Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing.

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators.

Molecular mechanisms driving clonal aggressiveness in leukemia are not fully understood. We tracked and analyzed two mouse MLL-rearranged leukemic clones independently evolving towards higher aggressiveness. More aggressive subclones lost their growth differential ex vivo but restored it upon secondary transplantation, suggesting molecular memory of aggressiveness. Development of aggressiveness was associated with clone-specific gradual modulation of chromatin states and expression levels across the genome, with a surprising preferential trend of reversing the earlier changes between normal and leukemic progenitors. To focus on the core aggressiveness program, we identified genes with consistent changes of expression and chromatin marks that were maintained in vivo and ex vivo in both clones. Overexpressing selected core genes (Smad1 as aggressiveness driver, Irx5 and Plag1 as suppressors) affected leukemic progenitor growth in the predicted way and had convergent downstream effects on central transcription factors and repressive epigenetic modifiers, suggesting a broader regulatory network of leukemic aggressiveness.

Single-cell analyses of metastatic bone marrow in human neuroblastoma reveals microenvironmental remodeling and metastatic signature.

Neuroblastoma is an aggressive pediatric cancer with a high rate of metastasis to the BM. Despite intensive treatments including high-dose chemotherapy, the overall survival rate for children with metastatic neuroblastoma remains dismal. Understanding the cellular and molecular mechanisms of the metastatic tumor microenvironment is crucial for developing new therapies and improving clinical outcomes. Here, we used single-cell RNA-Seq to characterize immune and tumor cell alterations in neuroblastoma BM metastases by comparative analysis with patients without metastases. Our results reveal remodeling of the immune cell populations and reprogramming of gene expression profiles in the metastatic niche. In particular, within the BM metastatic niche, we observed the enrichment of immune cells, including tumor-associated neutrophils, macrophages, and exhausted T cells, as well as an increased number of Tregs and a decreased number of B cells. Furthermore, we highlighted cell communication between tumor cells and immune cell populations, and we identified prognostic markers in malignant cells that are associated with worse clinical outcomes in 3 independent neuroblastoma cohorts. Our results provide insight into the cellular, compositional, and transcriptional shifts underlying neuroblastoma BM metastases that contribute to the development of new therapeutic strategies.

Clonal Parabacteroides from Gut Microfistulous Tracts as Transmissible Cytotoxic Succinate-Commensal Model of Crohn's Disease Complications.

Crohn's disease (CD) has been traditionally viewed as a chronic inflammatory disease that cause gut wall thickening and complications, including fistulas, by mechanisms not understood. By focusing on Parabacteroides distasonis (presumed modern succinate-producing commensal probiotic), recovered from intestinal microfistulous tracts (cavernous fistulous micropathologies CavFT proposed as intermediate between 'mucosal fissures' and 'fistulas') in two patients that required surgery to remove CD-damaged ilea, we demonstrate that such isolates exert pathogenic/pathobiont roles in mouse models of CD. Our isolates are clonally-related; potentially emerging as transmissible in the community and mice; proinflammatory and adapted to the ileum of germ-free mice prone to CD-like ileitis (SAMP1/YitFc) but not healthy mice (C57BL/6J), and cytotoxic/ATP-depleting to HoxB8-immortalized bone marrow derived myeloid cells from SAMP1/YitFc mice when concurrently exposed to succinate and extracts from CavFT-derived E. coli , but not to cells from healthy mice. With unique genomic features supporting recent genetic exchange with Bacteroides fragilis -BGF539, evidence of international presence in primarily human metagenome databases, these CavFT Pdis isolates could represent to a new opportunistic Parabacteroides species, or subspecies (' cavitamuralis' ) adapted to microfistulous niches in CD.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is 1) strictly dependent on pyrimidine nucleotide depletion, 2) independent of canonical antigen presentation pathway transcriptional regulators, and 3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Regulation of EZH2 Expression by INPP4B in Normal Prostate and Primary Prostate Cancer.

