Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Procoagulant platelets promote immune evasion in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive tumor entity, in which immune checkpoint (IC) molecules are primarily synthesized in the tumor environment. Here, we report that procoagulant platelets bear large amounts of such immunomodulatory factors and that the presence of these cellular blood components in TNBC relates to pro-tumorigenic immune cell activity and impaired survival. Mechanistically, tumor-released nucleic acids attract platelets into the aberrant tumor microvasculature where they undergo procoagulant activation, thus delivering specific stimulatory and inhibitory IC molecules. This concomitantly promotes pro-tumorigenic myeloid leukocyte responses and compromises anti-tumorigenic lymphocyte activity, ultimately supporting tumor growth. Interference with platelet-leukocyte interactions prevented immune cell misguidance and suppressed tumor progression, nearly as effective as systemic IC inhibition. Hence, our data uncover a self-sustaining mechanism of TNBC in utilizing platelets to misdirect immune cell responses. Targeting this irregular multicellular interplay might represent a novel immunotherapeutic strategy in TNBC without side effects of systemic IC inhibition.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

The fusion oncogene VAV1-MYO1F triggers aberrant T-cell receptor signaling in vivo and drives peripheral T-cell lymphoma in mice.

VAV1-MYO1F is a recently identified gain-of-function fusion protein of the proto-oncogene Vav guanine nucleotide exchange factor 1 (VAV1) that is recurrently detected in T-cell non-Hodgkin's lymphoma (T-NHL) patients. However, the pathophysiological functions of VAV1-MYO1F in lymphomagenesis are insufficiently defined. Therefore, we generated transgenic mouse models to conditionally express VAV1-MYO1F in T-cells in vivo. We demonstrate that VAV1-MYO1F triggers cell autonomous activation of T-cell signaling with an activation of the ERK, JNK, and AKT pathways. VAV1-MYO1F expression induces a T-cell activation phenotype with high surface expression of CD25, ICOS, CD44, PD-1, and decreased CD62L as well as aberrant T-cell differentiation, proliferation, and neoplastic transformation. Consequently, the VAV1-MYO1F expressing T-cells induce a malignant T lymphoproliferative disease with 100% penetrance in vivo that mimics key aspects of human peripheral T-cell lymphoma. These results demonstrate that the human T-cell oncogene VAV1-MYO1F is sufficient to trigger oncogenic T-cell signaling and neoplastic transformation, and moreover, it provides a new clinically relevant mouse model to explore the pathogenesis of and treatment concepts for human T-cell lymphoma.

Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas With EWSR1 and CIC::DUX4 Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers.

Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.

Proliferation and apoptosis after whole-body irradiation: longitudinal PET study in a mouse model.

PURPOSE: A reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose. METHODS: Four groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results. RESULTS: The hematopoietic bone marrow showed a significantly increased [18F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [18F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [18F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data. CONCLUSION: [18F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [18F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [18F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.

Sensitive Positron Emission Tomography Imaging of PD-L1 Expression in Human Breast and Lung Carcinoma Xenografts Using the Radiometalated Peptide Ga-68-TRAP-WL12.

Noninvasive imaging of the immune checkpoint protein programmed death ligand 1 (PD-L1; synonyms: CD274, B7-H1) holds great promise to improve patient selection and, thus, response rates for immune checkpoint therapy (ICT) with monoclonal antibodies targeting the PD1/PD-L1 axis. The PD-L1 specific peptide WL12 (cyclo(AcY-(NMe)A-N-P-H-L-Hyp-W-S-W(Me)-(NMe)Nle-(NMe)Nle-O-C)-G-NH2) was functionalized with the Gallium-68 chelator TRAP by means of click chemistry (CuAAC). The resulting conjugate TRAP-WL12 was labeled with Gallium-68 within 16 min, with approximately 90% radiochemical yield and 99% radiochemical purity, affording Ga-68-TRAP-WL12 with molar activities typically exceeding 100 MBq/nmol. This radiotracer was characterized by positron emission tomography (PET) imaging and ex vivo biodistribution in murine xenografts of nontransfected PD-L1 expressing tumor cell lines, MDA-MB-231 (human breast carcinoma), and H2009 (human lung adenocarcinoma). It showed a favorable biodistribution profile with rapid renal clearance and low background (tumor-to-blood ratio = 26.6, 3 h p.i.). Conjugation of the Ga-68-TRAP moiety to WL12 successfully mitigated the nonspecific uptake of this peptide in organs, particularly the liver. This was demonstrated by comparing Ga-68-TRAP-WL12 with the archetypical Ga-68-DOTA-WL12, for which tumor-to-liver ratios of 1.4 and 0.5, respectively, were found. Although immunohistochemistry (IHC) revealed a low PD-L1 expression in MDA-MB-213 and H2009 xenografts that corresponds well to the clinical situation, PET showed high tumor uptakes (6.6 and 7.3% injected activity per gram of tissue (iA/g), respectively) for Ga-68-TRAP-WL12. Thus, this tracer has the potential for routine clinical PD-L1 PET imaging because it detects even very low PD-L1 expression densities with high sensitivity and may open an avenue to replace PD-L1 IHC of biopsies as the standard means to select potential responders for ICT.

Erratum: PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis.

This corrects the article DOI: 10.1038/nature24649.

Evolutionary routes and KRAS dosage define pancreatic cancer phenotypes.

The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.

Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure.

Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin-mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c+ cells to limit cisplatin toxicity is specifically attributed to CD64+ MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.

How to use AI in pathology.

