Hepatobiliary Tumor Organoids Reveal HLA Class I Neoantigen Landscape and Antitumoral Activity of Neoantigen Peptide Enhanced with Immune Checkpoint Inhibitors.

Neoantigen-directed therapy lacks preclinical models recapitulating neoantigen characteristics of original tumors. It is urgent to develop a platform to assess T cell response for neoantigen screening. Here, immunogenic potential of neoantigen-peptides of tumor tissues and matched organoids (n = 27 pairs) are analyzed by Score tools with whole genome sequencing (WGS)-based human leukocyte antigen (HLA)-class-I algorithms. The comparisons between 9203 predicted neoantigen-peptides from 2449 mutations of tumor tissues and 9991 ones from 2637 mutations of matched organoids demonstrate that organoids preserved majority of genetic features, HLA alleles, and similar neoantigen landscape of original tumors. Higher neoantigen load is observed in tumors with early stage. Multiomics analysis combining WGS, RNA-seq, single-cell RNA-seq, mass spectrometry filters out 93 candidate neoantigen-peptides with strong immunogenic potential for functional validation in five organoids. Immunogenic peptides are defined by inducing increased CD107aCD137IFN-γ expressions and IFN-γ secretion of CD8 cells in flow cytometry and enzyme-linked immunosorbent assay assays. Nine immunogenic peptides shared by at least two individuals are validated, including peptide from TP53R90S . Organoid killing assay confirms the antitumor activity of validated immunogenic peptide-reactive CD8 cells, which is further enhanced in the presence of immune checkpoint inhibitors. The study characterizes HLA-class-I neoantigen landscape in hepatobiliary tumor, providing practical strategy with tumor organoid model for neoantigen-peptide identification in personalized immunotherapy.

Single-Cell Transcriptome Analysis Uncovers Intratumoral Heterogeneity and Underlying Mechanisms for Drug Resistance in Hepatobiliary Tumor Organoids.

Molecular heterogeneity of hepatobiliary tumor including intertumoral and intratumoral disparity always leads to drug resistance. Here, seven hepatobiliary tumor organoids are generated to explore heterogeneity and evolution via single-cell RNA sequencing. HCC272 with high status of epithelia-mesenchymal transition proves broad-spectrum drug resistance. By examining the expression pattern of cancer stem cells markers (e.g., PROM1, CD44, and EPCAM), it is found that CD44 positive population may render drug resistance in HCC272. UMAP and pseudo-time analysis identify the intratumoral heterogeneity and distinct evolutionary trajectories, of which catenin beta-1 (CTNNB1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and nuclear paraspeckle assembly transcript 1 (NEAT1) advantage expression clusters are commonly shared across hepatobiliary organoids. CellphoneDB analysis further implies that metabolism advantage organoids with enrichment of hypoxia signal upregulate NEAT1 expression in CD44 subgroup and mediate drug resistance that relies on Jak-STAT pathway. Moreover, metabolism advantage clusters shared in several organoids have similar characteristic genes (GAPDH, NDRG1 (N-Myc downstream regulated 1), ALDOA, and CA9). The combination of GAPDH and NDRG1 is an independent risk factor and predictor for patient survival. This study delineates heterogeneity of hepatobiliary tumor organoids and proposes that the collaboration of intratumoral heterogenic subpopulations renders malignant phenotypes and drug resistance.