Highlights d PDAC regional heterogeneity stems from sub-tumor microenvironments (subTMEs) d SubTMEs exhibit distinct immune phenotypes and CAF differentiation states d SubTMEs execute distinct tumor-promoting and chemoprotective functions d Intratumoral subTME co-occurrence links stromal heterogeneity to patient outcome

Epithelial-mesenchymal transition (EMT) is a pivotal process in development and disease. In carcinogenesis, various signaling pathways are known to trigger EMT by inducing the expression of EMT transcription factors (EMT-TFs) like SNAIL1, ultimately promoting invasion, metastasis and chemoresistance. However, how EMT is executed downstream of EMT-TFs is incompletely understood. Here, using human colorectal cancer (CRC) and mammary cell line models of EMT, we demonstrate that SNAIL1 critically relies on bone morphogenetic protein (BMP) signaling for EMT execution. This activity requires the transcription factor SMAD4 common to BMP/TGFβ pathways, but is TGFβ signaling-independent. Further, we define a signature of BMP-dependent genes in the EMT-transcriptome, which orchestrate EMT-induced invasiveness, and are found to be regulated in human CRC transcriptomes and in developmental EMT processes. Collectively, our findings substantially augment the knowledge of mechanistic routes whereby EMT can be effectuated, which is relevant for the conceptual understanding and therapeutic targeting of EMT processes.

Transforming growth factor beta (TGFβ) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFβ signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4, which codes for a transcription factor (TF) central to canonical TGFβ and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4-mutant (SMAD4mut) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4mut CRC cell lines. We show that SMAD4mut cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4mut cells was found to be entirely independent of TGFβ/BMP receptor activity. Global assessment of the SNAIL1-dependent transcriptome confirmed the manifestation of an EMT gene regulatory program in SMAD4mut cells highly related to established EMT signatures. Finally, analyses of human tumor transcriptomes showed that SMAD4 mutations are not underrepresented in mesenchymal tumor samples and that expression patterns of EMT-associated genes are similar in SMAD4mut and SMAD4 wild-type (SMAD4wt) cases. Altogether, our findings suggest that alternative TFs take over the gene regulatory functions of SMAD4 downstream of EMT-TFs, arguing for considerable plasticity of gene regulatory networks operating in EMT execution. Further, they establish that EMT is not categorically precluded in SMAD4mut tumors, which is relevant for their diagnostic and therapeutic evaluation.

Pancreatic ductal adenocarcinoma (PDAC) remains resistant to most treatments and demonstrates a complex pathobiology. Here, we deconvolute regional heterogeneity in the human PDAC tumor microenvironment (TME), a long-standing obstacle, to define precise stromal contributions to PDAC progression. Large scale integration of histology-guided multiOMICs with clinical data sets and functional in vitro models uncovers two microenvironmental programs in PDAC that were anchored in fibroblast differentiation states. These sub-tumor microenvironments (subTMEs) co-occurred intratumorally and were spatially confined, producing patient-specific cellular and molecular heterogeneity associated with shortened patient survival. Each subTME was uniquely structured to support discrete aspects of tumor biology: reactive regions rich in activated fibroblast communities were immune-hot and promoted aggressive tumor progression while deserted regions enriched in extracellular matrix supported tumor differentiation yet were markedly chemoprotective. In conclusion, PDAC regional heterogeneity derives from biologically distinct reactive and protective TME elements with a defined, active role in PDAC progression.

Pancreatic ductal adenocarcinoma (PDAC) remains mostly untreatable while its incidence is on the rise. PDAC stroma should be a reservoir for novel therapeutic targets. Yet, stromal cellular complexity and an extensive intratumoral heterogeneity in human patients have left the functions of PDAC stroma poorly understood and likely hamper its targeting. Here, we set out to deconvolute regional heterogeneity in human PDAC stroma and assess its role in disease progression. Using resected & advanced biospecimens with known patient outcome, we mapped the stromal and epithelial landscapes of PDAC by combining integrative histopathology, quantitative image analysis of a 30-marker IHC panel, genomics and proteomics, machine learning, scRNAseq of PDAC-derived fibroblasts, and controlled functional assays. This histology-guided multidimensional approach revealed two overarching types of sub-tumormicroenvironments (subTMEs) in PDAC, present across stages and primary and metastatic sites. These subTMEs co-occurred intratumourally but were spatially confined, producing patient-specific cellular and molecular heterogeneity in PDAC stroma. SubTME-resolved IHC characterization of PDAC specimens and multiplexed functional analyses with subTME-specific patient-derived fibroblasts and organoids then identified how both microenvironmental programs were uniquely structured to support crucial aspects of tumour biology. Forming a complementary stromal support system, reactive subTME regions rich in fibroblasts and immune cells were vascularized and promoted features of aggressive tumour progression, while protective subTME regions enriched in ECM supported tumour differentiation yet were markedly chemoprotective and translated into poor treatment response during 1st line chemotherapy. Consequently, tumours benefited from concomitant presence of both subTMEs and patients with phenotypically heterogenous stroma displayed shortened survival. Our patient-matched multidimensional data were then used to train a Random Forest model for prediction of subTME presence from bulk RNAseq samples. This again stratified PDAC patients in the independent TCGA cohort into distinct clinical outcomes by stromal phenotypic heterogeneity. In conclusion, we have delineated human PDAC stroma to subtype its heterogeneity into phenotypically distinct reactive and protective elements. This defines the active role of two overarching sub-microenvironment types in PDAC progression and sets a path towards developing novel stroma-instructed therapies. Citation Format: Barbara T. Grünwald, Antoine Devisme, Foram Vyas, Geoffroy Andrieux, Gun Ho Jang, Kazeera Aliar, Curtis McCloskey, Andrew Macklin, Joan Miguel Romero, Laura Tamblyn, Nikolina Radulovich, Peter Bronsert, Grainne O’Kane, Julie Wilson, Jennifer Knox, Sandra Fischer, Thomas Kislinger, Melanie Boerries, Steven Gallinger, Rama Khokha. Stromal phenotypic heterogeneity fosters pancreatic cancer progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-002.