Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited diagnostic biomarkers. Carbohydrate antigen 19-9 (CA19-9) is a widely used clinical biomarker and is generally considered to correlate with PDAC malignancy. However, the relationship between CA19-9 expression levels and tumor aggressiveness remains underexplored. In this study, we report a biphasic relationship between CA19-9 expression levels and PDAC malignancy, where both negative (<5 U/mL) and high (>37 U/mL) CA19-9 levels are associated with increased tumor aggressiveness. We defined CA19-9 negative PDAC as tumors that lack CA19-9 expression intracellulary, on the cell membrane, and in secreted form. In PDAC cell lines and patient-derived organoids, CA19-9 negativity, confirmed by immunofluorescence, flow cytometry and ELISA, correlated with more aggressive behaviors. In PDAC patients, tumors from those with serum CA19-9 levels below 5 U/mL exhibited stronger metabolically activity, more immunosuppressive tumor microenvironment, and worse survival than CA19-9 positive tumors, with over 90 % showing absent CA19-9 expression by immunohistochemistry (IHC). Glycoproteomics profiling identified CD44 as a highly expressed biomarker in CA19-9 negative PDAC. Elevated CD44 expression effectively distinguished CA19-9 negative PDAC from both CA19-9 positive PDAC and CA19-9 negative benign pancreatic diseases, suggesting its potential as a diagnostic tool. Furthermore, we developed a radionuclide-labeled CD44 antibody 89Zr-1M2E3, which specifically recognized CA19-9 negative PDAC tumors in preclinical models using PET-CT imaging. These findings highlight CD44 as a promising biomarker and therapeutic target for diagnosing and treating CA19-9 negative PDAC.

Malignant tumors notably decrease life expectancy. Despite advances in cancer diagnosis and treatment, the mechanisms underlying tumorigenesis, progression and drug resistance have not been fully elucidated. An emerging method to study tumors is tumor organoids, which are a three‑dimensional miniature structure. These retain the patient‑specific tumor heterogeneity while demonstrating the histological, genetic and molecular features of original tumors. Compared with conventional cancer cell lines and animal models, patient‑derived tumor organoids are more advanced at physiological and clinical levels. Their synergistic combination with other technologies, such as organ‑on‑a‑chip, 3D‑bioprinting, tissue‑engineered cell scaffolds and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9, may overcome limitations of the conventional 3D organoid culture and result in the development of more appropriate model systems that preserve the complex tumor stroma, inter‑organ and intra‑organ communications. The present review summarizes the evolution of tumor organoids and their combination with advanced technologies, as well as the application of tumor organoids in basic and clinical research.

The ability to establish organoids composed exclusively of tumour rather than healthy cells is essential for their implementation into clinical practice. Organoids have recently emerged as a powerful tool to expand patient material in culture and generate modifiable 3D models derived from humans or animal models. For translational research, they enable the creation of model systems for an ever-increasing number of cell types and diseases. And in personalised medicine, they potentially allow for functional drug testing with high predictive power in certain settings. We found that using biopsy material from untreated, early-stage primary breast cancer patients poses significant challenges for consistently culturing tumour cells as organoids. Specifically, we observed frequent outgrowth of genetically normal, non-cancerous epithelial cells. We analysed >100 biopsy samples from early-stage breast cancer and present our large collection of >70 organoid lines. We also show methods of assessing successful tumour cell culture in a time, and cost-efficient manner, proving a high rate (>85%) of normal cell overgrowth in early-stage breast cancer organoids. Finally, we show a number of successful attempts to culture cancer organoids from mastectomy-derived tissue of advanced, metastatic breast cancer. We conclude that the usefulness of organoids from early breast cancer for translational research and personalised medicine, especially guidance of adjuvant or post-surgical maintenance therapy, is strongly limited by the low success rate of culturing cancerous cells under organoid conditions.

Docetaxel resistance in gastric cancer poses a major therapeutic challenge. In this study, we established docetaxel-sensitive and -resistant gastric cancer organoids and performed single-cell RNA sequencing to identify cellular and molecular alterations. We observed significant shifts in cell populations, with increased secretory, immune-chemotactic, and transitional gastric cancer cells in the resistant group. Key resistance-related genes, including FOS, IFI27, and PTTG1IP, were upregulated in resistant organoids and gastric cancer patients. A pseudo-time trajectory analysis revealed that resistant cells predominantly occupied terminal differentiation stages. Knocking down FOS, IFI27, and PTTG1IP enhanced docetaxel sensitivity in both cell lines and organoids, regulating ROS production, autophagy, and apoptosis. In vivo, silencing these genes reduced tumor growth in response to docetaxel. These findings suggest that targeting FOS, IFI27, and PTTG1IP could overcome resistance and improve treatment outcomes for gastric cancer patients.

