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Fibroblast heterogeneity is increasingly recognised across cancer conditions. Given their important contribution to disease progression, mapping fibroblasts’ heterogeneity is critical to devise effective anti-cancer therapies. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in pancreatic ductal adenocarcinoma (PDAC). Whether CAF phenotypes are differently specified by PDAC cell lineages remains to be elucidated. Here, we reveal an important role for the MAPK signalling pathway in defining PDAC CAF phenotypes. We show that epithelial MAPK activity promotes the myofibroblastic differentiation of CAFs by sustaining the expression and secretion of TGF-β1. We integrate single-cell profiling of post-perturbation transcriptional responses from mouse models with cellular and spatial profiles of human tissues to define a MAPK high CAF (mapCAF) phenotype. We show that this phenotype associates with basal-like tumour cells and reduced frequency of CD8 + T cells. In addition to elevated MAPK activity, this mapCAF phenotype is characterized by TGF-β signalling, hypoxia responsive signatures, and immunoregulatory gene programs. Furthermore, the mapCAF signature is enriched in myofibroblastic CAFs from various cancer conditions and correlates with reduced response to immune checkpoint inhibition in melanoma. Altogether, our data expand our knowledge on CAF phenotype heterogeneity and reveal a potential strategy for targeting myofibroblastic CAFs in vivo. Phenotypic and functional heterogeneity of cancer associated fibroblasts (CAFs) has been reported in pancreatic ductal adenocarcinoma (PDAC). Here the authors show that epithelial MAPK activity promotes myofibroblastic differentiation of CAFs. Furthermore, the epithelial basal-like subtype is associated with a CAF phenotype characterized by elevated MAPK activity and TGF-β signalling, associated with T cell exclusion in PDAC.

Thymic stromal lymphopoietin (TSLP) is highly expressed by bronchial epithelial cells and skin keratinocytes in allergic diseases. TSLP acts as a master switch for allergic inflammation through the activation of dendritic cells and mast cells for initiating inflammatory type 2 T-helper lymphocyte responses. To elucidate the immunological cascades of epithelium/keratinocyte-eosinophil-mediated allergic inflammation, we examined the modulating effects of TSLP on human eosinophils. Expression of TSLP receptor complex was detected by RT-PCR, flow cytometry, and Western blot. Adhesion molecules, cytokine, and chemokines were quantitated by flow cytometry or ELISA. Intracellular signal transduction molecules were measured by Western blot and flow cytometry. We observed that human eosinophils constitutively expressed functional heterodimeric TSLP receptor complex comprising TSLP-binding chain TSLPR and IL-7Ralpha chain. TSLP could significantly delay eosinophil apoptosis, up-regulate cell surface expression of adhesion molecule CD18 and intercellular adhesion molecule-1, but down-regulate L-selectin, enhance eosinophil adhesion onto fibronectin, and induce the release of inflammatory cytokine IL-6 and chemokines CXCL8, CXCL1, and CCL2 (all P < 0.05). All these effects were concentration dependent and TSLP specific. TSLP regulated the above effects through the activation of extracellular signal-regulated protein kinase, p38 mitogen-activated protein kinase, and NF-kappaB signaling pathway, but not signal transducer and activator of transcription 5 and 3, which were usually activated in other effector cells upon TSLP stimulation. Collectively, the above findings elucidate the proallergic mechanisms of TSLP via the activation of distinct intracellular signaling pathways in eosinophils.

GATA6 promotes epithelial phenotypes and limits epithelial-to-mesenchymal (EMT) transition in pancreatic ductal adenocarcinoma (PDAC). Here we show that GATA6 defines a tumor cell state that induces MHCI expression and anti-tumor cytotoxicity upon therapy. In human PDAC, GATA6 expression correlates with immune cell infiltration, and spatial analysis reveals interaction between GATA6 + tumor cells and CD8 + T cells. In murine PDAC, MEK inhibition (MEKi) enriches antigenicity-related gene sets in GATA6 high cells, while GATA6 knockout or degradation impairs MEKi-induced MHCI upregulation. High-GATA6 tumors respond to MEKi with increased MHCI, enhancing T-cell cytotoxicity, whereas GATA6 loss abolishes this effect. Treatment-induced EMT reduces GATA6 + populations and MHCI expression, which is restored by combining MEKi with HDAC inhibitors, enhancing GATA6 + tumor cells, MHCI, CD8 + T cell infiltration, tumor suppression, and survival. These findings suggest that therapeutic strategies promoting a GATA6-driven tumor cell state improve immune recognition of PDAC cells and potentiate anti-tumor cytotoxic effects. GATA6 maintains epithelial traits and limits EMT in pancreatic cancer. Combined MEK and HDAC inhibition restores GATA6+ tumor cells, boosting MHCI expression and CD8+ T cellmediated anti-tumor immunity.

