Basal-like and Classical cells coexistence in pancreatic cancer revealed by single cell analysis

Abstract

26Pancreatic ductal adenocarcinoma (PDAC) is composed of stromal, immune and epithelial 27 cells. Transcriptomic analysis of the epithelial compartment allows a binary classification into 28 mainly two phenotypic subtypes, classical and basal-like. However, little is known about the 29 intra-tumor heterogeneity of the epithelial component. Growing evidences suggest that this 30 two side phenotypic segregation is not so clear and that both could coexist in a single tumor. In 31 order to elucidate this hypothesis, we performed single-cell transcriptomic analyses using 32 combinational barcoding on epithelial cells from 6 different classical PDAC obtained by 33 Endoscopic Ultrasound (EUS) with Fine Needle Aspiration (FNA). In order to purify the 34 epithelial compartment, PDAC were grown as Biopsy Derived Pancreatic Cancer Organoids. 35 Single cell transcriptomic analysis allowed the identification of 4 main cell clusters present in 36 different proportions in all tumors. Remarkably, although these tumors were classified as 37 Classical, one of the clusters corresponded to a basal-like. These results depict the 38 unanticipated high heterogeneity of pancreatic cancers and demonstrated that basal-like cells 39 with a high aggressive phenotype are more widespread than expected. 40 41 42With a survival rate of 5 years in less than 8% of the cases (Siegel et al., 2018) pancreatic 43 ductal adenocarcinoma (PDAC) is still one of the most lethal cancers. A principal problem 44 facing this disease is its heterogeneity that results as a consequence of the combination of 45 genetic, epigenetic, and micro-environmental factors (Lomberk et al., 2019(Lomberk et al., , 2018 Yachida and 46 Iacobuzio-Donahue, 2013). Recently, two main PDAC subtypes have been identified by 47 molecular characterization: 1-classical, that are more frequently resectables, presenting a 48 higher level of differentiation, often associated with fibrosis and inflammation; and 2-basal-49 like, with a poorest clinical outcome and a loss of differentiation (Moffitt et al., 2015; Nicolle 50 et al., 2017). This well-established binary classification could be controverted if cells from a 51 unique tumor contain both phenotypes at the same time. In fact, in addition to the tumor 52 differences between patients that argues in favor of stratification for personalizing PDAC 53 treatments, it is also absolutely necessary to consider the intra-tumor differences since they are 54 playing key roles in the evolution of tumors (i.e.: they can conduce to clonal selection of 55 resistant cells and to the relapse so frequently observed after the first line of chemotherapy). 56Single-cell analysis by transcriptomics is nowadays a powerful strategy to determine the intra-57 tumor heterogeneity, however we need to bypass two main challenging difficulties: The first 58 one is to obtain pure epithelial transformed cells. To do that, we specifically amplified these 59 cells by a few passages in three dimensional (3D) ex vivo culture (Tiriac et al., 2019). Three ...

Single-cell analysis of patient-derived PDAC organoids reveals cell state heterogeneity and a conserved developmental hierarchy

“…To categorize transcriptional heterogeneity in PDAC, an established classification scheme distinguishes 'classical' from 'basal-like' tumors. In line with recent results from other groups 17,39 , our cohort comprised PDAC organoids with uniformly 'classical' gene expression, but also a subset of organoids with heterogeneous expression of subtype marker genes across cells. The latter were associated with a poorer prognosis of the patient, as determined by histopathological grading of the original tumor.…”

Intraductal Transplantation Models of Human Pancreatic Ductal Adenocarcinoma Reveal Progressive Transition of Molecular Subtypes

“…Recent single-cell analyses have demonstrated the coexistence of basal-like and classical subtypes within a single tumor with the relative proportions of these cells determining the overarching subtype at the bulk transcriptome level (19,38). In another recent study, evolutionary analysis of mixed phenotype tumors, comprising classical and basal-like features, from autopsy patients revealed that the basallike subtype represented a subclone contained within an otherwise classical tumor (18).…”

Establishment of a pancreatic adenocarcinoma molecular gradient (PAMG) that predicts the clinical outcome of pancreatic cancer

“…We have made similar observations in this study. VIM, which is mainly expressed in basal-like subtype, was detected by immunohistochemistry in almost all classical tumors, with variable levels of expression [17]. We detected few VIM+ cells in tumors presenting an intermediate PAMG.…”

“…the infiltrated Immunogenic subtype), and delineated a consensual basal-like versus classical dichotomy. However, this two-subtype classification system of PDAC has recently been challenged by several studies showing the coexistence of basal-like and classical cells in the same tumors as well as to the likely existence of intermediate cellular phenotypes [15,17]. In this study, we have used a gradient system that takes this into consideration to classify PDAC.…”