A systems-level approach to understanding stromal and neoplastic heterogeneity in PDAC

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, with the poorest survival rate. It is defined by its highly heterogeneous nature (involving both stromal and epithelial compartments) and a dense stromal compartment with relatively few neoplastic cells. Different cellular states of PDAC have been identified to date, and it has been shown that they are often determined by the integration of extrinsic and intrinsic factors. Considering the volatile and heterogeneous environment of the PDAC tissues, co-existence of diverse cell states has been consistently reported. Oncogenic dosage variation is a major determinant of malignant progression, and it contributes to cell state transition and phenotypic heterogeneity. Whether and how genetic factors integrate environmental cues to define cell states remains to be elucidated. Moreover, the cancer phenotype is achieved due to the interaction of its different cellular component. Therefore, it is equally important to understand the non-cell autonomous consequences of cell plasticity. Fibroblasts represent the predominant cell type within the PDAC microenvironment, and considering their significant role in driving disease progression, understanding the diversity of fibroblast subtypes is essential for developing effective anti- cancer therapies. In Chapter 1 of this thesis, we investigated the phenotype heterogeneity of cancer-associated fibroblasts (CAFs) in the context of a specific tumor cells subtype. First, we integrated bulk with spatial profiles of human PDAC tissues to identify a population of fibroblasts with elevated MAPK activity anchored to neoplastic cells showing an aggressive phenotype. We then used an in vivo post-perturbational pathway mapping analysis leveraging a mouse model of basal-like PDAC to characterize this population of myofibroblasts. In addition to elevated MAPK activity, the mapCAFs (MAPKhigh CAFs) display hypoxia- and immune-related gene programs. From this data, we were able to extract a stromal-specific mapCAFs transcriptional signature, which we used mapped onto cellular and spatial transcriptional data of human PDAC. In human tissues, mapCAFs niches were populated by basal-like tumor cells and presented scarce infiltration by cytotoxic T cells. Accordingly, pharmacologic depletion of mapCAFs using MEK1/2 inhibitors favored infiltration of CD8+ T cells in basal-like mouse tumor models. Finally, we found this population in the myCAF compartment of additional tumor types and high levels of the mapCAF signature correlated with reduced response to immune checkpoint inhibition in melanoma. In Chapter 2, we investigated mechanisms of genomic plasticity which afford cancer cells enhanced fitness in harsh microenvironmental conditions. Oncogene amplifications on circular 3 extrachromosomal DNA (ecDNA) are an important source of intra-tumor heterogeneity and phenotypic plasticity. In our laboratory, we have identified ecDNA amplification as a relatively frequent event in early stage and treatment naive PDAC (about 30% of the cases). MYC amplification on ecDNA (ecMYC) is the most recurrent event, yet oncogene expression from ecDNA was found to be heavily dependent on the regulatory landscape of the circular amplicon. Leveraging patient-derived organoids (PDOs), we found that ecMYC were under positive selection upon withdrawal of WNT agonists from the culture medium, suggesting increased MYC as a mechanism of WNT-gated survival in PDAC. ecMYC accumulation was also associated with morphological changes in PDOs which transitioned from cystic structure towards solid or cribriform growth. These functional and morphological changes were rapidly reversible upon neutralization of the selection pressure. To investigate the spatial context of ecDNA driven MYC amplifications in vivo, we integrated spatial transcriptomics (ST) and cytogenetics on formalin-fixed paraffin embedded tissue sections from ecDNA-positive and ecDNA-negative PDAC samples. We used the per cell distribution of MYC copy number states to define ecDNA+ tissue areas. In line with in vitro data, ecDNA+ cells displayed a WNT unresponsive state, defined as low to absent LGR5 expression in neoplastic cells. That was associated with reduced expression of canonical WNT ligands by surrounding stromal cells. Moreover, ecDNA high tissue areas displayed morphological pattern consistent with cribriform/ solid and individual growths. Finally, ecDNA+ tissue areas were characterized by reduced infiltration of CD8+ T cells. Altogether, our results showed that MAPK hyperactivation in the stromal compartment is a non-cell-autonomous consequence of PDAC cells plasticity. We traced a subpopulation of myCAF with elevated level of MAPK activity (mapCAF) and their association with basal-like tumor niches. Our findings also suggest that the presence of mapCAF could serve as a predictive marker for immunotherapy response in PDAC and other malignancies, highlighting their potential as therapeutic targets. Moreover, we showed that ecDNAs are key elements of genomic plasticity in PDAC, where they drive adaptation to environmental stressors by amplifying oncogenes and creating heterogeneity.