fibroblasts (CAFs). These data will provide a resource for future studies aimed at further characterizing and targeting specific cell populations in PDA. ResultsCellular heterogeneity during PDA progression. We sought to determine the composition of single cells during the progression of PDA GEMMs. Normal mouse pancreas; 40-day-old Kras LSL−G12D/+ Ink4a fl/fl Ptf1a Cre/+ (KIC) (5) mouse pancreas, termed "early KIC" (with the early lesion initially confirmed by ultrasound; Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci. insight.129212DS1); and 60-day-old KIC pancreas, termed "late KIC" (Figure 1A) were freshly isolated and enzymatically digested followed by single-cell cDNA library generation using the 10× Genomics platform (6). Libraries were subsequently sequenced at a depth of more than 10 5 reads per cell. We performed stringent filtering, normalization, and graph-based clustering, which identified distinct cell populations in the normal pancreas and both stages of PDA.In the normal mouse pancreas, 2354 cells were sequenced and classified into appropriate cell types based on the gene expression of known markers: acinar cells, islet and ductal cells (Supplemental Figure 2), macrophages, T cells, and B cells, as well as 3 distinct populations of fibroblasts (Figure 1, B and E) were noted. In the early KIC lesion (3524 cells sequenced), the emergence of an expanded ductal population was observed (9.9% of cells), expressing known ductal markers, such as Krt18 and Sox9 (7), and displaying early neoplastic changes (Figure 1, A, C, and F, and Supplemental Figure 3). The acinar cell population was substantially reduced, while there was a marked increase in total macrophages and fibroblasts. Of note, the same 3 populations of fibroblasts seen in the normal pancreas were identified in the early KIC lesion. Additionally, endothelial cells were observed at this stage. This indicates that the expansion of fibroblasts and macrophages is an early event during PDA development. We next characterized the late KIC pancreas (804 cells sequenced) and noted the absence of normal exocrine (acinar) and endocrine (islet) cells (Figure 1, D and G). Instead, 2 distinct populations of cancer cells were present, suggesting phenotypic cancer cell heterogeneity as a late event in the course of the disease. We also observed the presence of only 2 distinct fibroblast populations, which had a similar percentage in relation to total cells. Noticeably, macrophages became a predominant cell population in the late KIC tumor. Moreover, we observed lymphocytes at this stage. The cellular heterogeneity in cancer cells and stromal cells in the early and late KIC lesions highlighted the dynamic cellular changes that occur during PDA progression.Cancer cells enriched with mesenchymal markers emerge in advanced PDA. Gene expression analysis of epithelial markers (Cdh1, Epcam, Gjb1, and Cldn3) and mesenchymal markers (Cdh2, Cd44, Axl, Vim, and S100a4) revealed that the early KIC neoplastic cell...

Background & AimsPancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression. The progression of PDA is a highly complex and dynamic process featuring changes in cancer cells and stromal cells; however, a comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression.MethodsWe employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models.ResultsOur data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression. We also found distinct populations of macrophages with increasing inflammatory features during PDA progression. In addition, we noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts are dynamically regulated by epigenetic mechanisms.ConclusionThis study systematically outlines the landscape of cellular heterogeneity during the progression of PDA. It strongly improves our understanding of the PDA biology and has the potential to aid in the development of therapeutic strategies against specific cell populations of the disease.

e15739 Background: Pancreatic ductal adenocarcinoma (PDA) is a major cause of cancer-related death with limited therapeutic options available. This highlights the need for improved understanding of the biology of PDA progression. The progression of PDA is a highly complex and dynamic process featuring changes in cancer cells and stromal cells; however, a comprehensive characterization of PDA cancer cell and stromal cell heterogeneity during disease progression is lacking. In this study, we aimed to profile cell populations and understand their phenotypic changes during PDA progression. Methods: We employed single-cell RNA sequencing technology to agnostically profile cell heterogeneity during different stages of PDA progression in genetically engineered mouse models. Results: Our data indicate that an epithelial-to-mesenchymal transition of cancer cells accompanies tumor progression. We also found distinct populations of macrophages with increasing inflammatory features during PDA progression. In addition, we noted the existence of three distinct molecular subtypes of fibroblasts in the normal mouse pancreas, which ultimately gave rise to two distinct populations of fibroblasts in advanced PDA, supporting recent reports on intratumoral fibroblast heterogeneity. Our data also suggest that cancer cells and fibroblasts are dynamically regulated by epigenetic mechanisms. Conclusions: This study systematically outlines the landscape of cellular heterogeneity during the progression of PDA. It strongly improves our understanding of the PDA biology and has the potential to aid in the development of therapeutic strategies against specific cell populations of the disease.

Despite adequate dietary management, patients with Classic Galactosemia continue to have increased risks of cognitive deficits, speech dyspraxia, primary ovarian insufficiency, and abnormal motor development. A recent evaluation of a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model revealed reduced fertility and growth restriction. These phenotypes resemble those seen in human patients. In this study, we further assess the fidelity of this new mouse model by examining the animals for the manifestation of a common neurological sequela in human patients: cerebellar ataxia. The balance, grip strength, and motor coordination of GALT-deficient and wild-type mice were tested using a modified rotarod. The results were compared to composite phenotype scoring tests, typically used to evaluate neurological and motor impairment. The data demonstrated abnormalities with varying severity in the GALT-deficient mice. Mice of different ages were used to reveal the progressive nature of motor impairment. The varying severity and age-dependent impairments seen in the animal model agree with reports on human patients. Finally, measurements of the cerebellar granular and molecular layers suggested that mutant mice experience cerebellar hypoplasia, which could have resulted from the down-regulation of the PI3K/Akt signaling pathway.