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Heterogeneity, inherent and acquired drug resistance in patient-derived organoid models of primary liver cancer

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Abstract

Purpose

We aimed to elucidate the applicability of tumor organoids for inherent drug resistance of primary liver cancer (PLC) and mechanisms of acquired drug resistance.

Methods

PLC tissues were used to establish organoids, organoid-derived xenograft (ODX) and patient-derived xenograft (PDX) models. Acquired drug resistance was induced in hepatocellular carcinoma (HCC) organoids. Gene expression profiling was performed by RNA-sequencing.

Results

Fifty-two organoids were established from 153 PLC patients. Compared with establishing PDX models, establishing organoids of HCC showed a trend toward a higher success rate (29.0% vs. 23.7%) and took less time (13.0 ± 4.7 vs. 25.1 ± 5.4 days, p = 2.28 × 10−13). Larger tumors, vascular invasion, higher serum AFP levels, advanced stages and upregulation of stemness- and proliferation-related genes were significantly associated with the successful establishment of HCC organoids and PDX. Organoids and ODX recapitulated PLC histopathological features, but were enriched in more aggressive cell types. PLC organoids were mostly resistant to lenvatinib in vitro but sensitive to lenvatinib in ODX models. Stemness– and epithelial–mesenchymal transition (EMT)–related gene sets were found to be upregulated, whereas liver development– and liver specific molecule–related gene sets were downregulated in acquired sorafenib-resistant organoids. Targeting the mTOR signaling pathway was effective in treating acquired sorafenib-resistant HCC organoids, possibly via inducing phosphorylated S6 kinase. Genes upregulated in acquired sorafenib-resistant HCC organoids were associated with an unfavorable prognosis.

Conclusions

HCC organoids perform better than PDX for drug screening. Acquired sorafenib resistance in organoids promotes HCC aggressiveness via facilitating stemness, retro-differentiation and EMT. Phosphorylated S6 kinase may be predictive for drug resistance in HCC.

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Data availability

The original RNA-Seq data of tumor tissues are deposited in the Sequence Read Archive (SRA) database with accession PRJNA841062. The original RNA-Seq data in this study are deposited in the Gene Expression Omnibus (GEO) database with accession GSE182593. Other data are available from the corresponding author upon reasonable request.

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Funding

This work was supported by the National Key Basic Research Program of China [grant number 2015CB554006 (GC)], the National Natural Science Foundation of China [grant numbers 91529305 (GC), 81520108021 (GC), 81673250 (GC), 81521091 (GC), 82003538 (WL), and 81502882 (XC)], the Shanghai Yangfan Program [grant numbers 20YF1458800 (WL)] and the “3-year public health promotion” program of Shanghai Municipal Health Commission [grant numbers GWV-10.1-XK17 (GC)].

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Author notes

  1. Linfeng Xian, Pei Zhao, Xi Chen, Zhimin Wei, Hongxiang Ji, Jun Zhao and Wenbin Liu contributed equally to this work.

Authors and Affiliations

  1. Department of Epidemiology, Faculty of Navy Medicine, Second Military Medical University, Shanghai, 200433, China

    Linfeng Xian, Xi Chen, Zhimin Wei, Wenbin Liu, Zishuai Li, Xiong Zhou, Junyan Fan, Jianhua Yin, Xiaojie Tan & Guangwen Cao

  2. Guangxi Medical University Cancer Hospital, Nanning, 530022, China

    Pei Zhao & Hongping Yu

  3. Department of Liver Cancer Surgery, Third Affiliated Hospital, Second Military Medical University, Shanghai, 200433, China

    Hongxiang Ji, Jun Zhao & Donghong Liu

  4. Department of Chronic Diseases, Center of Disease Control and Prevention of Yangpu District, Shanghai, 200090, China

    Xue Han

  5. Department of Pathology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 200433, China

    Youwen Qian & Hui Dong

  6. Department of Nutrition, School of Public Health, Anhui Medical University, Hefei, 230032, China

    Xiaoqiong Zhu

  7. Shanghai East Hospital, Key Laboratory of Arrhythmias, Ministry of Education, Tongji University School of Medicine, Tongji University, Shanghai, 200120, China

    Dongming Jiang & Guangwen Cao

  8. School of Public Health, Guangxi Medical University, Nanning, 530022, China

    Hongping Yu

  9. Key Laboratory of Signaling Regulation and Targeting Therapy of Liver Cancer, Ministry of Education, Shanghai, 530022, China

    Guangwen Cao

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  1. Linfeng Xian
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Cao G conceived and supervised the study. Xian L, Zhao P, and Wei Z were responsible for the culture and maintenance of tumor organoids. Chen X performed the bioinformatics analyses. Ji H and Zhao J did surgical treatments and provided suitable clinical specimens. Liu D, Li Z, Liu W, Zhou X, Fan J, Zhu X, Yin J, and Tan X took part in the cell experiments and animal care. Qian Y and Dong H took part in the histology and immunohistochemistry assays. Chen X, Han X, and Yu H conducted the statistical analyses. Cao G interpreted the data and drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Guangwen Cao.

