Tumor organoid modeling has been recognized as a state-of-the-art system for in vitro research on cancer biology and precision oncology. Organoid culture technologies offer distinctive advantages, including faithful maintenance of physiological and pathological characteristics of human disease, self-organization into three-dimensional multicellular structures, and preservation of genomic and epigenomic landscapes of the originating tumor. These features effectively position organoid modeling between traditional cell line cultures in two dimensions and in vivo animal models as a valid, versatile, and robust system for cancer research. Here, we review recent advances in genomic and epigenomic characterization of tumor organoids and the novel findings obtained, highlight significant progressions achieved in organoid modeling of gene–drug interactions and genotype–phenotype associations, and offer perspectives on future opportunities for organoid modeling in basic and clinical cancer research.

Head and neck squamous cell carcinoma (HNSCC) remains a significant health burden because of tumor heterogeneity and treatment resistance, emphasizing the need for improved biological understanding and tailored therapies. In this study, we enrolled 31 patients with HNSCC for the establishment of patient-derived tumor organoids (PDO), which faithfully maintained the genomic features and histopathologic traits of the primary tumors. Long-term culture preserved key characteristics, affirming PDOs as robust representative models. PDOs demonstrated predictive capability for cisplatin treatment responses, with ex vivo drug sensitivity correlating with patient outcomes. Bulk and single-cell RNA sequencing unveiled molecular subtypes and intratumor transcriptional heterogeneity (ITH) in PDOs, paralleling patient tumors. Notably, a hybrid epithelial–mesenchymal transition–like ITH program was associated with cisplatin resistance and poor patient survival. Functional analyses identified amphiregulin as a potential regulator of the hybrid epithelial–mesenchymal state. Moreover, amphiregulin contributed to cisplatin resistance via EGFR pathway activation, corroborated by clinical samples. In summary, HNSCC PDOs serve as reliable and versatile models, offer predictive insights into ITH programs and treatment responses, and uncover potential therapeutic targets for personalized medicine. Significance: Profiling of patient-derived organoids uncovers intertumoral heterogeneity and a hybrid epithelial–mesenchymal transition program conferring cisplatin resistance and highlights amphiregulin as a regulator of cellular plasticity and potential therapeutic target for HNSCC treatment.

Upper aerodigestive squamous cell carcinoma (UASCC) presents significant challenges in clinical management due to its aggressive nature. Here, we elucidate the role of MLL3 mutations as early, clonal genomic events in UASCC tumorigenesis, highlighting their role as foundational drivers of cancer development. Utilizing CRISPR-edited, cross-species organoid modeling, we demonstrate that loss of MLL3 contributes to early squamous neoplastic evolution. Furthermore, we identify an MLL3/GRHL2 protein complex that regulates the UASCC epigenome, particularly impacting immune response pathways. Notably, a novel MLL3/GRHL2-IRF1 axis promotes the expression of Th1 chemokines, enhancing anti-tumor immunity by facilitating T cell infiltration into the tumor microenvironment. Consequently, MLL3 regulates the in vivo efficacy of immune checkpoint blockade (ICB) therapy, corroborated by the strong association between MLL3 expression and human patients’ clinical response to ICB therapy. Our work underscores the significance of MLL3 in UASCC pathogenesis and highlights the interplay between MLL3/GRHL2 and immune response pathways as potential therapeutic targets for UASCC treatment.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily.

Transcriptional intratumoral heterogeneity (ITH) is a hallmark of aggressive cancers. Investigation into the ITH programs that drive tumor metastasis and immune evasion could help identify potential treatment and prevention approaches. Through single-cell RNA sequencing analysis of upper aerodigestive squamous cell carcinoma (UASCC) cells and patient tumors, we uncovered a hybrid epithelial–mesenchymal transition (hEMT) ITH program linked to metastatic dissemination. The transcription factor ETS1 was identified as a master regulator of the hEMT program, directly activating prometastatic genes and promoting distant spread in vivo. Unexpectedly, ETS1 also orchestrated an immune-cold tumor microenvironment by transcriptionally activating STAT1 and CD274 (PD-L1), suppressing T lymphocyte infiltration, and elevating immune checkpoint molecules. Clinically, high ETS1 expression in tumors strongly correlated with poor survival and resistance to immune checkpoint blockade across multiple cohorts. Drug screening demonstrated that ETS1-high cancers were vulnerable to HSP90 inhibitors (e.g., alvespimycin), which suppress ETS1 by disrupting hypoxia-inducible factor 1 alpha–mediated transcriptional activation. Together, this work reveals ETS1 as a dual driver of tumor distal metastasis and immune evasion in UASCC, while nominating HSP90 inhibition as a tailored treatment strategy for ETS1-driven tumors. These findings provide a roadmap for targeting aggressive ITH subsets and overcoming immunotherapy resistance. Significance:Transcriptional intratumoral heterogeneity drives metastasis and immune evasion via an ETS1-regulated hybrid EMT program, which can be targeted with HSP90 inhibitors to treat aggressive, immunotherapy-resistant tumors.

HSP90 inhibition suppresses ETS1-driven cell migration in UASCC cells.