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The fallopian tube (FT) is an important reproductive organ in females. Ample evidence suggests that the distal end of FT is the original site of high-grade serous ovarian carcinoma (HGSC). FT may suffer from repeated injury and repair stimulated by follicular fluid (FF); however, this hypothesis has not been examined. In fact, the molecular mechanism of homeostasis, differentiation, and the transformation of fallopian tube epithelial cells (FTECs) resulting from the stimulation of FF are still enigmatic. In this study, we examined the effects of FF along with factors present in the FF on a variety of FTEC models, including primary cell culture, ALI (air–liquid interface) culture, and 3D organ spheroid culture. We found that FF plays a similar role to estrogen in promoting cell differentiation and organoid formation. Moreover, FF significantly promotes cell proliferation and induces cell injury and apoptosis in high concentrations. These observations may help us to investigate the mechanisms of the initiation of HGSC.

Background Pancreatic cancer is a highly lethal malignancy with limited treatment response. Despite advancements in treatment, systemic chemotherapy remains the primary therapeutic approach for over 80% of patients, with no established biomarkers to guide drug selection. Traditional two-dimensional (2D) culture models fail to replicate the tumor microenvironment, necessitating the development of more advanced models, such as three-dimensional (3D) organoid models. Methods We established 3D organoid cultures using patient-derived conditionally reprogrammed cell (CRC) lines, originally cultured under 2D conditions. These CRC organoids were developed using a Matrigel-based platform without organoid-specific medium components to preserve the intrinsic molecular subtypes of the cells. Morphological, molecular, and drug sensitivity analyses were performed to compare the clinical responses of 3D CRC organoids with those of their 2D counterparts and clinical responses. Results The 3D CRC organoids retained the molecular characteristics, transcriptomic and mutational profiles of the parental tumors and displayed distinct morphologies corresponding to cancer stages and differentiation. Drug response profiling of gemcitabine plus nab-paclitaxel (Abraxane) and FOLFIRINOX demonstrated that the 3D organoids more accurately mirrored patient clinical responses than the 2D cultures. Notably, the IC50 values for the 3D organoids were generally higher, reflecting the structural complexity and drug penetration barriers observed in vivo. Conclusion Matrigel-based 3D organoid culture models provide a robust platform for pre-clinical drug evaluation, overcoming the limitations of 2D models. Although time- and resource-intensive, integrating both 2D and 3D platforms enables efficient initial screening and validation. This approach holds promise for identifying predictive biomarkers and advancing precision medicine in pancreatic cancer treatment.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self‐assembled three‐dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high‐throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient‐derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

Cancer organoids are three-dimensional in vitro models that closely replicate the genetic, phenotypic, and heterogeneity characteristics of original tumors, making them valuable tools in cancer research. However, the lack of standardized protocols limits their broader application. This study evaluates the role of enzymatic isolation in generating patient-derived organoids (PDOs) from colorectal cancer tissues by comparing four enzymatic methods: TrypLE, Trypsin–EDTA (T/E), Collagenase, and Hyaluronidase. Colorectal cancer tissues were processed using these enzymes, and cell viability, dissociation efficiency, and isolation quality were assessed via Trypan Blue exclusion assay and 7-AAD staining with flow cytometry. Cancer stem cells marked by LGR5 and CD133 were quantified via flow cytometry, while organoid generation and growth were monitored over 11 days using confocal microscopy. TrypLE and T/E demonstrated superior preservation of cell viability but limited dissociation efficiency, yielding lower cell count per milligram of tissue. In contrast, Collagenase and Hyaluronidase demonstrated superior tissue dissociation, yielding higher total cell counts and the highest proportions of LGR5 positive and CD133 positive stem cell populations. Collagenase produced the highest organoid counts, while Hyaluronidase supported the largest organoid expansion, with both enzymes generating larger organoid surface areas and a greater number of organoids compared to TrypLE and T/E. These results highlight Collagenase and Hyaluronidase as optimal choices for PDO generation, providing a framework for optimizing dissociation protocols. This study underscores the critical influence of enzymatic dissociation methods on the establishment and reliability of colorectal cancer patient-derived organoids, providing a foundation for optimizing PDO protocols and advancing their translational application in precision oncology.

