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The obligate intracellular bacterium is the leading cause of bacterial sexually transmitted infections. Once internalized in host cells, undergoes a biphasic developmental cycle within a membrane-bound compartment, known as the inclusion. Successful establishment of the intracellular niche relies on bacterial Type III effector proteins, such as Inc proteins. and systems have contributed to elucidating the intracellular lifestyle of , but additional models combining the archetypal environment of infection with the advantages of systems are needed. Organoids are three-dimensional structures that recapitulate the microanatomy of an organ's epithelial layer, bridging the gap between and systems. Organoids are emerging as relevant model systems to study interactions between bacterial pathogens and their hosts. Here, we took advantage of recently developed murine endometrial organoids (EMOs) and present a -murine EMO infection model system. Confocal microscopy of EMOs infected with fluorescent protein-expressing bacteria revealed that inclusions are formed within the cytosol of epithelial cells. Moreover, infection with a strain that allows for the tracking of RB to EB transition indicated that the bacteria undergo a full developmental cycle, which was confirmed by harvesting infectious bacteria from infected EMOs. Finally, the inducible gene expression and cellular localization of a Inc protein within infected EMOs further demonstrated that this model is compatible with the study of Type III secreted effectors. Altogether, we describe a novel and relevant system for the study of -host interactions.

Ductal carcinoma (DCIS) is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in around 40% of patients. While immune infiltrates have been observed in these early cancer lesions, their potential prognostic value is still unclear. Regulatory T (Treg) cells accumulate in advanced breast cancers, and predict poor outcome. We have shown before that ablation of Treg cells in established tumors leads to significant decrease in primary and metastatic tumor burden. In this work, we sought to investigate Treg cell function in the progression from non-invasive to invasive breast cancer lesions. To this end, we used the murine mammary tumor virus polyoma middle T (MMTV-PyMT) murine model of spontaneous, stage-wise breast carcinogenesis crossed to Foxp3 knock in mice, allowing Treg cell ablation by administration of diphtheria toxin. Transient targeting of Treg cells at the carcinoma stage resulted in a significant increase in the number of tumor-bearing mammary glands and size of growing tumors compared with control mice. Whole mammary gland mounts and histological examination confirmed larger emergent tumor area in Treg cell-ablated mice, and revealed that these tumors were characterized by a more advanced tumor staging, with presence of early invasion, increased desmoplasia and collagen deposition. Furthermore, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Interestingly, Treg cell ablation resulted in increased inflammatory cytokines IL-4 and IL-5 with a concomitant reduction in classically activated tumor associated macrophages. This TH2-biased immune regulatory mammary inflammation was consistent with the enhancement in tumor promotion that we observed. Overall, our study demonstrates that Treg cells oppose breast cancer progression at early stages, raising a cautionary note regarding the consideration of immune intervention targeted at boosting immune responses for DCIS.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1 – 4 . However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer—an aggressive malignancy that is refractory to standard treatments and current immunotherapies 5 – 8 —induces endoplasmic reticulum stress and activates the IRE1α–XBP1 arm of the unfolded protein response 9 , 10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N -linked protein glycosylation defects in T cells, which triggered IRE1α–XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N -linked protein glycosylation, abrogating IRE1α–XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α–XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts. In human and mouse models of ovarian cancer, endoplasmic reticulum stress and the activation of the IRE1α–XBP1 pathway decreases the metabolic fitness of T cells and limits their anti-tumour functions.

Metastasis causes most cancer-related deaths in colorectal carcinoma (CRC), and microbiome markers may have prognostic value. We hypothesized that primary tumor microbiomes predict distant metastases. We analyzed 5-year metastasis-free survival (MFS) in a retrospective cohort of 900 ORIEN CRC tumor microbiomes (RNAseq). ORIEN findings were validated on an independent cohort using 16S rDNA sequencing and pathobiont-specific qPCR. Microbiome alpha diversity was higher in primary tumors than metastases and positively correlated with metastasis risk. Microbiome beta diversity distinguished primary vs. metastasis and predicted 5-year MFS. High primary tumor abundance of B. fragilis and low F. nucleatum were associated with short MFS. Enterobacteriaceae, including E. coli, were enriched in metastases. qPCR identified increased enterotoxigenic B. fragilis and pks  +  E. coli detection in CRC metastasizers. Microbial co-occurrence analysis identified a 3-species clique that predicts metastasis (OR 1.9 [1.4–2.6]). Results suggest that primary tumor microbiomes and specific pathobionts are precision markers for metastasis risk.

