunity. These immunomodulatory effects were already established following the first cisplatin dose, indicating that early cisplatin-mediated events may determine (immuno)therapeutic outcome. Furthermore, a single cisplatin dose sufficed to alleviate anti-PD-1 resistance in a 4T1-based model, providing add-on disease reduction without toxic side effects as seen upon multiple cisplatin dosing. Overall, these results highlight cisplatin as immunotherapeutic ally in TNBC, providing durable immunostimulation, even after a single dose