Depletion of slow-cycling PDGFRα+ADAM12+ mesenchymal cells promotes antitumor immunity by restricting macrophage efferocytosis.

The capacity to survive and thrive in conditions of limited resources and high inflammation is a major driver of tumor malignancy. Here we identified slow-cycling ADAM12+PDGFRα+ mesenchymal stromal cells (MSCs) induced at the tumor margins in mouse models of melanoma, pancreatic cancer and prostate cancer. Using inducible lineage tracing and transcriptomics, we demonstrated that metabolically altered ADAM12+ MSCs induced pathological angiogenesis and immunosuppression by promoting macrophage efferocytosis and polarization through overexpression of genes such as Gas6, Lgals3 and Csf1. Genetic depletion of ADAM12+ cells restored a functional tumor vasculature, reduced hypoxia and acidosis and normalized CAFs, inducing infiltration of effector T cells and growth inhibition of melanomas and pancreatic neuroendocrine cancer, in a process dependent on TGF-ß. In human cancer, ADAM12 stratifies patients with high levels of hypoxia and innate resistance mechanisms, as well as factors associated with a poor prognosis and drug resistance such as AXL. Altogether, our data show that depletion of tumor-induced slow-cycling PDGFRα+ MSCs through ADAM12 restores antitumor immunity.

Borderline Hepatocellular Adenomas: A Practical Diagnostic Approach Based on Pathologic and Molecular Features.

Borderline hepatocellular adenomas (BL-HCA) are characterized by focal architectural/cytologic atypia and reticulin loss, features that are insufficient for a definitive diagnosis of hepatocellular carcinoma (HCC). The diagnosis and management of BL-HCA are challenging as their biological behavior, especially in terms of malignant potential, is still debated. We aimed to compare the clinicopathologic and molecular features of BL-HCA with those of typical HCA (T-HCA), HCA with malignant transformation (HCC on HCA), and HCC to assess the risk of malignancy. One hundred six liver resection specimens were retrospectively selected from 2 reference centers, including 39 BL-HCA, 42 T-HCA, 12 HCC on HCA, and 13 HCC specimens. Somatic mutations, including TERT promoter mutations associated with HCA malignant transformation and the gene expression levels of 96 genes, were investigated in 93 frozen samples. Additionally, TERT promoter mutations were investigated in 44 formalin-fixed, paraffin-embedded samples. The clinical features of patients with BL-HCA were similar to those of patients with T-HCA, patients being mainly women (69%) with a median age of 37 years. The median tumor size was 7.5 cm, 64% of patients had a single nodule, and no recurrence was observed. Compared with T-HCA, BL-HCA was significantly enriched in ß-catenin-mutated HCA in exon 3 (41% vs 6%; P < .001). Unsupervised statistical analysis based on gene expression showed that BL-HCA overlapped with T-HCA and HCC on HCA, favoring a molecular continuum of the tumors. TERT promoter mutations were observed only in HCC on HCA (42%) and in HCC (38%). In conclusion, these results suggest that despite their worrisome morphologic features, the clinicopathologic and molecular features of BL-HCA are much closer to those of T-HCA than those of HCC on HCA or HCC. This strongly supports the usefulness of combining morphologic and molecular analyses in a practical diagnostic approach for guiding the management of BL-HCA.

Periostin- and podoplanin-positive cancer-associated fibroblast subtypes cooperate to shape the inflamed tumor microenvironment in aggressive pancreatic adenocarcinoma.

Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The histone acetyltransferase hMOF promotes vascular invasion in hepatocellular carcinoma.

BACKGROUND & AIMS: Vascular invasion is a major prognostic factor in hepatocellular carcinoma (HCC). We previously identified histone H4 acetylated at lysine 16 (H4K16ac), a histone modification involved in transcription activation, as a biomarker of microvascular invasion (mVI) in HCC. This study aimed to investigate the role of hMOF, the histone acetyltransferase responsible for H4K16 acetylation, in the process of vascular invasion in HCC. METHODS: hMOF expression was assessed by RT-qPCR and immunohistochemistry in a retrospective series of HCC surgical samples, and correlated with the presence of mVI. The functional role of hMOF in HCC vascular invasion was investigated in vitro in HCC cell lines using siRNA, transcriptomic analysis and transwell invasion assay, and in vivo using a Zebrafish embryo xenograft model. RESULTS: We found that hMOF was significantly upregulated at the protein level in HCC with mVI, compared with HCC without mVI (P < .01). Transcriptomic analysis showed that hMOF downregulation in HCC cell line lead to significant downregulation of key genes and pathways involved in vascular invasion. These results were confirmed by transwell invasion assay, where hMOF downregulation significantly reduced HCC cells invasion. Finally, hMOF downregulation significantly reduced tumour cell intravasation and metastasis in vivo. CONCLUSIONS: Altogether, these results underpin a critical role for hMOF in vascular invasion in HCC, via transcription activation of key genes involved in this process. These data confirm the major role of epigenetic alterations in HCC progression, and pave the way for future therapies targeting hMOF in HCC.