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue. Tumors contain a mixture of many different types of cells, including cancer cells and non-cancer cells. The interactions between these two groups of cells affect how the cancer cells use nutrients, which, in turn, affects how fast these cells grow and divide. Furthermore, different cell types may use nutrients in diverse ways to make other molecules – known as metabolites – that the cell needs to survive. Fibroblasts are a subset of non-cancer cells that are typically found in tumors and can help them form. Separating fibroblasts from cancer cells in a tumor takes a lot longer than the chemical reactions in each cell of the tumor that produce and use up nutrients, also known as the cell’s metabolism. Therefore, measuring the levels of glucose (the sugar that is the main energy source for cells) and other metabolites in each tumor cell after separating them does not necessarily provide accurate information about the tumor cell’s metabolism. This makes it difficult to study how cancer cells and fibroblasts use nutrients differently. Lau et al. have developed a strategy to study the metabolism of cancer cells and fibroblasts in tumors. Mice with tumors in their pancreas were provided glucose that had been labelled using biochemical techniques. As expected, when the cell processed the glucose, the label was transferred into metabolites that got used up very quickly. But the label also became incorporated into larger, more stable molecules, such as proteins. Unlike the small metabolites, these larger molecules do not change in the time it takes to separate the cancer cells from the fibroblasts. Lau et al. sorted cells from whole pancreatic tumors and analyzed large, stable molecules that can incorporate the label from glucose in cancer cells and fibroblasts. The experiments showed that, in cancer cells, these molecules were more likely to have labeling patterns that are characteristic of two specific enzymes called pyruvate carboxylase and malic enzyme 1. This suggests that these enzymes are more active in cancer cells. Lau et al. also found that pancreatic cancer cells needed these two enzymes to metabolize glucose and to grow into large tumors. Pancreatic cancer is one of the most lethal cancers and current therapies offer limited benefit to many patients. Therefore, it is important to develop new drugs to treat this disease. Understanding how cancer cells and non-cancer cells in pancreatic tumors use nutrients differently is important for developing drugs that only target cancer cells.

Purpose Depending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques. Methods Tumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models. Results Three series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes. Conclusion We report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high‐risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR‐GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR‐GFP vectors were transduced into patient‐derived squamocolumnar junction organoids, and the presence of GFP‐positive cells was evaluated. Single‐cell RNA sequencing of GFP‐positive and GFP‐negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP‐positive and GFP‐negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP‐positive cells, whereas keratinization and mitophagy/autophagy‐related pathways were upregulated in GFP‐negative cells. siRNA‐mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV‐related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18‐infected cells downregulated stem cell‐related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18‐infected cervical lesions. Using a combination of squamocolumnar junction organoids and HPV18LCR‐GFP lentivirus, we successfully identified three molecules that may be associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. This novel approach could be a breakthrough in identifying the mechanism of initial HPV18 replication and the cellular origin of HPV18‐associated cervical cancer.

Background Organoids have emerged as powerful tools in reproductive medicine and bioengineering, offering three-dimensional (3D) models that closely mimic native tissues. However, the development of protocols for generating healthy epithelial ovarian organoids (OvaOs) remains significantly underexplored, as most studies focus on ovarian cancer models. This work presents an effective protocol for generating healthy bovine OvaOs as a physiological and translational model for ovarian research, mimicking the anatomical and functional similarities between bovine and human ovarian surface epithelium (OSE). Results Healthy bovine OvaOs were successfully derived using a mechanical-enzymatic method with a predominant mechanical approach, which proved superior to exclusively enzymatic techniques that failed to yield an adequate number of OSE cells. The biological potential of the resulting OvaOs to establish long-term organoid lines was demonstrated by their exponential growth over a 21-day culture period, extensive passaging capacity, and high viability after freeze-thaw cycles. Histological analyses confirmed that healthy bovine OvaOs recapitulated OSE tissue characteristics, including the expression of Cytokeratin 18, Vimentin, and CD44, while the absence of Paired box gene-8 (PAX8) expression excluded contamination by fimbrial cells. Conclusions This study describes an effective mechanical protocol for deriving healthy OvaOs from bovine ovaries. These 3D models faithfully replicate the biological features of bovine OSE, with sustained viability across long-term cultures, passaging, and freeze-thaw cycles. These findings underscore their potential as translational models for advancing ovarian physiology research and adapting protocols to human ovarian tissue.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.