The presence of cancer stem cells (CSCs) is one of the primary causes of recurring therapy resistance because they have two main capacities: self-renewal and avoiding apoptotic pathways. Despite their relevance, no full bibliometric analysis has yet been done in this topic. The goal of this work is to use bibliometric analysis to map the fundamental and emergent areas in therapeutics targeting colon cancer stem cells. To perform bibliometric analysis on colon cancer stem cells (CCSCs) literature, spanning roughly the last 40 years, in order to establish a firm base for future projections by emphasizing the findings of the most notable research. All information pertinent to CCSCs was accessed from Web of Science Core Collection database. In order to identify and analyze the research hotspots and trends related to this topic, Biblioshiny (RStudio) and VOSviewer were utilized to ascertain the countries/regions, institutions, journals, authors, references, and keywords involved. The targeted time span covered 1735 research-, and review articles. The most frequent keywords were "colorectal cancer," "cancer stem cells," and "colon cancer," while the most trending keywords in the last few years were "protein stability," "spheroid formation," "ubiquitination," "exosomes," "patient-derived organoids," and "gut microbiota." Over the past 40 years, there has been a significant advancement in researchers' understanding of colon cancer stem cells. In addition, the cluster map of co-cited literature showed that colon cancer stem cell research has emerged as a research hotspot. It was also anticipated that the main focus of the future efforts appears to involve clinical applications of cell-targeted colon cancer therapy. These results provide researchers with a comprehensive understanding of this field and provide insightful ideas for further research.

The combination of gemcitabine and cisplatin serves as a cornerstone in bladder cancer (BC) treatment, yet chemotherapy resistance continues to pose a significant challenge. This study utilizes a novel BC organoid model integrated with drug sensitivity assays to uncover the mechanisms underlying resistance and identify potential therapeutic targets. Our findings reveal that AP1M2 expression is markedly upregulated in gemcitabine- and cisplatin-resistant BC cells and tissues. Elevated AP1M2 levels contribute to enhanced chemotherapy resistance and tumor cell proliferation by facilitating the DNA damage response and increasing RAD54B expression. Mechanistically, AP1M2 interacts with the RNA-binding protein PUM1 to stabilize RAD54B mRNA, thereby supporting DNA repair and survival under chemotherapeutic stress. Notably, inhibition of AP1M2/PUM1-mediated RAD54B expression sensitized BC xenografts to gemcitabine-cisplatin treatment in vivo. These findings unveil a novel mechanism of chemotherapy resistance in BC and highlight the AP1M2/PUM1/RAD54B pathway as a promising therapeutic target to counter resistance and enhance treatment outcomes.

Anaplastic thyroid cancer (ATC) is a highly malignant and lethal tumor with poor prognosis, but there is a lack of effective treatment strategies. In our study, we screened a drug library and identified that TRx0237, a tau protein inhibitor, showed inhibitory effect on ATC cells. Further research demonstrated that the inhibitory effect of TRx0237 was mainly through the induction of apoptosis via reactive oxygen species (ROS)-mediated endoplasmic reticulum stress pathway. Meanwhile, the pro-apoptosis effect and mechanism of TRx0237 on ATC were verified in xenograft and ATC patient-derived organoids. In addition, TRx0237 significantly upregulated the expression of PD-L1 in ATC, and synergistically enhanced the effect of anti-PD-1 therapy in xenograft and organoids model. Therefore, our study suggests that TRx0237 showed anticancer effects by inducing apoptosis and improving the efficacy of anti-PD-1 immunotherapy. TRx0237 is a potential agent for the treatment of ATC.

Many cancer therapies fail in clinical trials despite showing potent efficacy in preclinical studies. One of the key reasons is the adopted preclinical models cannot recapitulate the complex tumor microenvironment (TME) and reflect the heterogeneity and patient specificity in human cancer. Cancer-on-a-chip (CoC) microphysiological systems can closely mimic the complex anatomical features and microenvironment interactions in an actual tumor, enabling more accurate disease modeling and therapy testing. This review article concisely summarizes and highlights the state-of-the-art progresses in CoC development for modeling critical TME compartments including the tumor vasculature, stromal and immune niche, as well as its applications in therapying screening. Current dilemma in cancer therapy development demonstrates that future preclinical models should reflect patient specific pathophysiology and heterogeneity with high accuracy and enable high-throughput screening for anticancer drug discovery and development. Therefore, CoC should be evolved as well. We explore future directions and discuss the pathway to develop the next generation of CoC models for precision cancer medicine, such as patient-derived chip, organoids-on-a-chip, and multi-organs-on-a-chip with high fidelity. We also discuss how the integration of sensors and microenvironmental control modules can provide a more comprehensive investigation of disease mechanisms and therapies. Next, we outline the roadmap of future standardization and translation of CoC technology toward real-world applications in pharmaceutical development and clinical settings for precision cancer medicine and the practical challenges and ethical concerns. Finally, we overview how applying advanced artificial intelligence tools and computational models could exploit CoC-derived data and augment the analytical ability of CoC.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers. Surgical resection combined with appropriate chemotherapy currently offers the best chance for long-term survival and potential cure. However, effective treatment is hindered by the limited chemotherapy options and the absence of reliable clinical tools to guide chemotherapy selection. Patient-derived organoids (PDOs) have emerged as a promising technology with the potential in precision medicine for PDAC. This review provides an overview of pancreatic organoid genesis, explores the role of PDOs in elucidating PDAC biology within clinically relevant contexts, and concludes by examining current literature on the utility of PDOs as biomarkers for personalized treatment strategies.

