A new Neu-a syngeneic model of spontaneously metastatic HER2-positive breast cancer.

Metastatic disease results from the dissemination of tumor cells beyond their organ of origin to grow in distant organs and is the primary cause of death in patients with advanced breast cancer. Preclinical murine models in which primary tumors spontaneously metastasize are valuable tools for studying metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. The NT2.5-lung metastasis (-LM) cell line was derived from serial passaging of tumor cells that were macro-dissected from spontaneous lung metastases after orthotopic mammary implantation of parental NT2.5 cells. Within one week of NT2.5-LM implantation, metastases are observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. We demonstrate that NT2.5-LM metastases are positive for NeuN-the murine equivalent of human epidermal growth factor 2 (HER2). We further demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT), suggestive of their enhanced metastatic potential. Genomic analyses support these findings and reveal enrichment in EMT-regulating pathways. In addition, the metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. The new NT2.5-LM model provides certain advantages over the parental NT2/NT2.5 model, given its more rapid and spontaneous development of metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses.

Evaluation of markers of immunity in different metastatic immune microenvironments suggests more suppression within breast to liver metastases in breast cancer.

PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.

Mimicking the breast metastatic microenvironment: characterization of a novel syngeneic model of HER2+ breast cancer.

Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.

DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) superfamily member tumor necrosis factor-like protein 1A (TL1A) has been associated with the susceptibility and severity of inflammatory bowel diseases. However, the function of the tumor necrosis factor-like protein 1A and its receptor death receptor 3 (DR3) in the development of intestinal inflammation is incompletely understood. We investigated the role of DR3 expressed by intestinal epithelial cells (IECs) during intestinal homeostasis, tissue injury, and regeneration. METHODS: Clinical phenotype and histologic inflammation were assessed in C57BL/6 (wild-type), Tl1a-/- and Dr3-/- mice in dextran sulfate sodium (DSS)-induced colitis. We generated mice with an IEC-specific deletion of DR3 (Dr3ΔIEC) and assessed intestinal inflammation and epithelial barrier repair. In vivo intestinal permeability was assessed by fluorescein isothiocyanate dextran uptake. Proliferation of IECs was analyzed by bromodeoxyuridine incorporation. Expression of DR3 messenger RNA was assessed by fluorescent in situ hybridization. Small intestinal organoids were used to determine ex vivo regenerative potential. RESULTS: Dr3-/- mice developed more severe colonic inflammation than wild-type mice in DSS-induced colitis with significantly impaired IEC regeneration. Homeostatic proliferation of IECs was increased in Dr3-/- mice, but blunted during regeneration. Cellular localization and expression of the tight junction proteins Claudin-1 and zonula occludens-1 were altered, leading to increased homeostatic intestinal permeability. Dr3ΔIEC mice recapitulated the phenotype observed in Dr3-/- mice with increased intestinal permeability and IEC proliferation under homeostatic conditions and impaired tissue repair and increased bacterial translocation during DSS-induced colitis. Impaired regenerative potential and altered zonula occludens-1 localization also were observed in Dr3ΔIEC enteroids. CONCLUSIONS: Our findings establish a novel function of DR3 in IEC homeostasis and postinjury regeneration independent of its established role in innate lymphoid cells and T-helper cells.

BATF3 Protects Against Metabolic Syndrome and Maintains Intestinal Epithelial Homeostasis.

The intestinal immune system and microbiota are emerging as important contributors to the development of metabolic syndrome, but the role of intestinal dendritic cells (DCs) in this context is incompletely understood. BATF3 is a transcription factor essential in the development of mucosal conventional DCs type 1 (cDC1). We show that Batf3-/- mice developed metabolic syndrome and have altered localization of tight junction proteins in intestinal epithelial cells leading to increased intestinal permeability. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function in obese Batf3-/- mice. High-fat diet further enhanced the metabolic phenotype and susceptibility to dextran sulfate sodium colitis in Batf3-/- mice. Antibiotic treatment of Batf3-/- mice prevented metabolic syndrome and impaired intestinal barrier function. Batf3-/- mice have altered IgA-coating of fecal bacteria and displayed microbial dysbiosis marked by decreased obesity protective Akkermansia muciniphila, and Bifidobacterium. Thus, BATF3 protects against metabolic syndrome and preserves intestinal epithelial barrier by maintaining beneficial microbiota.

Entinostat Decreases Immune Suppression to Promote Antitumor Responses in a HER2+ Breast Tumor Microenvironment.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

Clocking cancer: the circadian clock as a target in cancer therapy.

Disruption of the cellular pathway modulating endogenous 24-h rhythms, referred to as "the circadian clock", has been recently proven to be associated with cancer risk, development, and progression. This pathway operates through a complex network of transcription-translation feedback loops generated by a set of interplaying proteins. The expression of core circadian clock genes is frequently dysregulated in human tumors; however, the specific effects and underlying mechanisms seem to vary depending on the cancer types and are not fully understood. In addition, specific oncogenes may differentially induce the dysregulation of the circadian clock in tumors. Pharmacological modulation of clock components has been shown to result in specific lethality in certain types of cancer cells, and thus holds great promise as a novel anti-cancer therapeutic approach. Here we present an overview of the rationale and current evidence for targeting the clock in cancer treatment.

Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease, modulating the location and severity of inflammation and fibrosis. TL1A expression is increased in inflamed mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice causes spontaneous ileitis, and exacerbates induced proximal colitis and fibrosis. Intestinal fibroblasts express Death-receptor 3 (DR3; the only know receptor for TL1A) and stimulation with TL1A induces activation in vitro. However, the contribution of direct TL1A-DR3 activation on fibroblasts to fibrosis in vivo remains unknown. TL1A overexpressing naïve T cells were transferred into Rag-/- , Rag-/- mice lacking DR3 in all cell types (Rag-/-Dr3-/-), or Rag-/- mice lacking DR3 only on fibroblasts (Rag-/-Dr3∆Col1a2) to induce colitis and fibrosis, assessed by clinical disease activity index, intestinal inflammation, and collagen deposition. Rag-/- mice developed overt colitis with intestinal fibrostenosis. In contrast, Rag-/-Dr3-/- demonstrated decreased inflammation and fibrosis. Despite similar clinical disease and inflammation as Rag-/-, Rag-/-Dr3∆Col1a2 exhibited reduced intestinal fibrosis and attenuated fibroblast activation and migration. RNA-Sequencing of TL1A-stimulated fibroblasts identified Rho signal transduction as a major pathway activated by TL1A and inhibition of this pathway modulated TL1A-mediated fibroblast functions. Thus, direct TL1A signaling on fibroblasts promotes intestinal fibrosis in vivo. These results provide novel insight into profibrotic pathways mediated by TL1A paralleling its pro-inflammatory effects.