Soluble factors in malignant ascites promote the metastatic adhesion of gastric adenocarcinoma cells.

BACKGROUND: Adenocarcinoma of the proximal stomach is the fastest rising malignancy in North America. It is commonly associated with peritoneal accumulation of malignant ascites (MA), a fluid containing cancer and inflammatory cells and soluble proteins. Peritoneal metastasis (PM) is the most common site of gastric cancer (GC) progression after curative-intent surgery and is the leading cause of death among GC patients. METHODS/RESULTS: Using a panel of gastric adenocarcinoma cell lines (human: MKN 45, SNU-5; murine: NCC-S1M), we demonstrate that prior incubation of GC cells with MA results in a significant (> 1.7-fold) increase in the number of cells capable of adhering to human peritoneal mesothelial cells (HPMC) (p < 0.05). We then corroborate these findings using an ex vivo PM model and show that MA also significantly enhances the ability of GC cells to adhere to strips of human peritoneum (p < 0.05). Using a multiplex ELISA, we identify MIF and VEGF as consistently elevated across MA samples from GC patients (p < 0.05). We demonstrate that agents that block the effects of MIF or VEGF abrogate the ability of MA to stimulate the adhesion of GC cells to adhere to human peritoneum and promote both ex vivo and in vivo metastases. CONCLUSION: Agents targeting MIF or VEGF may be relevant to the treatment or prevention of PM in GC patients.

Relation between mismatch repair status, chemoresponse, survival and anatomic location in gastroesophageal adenocarcinoma.

BACKGROUND: It has recently been reported that mismatch repair (MMR) status and microsatellite instability (MSI) status in gastroesophageal carcinomas predict surgical, chemotherapeutic and immunotherapeutic outcomes; however, there is extensive variability in the reported incidence and clinical implications of MMR/MSI status in gastroesophaegal adenocarcinomas. We characterized a Canadian surgical patient cohort with respect to MMR status, clinicopathologic correlates and anatomic tumour location. METHODS: We investigated MMR and BRAF V600E status of gastroesophaegal adenocarcinomas in patients who underwent gastrectomy or esophagectomy with extended (D2) lymphadenectomy at a single centre between 2011 and 2019. We correlated patterns of MMR expression in the overall cohort and in anatomic location-defined subgroups with treatment response and overall survival using multivariate analysis. RESULTS: In all, 226 cases of gastroesophaegal adenocarcinoma (63 esophageal, 98 gastroesophageal junctional and 65 gastric) were included. The MMR-deficient (dMMR) immunophenotype was found in 28 tumours (12.3%) (15 junctional [15.3%], 13 gastric [20.0%] and none of the esophageal). The majority (25 [89%]) of dMMR cases showed MLH1/PMS2 loss without concurrent BRAF V600E mutation. Two MSH2/ MSH6-deficient gastric tumours and 1 MSH6-deficient junctional tumour were detected. The pathologic response to preoperative chemotherapy was comparable in the dMMR and MMR-proficient (pMMR) cohorts. However, dMMR status was associated with significantly longer median overall survival than pMMR status (5.8 yr v. 2.4 yr, hazard ratio [HR] 1.91, 95% confidence interval [CI] 1.06-3.46), particularly in junctional tumours (4.6 yr v. 1.9 yr, HR 2.97, 95% CI 1.27-6.94). CONCLUSION: Our study shows that MMR status has at least prognostic value, which supports the need for biomarker testing in gastroesophageal adenocarcinomas, including junctional adenocarcinomas. This highlights the clinical significance of determining the MMR status in all adenocarcinomas of the upper gastrointestinal tract. Response to induction chemotherapy, however, was not influenced by MMR status.

The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.

Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis.

Deficiency of the chromatin regulator BRPF1 causes abnormal brain development.

Epigenetic mechanisms are important in different neurological disorders, and one such mechanism is histone acetylation. The multivalent chromatin regulator BRPF1 (bromodomain- and plant homeodomain-linked (PHD) zinc finger-containing protein 1) recognizes different epigenetic marks and activates three histone acetyltransferases, so it is both a reader and a co-writer of the epigenetic language. The three histone acetyltransferases are MOZ, MORF, and HBO1, which are also known as lysine acetyltransferase 6A (KAT6A), KAT6B, and KAT7, respectively. The MORF gene is mutated in four neurodevelopmental disorders sharing the characteristic of intellectual disability and frequently displaying callosal agenesis. Here, we report that forebrain-specific inactivation of the mouse Brpf1 gene caused early postnatal lethality, neocortical abnormalities, and partial callosal agenesis. With respect to the control, the mutant forebrain contained fewer Tbr2-positive intermediate neuronal progenitors and displayed aberrant neurogenesis. Molecularly, Brpf1 loss led to decreased transcription of multiple genes, such as Robo3 and Otx1, important for neocortical development. Surprisingly, elevated expression of different Hox genes and various other transcription factors, such as Lhx4, Foxa1, Tbx5, and Twist1, was also observed. These results thus identify an important role of Brpf1 in regulating forebrain development and suggest that it acts as both an activator and a silencer of gene expression in vivo.

