ARP2/3 complex affects myofibroblast differentiation and migration in pancreatic ductal adenocarcinoma

Abstract

The ARP2/3 complex, which orchestrates actin cytoskeleton organization and lamellipodia formation, has been implicated in the initiation of pancreatic ductal adenocarcinoma (PDAC). This study aims to clarify its impact on the activity of cancer-associated fibroblasts (CAFs), key players in PDAC progression, and patient outcomes. Early pancreatic carcinogenesis was modeled in p48^Cre; LSL-Kras^G12D mice with caerulein-induced pancreatitis, complemented by in vitro studies on human immortalized pancreatic stellate cells (PSCs) and primary PDAC-derived CAFs. Data were gained from microarray analysis, RNA sequencing (RNA-seq), and single-cell RNA sequencing (sc-RNA-seq), with subsequent bioinformatics analysis. We uncovered a specific transcriptional signature associated with fibroblast migration in early pancreatic carcinogenesis and linked it to poor survival in patients with PDAC. A pivotal role of the ARP2/3 complex in CAF migration was identified. Inhibition of the ARP2/3 complex markedly decreased CAF motility and induced significant morphological changes in vitro. Furthermore, its inhibition also hindered TGFβ1-mediated myofibroblastic CAF differentiation but had no effect on IL-1-mediated inflammatory CAF differentiation. Our findings position the ARP2/3 complex as central to the migration and differentiation of myofibroblastic CAF. Targeting this complex presents a promising new therapeutic avenue for PDAC treatment.

Keywords: cancer‐associated fibroblasts; myCAFs; pancreatic carcinogenesis; transcriptional signature; transforming growth factor β.

FIGURE 1

Signature of fibroblast migration is enriched in early pancreatic carcinogenesis. (A) Volcano plot illustrating the differential expression of gene sets involved in the fibroblast biological process, highlighting the significance of fibroblast migration in early pancreatic carcinogenesis. (B), (C) Gene set enrichment analysis (GSEA) and time‐dependent heatmap demonstrate the impact of fibroblast migration in kras^G12D mice, an established model of early pancreatic carcinogenesis. (D) Gene set variation analysis (GSVA) reveals distinct gene expression patterns between wild‐type (WT) and kras^G12D mice, providing insights into the role of fibroblast migration in disease progression. (E) Smooth curve representation of the GSVA analysis in (D), further elucidating the divergence in gene expression tendencies between WT and kras^G12D mice.

FIGURE 2

Molecular signature of fibroblast migration correlates with poor prognosis in human PDAC. (A) GSVA analysis using public data (GSE28735) in paired normal human pancreas (n = 45) and PDAC tissue (n = 45), showcasing the relevance of fibroblast migration human PDAC. (B) Kaplan–Meier survival curve depicting the survival outcomes of PDAC patients with high‐ and low‐risk scores for fibroblast migration signature. In TCGA cohort, high‐risk (N = 81) vs. low‐risk (N = 97) patients exhibit a median survival of 592 vs. 661 days (log‐rank test: p <.05). (C) Impact of fibroblast migration on PDAC tumor stage (TNM) in the cancer genome atlas (TCGA) datasets, underscoring its association with disease progression.

FIGURE 3

ARP2/3 complex affects CAFs' migration capacity. (A) Representative images displaying ARP2 and ARP3 expression differences between normal and PDAC tissue. Scale bars: 50 μm. (B) Transwell‐based cell migration assay comparing CAFs treated with or without the Arp2/3 complex inhibitor CK666. Scale bars: 100 μm (left). (C) Schematic picture of morphological changes in CAFs induced by treatment with the Arp2/3 complex inhibitor CK666, emphasizing its impact on cell shape and motility (right). (D) Photomicrographs of CAFs cultured in 2D condition, either treated or untreated with CK666. (E) Quantitative analysis of leading edge length (left), number of spontaneous protruding lamellipodia per cell (middle), and the percentage of cells with multiple protruding lamellipodia (right) in 2D‐cultured cells. (F) Fluorescence confocal images stained with phalloidin. Scale bars: 50 μm. (G) Aggregated trajectories between human CAFs and CAFs with CK666 treatment (left). Distance and velocity between human CAFs and CAFs with CK666 treatment (right).

FIGURE 4

ARP2/3 complex is coupled with myofibroblast differentiation. (A), (B) This UMAP plot visually represents the presence of two distinct fibroblast populations (iCAFs and myCAFs) by using the published markers. (C) The dot plot illustrates the differential expression of the Arp2/3 complex signature in iCAFs and myCAFs, shedding light on their distinct molecular characteristics (lower left). (D) Immunofluorescence co‐staining of PDAC tumor tissue with ARP3 (green), αSMA (red) and DAPI (blue). One representative image is shown (n = 8). Scale bars: 100 μm. (E) Correlation analysis of αSMA+ and ARP3+ cells was conducted using the “Colocalization Finder” tool in ImageJ 1.5.3. (F) This model provides insight into the signaling pathways that influence the formation of iCAFs and myCAFs in PDAC: TGFβ signaling plays a key role in myCAF formation, IL1 signaling drives iCAF development. (G) The results from qRT‐PCR analysis reveal the expression levels of myCAF markers (ACTA2, TAGLN, and MYL9) when treated with TGFβ1 (20 ng/mL) and iCAF markers (CXCL1, IL6, and CCL2) when treated with IL1α (1 ng/mL) in human PSCs, both in the presence and absence of CK666. These findings are derived from three independent experiments and are presented as mean ± SEM. *, indicates statistical significance with p <.05, as determined by a Student t‐test.