In the nearly 50 years since the original models of cancer first hit the stage, mouse models have become a major contributor to virtually all aspects of cancer research, and these have evolved well beyond simple transgenic or xenograft models to encompass a wide range of more complex models. As the sophistication of mouse models has increased, an explosion of new technologies has expanded the potential to both further develop and apply these models to address major challenges in cancer research. In the current era, cancer modeling has expanded to include nongermline genetically engineered mouse models (GEMMs), patient-derived models, organoids, and adaptations of the models better suited for cancer immunology research. New technologies that have transformed the field include the application of CRISPR-Cas9-mediated genome editing, in vivo imaging, and single-cell analysis to cancer modeling. Here, we provide a historical perspective on the evolution of mouse models of cancer, focusing on how far we have come in a relatively short time and how new technologies will shape the future development of mouse models of cancer.

Neoadjuvant chemotherapy (NAC) can improve the survival outcomes of patients with pancreatic cancer, but for borderline resectable pancreatic cancer (BRPC) the proportion of conversion to surgery remains unsatisfactory. This single-arm pilot study aimed to assess the clinical efficacy and safety of NAC based on patient-derived organoids (PDOs) for BRPC.

Biopsy samples from BRPC patients were collected for generating PDOs. Gemcitabine plus nab-paclitaxel as NAC was initially administrated for one cycle, and then the treatment regimen was adjusted based on the PDO drug sensitivity testing. The primary endpoint was the objective response rate (ORR). Secondary endpoints included R0 resection rate, NAC-related adverse events (AEs), and postoperative complications. Exploratory objectives were to assess the chemoresistance to gemcitabine.

Totally 19 of 25 patients were eligible for the study, among whom 16 achieved partial response and received surgical resection, with the ORR of 84.2% (16/19). The R0 resection rate was 81.3% (13/16). During NAC, 8 (42.1%, 8/19) patients experienced different grades of AEs, mainly including grade 2 myelosuppression (26.3%), cutaneous pruritus (5.3%), and diarrhea (5.3%). scRNA-seq analysis of duct cells showed that the transcriptome in aneuploid cells may affect gemcitabine resistance via multiple pathways, among which upregulation of drug-resistant genes ( OLFM4, AGR2, MUC5AC, MUC1, HMGA1, REG4, IL17RB, GCNT3, AKR1B10, ITGA6, HMGCS2 , and SQLE ) and downregulation of sensitive genes ( SIK1, HEXIM1, SPINT2, GADD45 , and TIMP2 ) played crucial roles. Changes in the interactions between cancer cells and other cell groups may also involve in gemcitabine resistance.

PDO-based NAC shows a promising resectable rate in BRPC patients, with good tolerance. Potential drug-resistant and sensitive genes and cell-cell interaction changes may participate in the development of gemcitabine resistance.

Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5-year survival (∼13%). Thus, new, effective, and ideally, less toxic therapies are desperately needed. Epidemiologic studies have found that patients with PDAC prescribed H1-antihistamines have improved survival. Expression of the histamine H1 receptor (HRH1), a G protein-coupled receptor which is blocked by approved H1-antihistamines, is increased by ∼20-fold in PDAC tumors compared with normal pancreas. Here, we used bioinformatic and molecular biological techniques to identify the cellular localization of HRH1 in the PDAC tumor microenvironment, assess functional responses to HRH1 activation, and define its potential biological roles in PDAC. We found that HRH1 is primarily expressed in cancer cells of PDAC tumors in humans and KPC mice (mice engineered to develop PDAC) and signals via G protein q/11 to increase intracellular Ca2+. HRH1 activation increases migration and invasion by PDAC cancer cells. Orally administered fexofenadine, an H1-antihistamine, was bioavailable in the tumors of KPC mice and yielded smaller pancreatic tumor tissue weights and lower expression of immunomodulatory (interleukin 6 and PD-1) and fibrotic (Col1A1) genes than in vehicle-control KPC mice. Thus, PDAC cancer cells express HRH1, which is functional in vitro and in vivo, suggesting that the repurposing of approved H1-antihistamines may be an efficacious and safe therapeutic approach for patients with PDAC. SIGNIFICANCE STATEMENT: Pancreatic ductal adenocarcinoma (PDAC) has a ∼13% 5-year survival rate, highlighting the need for new therapies. The HRH1 (histamine) receptor, associated with poorer survival, is upregulated in PDAC tumors. This study found that HRH1 is functional in PDAC cells, increasing intracellular Ca2+ via Gq/11 and promoting tumorigenic responses. KPC mice treated with an H1-antihistamine have reduced pancreas weight and lower proinflammatory and fibrotic markers in PDAC tumors. Thus, HRH1 may be a potential target for repurposing approved H1-antihistamines to treat PDAC.

Bladder cancer is one of the most common malignancies of the urinary system and there's a significant unmet need for new effective therapeutics for bladder cancer. The limited number of available models to study malignant bladder tumors is one of the obstructions in developing bladder cancer therapeutics. Patient-derived xenograft (PDX) and organoid (PDO) models are more representatives of human cancer than cell lines and cell line-derived xenograft (CDX) and are likely to be more promising and efficient in predicting drug response and finding new therapeutics.

Three pairs of patient-derived xenograft (PDX) models of bladder cancer and their corresponding PDX-derived organoids (PDXOs) were successfully established. These models were utilized to assess the efficacy of abemaciclib. The sensitivity of the drug was determined through the Cell Counting Kit-8 (CCK8) assay in PDXO cultures, corroborated by the EdU incorporation assay. Additionally, the in vivo tumor growth was monitored in the matched PDX models.