Abstract Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5–5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function.sup.1-4. However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer--an aggressive malignancy that is refractory to standard treatments and current immunotherapies.sup.5-8--induces endoplasmic reticulum stress and activates the IRE1[alpha]-XBP1 arm of the unfolded protein response.sup.9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1[alpha]-XBP1 activation that suppressed mitochondrial activity and IFN[gamma] production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1[alpha]-XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1[alpha]-XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts.

The abnormal tumor vasculature can present a barrier to the infiltration of anti-tumor immune cells, which impairs immune surveillance and response to immunotherapy. Here, we show that targeting the epigenetic factor DNA methyltransferase 1 in endothelial cells (ECs) reduces angiogenesis while imparting profound changes to the tumor immune microenvironment (TIME), including increased proportions of CD4+ memory T-cells and NK cells. Depleting CD4+ T-cells, or blocking lymphocyte egress from the lymph nodes with FTY720, rescues tumor growth in mice with conditional deletion of Dnmt1 in ECs (Dnmt1iECKO) and dramatically shortens overall survival, whereas NK cells are dispensable. Tumors implanted in Dnmt1iECKO mice show reduced vascular branching, elevated expression of Vcam1, increased vessel-associated T-cells, and a shift in vascular specification including increased proportions of immune-permissive post-capillary venules (PCVs) and interferon-stimulated ECs (IFN-ECs). Deleting Dnmt1 in EC cultures strikingly potentiates responses to combinations of IFNy; and TNFa; and, notably, up-regulates important T-cell co-stimulatory molecules for memory CD4+ T-cells, including Icosl, Cd40, and Tnfsf4. Finally, immune checkpoint blockade (ICB) administered to Dnmt1iECKO mice with experimental melanoma lung metastasis reduces tumor burden, with some mice showing tumor eradication. Our findings identify endothelial Dnmt1 as a key regulator of vascular-mediated anti-tumor immunity, providing a rationale for integrating epigenetic modulation of the vasculature with cancer immunotherapy regimens.

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we demonstrate that subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across four immunologically and hormonally distinct murine BC models. Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4 and CD8 T cells. When layered onto this immunogenic milieu, αCD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 33% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Efficacy required both CD4 and CD8 T cells, and complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T cell activation. Together, these findings establish T-FUS as an immune-potentiating partner for CD40 agonism, capable of driving durable, robust BC regression and immunological memory. This work positions T-FUS+CD40 agonism as a clinically scalable, in situ vaccination-like strategy with potential to benefit breast cancers, including luminal subtypes, that remain largely refractory to immune checkpoint blockade.

Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naïve LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naïve LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

Ovarian cancer remains the most lethal gynecologic malignancy, due in part to the establishment of a profoundly immunosuppressive tumor microenvironment (TME). While TLR5 signaling has previously been implicated in promoting myeloid cell recruitment to the ovarian TME, the upstream source of ligand and its systemic effects on hematopoiesis remain poorly understood(1,2). Here, we show that ovarian cancer disrupts gut barrier integrity, leading to systemic translocation of TLR5 ligands into the peritoneum, blood, and bone marrow. This translocation correlates with enhanced expansion of myeloid progenitors in the bone marrow of wild-type (WT) but not TLR5-deficient (TLR5 KO) mice, leading to enhanced accumulation of monocytes into the tumor microenvironment. Pharmacologic blockade of TLR5 in tumor-bearing mice alters the composition of tumor-associated myeloid populations, increasing the frequency of monocytes and CCR2-expressing macrophages In the bone marrow of tumor-bearing WT mice. In the bone marrow, blockade of TLR5 signaling led to expansion of granulocyte-monocyte progenitors (GMPs), a phenotype recapitulated in a competitive chimera model. In vitro, stimulation of WT bone marrow cells with purified TLR5 ligands led to enhanced colony formation and skewed differentiation toward granulocyte-macrophage lineages. These data reveal that chronic TLR5 signaling, driven by tumor-induced gut leakage, promotes expansion of myeloid cells within the bone marrow and is a host-intrinsic mechanism driving accumulation of immature monocytes and macrophages into the tumor microenvironment.