Tumor Heterogeneity in Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma is one of the deadliest malignancies worldwide, mainly due to frequent diagnosis at an advanced stage and its strong chemoresistance. Tumor heterogeneity is evident at the histological level, both between tumors and even within a tumor. Recent high-throughput analyses have confirmed that intertumor heterogeneity is greater than intratumor heterogeneity that is mostly driven by successive catastrophic genetic events in the early stage and by epigenetic modifications in the metastatic stage. While this heterogeneity may complicate the search for a universal cure, these analyses have distinguished several subtypes at the genomic, transcriptomic, and metabolomic levels that offer, for some, new therapeutic opportunities.

Gly388Arg FGFR4 Polymorphism Is Not Predictive of Everolimus Efficacy in Well-Differentiated Digestive Neuroendocrine Tumors.

INTRODUCTION: Preclinical data suggest that the single nucleotide polymorphism substituting a glycine for an arginine in codon 388 of the FGFR4 transmembrane domain may increase the proliferation of xenografted neuroendocrine cell lines and decrease their sensitivity to everolimus by modulating STAT3 signaling and the mTOR pathway. AIM: To evaluate the prognostic and predictive values of this polymorphism on everolimus efficacy in patients treated for digestive neuroendocrine tumor (NET). PATIENTS AND METHODS: This monocentric retrospective cohort included patients with small bowel NET (SBNET) and pancreatic NET (PNET) treated with everolimus (2006-2013). The patients were genotyped by classical sequencing, and mTOR pathway activity was assessed by immunochemistry on formalin-fixed paraffin-embedded samples (PTEN/pPTEN/pAKT/pmTOR/pS6/p4EBP1). RESULTS: Forty-one patients (21 males, median age 57 years) with PNET (n = 28), SBNET (n = 12) or NET of unknown origin (n = 1), grade 1 (n = 8), 2 (n = 27), 3 (n = 3) or unknown grade (n = 3), were studied. At least one 388Arg allele was found in 14/23 PNET and 10/11 SBNET. Progression-free survival in the whole population and the PNET subgroup was not influenced by the presence of one or two 388Arg alleles [HR = 1.31 (0.58-2.99), p = 0.52 and HR = 1.11 (0.45-2.73), p = 0.82, respectively]. Similarly, overall survival was not influenced. Finally, mTOR pathway molecule expression was not modified by the presence of at least one 388Arg allele. CONCLUSION: The Gly388Arg FGFR4 polymorphism does not seem to have a prognostic value in digestive NET. In addition, it neither predicts the response to everolimus nor modifies the activation of the mTOR pathway.

Role of the fatty pancreatic infiltration in pancreatic oncogenesis.

Although pancreatic precancerous lesions are known to be related to obesity and fatty pancreatic infiltration, the mechanisms remain unclear. We assessed the role of fatty infiltration in the process of pancreatic oncogenesis and obesity. A combined transcriptomic, lipidomic and pathological approach was used to explore neoplastic transformations. Intralobular (ILF) and extralobular (ELF) lipidomic profiles were analyzed to search for lipids associated with pancreatic intraepithelial neoplasia (PanINs) and obesity; the effect of ILF and ELF on acinar tissue and the histopathological aspects of pancreatic parenchyma changes in obese (OB) and non-obese patients. This study showed that the lipid composition of ILF was different from that of ELF. ILF was related to obesity and ELF-specific lipids were correlated to PanINs. Acinar cells were shown to have different phenotypes depending on the presence and proximity to ILF in OB patients. Several lipid metabolic pathways, oxidative stress and inflammatory pathways were upregulated in acinar tissue during ILF infiltration in OB patients. Early acinar transformations, called acinar nodules (AN) were linked to obesity but not ELF or ILF suggesting that they are the first reversible precancerous pancreatic lesions to occur in OB patients. On the other hand, the number of PanINs was higher in OB patients and was positively correlated to ILF and ELF scores as well as to fibrosis. Our study suggests that two types of fat infiltration must be distinguished, ELF and ILF. ILF plays a major role in acinar modifications and the development of precancerous lesions associated with obesity, while ELF may play a role in the progression of PDAC.