The chromatin regulator Brpf1 regulates embryo development and cell proliferation.

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs.

HiChIP-Based Epigenomic Footprinting Identifies a Promoter Variant of UXS1 That Confers Genetic Susceptibility to Gastroesophageal Cancer.

Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNV) associated with the risk of gastroesophageal cancer (GEC). The majority of the identified SNVs map to noncoding regions of the genome. Uncovering the causal SNVs and genes they modulate could help improve GEC prevention and treatment. Herein, we used HiChIP against histone 3 lysine 27 acetylation (H3K27ac) to simultaneously annotate active promoters and enhancers, identify the interactions between them, and detect nucleosome-free regions (NFR) harboring potential causal SNVs in a single assay. The application of H3K27ac HiChIP in GEC relevant models identified 61 potential functional SNVs that reside in NFRs and interact with 49 genes at 17 loci. The approach led to a 67% reduction in the number of SNVs in linkage disequilibrium at these 17 loci, and at 7 loci, a single putative causal SNV was identified. One SNV, rs147518036, located within the promoter of the UDP-glucuronate decarboxylase 1 (UXS1) gene, seemed to underlie the GEC risk association captured by the rs75460256 index SNV. The rs147518036 SNV creates a GABPA DNA recognition motif, resulting in increased promoter activity, and CRISPR-mediated inhibition of the UXS1 promoter reduced the viability of the GEC cells. These findings provide a framework that simplifies the identification of potentially functional regulatory SNVs and target genes underlying risk-associated loci. In addition, the study implicates increased expression of the enzyme UXS1 and activation of its metabolic pathway as a predisposition to gastric cancer, which highlights potential therapeutic avenues to treat this disease. Significance: Epigenomic footprinting using a histone posttranslational modification targeted 3D genomics methodology elucidates functional noncoding sequence variants and their target genes at cancer risk loci.

Constructing 3D In Vitro Models of Heterocellular Solid Tumors and Stromal Tissues Using Extrusion-Based Bioprinting.

Malignant tumor tissues exhibit inter- and intratumoral heterogeneities, aberrant development, dynamic stromal composition, diverse tissue phenotypes, and cell populations growing within localized mechanical stresses in hypoxic conditions. Experimental tumor models employing engineered systems that isolate and study these complex variables using in vitro techniques are under development as complementary methods to preclinical in vivo models. Here, advances in extrusion bioprinting as an enabling technology to recreate the three-dimensional tumor milieu and its complex heterogeneous characteristics are reviewed. Extrusion bioprinting allows for the deposition of multiple materials, or selected cell types and concentrations, into models based upon physiological features of the tumor. This affords the creation of complex samples with representative extracellular or stromal compositions that replicate the biology of patient tissue. Biomaterial engineering of printable materials that replicate specific features of the tumor microenvironment offer experimental reproducibility, throughput, and physiological relevance compared to animal models. In this review, we describe the potential of extrusion-based bioprinting to recreate the tumor microenvironment within in vitro models.

Bioprinted Multicomponent Hydrogel Co-culture Tumor-Immune Model for Assessing and Simulating Tumor-Infiltrated Lymphocyte Migration and Functional Activation.

The immune response against a tumor is characterized by the interplay among components of the immune system and neoplastic cells. Here, we bioprinted a model with two distinct regions containing gastric cancer patient-derived organoids (PDOs) and tumor-infiltrated lymphocytes (TILs). The initial cellular distribution allows for the longitudinal study of TIL migratory patterns concurrently with multiplexed cytokine analysis. The chemical properties of the bioink were designed to present physical barriers that immune T-cells must breech during infiltration and migration toward a tumor with the use of an alginate, gelatin, and basal membrane mix. TIL activity, degranulation, and regulation of proteolytic activity reveal insights into the time-dependent biochemical dynamics. Regulation of the sFas and sFas-ligand present on PDOs and TILs, respectively, and the perforin and granzyme longitudinal secretion confirms TIL activation when encountering PDO formations. TIL migratory profiles were used to create a deterministic reaction-advection diffusion model. The simulation provides insights that decouple passive from active cell migration mechanisms. The mechanisms used by TILs and other adoptive cell therapeutics as they infiltrate the tumor barrier are poorly understood. This study presents a pre-screening strategy for immune cells where motility and activation across ECM environments are crucial indicators of cellular fitness.