AI plays an important role in pathology, both in clinical practice supporting pathologists in their daily work, and in research discovering novel biomarkers for improved patient care. Still, AI is in its starting phase, and many pathology labs still need to transition to a digital workflow to be able to enjoy the benefits of AI. In this perspective, we explain the major benefits of AI in pathology, highlight key requirements that need to be met and example how to use it in a typical workflow.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Experimental investigation of skin toxicity after immune checkpoint inhibition in combination with radiation therapy.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, structured knowledge to mitigate a patient's specific risk of developing adverse events are limited. Nevertheless, there is an exponential growth of clinical studies combining conventional therapies such as radiation therapy (RT) with ICIs. Cutaneous reactions are among the most common adverse events after monotherapy with either ICIs or RT. So far, little is known about interindividual differences for the risk of developing severe tissue toxicity after the combination of RT with ICIs, and the underlying biological mechanisms are ill defined. We used experimental models of RT-induced skin injury to analyze skin toxicity after simultaneous application of ICIs. We compared different RT regimens such as fractionated or stereotactic RT with varying dose intensity. Strikingly, we found that simultaneous application of RT and ICIs did not significantly aggravate acute skin injury in two different mouse strains. Detailed examination of long-term tissue damage of the skin revealed similar signs of epidermal hyperplasia, dermal fibrosis, and adnexal atrophy. In summary, we here present the first experimental study demonstrating the excellent safety profiles of concurrent treatment with RT and ICIs. These findings will help to interpret the development of adverse events of the skin after radioimmunotherapy and guide the design of new clinical trials and clinical decision-making in individual cases. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis.

T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules. Although these oncogenic alterations are thought to drive TCR pathways to induce chronic proliferation and cell survival programmes, it remains unclear whether T cells contain tumour suppressors that can counteract these events. Here we show that the acute enforcement of oncogenic TCR signalling in lymphocytes in a mouse model of human T cell lymphoma drives the strong expansion of these cells in vivo. However, this response is short-lived and robustly counteracted by cell-intrinsic mechanisms. A subsequent genome-wide in vivo screen using T cell-specific transposon mutagenesis identified PDCD1, which encodes the inhibitory receptor programmed death-1 (PD-1), as a master gene that suppresses oncogenic T cell signalling. Mono- and bi-allelic deletions of PDCD1 are also recurrently observed in human T cell lymphomas with frequencies that can exceed 30%, indicating high clinical relevance. Mechanistically, the activity of PD-1 enhances levels of the tumour suppressor PTEN and attenuates signalling by the kinases AKT and PKC in pre-malignant cells. By contrast, a homo- or heterozygous deletion of PD-1 allows unrestricted T cell growth after an oncogenic insult and leads to the rapid development of highly aggressive lymphomas in vivo that are readily transplantable to recipients. Thus, the inhibitory PD-1 receptor is a potent haploinsufficient tumour suppressor in T cell lymphomas that is frequently altered in human disease. These findings extend the known physiological functions of PD-1 beyond the prevention of immunopathology after antigen-induced T cell activation, and have implications for T cell lymphoma therapies and for current strategies that target PD-1 in the broader context of immuno-oncology.

AP1/Fra1 confers resistance to MAPK cascade inhibition in pancreatic cancer.

Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic KrasG12D, to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for KrasG12D-induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF-MEK-ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies.

CXCL9 inhibits tumour growth and drives anti-PD-L1 therapy in ovarian cancer.

BACKGROUND: Response to immune checkpoint blockade (ICB) in ovarian cancer remains disappointing. Several studies have identified the chemokine CXCL9 as a robust prognosticator of improved survival in ovarian cancer and a characteristic of the immunoreactive subtype, which predicts ICB response. However, the function of CXCL9 in ovarian cancer has been poorly studied. METHODS: Impact of Cxcl9 overexpression in the murine ID8-Trp53-/- and ID8-Trp53-/-Brca2-/- ovarian cancer models on survival, cellular immune composition, PD-L1 expression and anti-PD-L1 therapy. CXCL9 expression analysis in ovarian cancer subtypes and correlation to reported ICB response. RESULTS: CXCL9 overexpression resulted in T-cell accumulation, delayed ascites formation and improved survival, which was dependent on adaptive immune function. In the ICB-resistant mouse model, the chemokine was sufficient to enable a successful anti-PD-L1 therapy. In contrast, these effects were abrogated in Brca2-deficient tumours, most likely due to an already high intrinsic chemokine expression. Finally, in ovarian cancer patients, the clear-cell subtype, known to respond best to ICB, displayed a significantly higher proportion of CXCL9high tumours than the other subtypes. CONCLUSIONS: CXCL9 is a driver of successful ICB in preclinical ovarian cancer. Besides being a feasible predictive biomarker, CXCL9-inducing agents thus represent attractive combination partners to improve ICB in this cancer entity.

Single-Nucleus and In Situ RNA-Sequencing Reveal Cell Topographies in the Human Pancreas.

BACKGROUND & AIMS: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell sequencing approaches. METHODS: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. RESULTS: We created the first comprehensive atlas of human pancreas cells including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus sequencing data showed a different cellular composition of the endocrine tissue, highlighting the tissue dynamics occurring during development. By applying spatial cartography, involving cell proximity mapping through in situ sequencing, we found evidence of specific cell type neighborhoods, dynamic topographies in the endocrine and exocrine pancreas, and principles of morphologic organization of the organ. Furthermore, similar analyses in chronic pancreatitis biopsy samples showed the presence of acinar-REG+ cells, a reciprocal association between macrophages and activated stellate cells, and a new potential role of tuft cells in this disease. CONCLUSIONS: Our human pancreas cell atlas can be interrogated to understand pancreatic cell biology and provides a crucial reference set for comparisons with diseased tissue samples to map the cellular foundations of pancreatic diseases.

Mesenchymal Plasticity Regulated by Prrx1 Drives Aggressive Pancreatic Cancer Biology.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology. METHODS: To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis. RESULTS: Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC. CONCLUSIONS: Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.