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

The tumor stroma influences immune cell distribution and recruitment in a PDAC-on-a-chip model

: The dense tumor stroma of pancreatic ductal adenocarcinoma (PDAC) and its secreted immune active molecules provide a barrier for chemotherapy treatment as well as for immune cell infiltration to the tumor core, providing a challenge for immunotherapeutic strategies. Consequently, the investigation of processes underlying the interaction between the tumor stroma, particularly activated pancreatic stellate cells (PSCs), and immune cells may offer new therapeutic approaches for PDAC treatment. In this study, we established a 3D PDAC model cultured under flow, consisting of an endothelial tube, PSCs and PDAC organoids. This was applied to study the role of the tumor microenvironment (TME) on immune cell recruitment and its effect on partly preventing their interaction with pancreatic cancer cells. We observed that stromal cells form a physical barrier, partly shielding the cancer cells from migrating immune cells, as well as a biochemical microenvironment, that seems to attract and influence immune cell distribution. In addition, stromal targeting by Halofuginone led to an increase in immune cell infiltration. We propose that the here developed model setups will support the understanding of the cellular interplay influencing the recruitment and distribution of immune cells, and contribute to the identification of key players in the PDAC immunosuppressive TME as well as support the discovery of new strategies to treat this immune unresponsive tumor

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling

The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function