Immunoevolution of mouse pancreatic organoid isografts from preinvasive to metastatic disease

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients' survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

Objective: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. Design: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients’ tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. Results: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. Conclusions: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms

Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients

Malignant and fibroblast programs in aggressive PDAC molecular subtypes

Pancreatic Ductal Adenocarcinoma (PDAC) is the deadliest cancer worldwide. Late diagnosis and a complex biology make this a difficult-to-treat disease. PDAC displays extensive heterogeneity both in the malignant and non-malignant compartments. Of the non-malignant cells, cancer-associated fibroblasts (CAFs) are the most abundant cell type which critically influences tumour biology and response to treatments. The different PDAC cell states are shaped by the integration of cell intrinsic and cell extrinsic inputs. It is now well established that the transcriptional cell state (basal-like/squamous) displaying suppression of pancreatic endodermal gene programs has the most aggressive biological behaviour. Whether and how basal-like/squamous cells instruct the CAFs differently from the less aggressive classical phenotype remains to be elucidated. In this thesis work, we sought to disclose novel determinants of cell lineage specification as well as to identify subtype-specific stromal phenotypes. Receptor-Tyrosine Kinases (RTKs) are a large family of metazoan-specific plasma-membrane receptors that control several cellular processes including cell fate determination. Therefore, we focused on the potential role of RTKs in defining or sustaining aggressive molecular phenotypes of neoplastic cells. We found that the expression of FGFR4 was significantly elevated in the classical PDAC subtype and associated with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligned with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlated with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably led to the enrichment of basal-like/squamous gene programs and associated with either partial or full switch of phenotype. Whole-genome sequencing of advanced PDAC tissues and functional interrogation of organoid-based xenotransplantation have suggested RAS hyperactivation as a determinant of the basal-like/squamous subtype. Here, we used pathway mapping analysis and context-dependent pathway response signatures to infer MAPK activity and dependency in heterogeneous expression data from models and patients' samples. In situ expression analyses complemented this approach, which undisclosed an important role for the MAPK signalling pathway in the definition of PDAC CAFs phenotypes. We found that the epithelial activity of MAPK did not discriminate basal-like from classical tumours. Conversely, hyperactivation of MAPK signalling occurred in myCAFs populating basal-like/squamous tumour niches. Short-term inhibition of MAPK was invariably associated with a dramatic change of the myCAFs/iCAFs ratio in mouse PDAC tissue due to a unique dependency of myCAF on a proficient MAPK signalling. Gene expression signatures of MAPKhigh CAFs (sMEK) from mouse tumours suggested metabolic rewiring and immunoregulatory function. Finally, we found that the sMEK signature correlated with poor prognosis in several cancer conditions, including PDAC, and with reduced response to immunecheckpoint inhibition in bladder cancer

Modeling Cell Communication in Cancer With Organoids: Making the Complex Simple

Homotypic and heterotypic interactions between cells are of crucial importance in multicellular organisms for the maintenance of physiological functions. Accordingly, changes in cell-to-cell communication contribute significantly to tumor development. Cancer cells engage the different components of the tumor microenvironment (TME) to support malignant proliferation, escape immune control, and favor metastatic spreading. The interaction between cancerous and non-cancerous cell types within tumors occurs in many ways, including physical contact and paracrine signaling. Furthermore, local and long-range transfer of biologically active molecules (e.g., DNA, RNA, and proteins) can be mediated by small extracellular vesicles (EVs) and this has been shown to influence many aspects of tumor progression. As it stands, there is a critical need for suitable experimental systems that enable modeling the cell-to-cell communications occurring in cancer. Given their intrinsic complexity, animal models represent the ideal system to study cell-to-cell interaction between different cell types; however, they might make difficult to assess individual contribution to a given phenotype. On the other hand, simplest experimental models (i.e., in vitro culture systems) might be of great use when weighing individual contributions to a given phenomenon, yet it is imperative that they share a considerable number of features with human cancer. Of the many culture systems available to the scientific community, patient-derived organoids already proved to faithfully recapitulate many of the traits of patients' disease, including genetic heterogeneity and response to therapy. The organoid technology offers several advantages over conventional monolayer cell cultures, including the preservation of the topology of cell-to-cell and cell-to-matrix interactions as observed in vivo. Several studies have shown that organoid cultures can be successfully used to study interaction between cancer cells and cellular components of the TME. Here, we discuss the potential of using organoids to model the interplay between cancer and non-cancer cells in order to unveil biological mechanisms involved in cancers initiation and progression, which might ultimately lead to the identification of novel intervention strategy for those diseases

