Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease

Abstract

ObjectiveThe aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas.DesignWe evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed.ResultsIn normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme.ConclusionIn larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Cell Lineage Infidelity in PDAC Progression and Therapy Resistance

“…In keeping with this observation, the MC2 methylation subtype described by Espinet and others ( Espinet et al, 2021 ) and aligning with the transcriptomic basal-like subtype is suggested to derive from ductal cells. Furthermore, a recent manuscript demonstrating the presence of a rare ΔNp63 + ductal cell population in the normal human pancreas raises the possibility that these might represent a cell of origin for tumours with basal-like/squamous features ( Martens et al, 2021 ). Moreover, cell reprogramming, as a driver of basal-like/squamous PDAC, might have a cell-dependent context.…”

A shifting adversary: control of pancreatic cancer transcriptomic subtypes

“…PDA derived from acinar cells is associated with PanIN lesions, both before and after progression to invasive carcinoma, while duct-derived carcinomas display far fewer attendant PanIN lesions [13] . Importantly, duct-derived carcinoma cells show a tendency towards basal-like gene expression, while acinar-derived tumors more closely resemble the classical subtype identified in human cohorts [29] . Definitively ascribing a cell of origin to advanced human malignancies is impossible, meaning the role of the cell of origin in human PDA subtypes will remain undetermined.…”

Neuroanatomical characterization of the Nmu-Cre knock-in mice reveals an interconnected network of unique neuropeptidergic cells

“…Then, the brains were dehydrated in increasing concentrations of 1propanol pH 9 (1-propanol in CUBIC-R+; 25, 50, 75, 2 x 100 %, 30 min each step, and a final overnight step at 100 %). The next day, dehydrated samples were incubated in increasing concentrations of ethyl cinnamate (ECi, #112372, Sigma-Aldrich, Germany; ECi in 1-propanol pH 9; 25, 50, 75, 2 x 100 %, 45 min each step) [53][54][55] and were kept in 100 % ECi until image acquisition in a Light-sheet Fluorescence Microscope (LSFM).…”

“…CUBIC-L induced limited swelling, which was minimized further during the first RI matching step with CUBIC-R+(M) 50,51 . After dehydration with pH-adjusted 1-propanol series, a last step using the organic solvent ECi was performed, taking advantage of the complementary RI matching effect of both CUBIC-R and ECi 54 . The combination of 1-propanol pH 9 and ECi preserved endogenous fluorescence [53][54][55] .…”