The tumor microenvironment drives transcriptional phenotypes and their plasticity in metastatic pancreatic cancer

Abstract

In pancreatic ductal adenocarcinoma (PDAC), the basal-like and classical transcriptional subtypes are associated with differential chemotherapy sensitivity and patient survival. These phenotypes have been defined using bulk transcriptional profiling, which can mask underlying cellular heterogeneity and the biologic mechanisms that distinguish these subtypes. Furthermore, few studies have interrogated metastases, which are the cause of mortality in most patients with this highly lethal disease. Using single-cell RNA-sequencing of metastatic needle biopsies and matched organoid models, we demonstrate intra-tumoral subtype heterogeneity at the single-cell level and define a continuum for the basal-like and classical phenotypes that includes hybrid cells that co-express features of both states. Basal-like tumors show enrichment of mesenchymal and stem-like programs, and demonstrate immune exclusion and tumor cell crosstalk with specific macrophage subsets. Conversely, classical tumors harbor greater immune infiltration and a relatively pro-angiogenic microenvironment. Matched organoid models exhibit a strong bias against the growth of basal-like cells in standard organoid media, but modification of culture conditions can rescue the basal-like phenotype. This study reframes the transcriptional taxonomy of PDAC, demonstrates how divergent transcriptional subtypes associate with unique tumor microenvironments, and highlights the importance of evaluating both genotype and transcriptional phenotype to establish high-fidelity patient-derived cancer models.

Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution

“…More striking was the differential expression of transcripts that define the "classical" subtype, which are expressed almost exclusively by the later passage PDO (Figure 4F, top). We thus observe a subtype admixture over time, which is consistent with findings by Raghavan et al [20], demonstrating that later passages of PDOs in culture undergo transcriptional reprogramming towards a "classical" phenotype, and reiterating the inherent plasticity of cellular subtypes. Of note, a recent assessment for "basal-like" and "classical" cells in PDAC tissues by subtype-specific protein markers has uncovered bi-phenotypic "hybrid" cells [57], while Ting and colleagues have shown evidence of subtype "drift" in PDAC tissues with various therapies [58], both lines of evidence supporting the plasticity of subtype states.…”

“…The dichotomous Moffitt classification schema of "basal-like" and "classical" subtypes was initially established using bulk RNA sequencing strategies applied to human tumor samples [18]. PDAC transcriptional subtypes are known to associate with specific microenvironmental niches [20], and primary tumors have been shown to undergo subtype switching in response to microenvironmental cues [41]. However, it is not well established whether cell lines can reliably classified into Moffitt subtypes [42] as freshly dissociated tumor tissue or PDO models, nor whether there would be an observable degree of subtype admixture when single-cell methods are applied to subclonal derivatives of parental cell lines.…”

“…In addition to the use of adherent cell lines, efforts at better recapitulating the biology of in vivo disease have led to the burgeoning adoption of patient-derived organoid (PDO) models as an ex vivo platform in the PDAC field. Encouragingly, early passages of PDAC PDOs maintain genomic and transcriptomic features of the tumor of origin, and harbor a gene signature of response to commonly used cytotoxic agents, enabling therapeutic prediction [17][18][19][20]. Nonetheless, whether the clonal architecture of PDOs remains stable over more prolonged ex vivo propagation, considering both the time in culture and the lack of extrinsic cues from the tumor microenvironment in vivo, remains less studied.…”

Cell Lineage Infidelity in PDAC Progression and Therapy Resistance

“…While the dichotomisation into two subtypes has the perceived advantage of simplifying biomarker and functional studies, there is increasing evidence that cells with classical and basal-like features co-exist in the same tumour ( Figure 2B ) ( Puleo et al, 2018 ; Porter et al, 2019 ; Chan-Seng-Yue et al, 2020 ; Hwang et al, 2020 ; Juiz et al, 2020 ; Nicolle et al, 2020 ). This evidence has been generated from analyses of both human tissues ( Puleo et al, 2018 ; Porter et al, 2019 ; Chan-Seng-Yue et al, 2020 ; Hwang et al, 2020 ; Raghavan et al, 2021 ) and ex vivo cultures ( Porter et al, 2019 ; Juiz et al, 2020 ; Nicolle et al, 2020 ; Raghavan et al, 2021 ) and implies that molecular classification systems should account for this phenotypic heterogeneity for a better prediction of patient outcomes. Accordingly, Nicolle and others have recently shown the benefit of classifying patients based on a continuum of phenotypes rather than on two non-overlapping subtypes ( Nicolle et al, 2020 ).…”

“…Single cell and spatial transcriptomics promise to provide further insights into the evolution of PDAC. Recent studies have used single-cell RNA sequencing of either biopsies or organoids coupled with multiplex immunofluorescence to reveal that classical and basal programmes co-exist even at the cellular level ( Juiz et al, 2020 ; Raghavan et al, 2021 ). Finally, these techniques might help elucidate better the role of the microenvironment in influencing PDAC subtypes.…”