Anti-EGFR liposomal drug delivery system loaded with AGY2 potentiates the anti-cancer effect of AGY2 against EGFR expressed pancreatic cancer.

Thermoreversible cell-derived extracellular matrix only hydrogel (CEOgel): Development, characterization, and applications.

Reliable evaluation of chemotherapy efficacy is essential for optimizing treatment strategies in ovarian cancer. Organoids provide a physiologically relevant model to capture tumor heterogeneity, yet practical tools for functional assessment remain limited. Here, we report a wash-free near-infrared β-galactosidase-activated fluorescent probe Hcy-SO3-β-Gal for noninvasive imaging of ovarian cancer organoids. The probe remains quenched in its native form and is rapidly activated upon enzymatic cleavage by β-galactosidase, an enzyme associated with therapy-induced senescence and stress responses in cancer cells. The wash-free property enables real-time imaging with minimal background interference, facilitating dynamic monitoring of cellular responses to chemotherapy. Applied to organoid cultures, the probe provided sensitive readouts that correlated strongly with standard viability assays, while offering advantages of operational simplicity and high-throughput compatibility. This work introduces a practical imaging platform that integrates molecular specificity with translational potential, supporting precision evaluation of chemotherapy efficacy and informing personalized therapeutic strategies.

5-Fluorouracil (5-FU) is a first-line chemotherapy commonly used to treat colorectal cancer (CRC). However, the development of acquired resistance to 5-FU remains a significant clinical challenge, and the underlying epigenetic mechanisms are not fully understood. In this study, we demonstrate that euchromatic histone lysine methyltransferase 2 (EHMT2) is significantly upregulated in CRC patients with poor responses to 5-FU, directly correlating with lower overall survival rates. Using established 5-FU resistant (5-FUR) HCT116 and HT29 cell lines, RNA-sequencing confirmed robust EHMT2 overexpression compared with wild-type cells. Mechanistically, siRNA-mediated knockdown of EHMT2 restored 5-FU sensitivity by upregulating protein phosphatase 1B (PPM1B), a key downstream target. This EHMT2-PPM1B axis disruption effectively induced G1 phase cell cycle arrest and triggered apoptosis in 5-FUR cells, fundamentally impairing their proliferation. Furthermore, we validated the therapeutic potential of targeting this pathway using in vivo and ex vivo models. Combination treatment with 5-FU and the specific pharmacological EHMT2 inhibitor (BIX-01294) synergistically suppressed tumor growth in a 5-FUR cell-derived xenograft mouse model. Importantly, these therapeutic effects were faithfully recapitulated in 5-FUR patient-derived colorectal cancer organoid (PDO) models. Together, our findings elucidate a critical epigenetic mechanism where EHMT2 promotes 5-FU drug resistance. Targeting EHMT2 represents a promising and translatable therapeutic strategy for overcoming chemoresistance and improving clinical outcomes in CRC patients.

A novel chalcone analog inhibits cancer stemness in CD44+ and CD133+ populations via suppression of ERK signaling.

Lineage plasticity and tumor heterogeneity limit the effectiveness of targeted therapies, yet the functional dependencies used to nominate therapeutic targets are often derived from homogeneous systems that fail to capture this complexity. Here, we establish a framework to resolve state-specific genetic vulnerabilities by integrating single-cell multiomics (RNA and ATAC) with pooled CRISPR-Cas9 screening across a large panel of patient-derived organoids (PDOs) from castrate-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC). We generate a single-cell multiome atlas spanning >190,000 cells across 22 PDOs, defining seven lineage states-including intermediate and plastic populations not resolved by bulk profiling-and demonstrate that these lineage programs robustly classify independent transcriptomic datasets from prostate cancer patient tumors. By systematically coupling this atlas to subtype-resolved CRISPR screens, we construct a functional dependency map linking cell state in heterogeneous 3D human tumor models. We show that intratumoral heterogeneity fundamentally reshapes the interpretation of gene essentiality, whereby gene-level depletion reflects the composite behavior of co-existing subpopulations, and identify a general principle in which resistant "limiting" populations disproportionately determine aggregate fitness effects. This framework reveals both canonical and previously unrecognized lineage-restricted dependencies within highly plastic tumor and NEPC states, including a therapeutically targetable dependency on the aryl hydrocarbon receptor (AHR) in a novel hybrid stem-like/ASCL1 population. Together, these data establish an extensive multi-dimensional prostate cancer resource, identify novel lineage-resolved biology, and provide a generalizable strategy for interpreting functional genomics in heterogeneous human tumors.

