Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most frequent cause of cancer-related mortality worldwide. The multikinase inhibitor sorafenib is the only treatment option for advanced HCC. Due to tumor heterogeneity, its efficacy greatly varies between patients and is limited due to adverse effects and drug resistance. Current in vitro models fail to recapitulate key features of HCCs. We report the generation of long-term organoid cultures from tumor needle biopsies of HCC patients with various etiologies and tumor stages. HCC organoids retain the morphology as well as the expression pattern of HCC tumor markers and preserve the genetic heterogeneity of the originating tumors. In a proof-of-principle study, we show that liver cancer organoids can be used to test sensitivity to sorafenib. In conclusion, organoid models can be derived from needle biopsies of liver cancers and provide a tool for developing tailored therapies.

Keywords: Biobank; cholangiocellular carcinoma; drug response; genetic heterogeneity; hepatocellular carcinoma; liver; needle biopsy; patient-derived organoids; patient-derived xenografts.

Graphical abstract

Figure 1

Establishment of Organoid Cultures from Needle Biopsies of Hepatocellular Carcinoma and Paired Non-tumor Liver Tissues (A) Schematic workflow of organoid generation from needle biopsies. (B) Representative biopsy pieces of tumor and liver tissue used for organoid generation. (C) Representative bright-field images of tumor and paired non-tumor liver tissue organoids from three different patients. Tumor organoids form compact spheroids, whereas liver organoids from non-tumor liver tissue grow as cystic structures. Organoids were imaged at the indicated passage numbers. Scale bar: 500 μm.

Figure 2

Clinical, Histopathological, and Molecular Features of HCC Biopsies Used for Organoid Generation (A) Color-coded table of patient characteristics of all biopsies (n = 38) used for organoid generation. Edmondson grade (Edmondson and Steiner, 1954) and the growth pattern were determined in each biopsy on H&E-stained sections by two experienced hepato-pathologists (M.S.M. and L.M.T.). Clinical data were extracted from the electronic patient information system of the hospital. Of note, only the Edmondson grade III was a significant determinant of successful organoid generation (p = 0.01, Fisher’s exact test, two-sided). For the Edmondson grade, calculations were performed per biopsy, whereas all other parameters were calculated per patient. ALD, alcoholic liver disease; AFP, alpha-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; HBV, hepatitis B virus; HCV, hepatitis C virus; MVI, macrovascular invasion; NAFLD, nonalcoholic fatty liver disease. (B and C) Unsupervised hierarchical clustering analysis. (B) Biopsy (organoid) cohort (this study) combined with all HCCs from TCGA cohort. (C) Biopsy (organoid) cohort (this study) combined with high-grade (Edmondson grades III and IV) HCCs from TCGA cohort.

Figure 3

Histopathological Characteristics of HCC and CCC Organoids and Their Primary Tumors (A) Histological sections of HCC and CCC organoids and their original tumors stained with H&E. The originating tumors display primarily a solid-trabecular architectural pattern with poor differentiation (Edmondson grades III and IV), features that are maintained in the corresponding HCC organoids. Arrowheads indicate pseudoglandular structures in HCC organoids and intracytoplasmic lumen in CCC organoids. (B) AFP expression detected by immunohistochemistry on organoids and original biopsies. (C) Expression of biliary markers KRT7 and KRT19 detected by immunohistochemistry on organoids and original biopsies. Organoids were imaged at the indicated passage numbers. Scale bars: 100 μm.

Figure 4

Histological Analysis of Xenografts Derived from HCC and CCC Organoids (A) Growth curves of the xenograft tumors. (B) Histological sections of xenograft tumors derived from HCC and CCC organoids stained with H&E. The HCC marker AFP and the biliary marker KRT7 were detected by immunohistochemistry. Scale bar: 100 μm. (C) Trichrome and Alcian blue-PAS staining on biopsy, derivative organoids, and xenograft of patient 20. Collagen-rich areas representing the desmoplastic stroma reaction are colored in blue in Trichrome-stained sections. Mucin production appears light blue in sections stained with Alcian blue-PAS (arrowheads). Organoids were imaged at the indicated passage numbers. Scale bars: 100 μm. (D) Statistics of the xenograft experiments.

Figure 5

Repertoire of Genetic Alterations Found in the HCC and CCC Organoids and Their Originating Tumors (A) Venn diagrams illustrate the number of somatic non-synonymous mutations present in each HCC biopsy and their derivative HCC organoids. The dashed line denotes CCC-derived tumors and corresponding organoids. (B) Repertoire of somatic non-synonymous mutations affecting cancer genes (Fujimoto et al., 2016, Kandoth et al., 2013, Lawrence et al., 2014, Cancer Genome Atlas Research Network, 2017). The effects of the mutations are color-coded according to the legend, with hotspots (Chang et al., 2016, Gao et al., 2017) colored in red. Multiple non-synonymous mutations in the same gene are indicated by an asterisk. Loss of heterozygosity of the wild-type allele of a mutated gene is represented by a diagonal bar, and mutations found to be clonal by ABSOLUTE (Carter et al., 2012) are indicated by a black box. (C) Contour plots illustrate the distribution of the cancer cell fractions (CCFs) of somatic mutations in the tumors and their corresponding organoids, with the increasing shades of red indicating higher number of somatic mutations at a given CCF.

Figure 6

Differential Drug Responses in Patient-Derived HCC and CCC Organoids under Sorafenib Treatment HCC and CCC organoids were exposed to sorafenib at the indicated concentration for 6 days. DMSO-treated tumor organoids were used as control. (A) Representative bright-field images of sorafenib-treated HCC organoids (patient 5-B). Scale bar: 200 μm. (B) Sorafenib reduces cell viability of HCC and CCC organoids in a dose-dependent manner. The dashed line represents the IC₅₀. Data are presented as the percentage of control DMSO-treated tumor organoids and are the mean of at least two independent experiments performed in duplicate. (C) Differential IC₅₀ (in μM) of HCC and CCC organoids shown as mean ± SEM. Patient numbers correspond to Table 1.