Cell lineage infidelity in PDAC progression and therapy resistance

Infidelity to cell fate occurs when differentiated cells lose their original identity and either revert to a more multipotent state or transdifferentiate into a different cell type, either within the same embryonic lineage or in an entirely different one. Whilst in certain circumstances, such as in wound repair, this process is beneficial, it can be hijacked by cancer cells to drive disease initiation and progression. Cell phenotype switching has been shown to also serve as a mechanism of drug resistance in some epithelial cancers. In pancreatic ductal adenocarcinoma (PDAC), the role of lineage infidelity and phenotype switching is still unclear. Two consensus molecular subtypes of PDAC have been proposed that mainly reflect the existence of cell lineages with different degrees of fidelity to pancreatic endodermal precursors. Indeed, the classical subtype of PDAC is characterised by the expression of endodermal lineage specifying transcription factors, while the more aggressive basal-like/squamous subtype is defined by epigenetic downregulation of endodermal genes and alterations in chromatin modifiers. Here, we summarise the current knowledge of mechanisms (genetic and epigenetic) of cell fate switching in PDAC and discuss how pancreatic organoids might help increase our understanding of both cell-intrinsic and cell-extrinsic factors governing lineage infidelity during the distinct phases of PDAC evolution.VC is supported by the Associazione Italiana Ricerca sul Cancro (AIRC; Grant No. 18178). VC and AM are also supported by the Marie Skłodowska-Curie Actions project PRECODE (Grant No: 861196). Work in the laboratory of FXR is supported, in part, by Grant RTI2018-101071-B-I00 from Ministerio de Ciencia, Innovación y Universidades (Madrid, Spain) (co-funded by the ERDF-EU)

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

ObjectiveThe dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression. DesignWe integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients’ tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype. ResultsIn PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro, SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3Ahigh tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment. ConclusionsHere, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms.publishe

Unveiling the mechanistic link between extracellular amyloid fibrils, mechano-signaling and YAP activation in cancer

: The tumor microenvironment is a complex ecosystem that plays a critical role in cancer progression and treatment response. Recently, extracellular amyloid fibrils have emerged as novel components of the tumor microenvironment; however, their function remains elusive. In this study, we establish a direct connection between the presence of amyloid fibrils in the secretome and the activation of YAP, a transcriptional co-activator involved in cancer proliferation and drug resistance. Furthermore, we uncover a shared mechano-signaling mechanism triggered by amyloid fibrils in both melanoma and pancreatic ductal adenocarcinoma cells. Our findings highlight the crucial role of the glycocalyx protein Agrin which binds to extracellular amyloid fibrils and acts as a necessary factor in driving amyloid-dependent YAP activation. Additionally, we reveal the involvement of the HIPPO pathway core kinase LATS1 in this signaling cascade. Finally, we demonstrate that extracellular amyloid fibrils enhance cancer cell migration and invasion. In conclusion, our research expands our knowledge of the tumor microenvironment by uncovering the role of extracellular amyloid fibrils in driving mechano-signaling and YAP activation. This knowledge opens up new avenues for developing innovative strategies to modulate YAP activation and mitigate its detrimental effects during cancer progression

Clinical and Genomic Characterization of Pancreatic Ductal Adenocarcinoma with Signet-Ring / Poorly Cohesive Cells

: Signet-ring cell/poorly cohesive cell (SRC) carcinoma is an aggressive variant of pancreatic ductal adenocarcinoma (PDAC). This study aimed to clarify its clinicopathological and molecular profiles based on a multi-institutional cohort of 20 cases. The molecular profiles were investigated using DNA and RNA sequencing. The clinicopathological parameters and molecular alterations were analyzed based on survival indices and using a validation/comparative cohort of 480 conventional PDAC patients. The primary findings were as follows: 1) clinicopathological features: SRC carcinomas are highly aggressive neoplasms with poor prognosis and the lungs are elective metastatic sites; 2) survival analysis: a higher SRC component was indicative of poorer prognosis. In particular, the most clinically significant threshold of SRC was 80%, showing statistically significant differences in both disease-specific and disease-free survival; 3) genomic profiles: SRC carcinomas are similar to conventional PDAC with the most common alterations affecting the classic PDAC drivers KRAS (70% of cases), TP53 (55%), SMAD4 (25%), and CDKN2A (20%). EGFR alterations, RET-CCDC6 fusion gene, and microsatellite instability (3 different cases, one alteration per case) represent novel targets for precision oncology. The occurrence of SMAD4 mutations was associated with poorer prognosis; 4) pancreatic SRC carcinomas are genetically different from gastric SRC carcinomas: CDH1, the classic driver gene of gastric SRC carcinoma, is not altered in pancreatic SRC carcinoma; 5) transcriptome analysis: the cases clustered into two groups, one classical/exocrine-like and the other squamous-like; 6) SRC carcinoma-derived organoids can be successfully generated, and their cultures preserve the histological and molecular features of parental SRC carcinoma. Although pancreatic SRC carcinoma shares similarities with conventional PDAC regarding the most important genetic drivers, it also exhibits important differences. A personalized approach for patients with this tumor type should consider the clinical relevance of histological determination of the SRC component and the presence of potentially actionable molecular targets