The phosphatases INPP4B and PTEN are tumor suppressors that are lost in nearly half of advanced metastatic cancers. The loss of PTEN in prostate epithelium initially leads to an upregulation of several tumor suppressors that slow the progression of prostate cancer in mouse models. We tested whether the loss of INPP4B elicits a similar compensatory response in prostate tissue and whether this response is distinct from the one caused by the loss of PTEN. Knockdown of INPP4B but not PTEN in human prostate cancer cell lines caused a decrease in EZH2 expression. In Inpp4b-/- mouse prostate epithelium, EZH2 levels were decreased, as were methylation levels of histone H3. In contrast, Ezh2 levels were increased in the prostates of Pten-/- male mice. Contrary to PTEN, there was a positive correlation between INPP4B and EZH2 expression in normal human prostates and early-stage prostate tumors. Analysis of single-cell transcriptomic data demonstrated that a subset of EZH2-positive cells expresses INPP4B or PTEN, but rarely both, consistent with their opposing correlation with EZH2 expression. Unlike PTEN, INPP4B did not affect the levels of SMAD4 protein expression or Pml mRNA expression. Like PTEN, p53 protein expression and phosphorylation of Akt in Inpp4b-/- murine prostates were elevated. Taken together, the loss of INPP4B in the prostate leads to overlapping and distinct changes in tumor suppressor and oncogenic downstream signaling.

Androgen blockade primes NLRP3 in macrophages to induce tumor phagocytosis.

Immune-based therapies induce durable remissions in subsets of patients across multiple malignancies. However, there is limited efficacy of immunotherapy in metastatic castrate-resistant prostate cancer (mCRPC), manifested by an enrichment of immunosuppressive (M2) tumor- associated macrophages (TAM) in the tumor immune microenvironment (TME). Therefore, therapeutic strategies to overcome TAM-mediated immunosuppression are critically needed in mCRPC. Here we discovered that NLR family pyrin domain containing 3 (NLRP3), an innate immune sensing protein, is highly expressed in TAM from metastatic PC patients treated with standard-of-care androgen deprivation therapy (ADT). Importantly, ex vivo studies revealed that androgen receptor (AR) blockade in TAM upregulates NLRP3 expression, but not inflammasome activity, and concurrent AR blockade/NLRP3 agonist (NLRP3a) treatment promotes cancer cell phagocytosis by immunosuppressive M2 TAM. In contrast, NLRP3a monotherapy was sufficient to enhance phagocytosis of cancer cells in anti-tumor (M1) TAM, which exhibit high de novo NLRP3 expression. Critically, combinatorial treatment with ADT/NLRP3a in a murine model of advanced PC resulted in significant tumor control, with tumor clearance in 55% of mice via TAM phagocytosis. Collectively, our results demonstrate NLRP3 as an AR-regulated "macrophage phagocytic checkpoint", inducibly expressed in TAM by ADT and activated by NLRP3a treatment, the combination resulting in TAM-mediated phagocytosis and tumor control.

The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG.

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.

Health Consequences of Thymus Removal in Adults.