In vitro PDXO cultures and in vivo PDX tumor models consistently demonstrated that abemaciclib had varying degrees of inhibitory effects across different bladder cancer (BC) patients. Notably, our study further revealed that treatment with abemaciclib significantly modified the expression patterns of CyclinD1/CDK4. This was achieved by not only diminishing their expression levels but also by shifting their expression from a membrane-associated localization to the nucleus.

Our research provided compelling evidence attesting to the reliability and potential of PDX and PDXO models in the realm of precision medicine. These models are instrumental in identifying patients who are likely to respond favorably to a specific drug treatment.

Pancreatic cancer (PC) is a major global health challenge, with rising incidence and mortality rates, particularly in high-socio-demographic index regions. Given its high mortality and significant morbidity, research on patient quality of life (QoL) has gained momentum, addressing symptom burdens, psychological distress, and treatment-related outcomes. Bibliometric analysis provides a valuable approach to mapping research trends, identifying key contributors, and forecasting future directions.

This study aimed to map global research on QoL in pancreatic cancer patients, highlighting key findings, challenges, and future directions through bibliometric analysis over the past two decades.

Data for this study were collected from the Web of Science Core Collection (WoSCC) database, using specific search strategies to retrieve relevant documents on the quality of life in pancreatic cancer patients. The data were analysed using the Bibliometrix R package to create knowledge maps and visualize research trends, collaborations, and emerging hotspots in the field.

A total of 819 articles on pancreatic cancer and quality of life were identified, with an average citation count of 47.13 per article, highlighting moderate academic impact. The research revealed a growing trend in collaborative efforts, with an average of 9.42 co-authors per article and 16 % international collaborations. The United States emerged as the leading contributor, with 203 publications and the highest citation count, followed by France and the United Kingdom.

This bibliometric analysis highlights the growing volume of pancreatic cancer and quality of life research, with a steady annual growth rate of 6.9 % and increasing collaboration, especially from the United States. However, despite the rising number of publications, a decline in citation impact for recent studies suggests a need for continued innovation in therapeutic strategies to improve clinical outcomes.

Pancreatic ductal adenocarcinoma (PDAC) is among the cancer types with poorest prognosis and survival rates primarily due to resistance to standard-of-care therapies, including gemcitabine (GEM) and olaparib. Particularly, wild-type (wt)BRCA tumours, the most prevalent in PDAC, are more resistant to DNA-targeting agents like olaparib, restraining their clinical application. Recently, we disclosed a monoterpene indole alkaloid derivative (BBIT20) as a new inhibitor of homologous recombination (HR) DNA repair with anticancer activity in breast and ovarian cancer. Since inhibition of DNA repair enhances the sensitivity of cancer cells to chemotherapy, we aimed to investigate the anticancer potential of BBIT20 against PDAC, particularly carrying wtBRCA.

In vitro and in vivo PDAC models, particularly human cell lines (including GEM-resistant PDAC cells), patient-derived organoids and xenograft mice of PDAC were used to evaluate the anticancer potential of BBIT20, alone and in combination with GEM or olaparib. Disruption of the BRCA1-BARD1 interaction by BBIT20 was assessed by co-immunoprecipitation, immunofluorescence and yeast two-hybrid assay.

The potent antiproliferative activity of BBIT20, superior to olaparib, was demonstrated in PDAC cells regardless of BRCA status, by inducing cell cycle arrest, apoptosis, and DNA damage, while downregulating HR. The disruption of DNA double-strand breaks repair by BBIT20 was further reinforced by non-homologous end joining (NHEJ) suppression. The inhibition of BRCA1-BARD1 heterodimer by BBIT20 was demonstrated in PDAC cells and confirmed in a yeast two-hybrid assay. In GEM-resistant PDAC cells, BBIT20 showed potent antiproliferative, anti-migratory and anti-invasive activity, overcoming GEM resistance by inhibiting the multidrug resistance P-glycoprotein, upregulating the intracellular GEM-transporter ENT1, and downregulating GEM resistance-related microRNA-20a and GEM metabolism enzymes as RRM1/2. Furthermore, BBIT20 did not induce resistance in PDAC cells. It inhibited the growth of patient-derived PDAC organoids, by inducing apoptosis, repressing HR, and potentiating olaparib and GEM cytotoxicity. The enhancement of olaparib antitumor activity by BBIT20 was confirmed in xenograft mice of PDAC. Notably, it hindered tumour growth and liver metastasis formation, improving survival of orthotopic xenograft mice of PDAC. Furthermore, its potential as a stroma-targeting agent, reducing fibrotic extracellular matrix and overcoming desmoplasia, associated with an enhancement of immune cell response by depleting PD-L1 expression in tumour tissues, renders BBIT20 even more appealing for combination therapy, particularly with immunotherapy.

These findings underscore the great potential of BBIT20 as a novel multifaceted anticancer drug candidate for PDAC treatment.

