Gallbladder cholangiocyte organoids

Abstract

Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.

Keywords: Wnt pathway; anti‐cancer drug screen; biliary tract cancer; gallbladder cancer; organoids.

FIGURE 1

Establishment of gallbladder cholangiocyte organoids and their potential applications. Gallbladder cholangiocytes (epithelial cells) are isolated from the mucosal layer of surgically resected gallbladder using mechanical scraping or enzymatic digestion. The cells are washed, passed through strainer and seeded into extracellular matrix domes. The domes are then overlaid with organoid growth media and incubated at 37°C temperature inside a CO2‐incubator for organoid development. Developed organoids can be subjected to various applications, such as gallbladder disease biology study, drug screening and biliary repair and regeneration. Created with BioRender.com.

FIGURE 2

Overview of the canonical and non‐canonical Wnt signaling pathway. In the canonical Wnt pathway; binding of WNT ligand to Frizzled and LRP5/6 receptors phosphorylates Dishevelled. This inhibits GSK‐3 kinase activity and prevents destruction complex (APC‐Axin‐GSK3‐CK1α) formation. This stabilizes β‐catenin in the cytosol. Unphosphorylated β‐catenin translocates to the nucleus, where it acts as co‐transcription factor with TCF/LEF to promote transcription of effectors for self‐renewal. RSPO1 binds with ZNRF3/RNF43 and forms a complex with LGR4/5/6 and stabilizes Frizzled. Alternatively, exogenous addition of GSK‐3 inhibitor (CHIR99021) can activate the canonical Wnt pathway. DKK1 inhibits β‐catenin‐dependent Wnt signaling pathway by binding to the LRP5/6 receptor. Competitive binding of DKK1, facilitates WNT binding to Frizzled and activates the non‐canonical Wnt pathway through Ror/Ryk receptor complex. This activates RhoA‐ROCK and JNK pathway. They regulate the cytoskeleton remodelling and planar cell polarity, respectively (Corda & Sala, 2017). DKK, Dickkopf‐1; RSPO1, Rspondin 1; ZNRF3/RNF43, zing and ring finger 3/ ring finger 43; LGR5, leucine‐rich‐repeat‐containing G coupled receptors 4/5/6; TCF, T‐cell factor; LEF, lymphoid enhancer factor; ROR/RYK, retinoic acid‐related orphan receptors/receptor‐like tyrosine kinase; JNK, Jun N‐terminal kinase; RhoA, RAS homolog gene‐family member A; ROCK, Rho‐associated coiled‐coil containing protein kinase; APC, adenomatous polyposis coli; GSK, glycogen synthase kinase 3; LRP5/6, lipoprotein receptor‐related protein‐5/6 receptor; CK1α, Casein kinase. Created with BioRender.com.

FIGURE 3

Schematic diagram of gallbladder cancer pathogenesis. Metabolic (Larsson & Wolk, 2007) and environmental stress (Ganesan et al., 2020) induced ROS generation and/or bacterial toxin CdtB result in DNA double strand breaks. In presence of functional TP53 and DNA repair system, DNA damage is repaired. Continuous exposure to oxidative stress causes repeated cycles of DNA damage and repair, resulting in prolonged chronic inflammatory condition. This ultimately results in dysfunctional DNA repair system leading to accumulation of mutational burden and thus oncogenic transformation of gallbladder cholangiocytes (Roa et al., 2022). CdtB, cytolethal distending toxin subunit B; DSB, double‐strand breaks; ROS, reactive oxygen species; ECM, extracellular matrix; As III, trivalent inorganic arsenic ions. Created with BioRender.com.