Spatially Resolved Single-Cell Assessment of Pancreatic Cancer Expression Subtypes Reveals Co-expressor Phenotypes and Extensive Intratumoral Heterogeneity

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intra-tumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in >90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intra-tumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.

High‐resolution and quantitative spatial analysis reveal intra‐ductal phenotypic and functional diversification in pancreatic cancer

“…Raghavan et al reported altered transcriptomic profiles of organoids when microenvironmental conditions are changed [25,33]. Here we demonstrated a shift, not only between but also within individual organoids.…”

“…Bulk NGS and, more recently, single-cell sequencing, spatial profiling, and AI-based histology have revealed the complexity of human PDAC [2][3][4][5][6]20,[24][25][26][27][28][29][30], with an emphasis on transcriptomic subtypes and the tumour microenvironment (TME) with different CAF subtypes and immune-suppressive cells. However, we perceived a lack of in situ assessment of point mutations (in particular those of KRAS that were reported to be amplified in subsets of aggressive patient tumours [3,10,36]) and, beyond GATA6, reliable markers that captured the two consensus transcriptomic subtypes in situ.…”

“…single ducts. The observation of intraductal heterogeneity was recently also reported by Williams et al [25]. In contrast to that valuable study, we integrated a systematic and quantitative approach with markers directly informed by the transcriptomic signatures from two of the seminal publications of PDAC transcriptomic subtypes [4,5].…”

Refining the Treatment of Pancreatic Cancer From Big Data to Improved Individual Survival

“… 8 , 73 Williams et al recently performed a spatially resolved single-cell assessment of Classical, Basal, and Hybrid phenotypes in resectable and metastatic patient samples using a quantitative multimarker protein panel (Classical protein biomarkers: CLDN18.2, TFF1, GATA6; Basal-like protein biomarkers: KRT17, KRT5, S100A2). 74 This analysis found that primary and metastatic samples exhibit considerable intratumoral subtype heterogeneity with very few tumors existing as purely Basal or purely Classical. While most tumors exhibited mixed Classica/Basal-like phenotypes, metastatic lesions exhibited a higher relative fraction of Basal-like to Classical cells when compared to primary tumors.…”

“… 8 To add to this complexity, a wealth of evidence now suggests that crosstalk between neoplastic and stromal cells—cancer associated fibroblasts (CAFs) and immune cell subsets—may shape subtype identity with distinct communities of neoplastic and stromal cells (ecotypes) co-evolving within the same patient tumors. 8 , 74 , 80–82 Precisely how these ecotypes evolve during cancer progression and in response to therapy are important questions for future research.…”