Leveraging Patient-Derived Organoids for Personalized Liver Cancer Treatment

Abstract

Primary liver cancer (PLC) is a primary cause of cancer-related death worldwide, and novel treatments are needed due to the limited options available for treatment and tumor heterogeneity. 66 surgically removed PLC samples were cultured using the self-developed 2:2 method, and the final success rate for organoid culture was 40.9%. Organoid performance has been evaluated using comprehensive molecular measurements, such as whole-exome and RNA sequencing, as well as anticancer drug testing. Multiple organoids and their corresponding tumor tissues contained several of the same mutations, with all pairs sharing conventional TP53 mutations. Regarding copy number variations and gene expression, significant correlations were observed between the organoids and their corresponding parental tumor tissues. Comparisons at the molecular level provided us with an assessment of organoid-to-tumor concordance, which, in combination with drug sensitivity testing provided direct guidance for treatment selection. Finally, we were able to determine an appropriate pharmacological regimen for a patient with ICC, demonstrating the clinical practicality in tailoring patient-specific drug regimens. Our study provides an organoid culture technology that can cultivate models that retain most of the molecular characteristics of tumors and can be used for drug sensitivity testing, demonstrating the broad potential application of organoid technology in precision medicine for liver cancer treatment.

Keywords: drug screening; organoid; personalized treatment; primary liver cancer.

Figure 1

Patient-derived primary liver cancer (PLC) organoid cultures. Representative H&E staining of the organoids and corresponding tumor tissues. H&E staining of HCC No. 35, and ICC No. 42 tissues and PLCOs (top and second row, scale bar: 100 µm) and brightfield microscopy images of second-generation (P2) organoids on day 3. No. 42 was well cultured to the fourth generation (P4), showing good growth and retaining the organoid structure.

Figure 2

Genomic alterations of PLCOs vs. tumor tissues based on whole-exome sequencing (WES). (A-D) Somatic mutational landscape (A), transition and transversion (B), distribution of major signatures (C), and a visualized heatmap of copy number variations (CNVs) (D) of all samples using WES. (E) The copy numbers of several genes associated with the development of liver cancer were compared between PLCOs and tumors, with p values for differences calculated using the Wilcoxon test. (F) Correlation analysis of PLCOs vs. tumor tissues at CNV level.

Figure 3

Expression profiles based on RNA sequencing. (A) PCA analysis of PLCOs and tumor tissues. (B) Correlation analysis of the expression levels between PLCOs and tumor tissues based on RNA sequencing. (C) Volcano plot of DEGs between PLCOs and tumors. (D) The GO and KEGG enrichment results of downregulated DEGs.

Figure 4

Transcriptomic patterns of all expressed genes in PLCOs compared with cancer cell lines and tumor tissues. (A) Heatmap of the expression pattern similarity of tumor-specific genes among samples. (B) Comparison of the similarity of the expression patterns of tumor-specific genes in various tissues compared to real tumor (malignant) cells. (C) The intersection between DEGs and cell type-specific genes.

Figure 5

Dose-response curves after 6 days of treatment with PLC-related compounds generated from the luminescent signal intensities.

Figure 6

PLCO reflects the clinical outcome of one patient with intrahepatic cholangiocarcinoma (ICC). (A)Treatment and procedure timeline of the patient. The black triangle indicates the time point at which CT was administered, as indicated by the images in the middle row, and the red triangle indicates the time point at which PLCOs were obtained and the results of organoid drug sensitivity. (B) H&E staining of ICC tissue and PLCO (top row, scale bar:100 µm) and brightfield microscopy image of PLCOs in the first and second generations (P1 and P2) aon day 3 showing good growth of PLCOs. (C) Continuous brightfield microscopy image of PLCOs treated with different concentrations of irinotecan plus cisplatin at 0, 4, and 7 days.