Metastasis is the major cause of cancer-related death, but whether metastatic lesions exhibit the same cellular composition as primary tumors has yet to be elucidated. To investigate the cellular heterogeneity of metastatic colorectal cancer (CRC), we established 72 patient-derived organoids (PDOs) from 21 patients. Combined bulk transcriptomic and single-cell RNA-sequencing analysis revealed decreased gene expression of markers for differentiated cells in PDOs derived from metastatic lesions. Paradoxically, expression of potential intestinal stem cell markers was also decreased. We identified OLFM4 as the gene most strongly correlating with a stem-like cell cluster, and found OLFM4+ cells to be capable of initiating organoid culture growth and differentiation capacity in primary PDOs. These cells were required for the efficient growth of primary PDOs but dispensable for metastatic PDOs. These observations demonstrate that metastatic lesions have a cellular composition distinct from that of primary tumors; patient-matched PDOs are a useful resource for analyzing metastatic CRC.

The mitotic spindle is assembled by the coordinated action of centrosomes and kinetochore microtubules. An evolutionally conserved protein family, transforming acidic coiled-coil (TACC), has been shown to be involved in this process. In humans, TACC3 is aberrantly expressed in a variety of human cancers, but its biological significance remains to be elucidated. Here, using a novel compound targeting TACC3, spindlactone (SPL), we show that the perturbation of TACC3 selectively inhibited the nucleation of centrosome microtubules in ovarian cancer cells. In contrast to centrosome microtubules, the kinetochore microtubules were robustly assembled, forming ectopic spindle poles that resulted in multipolar spindles. Interestingly, the extensive inhibition of TACC3 partially suppressed the nucleation of kinetochore microtubules. These dose-dependent effects of SPL were consistent with the results observed by the depletion of TACC3 and its binding partner, colonic and hepatic tumor overexpressed gene protein (TOGp). Although these proteins both have roles in the assembly of centrosome and kinetochore microtubules, their contributions were spatiotemporally different. Notably, SPL did not affect spindle assembly in normal cells. Furthermore, the oral administration of SPL significantly suppressed tumor growth in vivo. The unique mechanism of action of SPL not only enables it to be used as a tool to dissect the molecular basis of spindle assembly but also to provide a rationale for the use of TACC3 as a molecular target for cancer treatment. This rationale offers an opportunity to develop new strategies for cancer chemotherapy that overcome the limitations of microtubule toxins and expand their scope and clinical efficacy.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Drug resistance often stems from drug-tolerant persister (DTP) cells in cancer. These cells arise from various lineages and exhibit complex dynamics. However, effectively targeting DTP cells remains challenging. We used single-cell RNA sequencing (scRNA-Seq) data and machine learning (ML) models to identify DTP cells in patient-derived organoids (PDOs) and computationally screened candidate drugs targeting these cells in familial adenomatous polyposis (FAP), associated with a high risk of colorectal cancer. Three PDOs (benign and malignant tumor organoids and a normal organoid) were evaluated using scRNA-Seq. ML models constructed based on public scRNA-Seq data classified DTP versus non-DTP cells. Candidate drugs for DTP cells in a malignant tumor organoid were identified from public drug sensitivity data. From FAP scRNA-Seq data, a specific TC1 cell cluster in tumor organoids was identified. The ML model identified up to 36 % of TC1 cells as DTP cells, a higher proportion than those for other clusters. A viability assay using a malignant tumor organoid demonstrated that YM-155 and THZ2 exert synergistic effects with trametinib. The constructed ML model is effective for DTP cell identification based on scRNA-Seq data for FAP and provides candidate treatments. This approach may improve DTP cell targeting in the treatment of colorectal and other cancers.

Colorectal cancer (CRC) is a heterogenous disease, and patients have differences in therapeutic response. However, the mechanisms underlying interpatient heterogeneity in the response to chemotherapeutic agents remain to be elucidated, and molecular tumor characteristics are required to select patients for specific therapies. Patient‐derived organoids (PDOs) established from CRCs recapitulate various biological characteristics of tumor tissues, including cellular heterogeneity and the response to chemotherapy. Patient‐derived organoids established from CRCs show various morphologies, but there are no criteria for defining these morphologies, which hampers the analysis of their biological significance. Here, we developed an artificial intelligence (AI)‐based classifier to categorize PDOs based on microscopic images according to their similarity in appearance and classified tubular adenocarcinoma‐derived PDOs into six types. Transcriptome analysis identified differential expression of genes related to cell adhesion in some of the morphological types. Genes involved in ribosome biogenesis were also differentially expressed and were most highly expressed in morphological types showing CRC stem cell properties. We identified an RNA polymerase I inhibitor, CX‐5641, to be an upstream regulator of these type‐specific gene sets. Notably, PDO types with increased expression of genes involved in ribosome biogenesis were resistant to CX‐5461 treatment. Taken together, these results uncover the biological significance of the morphology of PDOs and provide novel indicators by which to categorize CRCs. Therefore, the AI‐based classifier is a useful tool to support PDO‐based cancer research.