Basic research on castration-resistant prostate cancer (CRPC) is limited by the lack of clinically relevant models. This study aimed to establish patient-derived xenografts (PDX) and PDX-derived organoids from clinical CRPC specimens to develop a bidirectional experimental platform for in vivo xenografts and ex vivo organoids. We established a new PDX library (KUCaP PDX series) using CRPC clinical specimens and derived prostate cancer organoids. Comprehensive biological characterization of clinical specimens, PDXs, PDX-derived organoids, and organoid-derived xenografts (ODXs) was performed to confirm the preservation of the original tumor features. Using our PDX library, we conducted genetic engineering and drug testing to explore novel therapeutic approaches. PDX-derived organoids were successfully established from all eight KUCaP PDX lines (100%). Four of the eight lines (50%) were maintained during the long-term culture experiments for over ten passages. Key features observed in the original clinical specimens, including genetic alterations and castration responsiveness, were maintained across the PDX, PDX-derived organoid, and ODX models. RNA sequencing revealed that transcriptomic profiles were consistently maintained across clinical specimens, PDXs, PDX-derived organoids, and ODXs. One PDX and organoid (KUCaP19) which exhibited a high homologous recombination deficiency (HRD) score, without any pathogenic homologous recombination repair (HRR) gene alterations, showed sensitivity to a poly ADP-ribose polymerase (PARP) inhibitor. In contrast, KUCaP12, which had no HRR alterations and a low HRD score, did not respond to PARP inhibition. We developed the KUCaP library as a novel experimental platform for CRPC research by integrating clinical specimens with PDX, organoid, and ODX models along with their genomic and transcriptomic data. These models largely retained the genetic profiles and responses to castration observed in the original tumors. The bidirectional use of personalized PDX and organoids will facilitate the elucidation of the molecular mechanisms of CRPC.

Patient-derived ovarian cancer organoids as platforms for predicting platinum resistance and screening tumor stem cell inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis and rising incidence. Late detection and limited responsiveness to standard treatment translates into a 5-year overall survival of less than 12%. The pathology contributes to a desmoplastic tumor microenvironment that creates a physical barrier, leading to a dense, hypoxic environment that promotes further tumorigenesis, limited immunogenicity, and chemoresistance, resulting in a still significant translational gap in PDAC research. Feasible techniques to further elucidate tumorigenesis are indispensable because of the frequently limited predictive value of current preclinical models. PDAC organoids offer a powerful tool that can be rapidly generated from resected tumors and biopsies. This review summarizes the current technical and scientific knowledge and highlights the importance of the tumor microenvironment, the use of realistic oxygen conditions, and the role of the hypoxia-inducible factors. Additionally, various protocols based on different media and scaffolds are displayed, and it is illustrated how PDAC organoids can help to improve both diagnosis and treatment options. Finally, critical bottlenecks in modeling PDAC tumor-stromal interactions are identified, and integrated co-culture platforms are proposed as a promising solution for translational applications.

Obesity exacerbates breast cancer metastasis, yet the underlying mechanisms remain incompletely understood. Here, we identify neuregulin 4 (NRG4), a ligand of Erb-B2 receptor tyrosine kinase 4 (ERBB4), as a key regulator of metastasis, through the ERBB4-YAP1 signaling axis. Using MMTV-PyMT and 4T1 breast cancer models, we demonstrate that obesity accelerates metastasis, while NRG4, secreted by inguinal white adipose tissue (iWAT), inhibits cancer cell migration and epithelial-mesenchymal transition (EMT). Mechanistically, NRG4 activates ERBB4, producing a cleaved pERBB4 fragment that interacts with phosphorylated YAP1 (pYAP1), restricting its nuclear translocation. RNA sequencing revealed that NRG4 suppressed the transcription of Mmp9 and Mmp12, which encode matrix metalloproteinases critical for extracellular matrix remodeling and invasion. Co- immunoprecipitation and promoter assay confirmed that YAP1 bound to TEAD1 and activated MMP9/MMP12 transcription in the absence of NRG4. Importantly, recombinant NRG4 (rNRG4) reduced the growth and invasiveness of breast cancer organoids. These findings establish NRG4 as a metastasis suppressor in obesity-associated breast cancer by inhibiting the ERBB4-YAP1 pathway and down-regulating matrix metalloproteinases. Our study highlights the therapeutic potential of targeting NRG4-ERBB4 signaling to mitigate obesity-driven breast cancer progression.