Pancreatic Ductal Adenocarcinoma Arising in Young and Old Patients Displays Similar Molecular Features.

Pancreatic ducal adenocarcinoma is classically diagnosed in the 7th decade, but approximately 10% of patients are diagnosed under 55 years (y.o.). While the genomic and transcriptomic landscapes of late-onset tumors (LOT) have been described, little is known about early-onset tumors (EOT). Ageing is known to impact DNA methylation and proteome integrity through carbonylation-related oxidative damages. We therefore aimed to assess the global molecular features of EOT. We compared 176 EOT (≤55 y.o.) and 316 LOT (≥70 y.o.) from three distinct surgical cohorts at the clinical/genomic/epigenomic/transcriptomic level. Furthermore, we assessed oxidative stress responses and oxidative proteome damages using 2D gel electrophoresis followed by mass spectrometry protein identification. There was no consistent clinical difference between EOT and LOT across the three cohorts. The mutational landscape of key driver genes and the global methylation profile were similar in the two groups. LOT did display age-related features such as enriched DNA repair gene signatures and upregulation of oxidative stress defenses together with increased proteome carbonylation. However, these age-related differences were more preeminent in non-tumor tissues while tumor proteome and proteome damages were fairly comparable. In conclusion, this multi-omics comparison showed that EOT harbor a comparable molecular profile to that of LOT.

Pharmacologic Normalization of Pancreatic Cancer-Associated Fibroblast Secretome Impairs Prometastatic Cross-Talk With Macrophages.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. METHODS: Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. RESULTS: Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. CONCLUSIONS: We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.

Prognostic Biomarkers in Pancreatic Cancer: Avoiding Errata When Using the TCGA Dataset.

Data from the Cancer Genome Atlas (TCGA) are now easily accessible through web-based platforms with tools to assess the prognostic value of molecular alterations. Pancreatic tumors have heterogeneous biology and aggressiveness ranging from the deadly adenocarcinoma (PDAC) to the better prognosis, neuroendocrine tumors. We assessed the availability of the pancreatic cancer TCGA data (TCGA_PAAD) from several repositories and investigated the nature of each sample and how non-PDAC samples impact prognostic biomarker studies. While the clinical and genomic data (n = 185) were fairly consistent across all repositories, RNAseq profiles varied from 176 to 185. As a result, 35 RNAseq profiles (18.9%) corresponded to a normal, inflamed pancreas or non-PDAC neoplasms. This information was difficult to obtain. By considering gene expression data as continuous values, the expression of the 5312 and 4221 genes were significantly associated with the progression-free and overall survival respectively. Considering the cohort was not curated, only 4 and 14, respectively, had prognostic value in the PDAC-only cohort. Similarly, mutations in key genes or well-described miRNA lost their prognostic significance in the PDAC-only cohort. Therefore, we propose a web-based application to assess biomarkers in the curated TCGA_PAAD dataset. In conclusion, TCGA_PAAD curation is critical to avoid important biological and clinical biases from non-PDAC samples.

Endothelial fatty liver binding protein 4: a new targetable mediator in hepatocellular carcinoma related to metabolic syndrome.

Metabolic syndrome (MS) is becoming the leading risk factor for hepatocellular carcinoma (HCC). HCC development related to MS may occur in advanced or non-advanced liver fibrosis, suggesting specific molecular pathways. Among these pathways, basal inflammatory state and adipokines production are involved. The aim of this study was to evaluate the role of fatty acid-binding protein 4 (FABP4). In this study, we demonstrate the specific overexpression of FABP4 in human HCC samples from patients with MS compared to other risk factors for chronic liver disease with FABP4 expression restricted to peritumoral endothelial cells. In vitro, glucose, insulin, VEGFA and hypoxia upregulated endothelial FABP4, which was reversed by metformin through mTOR pathway inhibition. FABP4 exerts oncogenic effects on hepatoma cell lines by upregulating the angiogenesis gene signature and pathways involved in the cell cycle, leading to increased cell proliferation and migration, and downregulating HIF1 pathway; effects were reversed in the presence of a specific FABP4 inhibitor (BMS309403). We showed the role of microvesicles as FABP4 vectors between endothelial and tumor cells. In vivo, BMS309403 significantly reduces tumor growth in heterotopic and orthotopic xenografted mice model. In conclusion, this study demonstrates the emerging oncogenic role of liver endothelial cells through FABP4 in HCC related to MS, and highlights new anti-neoplastic mechanism of metformin.