Activation of eosinophils by microbe-derived molecules via Toll-like receptors (TLR) potentially provides the link between microbe-induced innate immune responses and the exacerbation of allergic inflammation. We investigated the expression of TLRs and the effect of their ligands on human eosinophils. Expression of TLR1–9 was detected by Western blot and flow cytometry. Adhesion molecules, cytokines, superoxides, and eosinophlilic cationic protein (ECP) were assessed by flow cytometry, enzyme-linked immunosorbent assay, chemiluminescent method, and fluorescence immunoassay, respectively. Human eosinophils differentially expressed TLR1, -2, -4, -5, -6, -7, and -9. Peptidoglycan (PGN) (TLR2 ligand), flagellin (TLR5 ligand), and Imiquimod R837 (TLR7 ligand) could significantly upregulate cell surface expression of intercellular adhesion molecule (ICAM)-1 and CD18, and induce the release of IL-1β, IL-6, IL-8, growth-related oncogene (GRO)-α, and superoxides of eosinophils. Only PGN could induce the degranulation for ECP release. However, ds poly I-C (TLR3 ligand), LPS (TLR4 ligand), ssRNA (TLR8 ligand), and CpG-DNA (TLR9 ligand) were much less effective or inactive. PGN, flagellin, and R837 could activate both nuclear factor (NF)-κB and extracellular signal–regulated protein kinase (ERK). PGN could activate phosphatidylinositol 3-kinase (PI3K)-Akt, and R837 both PI3K-Akt and p38 mitogen-activated protein kinase (MAPK). The induction of the release of IL-1β, IL-6, IL-8, GRO-α, superoxides, and ECP by PGN, flagellin, and R837 was found to be differentially regulated by NF-κB, ERK, PI3K-Akt, and p38 MAPK. The above results therefore support that microbial infection may lead to the exacerbation of allergic inflammation.

Background Granulin-epithelin precursor (GEP) is a secretory growth factor, which has been demonstrated to control cancer growth, invasion, drug resistance and immune escape. Our previous studies and others also demonstrated its potential in targeted therapy. Comprehensive characterization of GEP partner on cancer cells are warranted. We have previously shown that GEP interacted with heparan sulfate on the surface of liver cancer cells and the interaction is crucial for GEP-mediated signaling transduction. This study aims to characterize GEP protein partner at the cell membrane with the co-immunoprecipitation and mass spectrometry approach. Methods The membrane fraction from liver cancer model Hep3B was used for capturing binding partner with the specific monoclonal antibody against GEP. The precipitated proteins were analyzed by mass spectrometry. After identifying the GEP binding partner, this specific interaction was validated in additional liver cancer cell line HepG2 by co-immunoprecipitation using GRP78 and GEP antibodies, respectively, as the bait. GRP78 transcript levels in hepatocellular carcinoma (HCC) clinical samples ( n  = 77 pairs) were examined by real-time quantitative RT-PCR. GEP and GRP78 protein expressions were investigated by immunohistochemistry on paraffin sections. Results We identified the GEP-binding protein as 78-kDa glucose-regulated protein (GRP78, also named heat shock 70-kDa protein 5, HSPA5). This interaction was validated in independent HCC cell lines. Increased GRP78 mRNA levels were demonstrated in liver cancer tissues compared with the paralleled liver tissues ( t -test, P  = 0.002). GRP78 and GEP transcript levels were significantly correlated (Spearman’s correlation, P  = 0.001), and the proteins were also detectable in the cytoplasm of liver cancer cells by immunohistochemical staining. Conclusions GRP78 and GEP are interacting protein partners in liver cancer cells and may play a role in GEP-mediated cancer progression in HCC.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein’s dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Background Hepatocellular carcinoma (HCC) is an aggressive cancer with high mortality and morbidity worldwide. The limited clinically relevant model has impeded the development of effective HCC treatment strategy. Patient-derived xenograft (PDX) models retain most of the characteristics of original tumors and were shown to be highly predictive for clinical outcomes. Notably, primary cell line models allow in-depth molecular characterization and high-throughput analysis. Combined usage of the two models would provide an excellent tool for systematic study of therapeutic strategies. Here, we comprehensively characterized the novel PDX and the paralleled primary HCC cell line model. Methods Tumor tissues were collected from HCC surgical specimens. HCC cells were sorted for in vivo PDX and in vitro cell line establishment by the expression of hepatic cancer stem cell marker to enhance cell viability and the rate of success on subsequent culture. The PDX and its matching primary cell line were authenticated and characterized in vitro and in vivo. Results Among the successful cases for generating PDXs and primary cells, HCC40 is capable for both PDX and primary cell line establishment, which were then further characterized. The novel HCC40-PDX and HCC40-CL exhibited consistent phenotypic characteristics as the original tumor in terms of HBV protein and AFP expressions. In common with HCC40-PDX, HCC40-CL was tumorigenic in immunocompromised mice. The migration ability in vitro and metastatic properties in vivo echoed the clinical feature of venous infiltration. Genetic profiling by short tandem repeat analysis and p53 mutation pattern consolidated that both the HCC40-PDX and HCC40-CL models were derived from the HCC40 clinical specimen. Conclusions The paralleled establishment of PDX and primary cell line would serve as useful models in comprehensive studies for HCC pathogenesis and therapeutics development for personalized treatment.