Improved platform for breast cancer circulating tumor cell enrichment and characterization with next-generation sequencing technology.

Circulating tumor cells (CTCs) represent cells shed from the primary tumor or metastatic sites and can be used to monitor treatment response and tumor recurrence. However, CTCs circulate in extremely low numbers making in-depth analysis beyond simple enumeration challenging when collected from peripheral blood. Furthermore, tumor heterogeneity, a hallmark of many tumors, especially breast cancer, further complicates CTC characterization. To overcome this limitation, we developed a platform based on the large-scale isolation of CTCs by apheresis, allowing us to collect CTCs in large numbers, which were preserved live in liquid nitrogen for further characterization. Flow cytometry followed by cell sorting (FACS) was performed using a combination of antibodies directed against cell surface markers of white blood cells (CD45) and epithelial tumor cells (CK8). Analysis of subpopulations CD45+/- and CK8+/- by bulk RNA sequencing (RNAseq) and the CD45-/CK8 positive population by single-cell RNAseq was performed. The CD45- population was enriched using CD45 magnetic beads separation and examined by IHC for pan-cytokeratin and immunofluorescence (IF) for specific markers, including the elusive circulating cancer stem cells (CSCs). CSC-rich mammospheres were grown in vitro for further analysis and treated to examine their response to chemotherapeutic agents. Finally, mammospheres were transplanted into the mammary fat pad and bone of immunodeficient mice to examine tumor growth in vivo. This platform enables the detection and collection of CTCs in early and late-stage breast cancer patients of every subtype. Markers including CD44/24, ALDH1 and CXCR4 were identified by IF and showed high expression following mammosphere culture, which responded predictably to chemotherapeutic agents. Mammospheres were also transplanted into nude mice and induced tumors in the mammary fat pad and bone following intra-tibial transplantation. Finally, bulk RNA analysis of the FACS isolated CD45+/- and CK8+/- cells showed a clear separation of CD45- away from CD45+ populations. Single-cell RNAseq of the FACS isolated CD45-/CK8+ cells showed the presence of 4-5 clusters, confirming the high degree of heterogeneity of CTCs. Our platform for large-scale isolation of CTCs using apheresis is suitable for an in-depth analysis of the cancer phenotype and may eventually allow evaluation in real-time of the disease process to optimize cancer regimens.

Protein-tyrosine phosphatase 1B modulates early endosome fusion and trafficking of Met and epidermal growth factor receptors.

The endoplasmic reticulum-localized non-receptor protein-tyrosine phosphatase 1B (PTP1B) is associated with oncogenic, metabolic, and cytokine-related signaling and functionally targets multiple receptor tyrosine kinases (RTKs) for dephosphorylation. Loss of PTP1B activity leads to enhanced ligand-dependent biological activity of the Met RTK among others. Here, we demonstrate that knockdown of PTP1B or expression of a PTP1B trapping aspartic acid-to-alanine substitution (D/A) mutant delayed ligand-induced degradation of the Met and EGF RTKs. Loss of PTP1B function abrogated trafficking of Met and EGF receptor to Rab5- and phosphatidylinositol 3-phosphate (Pl3P)-positive early endosomes and subsequent trafficking through the degradative pathway. Under these conditions, internalization of the Met and EGF receptors was unaltered, suggesting a block at the level of early endosome formation. We show that the N-ethylmaleimide-sensitive factor (NSF), an essential component of the vesicle fusion machinery, was hyperphosphorylated in PTP1B knockdown or PTP1B D/A-expressing cells and was a target for PTP1B. NSF knockdown phenocopied PTP1B knockdown, demonstrating a mechanism through which PTP1B regulates endocytic trafficking. Finally, we show that PTP1B dephosphorylated NSF and that this interaction was required for physiological RTK trafficking and appropriate attenuation of downstream signaling.

Gene-expression profiling of microdissected breast cancer microvasculature identifies distinct tumor vascular subtypes.