Culture platforms that closely mimic the spatial architecture, cellular diversity, and extracellular matrix composition of native tissues can serve as invaluable tools for a range of scientific discovery and biomedical applications. Organoids have emerged as a promising alternative to both traditional 2D cell culture and animal models, offering a physiologically relevant 3D culture system for studying human cell biology. Organoids provide a manipulable platform to investigate organ development and function as well as to model patient-specific phenotypes. This mini review examines various methods used for culturing organoids to model normal and disease conditions in gastrointestinal tissues. We focus on how the matrix composition and media formulations can impact cell signaling, altering the baseline cellular phenotypes as well as response to perturbations. We discuss future directions for optimizing organoid culture conditions to improve basic and translational potential.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. Inhibition of the KRAS-RAF-MEK-ERK pathway enhances autophagic flux and dependency, and concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK-MAPK pathway inhibitors can synergistically block PDAC growth. However, CQ is limited in terms of specificity and potency. To find alternative anti-autophagy strategies, in this study, we performed a CRISPR-Cas9 loss-of-function screen in PDAC cell lines that identified the lipid kinase phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) as a growth-promoting gene. PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition caused lysosomal dysfunction, reduced autophagic flux, and led to the accumulation of autophagy-related proteins. Furthermore, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and the RAS-MAPK pathway showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell-cycle arrest and induction of apoptosis following loss of inhibitor of apoptosis proteins. These findings indicate that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a therapeutic strategy for PDAC. Significance: PIKfyve inhibition effectively blocks autophagy in multiple models of KRAS-mutant pancreatic cancer and can synergize with inhibitors of members of the RAS-MAPK pathway, providing an effective combination strategy for pancreatic cancer.

Oral cancer poses significant health risks with an increasing incidence annually. Despite advancements in treatment methods, their efficacy is frequently constrained by cancer heterogeneity and drug resistance, leading to minimal improvement in the 5-year survival rate. Therefore, there is a critical need for new treatment methods leaded by representative preclinical research models. Compared to other models, organoids can more precisely simulate the tissue structure, genetic characteristics, and tumor microenvironment (TME) of in vivo tumors, exhibiting high tumor specificity. This makes organoid technology a valuable tool in investigating tumor development, mechanisms of metastasis, drug screening, prediction of clinical responses, and personalized patient treatment. Moreover, integrating organoid technology with other biotechnologies could expand its applications in tissue regeneration. Although organoid technology is increasingly utilized in oral cancer research, a systematic review in this field is absent. This paper is to bridge the gap by reviewing the development and current status of organoid research, highlighting its applications, future prospects, and challenges in oral cancer. It aims to provide novel insights into the role of organoids in precision treatment and regenerative medicine for oral cancer.

Pancreatic ductal adenocarcinoma (PDAC) contains an extensive stroma that modulates response to therapy, contributing to the dismal prognosis associated with this cancer. Evidence suggests that PDAC stromal composition is shaped by mutations within malignant cells, but most previous work has focused on preclinical models driven by KrasG12D and mutant Trp53. Elucidation of the contribution of additional known oncogenic drivers, including KrasG12V mutation and Smad4 loss, is needed to increase the understanding of malignant cell-stromal cell cross-talk in PDAC. In this study, we used single-cell RNA sequencing to analyze the cellular landscape of Trp53-mutant mouse models driven by KrasG12D or KrasG12V, in which Smad4 was wild type or deleted. KrasG12DSmad4-deleted PDAC developed a fibro-inflammatory rich stroma with increased malignant JAK/STAT cell signaling and enhanced therapeutic response to JAK/STAT inhibition. SMAD4 loss in KrasG12V PDAC differently altered the tumor microenvironment compared with KrasG12D PDAC, and the malignant compartment lacked JAK/STAT signaling dependency. Thus, malignant cell genotype affects cancer cell and stromal cell phenotypes in PDAC, directly affecting therapeutic efficacy. Significance: SMAD4 loss differentially impacts malignant cell-stromal cell signaling and treatment sensitivity of pancreatic tumors driven by KRASG12D or KRASG12V, highlighting the importance of understanding genotype-phenotype relationships for precision therapy.

Longitudinal imaging of 3D cell cultures like tumor organoids and spheroids offers crucial insights into cancer progression and treatment. However, spatial displacement during time-course imaging, caused by matrix detachment or experimental artifacts, can confound analyses. Existing computational methods struggle to address this issue. We present a new algorithm to evaluate data integrity and rectify mislabeling in longitudinal imaging of 3D cell culture. Our algorithm integrates permutation-based optimization with Procrustes analysis. By using X and Y coordinates of images, it accurately reorders, matches, and aligns object positions across time points, correcting for rotation, translation, and small movements. Validation with simulated data confirmed its accuracy and robustness. Applied to longitudinal imaging of tumor spheroids, our algorithm revealed frequent displacement amongst the spheroids between time points and corrected many mislabeled images. This computationally efficient and adaptable method needs no experimental adjustments and presents a readily accessible solution for data quality control.

Three-dimensional (3D) in vitro models, such as tumor organoids and spheroids embedded in an extracellular matrix, are increasingly vital for studying normal and disease biology, including drug responses. 1-3 A key advantage of these models is that imaging platforms can perform continuous longitudinal imaging to track phenotypic changes. However, common issues in 3D techniques, such as matrix shifts during experimental setup or image capture, can introduce technical artifacts that affect downstream analyses. Currently, no automated analytical approaches exist for assessing or correcting technical artifacts. Here, we introduce a robust, automated algorithm for assessing the quality of time-course image data and, in some cases, correcting object mislabeling to enable accurate tracking of individual spheroids over time. This approach relies only on image metadata, requiring no experimental modifications. It offers a readily implementable solution for improving data integrity and reproducibility and enhancing the reliability of longitudinal 3D cell culture studies.