The novel interleukin (IL)-1 family cytokine IL-33 has been shown to activate T helper 2 (Th2) lymphocytes, mast cells and basophils to produce an array of proinflammatory cytokines, as well as to mediate blood eosinophilia, IgE secretion and hypertrophy of airway epithelium in mice. In the present study, we characterized the activation of human eosinophils by IL-33, and investigated the underlying intracellular signaling mechanisms. IL-33 markedly enhanced eosinophil survival and upregulated cell surface expression of the adhesion molecule intercellular adhesion molecule (ICAM)-1 on eosinophils, but it suppressed that of ICAM-3 and L-selectin. In addition, IL-33 mediates significant release of the proinflammatory cytokine IL-6 and the chemokines CXCL8 and CCL2. We found that IL-33-mediated enhancement of survival, induction of adhesion molecules, and release of cytokines and chemokines were differentially regulated by activation of the nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we compared the above IL-33 activities with two structurally and functionally related cytokines, IL-1β and IL-18. IL-1β, but not IL-18, markedly upregulated cell surface expression of ICAM-1. IL-1β and IL-18 also significantly enhanced eosinophil survival, and induced the release of IL-6 and chemokines CXCL8 and CCL2 via the activation of the NF-κB, p38 MAPK and ERK pathways. Synergistic effects on the release of IL-6 were also observed in combined treatment with IL-1β, IL-18 and IL-33. Taken together, our findings provide insight into IL-33-mediated activation of eosinophils via differential intracellular signaling cascades in the immunopathogenesis of allergic inflammation.

Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry. Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining. TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.

Immune evasion is indispensable for cancer initiation and progression, although its underlying mechanisms in pancreatic ductal adenocarcinoma (PDAC) are not fully known. Here, we characterize the function of tumor-derived PGRN in promoting immune evasion in primary PDAC. Tumor- but not macrophage-derived PGRN is associated with poor overall survival in PDAC. Multiplex immunohistochemistry shows low MHC class I (MHCI) expression and lack of CD8 + T cell infiltration in PGRN-high tumors. Inhibition of PGRN abrogates autophagy-dependent MHCI degradation and restores MHCI expression on PDAC cells. Antibody-based blockade of PGRN in a PDAC mouse model remarkably decelerates tumor initiation and progression. Notably, tumors expressing LCMV-gp33 as a model antigen are sensitized to gp33-TCR transgenic T cell-mediated cytotoxicity upon PGRN blockade. Overall, our study shows a crucial function of tumor-derived PGRN in regulating immunogenicity of primary PDAC. Immune responses to pancreatic ductal adenocarcinoma can be inhibited by cancer cells. Here the authors show that high levels of progranulin in PDAC inhibits immune responses by reducing MHC class I antigen presentation through enhanced degradation of MHC class I via autophagy.