Kuo Qi ,Rong Liu

1The First Clinical Medical College,Inner Mongolia Medical University,Hohhot 010110,China;2Translational Cancer Research Center,Peking University First Hospital,Beijing 100034,China

Abstract Hepatobiliary tumors are of high grade of heterogeneity,which is recognized as a key contributor to drug resistance and poor disease prognosis.However,the intrinsic mechanism between heterogeneity and drug response in hepatobiliary tumor is still largely unknown.Using tumor organoid models,Wang and her colleagues have found that cooperation among distinct subpopulations might be a key mechanism for drug resistance in hepatobiliary tumor.

Keywords: Hepatobiliary tumors;organoid;drug resistance

The presence of tumor heterogeneity,including intertumoral and intratumoral disparity,usually results in marked differences in multiple aspects of individual patient tumors,including tumor growth,invasive capacity and drug sensitivity.Cancer stem cells (CSCs) have been considered to be one of the determining factors that contribute to intratumor heterogeneity (1). Different cell surface markers,such as prominin 1 (PROM1),CD44,CD24,epithelial cell adhesion molecule (EPCAM),and so on,have been identified by various studies to distinguish hepatobiliary tumor stem cell populations in the past decades,which indicate that heterogeneity exists even in CSCs.High-throughput sequencing technologies have provided a systematic approach for discovering genetic alterations,aberrant regulatory programs,and molecular subtypes for human tumors.However,traditional bulk sequencing methods are only able to provide averaged data at multiple cell levels,which apparently could hardly meet the requirements to deciphering the characteristics of different CSC subpopulations,yet single-cell sequencing provides an avenue to explore cell-to-cell differences.

Meanwhile,in order to investigate the heterogeneity of hepatobiliary tumors conveniently,anin vitroresearch model which can faithfully recapitulate thein vivophenotype is extremely important.The three-dimensional organoids have emerged as promising experimental tools that may largely recapitulate the cellular diversity seen in patient tumors.Importantly,patient-derived tumor organoids not only retain the histopathological characters of the original tumor,but also recapitulate drug responses in the clinic (2),which make tumor organoid an appropriate model for pre-clinical study.Recently,Wang and her collaborators (3) explored the heterogeneity and evolution of hepatobiliary tumor via single-cell RNA sequencing using organoids they have generated,which provides important insights into the intrinsic mechanisms between heterogeneity and drug response.

They have generated 7 organoids from different primary hepatobiliary tumors,including 4 hepatocellular carcinoma(HCC),2 intrahepatic cholangiocarcinoma (ICC),and 1 gallbladder cancer (GBC).These organoids maintained comparable histopathological features of their matched primary tumor tissues even after long-term expansionin vitro,which provide a guarantee for subsequent studies.Single-cell sequencing of these cultured organoids was then performed to dissect inter-and intra-tumoral transcriptomic heterogeneity.A single-cell atlas comprising 22,505 cells collected from seven organoids was established.Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) analysis revealed clustering of cells was primarily driven by organoids of origin (inter-tumoral heterogeneity).Further expression evaluation of gene sets related to tumor development,including cell proliferation,invasion and metastasis,revealed that one of the organoids,HCC272,has the highest partial epithelial-mesenchymal transition score (4),suggesting its potential tumoral malignancy.Meanwhile,combing the highly expressed genes in HCC272 was mainly enriched for malignancy-related functions,including HIF-1 signaling,mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways,and epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor resistance,and HCC272 exhibited broad resistance to tyrosine kinase inhibitors(TKIs).It is likely that constitutive activation of downstream pathways may be a part of factors that tumors produce drug resistance (3).

Since CSCs are a predominant cause of intratumoral heterogeneity,CSC-like markers were globally examined to explore the cancer stemness within hepatobiliary tumor organoids.It is shown that the percentage of cells with known CSC-like markers varied greatly among individual organoid,and HCC272 consisted of the highest proportion ofCD44+cells.Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis further revealed that Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway was activated inCD44+cells and HCC272 showed strong sensitivity to STAT3 inhibitor (cryptotanshinone),suggesting the large dependence on Jak-STAT3 signaling inCD44+cells to form the drug resistance of organoid HCC272.

UMAP analysis was utilized to identify heterogenous clusters with diverse gene expression pattern in the organoids,and thus each organoid is divided into several subgroups.Among all the genes analyzed,distinct distribution of two genes,glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or nuclear paraspeckle assembly transcript 1 (NEAT1) was observed in these organoids.On the one hand,trajectory and pseudo-time analysis of these clusters via monocle revealed thatGAPDH-enriched cluster was found located at the top of the trunk among all the subpopulations in HCC272 and other two organoids,suggesting that the reciprocal regulation ofGAPDHenriched cluster was necessary for their metabolism advantage.KEGG analysis identified hypoxia inducible factor-1 (HIF-1) signaling pathway to be enriched inGAPDH-enriched cluster,and expression network analysis further revealed the central role ofGAPDHin activating HIF-1 signaling pathway.On the other hand,a significantly higher level ofCD44and Jak-STAT related genes were found to exist inNEAT1highcluster,and NEAT1 is reported to regulate CD44 expression in CSCs (5),suggesting the existence of NEAT1-CD44-Jak/STAT axis in regulating drug resistance of the organoids.

Interestingly,CellPhoneDB analyses showed an apparently increased interactions of receptor-ligand pairs,predominantly EGFR-MIF etc.,betweenGAPDHhighandNEAT1highclusters,suggesting a close cell-cell communications among them.MIF,upregulated byHIF-1α,is secreted fromGAPDHhighcluster to activate EGFR pathway and downstream NEAT1-CD44-Jak/STAT axis,contributing to drug resistance.Meanwhile,the GRN and MIF secreted byNEAT1highconferred the capacity of drug resistance toGAPDHhighby activating EGFR pathway(Figure 1).Similar to a previous study that cooperation between distinct subclones has been found to gain a selective growth advantage in breast cancer (6),findings of this research uncovered a regulatory circuit among distinct clusters in hepatobiliary tumor organoids,which facilitates tumor drug resistance (3).

Figure 1 Molecular interactions between GAPDHhigh and NEAT1high clusters contribute to drug resistance.

Therefore,these findings are exciting in that they not only identified biological and transcriptomic heterogeneity,especially the diversification of CSC distribution and the complexity of cell evolution trajectory,in patient-derived hepatobiliary tumor organoids;but also revealed thatinterplay among distinct subpopulations might be a key mechanism for drug resistance in hepatobiliary tumor.It would be interesting to further confirm the reciprocal actions and mechanisms between these subpopulations in organoids model and clinical samples,and to reveal whether the cooperation is necessary for developing resistance to drug treatment in hepatobiliary tumors.

Acknowledgements

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Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.