BACKGROUND: The function of the thymus in human adults is unclear, and routine removal of the thymus is performed in a variety of surgical procedures. We hypothesized that the adult thymus is needed to sustain immune competence and overall health. METHODS: We evaluated the risk of death, cancer, and autoimmune disease among adult patients who had undergone thymectomy as compared with demographically matched controls who had undergone similar cardiothoracic surgery without thymectomy. T-cell production and plasma cytokine levels were also compared in a subgroup of patients. RESULTS: After exclusions, 1420 patients who had undergone thymectomy and 6021 controls were included in the study; 1146 of the patients who had undergone thymectomy had a matched control and were included in the primary cohort. At 5 years after surgery, all-cause mortality was higher in the thymectomy group than in the control group (8.1% vs. 2.8%; relative risk, 2.9; 95% confidence interval [CI], 1.7 to 4.8), as was the risk of cancer (7.4% vs. 3.7%; relative risk, 2.0; 95% CI, 1.3 to 3.2). Although the risk of autoimmune disease did not differ substantially between the groups in the overall primary cohort (relative risk, 1.1; 95% CI, 0.8 to 1.4), a difference was found when patients with preoperative infection, cancer, or autoimmune disease were excluded from the analysis (12.3% vs. 7.9%; relative risk, 1.5; 95% CI, 1.02 to 2.2). In an analysis involving all patients with more than 5 years of follow-up (with or without a matched control), all-cause mortality was higher in the thymectomy group than in the general U.S. population (9.0% vs. 5.2%), as was mortality due to cancer (2.3% vs. 1.5%). In the subgroup of patients in whom T-cell production and plasma cytokine levels were measured (22 in the thymectomy group and 19 in the control group; mean follow-up, 14.2 postoperative years), those who had undergone thymectomy had less new production of CD4+ and CD8+ lymphocytes than controls (mean CD4+ signal joint T-cell receptor excision circle [sjTREC] count, 1451 vs. 526 per microgram of DNA [P = 0.009]; mean CD8+ sjTREC count, 1466 vs. 447 per microgram of DNA [P<0.001]) and higher levels of proinflammatory cytokines in the blood. CONCLUSIONS: In this study, all-cause mortality and the risk of cancer were higher among patients who had undergone thymectomy than among controls. Thymectomy also appeared be associated with an increased risk of autoimmune disease when patients with preoperative infection, cancer, or autoimmune disease were excluded from the analysis. (Funded by the Tracey and Craig A. Huff Harvard Stem Cell Institute Research Support Fund and others.).

DHODH: a promising target in the treatment of T-cell acute lymphoblastic leukemia.

Patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) have a poor prognosis with few therapeutic options. With the goal of identifying novel therapeutic targets, we used data from the Dependency Map project to identify dihydroorotate dehydrogenase (DHODH) as one of the top metabolic dependencies in T-ALL. DHODH catalyzes the fourth step of de novo pyrimidine nucleotide synthesis. Small molecule inhibition of DHODH rapidly leads to the depletion of intracellular pyrimidine pools and forces cells to rely on extracellular salvage. In the absence of sufficient salvage, this intracellular nucleotide starvation results in the inhibition of DNA and RNA synthesis, cell cycle arrest, and, ultimately, death. T lymphoblasts appear to be specifically and exquisitely sensitive to nucleotide starvation after DHODH inhibition. We have confirmed this sensitivity in vitro and in vivo in 3 murine models of T-ALL. We identified that certain subsets of T-ALL seem to have an increased reliance on oxidative phosphorylation when treated with DHODH inhibitors. Through a series of metabolic assays, we show that leukemia cells, in the setting of nucleotide starvation, undergo changes in their mitochondrial membrane potential and may be more highly dependent on alternative fuel sources. The effect on normal T-cell development in young mice was also examined to show that DHODH inhibition does not permanently damage the developing thymus. These changes suggest a new metabolic vulnerability that may distinguish these cells from normal T cells and other normal hematopoietic cells and offer an exploitable therapeutic opportunity. The availability of clinical-grade DHODH inhibitors currently in human clinical trials suggests a potential for rapidly advancing this work into the clinic.

The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression.

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

Limited plasticity of monocyte fate and function associated with epigenetic scripting at the level of progenitors.

Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.

Prenylcysteine oxidase 1 like protein is required for neutrophil bactericidal activities.

The bactericidal function of neutrophils is dependent on a myriad of intrinsic and extrinsic stimuli. Using systems immunology approaches we identify microbiome- and infection-induced changes in neutrophils. We focus on investigating the Prenylcysteine oxidase 1 like (Pcyox1l) protein function. Murine and human Pcyox1l proteins share ninety four percent aminoacid homology revealing significant evolutionary conservation and implicating Pcyox1l in mediating important biological functions. Here we show that the loss of Pcyox1l protein results in significant reductions in the mevalonate pathway impacting autophagy and cellular viability under homeostatic conditions. Concurrently, Pcyox1l CRISPRed-out neutrophils exhibit deficient bactericidal properties. Pcyox1l knock-out mice demonstrate significant susceptibility to infection with the gram-negative pathogen Psuedomonas aeruginosa exemplified through increased neutrophil infiltrates, hemorrhaging, and reduced bactericidal functionality. Cumulatively, we ascribe a function to Pcyox1l protein in modulation of the prenylation pathway and suggest connections beween metabolic responses and neutrophil functionality.

