Single-nucleus and spatial transcriptomics of archival pancreatic cancer reveals multi-compartment reprogramming after neoadjuvant treatment

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a treatment-refractory disease. Characterizing PDAC by mRNA profiling remains particularly challenging. Previously identified bulk expression subtypes were influenced by contaminating stroma and have not yet informed clinical management, whereas single cell RNA-seq (scRNA-seq) of fresh tumors under-represented key cell types. Here, we developed a robust single-nucleus RNA-seq (snRNA-seq) technique for frozen archival PDAC specimens and used it to study both untreated tumors and those that received neoadjuvant chemotherapy and radiotherapy (CRT). Gene expression programs learned across untreated malignant cell and fibroblast profiles uncovered a clinically relevant molecular taxonomy with improved prognostic stratification compared to prior classifications. Moreover, in the increasingly-adopted neoadjuvant treatment context, there was a depletion of classical-like phenotypes in malignant cells in favor of basal-like phenotypes associated with TNF-NFkB and interferon signaling as well as the presence of novel acinar and neuroendocrine classical-like states, which may be more resilient to cytotoxic treatment. Spatially-resolved transcriptomics revealed an association between malignant cells expressing these basal-like programs and higher immune infiltration with increased lymphocytic content, whereas those exhibiting classical-like programs were linked to sparser macrophage-predominant microniches, perhaps pointing to susceptibility to distinct therapeutic strategies. Our refined molecular taxonomy and spatial resolution can help advance precision oncology in PDAC through informative stratification in clinical trials and insights into differential therapeutic targeting leveraging the immune system.

Single-Nucleus and In Situ RNA–Sequencing Reveal Cell Topographies in the Human Pancreas

“…Importantly, we detected acinar-REG þ cells in chronic pancreatitis samples, and these cells were also recently described in a single-cell study of pancreatic ductal adenocarcinoma. 48 In acinar-i cells, we find evidence suggesting that hydrolytic enzyme production may be reduced compared to acinar-s cells and speculate that this acinar cell state could be a protective adaptation to periods of intense zymogen production and increased endoplasmic reticulum stress. If so, acinar-i cells might be analogous to a subset of postulated metabolically stressed islet beta cells.…”

Applications of Single-Cell Omics in Tumor Immunology

“…More recently, Hwang et al. delineated the molecular taxonomy changes of TME in pancreatic ductal adenocarcinoma patients treated with or without neoadjuvant chemotherapy and radiotherapy by using the integrated single-nucleus RNA sequencing and spatially resolved transcriptomics analyses ( 81 ). They found that the basal-like or classical-like reprogramming of malignant cells was associated with distinct immune infiltration in tumors and further affected the treatment outcomes and clinical decisions.…”

Tumor cell heterogeneity and its transcriptional bases in pancreatic cancer: a tale of two cell types and their many variants

“…Independently of the underlying pathogenic routes, PDAC is characterized by a high level of intratumor heterogeneity (Hwang et al, 2020) that is not fully captured by current classification schemes (Collisson et al, 2011; Moffitt et al, 2015; Bailey et al, 2016; Collisson et al, 2019; Hayashi et al, 2020; Kalimuthu et al, 2020). In these different classifications (Box 1), which are based on transcriptomes from bulk tumors or relatively broad tumor regions or isolated tumor epithelial components, the PDAC subtype identified in fact reflects the most abundant population of cells in the sample analyzed.…”

“…It remains that the coexistence of these two main types of tumor cells (or their variants thereof) in PDACs represents a major element of complexity in the therapeutic response. Indeed, when malignant cell gene signatures associated with endodermal differentiation on the one hand and with squamous or quasi ‐mesenchymal properties on the other were used to score intratumor heterogeneity in bulk tumor datasets, it became clear that a high heterogeneity score was associated with worse overall survival (Hwang et al, 2020), suggesting that coexistence of multiple distinct tumor cell types lays the ground for swift therapy‐induced selection of most aggressive clones. Therefore, it is critical on the one hand to better understand the molecular circuitries dictating different tumor properties and on the other to determine the relationship between morphological features, molecular signatures, and biological properties: To what extent can different gene expression programs be precisely assigned to morphologically distinct, unambiguously identifiable cells with defined biological properties?…”

“…In some cases, these two components, glandular and poorly differentiated, may have a distinct regional distribution, suggesting they may belong to different tumor subclones, and be thus separately identifiable in multi‐regional transcriptomic analysis (Hayashi et al, 2020). Irrespective of their regional distribution, single‐cell and single‐nucleus RNA‐seq make it clear that well‐differentiated and squamous PDAC cells coexist in variable proportions in most PDACs (Chan‐Seng‐Yue et al, 2020; Hwang et al, 2020; Juiz et al, 2020). Therefore, the coexistence of these two cell types is in fact an essential and general property of this cancer.…”