INTRODUCTION: Angiogenesis represents a potential therapeutic target in breast cancer. However, responses to targeted antiangiogenic therapies have been reported to vary among patients. This suggests that the tumor vasculature may be heterogeneous and that an appropriate choice of treatment would require an understanding of these differences. METHODS: To investigate whether and how the breast tumor vasculature varies between individuals, we isolated tumor-associated and matched normal vasculature from 17 breast carcinomas by laser-capture microdissection, and generated gene-expression profiles. Because microvessel density has previously been associated with disease course, tumors with low (n = 9) or high (n = 8) microvessel density were selected for analysis to maximize heterogeneity for this feature. RESULTS: We identified differences between tumor and normal vasculature, and we describe two subtypes present within tumor vasculature. These subtypes exhibit distinct gene-expression signatures that reflect features including hallmarks of vessel maturity. Potential therapeutic targets (MET, ITGAV, and PDGFRß) are differentially expressed between subtypes. Taking these subtypes into account has allowed us to derive a vascular signature associated with disease outcome. CONCLUSIONS: Our results further support a role for tumor microvasculature in determining disease progression. Overall, this study provides a deeper molecular understanding of the heterogeneity existing within the breast tumor vasculature and opens new avenues toward the improved design and targeting of antiangiogenic therapies.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone.

INTRODUCTION: Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. METHODS: To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors. RESULTS: ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5. CONCLUSIONS: Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

Characterisation of the Stromal Microenvironment in Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways.

Inhibition of LPS-mediated TLR4 activation abrogates gastric adenocarcinoma-associated peritoneal metastasis.

Surgical resection, the cornerstone of curative intent treatment for gastric adenocarcinoma, is associated with a high rate of infection-related post-operative complications, leading to an increased incidence of metastasis to the peritoneum. However, the mechanisms underlying this process are poorly understood. Lipopolysaccharide (LPS), an antigen from Gram-negative bacteria, represents a potential mechanism via induction of local and systemic inflammation through activation of Toll-like receptor (TLR). Here, we use both a novel ex vivo model of peritoneal metastasis and in vivo animal models to assess gastric cancer cell adhesion to peritoneum both before and after inhibition of the TLR4 pathway. We demonstrate that activation of TLR4 by either LPS or Gram-negative bacteria (E. coli) significantly increases the adherence of gastric cancer cells to human peritoneal mesothelial cells, and that this increased adherence is abrogated by inhibition of the TLR4 signal cascade and downstream TAK1 and MEK1/2 pathways. We also demonstrate that the influence of LPS on adherence extends to peritoneal tissue and metastatic spread. Furthermore, we show that loss of TLR4 at the site of metastasis reduces tumor cell adhesion, implicating the TLR4 signaling cascade in potentiating metastatic adhesion and peritoneal spread. These results identify potential therapeutic targets for the clinical management of patients undergoing resection for gastric cancer.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

Chemogenomic profiling of breast cancer patient-derived xenografts reveals targetable vulnerabilities for difficult-to-treat tumors.

Subsets of breast tumors present major clinical challenges, including triple-negative, metastatic/recurrent disease and rare histologies. Here, we developed 37 patient-derived xenografts (PDX) from these difficult-to-treat cancers to interrogate their molecular composition and functional biology. Whole-genome and transcriptome sequencing and reverse-phase protein arrays revealed that PDXs conserve the molecular landscape of their corresponding patient tumors. Metastatic potential varied between PDXs, where low-penetrance lung micrometastases were most common, though a subset of models displayed high rates of dissemination in organotropic or diffuse patterns consistent with what was observed clinically. Chemosensitivity profiling was performed in vivo with standard-of-care agents, where multi-drug chemoresistance was retained upon xenotransplantation. Consolidating chemogenomic data identified actionable features in the majority of PDXs, and marked regressions were observed in a subset that was evaluated in vivo. Together, this clinically-annotated PDX library with comprehensive molecular and phenotypic profiling serves as a resource for preclinical studies on difficult-to-treat breast tumors.

Primary tumors induce neutrophil extracellular traps with targetable metastasis promoting effects.

Targeting the dynamic tumor immune microenvironment (TIME) can provide effective therapeutic strategies for cancer. Neutrophils are the predominant leukocyte population in mice and humans, and mounting evidence implicates these cells during tumor growth and metastasis. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of binding tumor cells to support metastatic progression. Here we demonstrate for the first time that circulating NET levels are elevated in advanced esophageal, gastric and lung cancer patients compared to healthy controls. Using pre-clinical murine models of lung and colon cancer in combination with intravital video microscopy, we show that NETs functionally regulate disease progression and that blocking NETosis through multiple strategies significantly inhibits spontaneous metastasis to the lung and liver. Further, we visualize how inhibiting tumor-induced NETs decreases cancer cell adhesion to liver sinusoids following intrasplenic injection - a mechanism previously thought to be driven primarily by exogenous stimuli. Thus, in addition to neutrophil abundance, the functional contribution of NETosis within the TIME has critical translational relevance and represents a promising target to impede metastatic dissemination.

Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.