Reversal of Lactate and PD-1-mediated Macrophage Immunosuppression Controls Growth of PTEN/p53-deficient Prostate Cancer.

PURPOSE: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC. EXPERIMENTAL DESIGN: Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC samples was performed using 10X Genomics platform. RESULTS: Coclinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/ß-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression. CONCLUSIONS: Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.

Comprehensive Next-Generation Sequencing Testing in a Patient with TEMPI Syndrome.

TEMPI syndrome is a new and poorly understood disease that is currently considered a type of plasma cell neoplasm with paraneoplastic manifestations. The TEMPI acronym defines the hallmarks of the syndrome: T for telangiectasia; E for erythrocytosis with elevated erythropoietin; M, monoclonal gammopathy; P, perinephric collections; and I, intrapulmonary shunting. Due to the marked erythrocytosis as the most common presenting feature, TEMPI is often misdiagnosed as polycythemia vera. However, unlike polycythemia vera, TEMPI is not associated with a JAK2 mutation. The pathogenesis of TEMPI syndrome is unknown, although a few hypothetical disease mechanisms have been previously discussed. Here we present a new case of TEMPI syndrome, discuss results of a next-generation sequencing (NGS) panel covering 1,425 known cancer-related genes, and review the current literature with focus on an update of the genetics of TEMPI syndrome. This is the first report of TEMPI that includes results of comprehensive NGS testing.

Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay.

Neutrophils are a vital component of the innate immune system and play an essential function in the recognition and clearance of bacterial and fungal pathogens. There is great interest in understanding mechanisms of neutrophil dysfunction in the setting of disease and deciphering potential side effects of immunomodulatory drugs on neutrophil function. We developed a high throughput flow cytometry-based assay for detecting changes to four canonical neutrophil functions following biological or chemical triggers. Our assay detects neutrophil phagocytosis, reactive oxygen species (ROS) generation, ectodomain shedding, and secondary granule release in a single reaction mixture. By selecting fluorescent markers with minimal spectral overlap, we merge four detection assays into one microtiter plate-based assay. We demonstrate the response to the fungal pathogen, Candida albicans and validate the assay's dynamic range using the inflammatory cytokines G-CSF, GM-CSF, TNFα, and IFNγ. All four cytokines increased ectodomain shedding and phagocytosis to a similar degree while GM-CSF and TNFα were more active in degranulation when compared to IFNγ and G-CSF. We further demonstrated the impact of small molecule inhibitors such as kinase inhibition downstream of Dectin-1, a critical lectin receptor responsible for fungal cell wall recognition. Bruton's tyrosine kinase (Btk), Spleen tyrosine kinase (Syk), and Src kinase inhibition suppressed all four measured neutrophil functions but all functions were restored with lipopolysaccharide co-stimulation. This new assay allows for multiple comparisons of effector functions and permits identification of distinct subpopulations of neutrophils with a spectrum of activity. Our assay also offers the potential for studying the intended and off-target effects of immunomodulatory drugs on neutrophil responses.

Dissecting the immune suppressive human prostate tumor microenvironment via integrated single-cell and spatial transcriptomic analyses.

The treatment of low-risk primary prostate cancer entails active surveillance only, while high-risk disease requires multimodal treatment including surgery, radiation therapy, and hormonal therapy. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we comprehensively describe the tumor microenvironment of localized prostate cancer in comparison with adjacent normal samples and healthy controls. Single-cell RNA sequencing and high-resolution spatial transcriptomic analyses reveal tumor context dependent changes in gene expression. Our data indicate that an immune suppressive tumor microenvironment associates with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We infer cell-to-cell relationships from high throughput ligand-receptor interaction measurements within undissociated tissue sections. Our work thus provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.