Longitudinal prediction of drug response in high-grade serous ovarian cancer organoid cultures aligning with clinical responses.

Patient-Derived Lung Cancer Organoids for Precision Oncology: A Comprehensive Analysis.

Macrophages are abundant in the colorectal tumour microenvironment and can alter drug response. Using mouse Apc / Kras / Trp53 ( AKP ) colorectal cancer organoids, we found that macrophages and/or macrophage-conditioned medium reduced sensitivity to the MEK inhibitor trametinib and the pan-RAS inhibitor RMC-6236. In contrast, macrophage-conditioned medium had little effect on regorafenib and increased sensitivity to dabrafenib, suggesting that resistance depends on the inhibitory profile of each drug. Secretome profiling identified PDGF-BB and GDF-15 as candidate mediators. Adding both ligands to organoid medium reproduced much of the conditioned-medium effect, whereas either ligand alone was insufficient. Inhibition of PDGFR or RET partially reduced drug resistance, suggesting that PDGF-BB and GDF-15 likely act through canonical signalling by that additional macrophage-derived signals also contribute. Kinome profiling pointed to increased tyrosine kinase signalling during trametinib treatment, with SRC family kinases emerging as a key downstream node. Consistent with this, SRC inhibition reduced the difference between control and conditioned-medium responses. The multi-kinase inhibitor masitinib-which targets several kinases along this resistance network-strongly restored sensitivity to trametinib and RMC-6236. Together, these data define a macrophage-driven resistance network in KRAS-mutant colorectal cancer organoids and support combined inhibition of RAS-pathway and tyrosine kinase signalling.

Dual inhibition of PKMYT1 and WEE1, key G2/M checkpoint kinases that phosphorylate CDK1 at T14 and Y15, offers a strategy for tumors with abrogated G1/S checkpoint and high replication stress. Building on our prior PKMYT1 chemotype, we designed 1,7-naphthyridinone derivatives by displacing crystallographic water (core 5'-N-Asp251) and adding a 7'-ring nitrogen to retain physicochemical properties. 5'-Site structure fine-tuning enhanced WEE1 engagement while preserving the PKMYT1-preferred hinge flip and superior kinome selectivity. Optimization identified compound 24 with single-digit nM PKMYT1 NanoBRET and sub-μM WEE1 NanoBRET potency, translating to pCDK1 T14 IC50 4.9 nM and pCDK1 Y15 0.186 μM in HCC1569 cells. Kinome profiling confirmed favorable selectivity. In colorectal cancer organoids, 24 outperformed our prior PKMYT1 inhibitor (6), RP-6306, and WEE1 inhibitor AZD1775, with efficacy correlating to improved WEE1 activity. Compound 24 also showed favorable in vitro ADME and early safety profiles, supporting dual checkpoint targeting in checkpoint-deficient cancers.

Hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR + /HER2 - ) breast cancer, the most common subtype, shows a low pathological complete response (pCR) rate and limited benefit from immunotherapy, highlighting the need for more effective strategies. Although immunotherapy has become increasingly important in cancer treatment, its efficacy in this subtype remains modest. CDK4/6 inhibitors, first-line treatments for advanced HR + /HER2- breast cancer, not only suppress tumor proliferation but may also reshape the immune microenvironment, offering new opportunities for immunotherapy. In this study, multiplex immunohistochemistry, drug testing of HR + /HER2- breast cancer organoids, single-cell sequencing, and primary cell coculture showed that the CDK4/6 inhibitor palbociclib promotes fibroblast senescence, thereby increasing IGF1 and FGF7 levels. These factors drive macrophage polarization toward an M2-like phenotype through STAT3 Tyr705 phosphorylation and ARG1 upregulation, resulting in arginine depletion and reduced lymphocyte viability. To counteract this immunosuppressive microenvironment, we selected the CSF1R inhibitor pexidartinib. Pexidartinib inhibited macrophage activity, suppressed STAT3 phosphorylation, reduced ARG1 expression, and increased lymphocyte viability, thereby enhancing the antitumor efficacy of palbociclib in HR + /HER2- breast cancer. These findings reveal a previously unrecognized immunosuppressive mechanism induced by CDK4/6 inhibition and support CSF1R blockade as a promising combination strategy.

