Clonal heterogeneity in human pancreatic ductal adenocarcinoma and its impact on tumor progression.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer characterized by profound desmoplasia and cellular heterogeneity, which cannot be fully resolved using traditional bulk sequencing approaches. To understand how this heterogeneity contributes to PDAC biology, we analyzed a large cohort of primary tumors (n = 62) profiled using single-cell RNA sequencing (443,451 single cells), including a subset of 11 tumors with matched spatial transcriptomics (53,236 spots), to map clonal, transcriptional and spatial diversity. Moreover, our sample set includes a unique cohort of matched samples from six tumors collected before and after treatment with chemotherapy. Our analysis revealed notable intratumoral heterogeneity, with multiple genetically distinct neoplastic clones coexisting within individual tumors. We observed clone-specific ligand-receptor programs that are modulated by genomic dosage: amplifications of ligands or receptors enriched corresponding interactions, while deletions suppressed them, with genetically encoded differences supported by spatial transcriptomics and multiplex immunofluorescence. Phylogenetic integration identified focal 8q24 (MYC) amplification as recurrently associated with classical-to-basal transitions occurring within tumor lineages at a frequency above chance; these transitions were accompanied by coordinated depletion of immune-response programs. Functional perturbation experiments in murine and primary human models showed that MYC knockdown reactivates classical transcriptional programs and restores immune-response pathways, supporting a role for MYC in maintaining a basal, immune-depleted state. Spatial analyses further revealed dispersed clones enriched for epithelial-to-mesenchymal transition activity and immune suppression, correlating with metastatic potential and colonization of lymph node niches. These dispersed clones shifted toward a basal phenotype, contributing to disease progression. Our findings highlight the critical role of clonal diversity, transcriptional plasticity and tumor microenvironment interactions in shaping human PDAC biology. This work provides a detailed view of the molecular and spatial heterogeneity of PDAC and offers potential avenues for therapeutic intervention targeting clonal evolution and the mechanisms driving metastasis.