hENT1 Testing in Pancreatic Ductal Adenocarcinoma: Are We Ready? A Multimodal Evaluation of hENT1 Status.

Gemcitabine is still one of the standard chemotherapy regimens for pancreatic ductal adenocarcinoma (PDAC). Gemcitabine uptake into tumor cells is mainly through the human equilibrative nucleoside transport 1 (hENT1). It was therefore proposed as a potential predictive biomarker of gemcitabine efficacy but reports are conflicting, with an important heterogeneity in methods to assess hENT1 expression. A multicenter cohort of 471 patients with a resected PDAC was used to assess simultaneously the predictive value of the 2 best described hENT1 antibodies (10D7G2 and SP120). Three additional antibodies and the predictive value of hENT1 mRNA were also tested on 251 and 302 patients, respectively. hENT1 expression was assessed in 54 patients with matched primary tumors and metastases samples. The 10D7G2 clone was the only hENT1 antibody whose high expression was associated with a prolonged progression free survival and overall survival in patients who received adjuvant gemcitabine. hENT1 mRNA level was also predictive of gemcitabine benefit. hENT1 status was concordant in 83% of the cases with the best concordance in synchronous metastases. The 10D7G2 clone has the best predictive value of gemcitabine benefit in PDAC patients. Since it is not commercially available, hENT1 mRNA level could represent an alternative to assess hENT1 status.

[Molecular characterisation defines several subtypes of pancreatic ductal adenocarcinoma]. / La caractérisation moléculaire définit plusieurs sous-types d'adénocarcinome canalaire du pancréas.

Multi-omics high throughput analyses lead to the description of multiple molecular subtypes of pancreatic adenocarcinoma with major prognostic impact for most of them. There is no consensual multilevel integrative classification yet like in colon or breast cancers. Genomic classifications have identified a tumor subtype (15% of the patients) with deficient homologous DNA repair-system leading to increase sensitivity to platinum-based therapies and possibly to PARP inhibitors and immunotherapies. Transcriptomic classifications are still debated but all have identified an aggressive subtype with a very poor prognosis, presumably unfit for a surgical approach. Finally, approaches based on metabolomic or proteomic profiling have identified subtypes with a particular sensitivity to compounds targeting the hallmarks metabolomics or oncogenic pathways of each subtype. These classifications were mostly based on tumor cell but the micro-environment is also very heterogeneous and several types of stroma will be described soon. Subtype determination in daily practice remains a major challenge as most technologies used to build these classifications are very expensive, requires dedicated bio-informatics analysis pipelines and are not adapted to routine samples that are mostly formal in fixed paraffin embedded biopsies, in which tumor cells are highly contaminated by the cell from the microenvironment and the clot.

Oenococcus oeni Exopolysaccharide Biosynthesis, a Tool to Improve Malolactic Starter Performance.

Oenococcus oeni is the lactic acid bacterium that most commonly drives malolactic fermentation (MLF) in wine. Though the importance of MLF in terms of wine microbial stability and sensory improvement is well established, it remains a winemaking step not so easy to control. O. oeni displays many adaptation tools to resist the harsh wine conditions which explain its natural dominance at this stage of winemaking. Previous findings showed that capsular polysaccharides and endogenous produced dextran increased the survival rate and the conservation time of malolactic starters. In this paper, we showed that exopolysaccharides specific production rates were increased in the presence of single stressors relevant to wine (pH, ethanol). The transcription of the associated genes was investigated in distinct O. oeni strains. The conditions in which eps genes and EPS synthesis were most stimulated were then evaluated for the production of freeze dried malolactic starters, for acclimation procedures and for MLF efficiency. Sensory analysis tests on the resulting wines were finally performed.