Despite advances in defined culture systems, current organoid models lack programmable control of transcriptomic states beyond fixed genetic constraints or broadly specific microenvironmental conditions. Here, a bottom-up biomaterial-based platform is introduced to program cell state changes in pancreatic cancer organoids by tuning minimal adhesion cues within a synthetic matrix. A Design of Experiments framework is used to systematically model the patient-specific transcriptome-wide impact of matrix-presented adhesion cues. Focusing on epithelial-mesenchymal transition (EMT) as a proof-of-concept cellular program, a multiobjective optimization approach is applied to identify patient-specific matrix compositions that enrich EMT-associated transcriptional programs. Organoids cultured in these optimized matrices exhibit transcriptomic signatures consistent with EMT enrichment and coordinated shift in EMT-associated regulatory signatures. Secretome profiling further reveals changes in cytokines previously linked to EMT-associated inflammatory, hypoxia, and TGF-β signaling. Together, these findings demonstrate that quantitative and targeted modulation of defined adhesion cues enables programmable control of transcriptomic states in pancreatic cancer organoids.

Hormone receptor (HR)-positive breast cancer accounts for approximately 60% of all breast cancer cases, for which endocrine therapy represents the mainstay of treatment; however, the development of therapeutic resistance substantially limits its clinical efficacy. Extracellular adenosine 5'-triphosphate (ATP) has been implicated as a key mediator of metastasis and chemotherapy resistance in multiple malignancies, including breast cancer, yet its role in endocrine resistance remains poorly defined. Here, we demonstrate that extracellular ATP upregulates glycogen phosphorylase L (PYGL) expression in ER-positive breast cancer cells following endocrine treatment, thereby promoting endocrine resistance. Mechanistically, extracellular ATP activates the P2Y12-AhR signaling axis, leading to increased PYGL expression, enhanced glycolytic activity, and subsequent resistance to endocrine therapy. Moreover, elevated PYGL expression was strongly associated with reduced endocrine therapy sensitivity in breast cancer organoids and clinical tumor specimens. Collectively, these findings identify extracellular ATP-driven PYGL activation as a critical mechanism underlying endocrine resistance and suggest that targeting this pathway may represent a promising strategy to improve endocrine therapy efficacy in breast cancer patients.

High-throughput experiments, unidirectional fluid replacement, real-time process monitoring, and simultaneous drug sensitivity and toxicity tests are hard to achieve on most existing tumor organoid chips. Here, we developed a gravity-driven organoid perfusion (GDOP) platform facilitating scalable throughput and supporting drug sensitivity and toxicity assessment on organoids. The unidirectional perfusion capability and optimized operational parameters of the GDOP chip were validated through fluid dynamics simulations. Using this platform, we successfully established uniform on-chip triple-negative breast cancer (TNBC) organoids, with endpoint detection results aligning closely with clinical diagnosis. Throughout the drug treatment process, we monitored and then analyzed the morphological and grayscale changes of the organoids. The sensitivity and toxicity tests revealed the optimal concentration range for the 3 chemotherapeutic drugs. In addition, on-chip brain organoids were established, which lays a feasible foundation for future drug toxicity tests of complex organoids. The GDOP platform, combined with its integrated evaluation method, provides a powerful and reliable approach for advancing organoid-based researches.

Immune evasion limits the efficacy of cancer immunotherapy, yet the metabolic basis underlying differential immune sensitivity remains poorly explored. Here, we established an integrated platform that couples mass spectrometry imaging (MSI) with a patient-derived tumor organoid/immune cell coculture system to delineate metabolic adaptations driving tumor immune resistance. By optimizing a pretreatment workflow that preserves morphological integrity, we achieved in situ MSI mapping of phospholipids, fatty acids, taurine, glutathione, and other metabolites within tumor organoids. Colorectal cancer (CRC) organoids segregate into immune-sensitive and immune-resistant subtypes according to their responses to cocultured peripheral blood mononuclear cells (PBMCs), and the resistant fraction exhibits significantly elevated B7-H3 expression. MSI of these organoids further revealed that phospholipid metabolism is markedly reprogrammed in immune-resistant organoids. This metabolic signature was corroborated by MSI of human CRC tissues, and dynamic MSI of organoids exposed to PBMCs showed that immune-resistant variants preserved a significantly larger phospholipid pool than did their sensitive counterparts, indicating superior membrane-lipid conservation under immune pressure. This integrated MSI-organoid immunity platform offers a broadly applicable framework for dissecting immune-resistance mechanisms and highlights phospholipid metabolism as a potential targetable axis to restore antitumor immunity in cancer treatment.

Development and In vitro antitumor evaluation of a novel anti-p16 antibody fragment-drug conjugate.