Preconditioning with lipopolysaccharide or lipoteichoic acid protects against Staphylococcus aureus mammary infection in mice

Staphylococcus aureus is one of the most causative agents of mastitis and is associated with chronic udder infections. The persistency of the pathogen is believed to be the result of an insufficient triggering of local inflammatory signaling. In this study, the preclinical mastitis model was used, aiming to evaluate if lipopolysaccharide (LPS) or lipoteichoic acid (LTA) preconditioning could aid the host in more effectively clearing or at least limiting a subsequent S. aureus infection. A prototypic Gram-negative virulence factor, i.e., LPS and Gram-positive virulence factor, i.e., LTA were screened whether they were able to boost the local immune compartment. Compared to S. aureusinduced inflammation, both toxins had a remarkable high potency to efficiently induce two novel selected innate immunity biomarkers i.e., lipocalin 2 (LCN2) and chitinase 3-like 1 (CHI3L1). When combining mammary inoculation of LPS or LTA prior to a local S. aureus infection, we were able to modulate the innate immune response, reduce local bacterial loads, and induce either LCN2 or CHI3L1 at 24 h post-infection. Clodronate depletion of mammary macrophages also identified that macrophages contribute only to a limited extend to the LPS/LTA-induced immunomodulation upon S. aureus infection. Based on histological neutrophil influx evaluation, concomitant local cytokine profiles and LCN2/CHI3L1 patterns, the macrophage-independent signaling plays a major role in the LPS-or LTA-pretreated S. aureus-infected mouse mammary gland. Our results highlight the importance of a vigilant microenvironment during the innate immune response of the mammary gland and offer novel insights for new approaches concerning effective immunomodulation against a local bacterial infection

Enterobactin Deficiency in a Coliform Mastitis Isolate Decreases Its Fitness in a Murine Model:A Preliminary Host–Pathogen Interaction Study

Iron is an essential nutrient for bacterial growth. Therefore, bacteria have evolved chelation mechanisms to acquire iron for their survival. Enterobactin, a chelator with high affinity for ferric iron, is secreted by Escherichia coli and contributes to its improved bacterial fitness. In this preliminary study, we evaluated enterobactin deficiency both in vitro and in vivo in the context of E. coli mastitis. Firstly, we showed that expression of lipocalin 2, a protein produced by the host that is able to both bind and deplete enterobactin, is increased upon E. coli infection in the cow’s mastitic mammary gland. Secondly, we demonstrated in vitro that enterobactin deficiency does not alter interleukin (IL)-8 expression in bovine mammary epithelial cells and its associated neutrophil recruitment. However, a significantly increased reactive oxygen species production of these neutrophils was observed. Thirdly, we showed there was no significant difference in bacterial in vitro growth between the enterobactin-deficient mutant and its wild-type counterpart. However, when further explored in a murine model for bovine mastitis, the enterobactin-deficient mutant vs. the wild-type strain revealed a significant reduction of the bacterial load and, consequently, a decrease in pro-inflammatory cytokines (IL-1a,−1b,−4,−6, and−8). A reduced neutrophilic influx was also observed immunohistochemically. These findings therefore identify interference of the enterobactin iron-scavenging mechanism as a potential measure to decrease the fitness of E. coli in the mastitic mammary gland

OMO-1 reduces progression and enhances cisplatin efficacy in a 4T1-based non-c-MET addicted intraductal mouse model for triple-negative breast cancer

c-MET is considered a driver of cancer progression, impacting tumor growth and tumor-supporting stroma. Here, we investigated the therapeutic efficacy of OMO-1, a potent and selective c-MET inhibitor, in an immunocompetent intraductal mouse model for triple-negative breast cancer (TNBC). OMO-1 reduced non-c-MET addicted 4T1 tumor progression dose dependently as monotherapeutic and provided additional disease reduction in combination with cisplatin. At the stromal level, OMO-1 significantly reduced neutrophil infiltration in 4T1 tumors, promoted immune activation, and enhanced cisplatin-mediated reduction of tumor-associated macrophages. OMO-1 treatment also reduced 4T1 tumor hypoxia and increased expression of pericyte markers, indicative for vascular maturation. Corroborating this finding, cisplatin delivery to the 4T1 primary tumor was enhanced upon OMO-1 treatment, increasing cisplatin DNA-adduct levels and tumor cell death. Although verification in additional cell lines is warranted, our findings provide initial evidence that TNBC patients may benefit from OMO-1 treatment, even in cases of non-c-MET addicted tumors

Comparative profiling of metastatic 4T1- vs. non-metastatic Py230-based mammary tumors in an intraductal model for triple-negative breast cancer

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models

Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer

Background: Murine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages. Methods: Intraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level. Results: Mice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2. Conclusions: The current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target

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

Chitinase 3-like 1 stimulates macrophage-mediated lymphatic remodeling in triple-negative breast cancer

Tumor cells often rely on lymphatic vessels as a transportation route to form distant metastases. Tumor-associated macrophages (TAMs) play an important role in this lymphatic spreading by attaching and integrating into the lymphatic vessel structure, allowing metastatic tumor cells to invade the lymphatics. This process is referred to as macrophage-mediated lymphatic remodeling and can be promoted by specific proteins with macrophage-stimulating and angiogenic properties. The role of chitinase 3-like 1 (CHI3L1), a potent activator of macrophages towards a protumorigenic and angiogenic phenotype, in the macrophage-mediated lymphatic remodeling process has not yet been investigated. Here, we show at first that recombinant (rm)CHI3L1 stimulates macrophages to express podoplanin (PDPN), a characteristic prerequisite marker for lymphatic integration. Additional in vitro analyses showed that rmCHI3L1-treated macrophages adhere more frequently to human-derived lymphatic endothelial cells (HDLECs) in 2D cocultures. Moreover, upon 3D coculturing with HDLEC-derived vessel-like structures on Matrigel® pads, rmCHI3L1-treated macrophages showed increased ability to cluster on and integrate into these in vitro lymphatics. Blocking of the CHI3L1 receptor IL-13Rα2 or PDPN with specific antibodies reduced the rmCHI3L1-stimulated macrophage adhesion to HDLECs and integration into vessel-like structures. Lymphatic TAM integration was further evaluated in primary tumors from a 4T1-based mouse model for triple-negative breast cancer (TNBC) through dual color immunohistochemistry for F4/80+ TAMs and LYVE-1+ lymphatic endothelial cells. The lymphatic integration was affected upon CHI3L1 blockade with chitin or anti-CHI3L1 antibodies. In conclusion, our data identify CHI3L1 as a stimulator of macrophage-mediated lymphatic remodeling and a candidate target to reduce lymphatic (TNBC) metastasis

Splenic hematopoietic and stromal cells in cancer progression

Tumor-derived secretory factors orchestrate splenic hematopoietic and stromal cells to fuel metastasis. The spleen acts as a reservoir site for hematopoietic stem and progenitor cells, which are rapidly exploited as myeloid-derived suppressor cells at the cost of tumor-reactive lymphoid cells. Splenic erythroid progenitor cells and mesenchymal stromal cells contribute directly and indirectly to both tumor immune escape and the metastatic cascade. Animal models provide valuable mechanistic insights, but their translation to a clinical setting highlights specific challenges and open issues. In this review, we envision the exploitation of the spleen as a source for novel biomarkers and therapeutic approaches