Tumoroids are cultures of patient-derived tumor cells, which are grown in 3D in the presence of an extracellular matrix extract and specific growth factors. Tumoroids can be generated from adult as well as pediatric cancers, including epithelial cancers, sarcomas, and brain cancers. Tumoroids retain multi-omic characteristics of their corresponding tumor and recapitulate interpatient and intratumor heterogeneity. Retrospective and prospective studies have demonstrated that tumoroids predict patient responses to anticancer therapies, making them a promising tool for precision oncology. However, several challenges remain before tumoroids can be fully integrated into clinical decision-making, including success rates of tumoroid establishment and turnaround times. This review discusses the current advances, challenges, and future directions of tumoroid-based models in cancer research and precision therapy.

Current clinical decision-making lacks reliable preclinical models to predict patient outcomes. Here, we establish patient-derived organoids (PDOs) as predictive tools in Crohn's disease (CD), a complex, heterogeneous disorder. Using a living biobank of adult stem cell-derived colonic-PDOs, we identified two molecular CD subtypes-Immune-Deficient Infectious CD ( IDICD ) and Stress and Senescence-Induced Fibrostenotic CD ( S2FCD )-each with distinct genomic, transcriptomic and functional profiles, along with paired therapeutics. By prospectively linking colonic PDO-derived phenotypes to real-world patient outcomes, we uncovered that while S2FCD associates with severe colonic disease, IDICD associates with severe ileal disease, prior ileocecal surgery, and future disease progression. This approach transforms PDOs from static descriptive models into dynamic tools that capture the past, present, and future of disease behavior and reveals their utility as patient-specific predictive platforms, extending their use beyond oncology to complex inflammatory diseases. Findings also suggest that colonic immune dysfunction may drive ileal-CD, independent of colonic involvement.

In this work, Penrose et al. demonstrate the potential of patient-derived organoids (PDOs) as predictive tools in Crohn's disease (CD) that capture the past, present, and future of disease behavior, thereby advancing PDO-informed precision medicine beyond oncology into complex inflammatory diseases.

A living PDO biobank identified two molecular CD subtypes with distinct functional phenotypes.PDO subtyping tracked severity of ileal disease, prior surgery and future disease progression.Colonic immune dysfunction may drive ileal-CD, independent of colonic involvement.Colonic CD-PDOs are dynamic platforms for outcome-deterministic therapeutic testing.

GATA6 expression is recognized as a favorable prognostic marker of pancreatic cancer, whereas TP53 is a poor prognostic marker. We evaluated treatment outcomes by genetic alterations in TP53 and GATA6 to determine the prognostic and predictive impact of co-alterations.

A single institution retrospective analysis was performed on patients diagnosed with pancreatic ductal adenocarcinoma between 2014 and 2023. TP53 genotype and GATA6 amplification status were included in an analysis of overall survival (OS) and progression-free survival (PFS). Previously published patient-derived organoids were used to investigate correlation between genetic status and drug sensitivity.

Patients with TP53 mutations had worse OS compared with the wild-type TP53 population. Patients with GATA6 amplification had better OS and a trend toward better PFS than the nonamplified population. Among patients with a TP53 mutation, patients with GATA6 co-alteration had longer OS compared with those who were not GATA6 amplified. In contrast, among patients who were TP53 wild-type, the presence or absence of a GATA6 amplification did not impact OS or PFS. GATA6 genotype was not associated chemotherapy drug response in an organoid pharmacotyping model.

We found that GATA6 amplification appeared to attenuate poor prognosis observed in TP53-mutant patients regardless of the type of standard chemotherapy received, suggesting the GATA6 amplification is a prognostic biomarker but not a predictive biomarker of standard-of-care chemotherapy.

Intratumour heterogeneity and phenotypic plasticity drive tumour progression and therapy resistance1,2. Oncogene dosage variation contributes to cell-state transitions and phenotypic heterogeneity3, thereby providing a substrate for somatic evolution. Nonetheless, the genetic mechanisms underlying phenotypic heterogeneity are still poorly understood. Here we show that extrachromosomal DNA (ecDNA) is a major source of high-level focal amplification in key oncogenes and a major contributor of MYC heterogeneity in pancreatic ductal adenocarcinoma (PDAC). We demonstrate that ecDNAs drive varying levels of MYC dosage, depending on their regulatory landscape, enabling cancer cells to rapidly and reversibly adapt to microenvironmental changes. In the absence of selective pressure, a high ecDNA copy number imposes a substantial fitness cost on PDAC cells. We also show that MYC dosage affects cell morphology and dependence of cancer cells on stromal niche factors. Our work provides a detailed analysis of ecDNAs in PDAC and describes a new genetic mechanism driving MYC heterogeneity in PDAC.

A new era of cancer management is underway in which treatments are being developed for the entire continuum of the disease process. The availability of genetically engineered and naturally occurring preclinical models serves as instructive platforms for evaluating therapeutic mechanisms. However, a major clinical challenge is that the entire malignancy process occurs across multiple scales including genetic mutations, malignant changes in cell behavior, dysregulated tumor microenvironments, and systemic adaptations in the host. A multidisciplinary group of investigators coalesced at the National Cancer Institute Oncology Models Forum with the overall goal to provide updates on the use of precision preclinical models of cancer. The benefits and limitations of preclinical models were discussed to identify strategies for maximizing opportunities in modeling that could inform future cancer prevention and treatment approaches. Our shared perspective is that the continuum of single cell, multicell, organoid, and in situ models are remarkable resources for the clinical challenges ahead. We provide a roadmap for parsing already available models and include preliminary recommendations for the application of next-generation preclinical modeling in cancer intervention.