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Ethical approval and consent to participate

This study protocol was reviewed and approved by Ethics Committee of Eastern Hepatobiliary Surgery Hospital, approval number 2019UE-023. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all participants included in the study. All animal experiments in this study were conducted in accordance with the guidelines of the animal ethical committee for animal experimentation in China. The experimental design was approved by the Institutional Animal Care and Use Committee of Second Military Medical University.

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All patients signed informed consent regarding publishing their data.

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The authors declare that they have no competing interests.

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Supplementary Information

Fig. S1

The success rate and average days of constructing PLC organoids and PDX model. (a) The success rate of establishing tumor organoids of each HCC and ICC. (b) The success rate of establishing tumor PDX models of HCC and ICC. (c) The duration of tumor organoids’ culture from tissue separation to the first passage was 13.0 ± 4.7 and 13.8 ± 3.4 days for HCC and ICC, respectively. (d) The duration of PDX construction from the first tumor implantation to the second implantation was 25.1 ± 5.4 and 33. 0 ± 5.7 days for HCC and ICC, respectively. (PNG 82 kb)

High resolution image (TIF 4351 kb)

Fig. S2

H&E staining images of organoids at different passages. Histological features of the first generation organoids (fist line) and the seventh generation organoids (second line) of HCC-118, ICC-6, and CHC-3. Scale bars, 50 μm (PNG 1743 kb)

High resolution image (TIF 9482 kb)

Fig. S3

Quality control of RNA-Seq data derived from HCC samples. The number of read pairs (marked on left Y-axis), rate of high quality (HQ) reads (calculated by NGSQCToolkit) (marked on right Y-axis), and the alignment rate (calculated by HISAT2) (marked on right Y-axis) for each sample were plotted. (PNG 41 kb)

High resolution image (TIF 3115 kb)

Fig. S4

Immunohistochemistry of tumor tissues, organoids, and ODX and PDX from PLC patients of major histotypes. (a) Expression of AFP in tumor tissues, tumor organoids, ODX, and PDX derived from HCC-25, HCC-118, ICC-6, and CHC-3 patients. (b) Expression of CK-19 in tissues, organoids, ODX, and PDX. (c) Expression of EpCAM in tissues, organoids, ODX and PDX. Scale bars, 50 μm. CK19, cytokeratin 19; PLC, primary liver cancer; HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; CHC, hepatocellular cholangiocarcinoma; PDX, patient-derived xenograft; and ODX, organoids-derived xenograft. (PNG 7243 kb)

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Fig. S5

The stemness- and epithelial–mesenchymal transition-related gene sets enriched in each of the four acquired sorafenib-resistant HCC organoids. (a) gene sets enriched in the organoid of HCC-52; (b) gene sets enriched in the organoid of HCC-118; (c) gene sets enriched in the organoid of HCC-10; and (d) gene sets enriched in the organoid of HCC-25. NES, normalized enrichment score; FDR, false discovery rate. (PNG 141 kb)

High resolution image (TIF 7914 kb)

Fig. S6

The heterogeneity of stemness- and epithelial–mesenchymal transition-related gene expression patterns in four HCC organoids with and without acquired sorafenib resistance. (a) Western blotting showed the expression patterns of N-cadherin, Vimentin, Claudin-1, CD44, ABCG2, and EpCAM in HCC-118, HCC-25, HCC-10, and HCC-52 parental and sorafenib-resistant organoids, respectively. (b) Immunohistochemistry showed the expression of CD44, EpCAM, N-cadherin, and Vimentin in the parental and acquired sorafenib-resistant organoids of HCC-118 and HCC-25. Scale bars, 50 μm. (PNG 1736 kb)

High resolution image (TIF 19323 kb)

Fig. S7

Association of the differentially expressed genes in acquired sorafenib-resistant HCC organoids with the prognosis (a) overall survival; and (b) recurrence-free survival. (PNG 37 kb)

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Xian, L., Zhao, P., Chen, X. et al. Heterogeneity, inherent and acquired drug resistance in patient-derived organoid models of primary liver cancer. Cell Oncol. 45, 1019–1036 (2022). https://doi.org/10.1007/s13402-022-00707-3

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