Targeting cancer-associated fibroblasts/tumor cells cross-talk inhibits intrahepatic cholangiocarcinoma progression via cell-cycle arrest

Background
Cancer-associated fibroblasts (CAFs) play a key role in the desmoplastic reaction, a hallmark of intrahepatic cholangiocarcinoma (iCCA). They likely contribute to tumor aggressiveness and therapy resistance, although the precise molecular mechanisms remain unclear. This study aimed to explore how targeting the interaction between human CAFs (hCAFs) and iCCA cells using Crenigacestat, a Notch1 inhibitor, could influence cancer progression.

Methods
We employed various in vitro models, including 2D cultures and newly developed 3D hetero-spheroids combining iCCA cells with hCAFs. Findings from these models were validated using a xenograft mouse model, followed by transcriptomic and bioinformatic analyses of the explanted tumors.

Results
The interaction between hCAFs and iCCA cells promoted enhanced migration in both KKU-M213 and KKU-M156 cell lines. Crenigacestat selectively inhibited migration stimulated by the CAF-cancer cell cross-talk. Hetero-spheroids, which incorporated both iCCA cells and hCAFs, were larger than homo-spheroids composed solely of iCCA cells. Crenigacestat significantly reduced the growth and invasion of hetero-spheroids, but had no impact on homo-spheroids. In xenograft models, tumors formed from hCAFs and KKU-M213 cells grew larger than those formed from KKU-M213 cells alone, but Crenigacestat treatment markedly reduced tumor size and suppressed fibrosis.

Transcriptomic analysis revealed that the hCAFs/KKU-M213 tumors expressed genes linked to increased tumor burden, and Crenigacestat inhibited several activated canonical pathways. Notably, the most affected pathway involved cell cycle regulation. Crenigacestat decreased the expression of *CCNE2*, leading to cell cycle arrest. In hetero-spheroids, cell accumulation in the G2/M phase was observed, but Crenigacestat treatment significantly reduced the number of cells in this phase in hetero-spheroids, with no effect on homo-spheroids.

Conclusions
The interaction between hCAFs and iCCA cells plays a critical role in cancer progression, presenting a novel therapeutic target. Crenigacestat effectively disrupts this cross-talk, inhibiting tumor growth and migration, and offers potential for future treatment strategies.