Organoids represent a significant advancement in disease modeling, demonstrated by their capacity to mimic the physiological/pathological structure and functional characteristics of the native tissue. Recently CRISPR/Cas9 technology has emerged as a powerful tool in combination with organoids for the development of novel therapies in preclinical settings. This review explores the current literature on applications of pooled CRISPR screening in organoids and the emerging role of these models in understanding cancer. We highlight the evolution of genome-wide CRISPR gRNA library screens in organoids, noting their increasing adoption in the field over the past decade. Noteworthy studies utilizing these screens to investigate oncogenic vulnerabilities and developmental pathways in various organoid systems are discussed. Despite the promise organoids hold, challenges such as standardization, reproducibility, and the complexity of data interpretation remain. The review also addresses the ideas of assessing tumor organoids (tumoroids) against established cancer hallmarks and the potential of studying intercellular cooperation within these models. Ultimately, we propose that organoids, particularly when personalized for patient-specific applications, could revolutionize drug screening and therapeutic approaches, minimizing the reliance on traditional animal models and enhancing the precision of clinical interventions.

The cryopreservation of cancer tissues to generate frozen libraries is a common practice used worldwide for storing patient samples for later applications. However, frozen samples stored by existing methods cannot be used for initiating living cell cultures, such as patient-derived tumor organoids (PDOs), which offer great potential for personalized treatment. To overcome this challenge, we developed a novel procedure for culturing PDOs using frozen live tumor tissues. We show that tumor specimens stored using this technique maintain their viability and can be successfully used to generate organoids even after long-term freezing, with an impressive success rate of 95.2 %. Importantly, we found that the structural features, tumor marker expression, and drug responses of organoids derived from frozen tissues are similar to those derived from fresh tissues. Moreover, organoids derived from frozen tissues can be routinely passaged and frozen, making them ideal for high-throughput drug screening at any time. Notably, cryopreserved tumor tissues can also be utilized in air-liquid interface (ALI) culture. This method allows for preserving the original tumor microenvironment, making it an invaluable resource for conducting tests on antitumor drug responses, including immune checkpoint inhibitors (ICIs). This innovation has the potential to enable the identification of potentially effective drugs for patients and facilitate the development of novel therapeutic drugs. Thus, we have established protocols for the long-term cryopreservation of cancer tissues to maintain their viability and microenvironment, which are useful for personalized therapy.

SOX9 is widely regarded as a key master regulator of gene transcription, responsible for the development and differentiation programs within tissue and organogenesis, particularly in the pancreas. SOX9 overexpression has been observed in multiple tumor types, including pancreatic cancer, and is discussed as a prognostic marker. In order to gain a more profound understanding of the role of SOX9 in pancreatic cancer, we have performed SOX9 knockdown in the COLO357 and PANC-1 cells using RNA interference, followed by full-transcriptome analysis of the siRNA-transfected cells. The molecular pathway enrichment analysis between SOX9-specific siRNA-transfected cells and control cells reveals the activation of processes associated with cellular signaling, cell differentiation, transcription, and methylation, alongside the suppression of genes involved in various stages of the cell cycle and apoptosis, upon the SOX9 knockdown. Alterations of the expression of transcription factors, epithelial-mesenchymal transition markers, oncogenes, tumor suppressor genes, and drug resistance-related genes upon SOX9 knockdown in comparison of primary and metastatic pancreatic cancer cells are discovered. The expression levels of genes comprising prognostic signatures for pancreatic cancer were also evaluated following SOX9 knockdown. Additional studies are needed to assess the properties and prognostic significance of SOX9 in pancreatic cancer using other biological models.

Pancreatic ductal adenocarcinoma (PDAC) tumor heterogeneity impedes the development of biomarker assays for early disease detection. We hypothesized that PDAC cell subpopulations could be identified by aberrant glycan signatures in both tumor tissue and blood samples. We used multiplexed glycan immunofluorescence to distinguish between PDAC and noncancer cell subpopulations within tumor tissue, and we developed hybrid glycan sandwich assays to determine whether the aberrant glycan signatures could be detected in blood samples. We found that PDAC cells were identified by signatures of glycans detected by four glycan-binding proteins (VVL, CA19-9, sTRA, and GM2) and that there are three types of glycan-defined PDAC tumors: sTRA type, CA19-9 type, and intermixed. In patient-matched tumor and blood samples, the PDAC tumor type could be determined by the aberrant glycans in the blood. As a result, the combined assays of aberrant glycan signatures were more sensitive and specific than any individual assay. Our results demonstrate a methodology to detect and stratify PDAC.

Gemcitabine combined with albumin-paclitaxel (AG) is a crucial therapeutic option for pancreatic ductal adenocarcinoma (PDAC). However, the response to chemotherapy is relatively poor, with rapid development of resistance. The aim of this study was to explore the mechanism of resistance to AG and to develop strategies that can sensitize the AG regimen.

