Background and Aims: Cholangiocarcinoma (CCA) is an aggressive, heterogeneous cancer with low survival rates. Patient-derived cholangiocarcinoma organoids (CCAOs) hold potential for understanding disease progression and developing novel treatment options, based on their ability to mimic the original tumor. However, a hallmark of cancer is the disturbance of morphological cues resulting in typical dysplastic tumor architecture, an aspect not well recapitulated in CCAOs. Currently, CCAO expansion protocols focus on stimulation of the canonical WNT/β-catenin pathway, however there is growing evidence that non-canonical WNT pathways also play a crucial role in cancer progression. This project aims to establish a novel in vitro 3D model for CCA, better recapitulating the in vivo tumor, by stimulating both canonical and non-canonical WNT pathways.
Method: Branching cholangiocarcinoma organoids (BRCCAOs) (n = 3 patients) were established with a two-step protocol. First, CCAOs were initiated and cultured under standard conditions in canonical WNT stimulating expansion medium. Next, expansion medium was replaced by medium that stimulates canonical WNT-signaling (through R-spondin) and non-canonical WNT-signaling (with Dickkopf-related protein 1, DKK1) simultaneously, after which a branching-like morphology could be observed. Tumor cell behavior in BRCCAOs and CCAOs was assessed and compared through immunohistochemical stainings, bulk RNA-sequencing, and drug response studies.
Results: BRCCAOs presented a distinct branching morphology, with the formation of peripheral branches with a lumen, variable in diameter, surrounded by compact layers of cells. This morphology displayed an architecture similar to in vivo tumors, while maintaining tumorigenic potential and showing a lack of defined cellular polarity through staining of zonula occludens-1 (ZO-1). Bulk RNA-sequencing of BRCCAOs unveiled significant upregulation of cancer-associated molecular pathways, including hypoxia, compared to CCAOs and a close correlation (coefficient 0.80±0.05) to the transcriptome of the original tumor tissue. BRCCAOs also exhibited patient-specific responses to a large panel of 166 anti-cancer drugs, including a universal strong resistant phenotype to multiple drugs that have previously failed in clinical trials for CCA patients (i.e. docetaxel, palbociclib, and irinotecan). Specifically, compared to CCAOs, BRCCAOs showed an approximately 10.000-fold (p < 0.0001) increase in chemo resistance against gemcitabine and cisplatin, first-line chemotherapy drugs for CCA, independent of patient variance.This resistant phenotype mimics patient response as clinically CCA patients experience only a modest increase in overall survival when receiving gemcitabine and cisplatin combinational therapy.
Conclusion: These results demonstrate that BRCCAOs better resemble in vivo CCA tumor tissue compared to conventional CCAOs, particularly with regards to morphology, transcriptome profile, and drug responses. Gemcitabine and cisplatin combinational therapy only provides CCA patients with a modest benefit in overall survival, and BRCCAOs appear to mimic this response more closely. This fosters new possibilities for personalized medicine applications.