We used organoid models, patient-derived xenografts, and genetically engineered mouse models in our study. Chromatin immunoprecipitation, double luciferase assay, co-immunoprecipitation, and far-western blotting analysis were performed to investigate the mechanism. The AVL9 inhibitors were identified through protein structure analysis and molecular docking analysis, and their efficacy was verified in patient-derived xenografts, patient-derived organoids-based xenograft, and KPC models.

Through multistrategy screening, we identified AVL9 as a key target for AG resistance in PDAC. Its tumor-promoting effects were confirmed in our clinical cohorts. Mechanistically, HIF-1α, a hypoxia-related transcription factor, drives the expression of AVL9. AVL9 acts as a scaffold that facilitates the binding of IκBα to SKP1, leading to enhanced ubiquitination and degradation of IκBα, which further activates the nuclear factor-κB pathway. The potential AVL9-targeting inhibitor, Edotecarin, was shown to reverse AG chemo-resistance in PDAC.

AVL9 expression is driven by HIF-1α in PDAC. The physical interaction of AVL9, IκBα, and SKP1 provides a novel molecular mechanism for the abnormal activation of the nuclear factor-κB pathway. Therefore, the AVL9-targeting drug Edotecarin could be a promising therapeutic strategy for sensitizing PDAC to AG.

Liver cancer poses a global health challenge with limited therapeutic options. Notably, the limited success of current therapies in patients with primary liver cancers (PLCs) may be attributed to the high heterogeneity of both hepatocellular carcinoma (HCCs) and intrahepatic cholangiocarcinoma (iCCAs). This heterogeneity evolves over time as tumor-initiating stem cells, or cancer stem cells (CSCs), undergo (epi)genetic alterations or encounter microenvironmental changes within the tumor microenvironment. These modifications enable CSCs to exhibit plasticity, differentiating into various resistant tumor cell types. Addressing this challenge requires urgent efforts to develop personalized treatments guided by biomarkers, with a specific focus on targeting CSCs. The lack of effective precision treatments for PLCs is partly due to the scarcity of ex vivo preclinical models that accurately capture the complexity of CSC-related tumors and can predict therapeutic responses. Fortunately, recent advancements in the establishment of patient-derived liver cancer cell lines and organoids have opened new avenues for precision medicine research. Notably, patient-derived organoid (PDO) cultures have demonstrated self-assembly and self-renewal capabilities, retaining essential characteristics of their respective in vivo tissues, including both inter- and intratumoral heterogeneities. The emergence of PDOs derived from PLCs serves as patient avatars, enabling preclinical investigations for patient stratification, screening of anticancer drugs, efficacy testing, and thereby advancing the field of precision medicine. This review offers a comprehensive summary of the advancements in constructing PLC-derived PDO models. Emphasis is placed on the role of CSCs, which not only contribute significantly to the establishment of PDO cultures but also faithfully capture tumor heterogeneity and the ensuing development of therapy resistance. The exploration of PDOs' benefits in personalized medicine research is undertaken, including a discussion of their limitations, particularly in terms of culture conditions, reproducibility, and scalability.

Matrigel is the most commonly used matrix for 3D organoid cultures. Research on the biomaterial basis of Matrigel for organoid cultures is a highly challenging field. Currently, many studies focus on Matrigel-based biological macromolecules or combinations to construct natural Matrigel and synthetic hydrogel scaffolds based on collagen, peptides, polysaccharides, microbial transglutaminase, DNA supramolecules, and polymers for organoid culture. In this review, we discuss the limitations of both natural and synthetic Matrigel, and describe alternative scaffolds that have been employed for organoid cultures. The patient-derived organoids were constructed in different cancer types and limitations of animal-derived organoids based on the hydrogel or Matrigel. The constructed techniques utilizing 3D bioprinting platforms, air-liquid interface (ALI) culture, microfluidic culture, and organ-on-a-chip platform are summarized. Given the potential of organoids for a wide range of therapeutic, tissue engineering and pharmaceutical applications, it is indeed imperative to develop defined and customized hydrogels in addition to Matrigel.

Patient-derived organoids represent a novel platform to recapitulate the cancer cells in the patient tissue. While cancer heterogeneity has been extensively studied by a number of omics approaches, little is known about the spatiotemporal kinase activity dynamics. Here we applied a live imaging approach to organoids derived from 10 pancreatic ductal adenocarcinoma (PDAC) patients to comprehensively understand their heterogeneous growth potential and drug responses. By automated wide-area image acquisitions and analyses, the PDAC cells were non-selectively observed to evaluate their heterogeneous growth patterns. We monitored single-cell ERK and AMPK activities to relate cellular dynamics to molecular dynamics. Furthermore, we evaluated two anti-cancer drugs, a MEK inhibitor, PD0325901, and an autophagy inhibitor, hydroxychloroquine (HCQ), by our analysis platform. Our analyses revealed a phase-dependent regulation of PDAC organoid growth, where ERK activity is necessary for the early phase and AMPK activity is necessary for the late stage of organoid growth. Consistently, we found PD0325901 and HCQ target distinct organoid populations, revealing their combination is widely effective to the heterogeneous cancer cell population in a range of PDAC patient-derived organoid lines. Together, our live imaging quantitatively characterized the growth and drug sensitivity of human PDAC organoids at multiple levels: in single cells, single organoids, and individual patients. This study will pave the way for understanding the cancer heterogeneity and promote the development of new drugs that eradicate intractable cancer.