Pancreatic ductal adenocarcinoma (PDAC) remains among the most lethal gastrointestinal cancers, characterized by rapid progression and resistance to therapy driven by significant metabolic reprogramming. Although alterations in glycolysis, glutamine, and lipid metabolism are well established, recent studies emphasize a more crucial factor: the emergence of context-specific metabolic dependencies within the nutrient-deprived tumor microenvironment. This review highlights that focusing on these dependencies, particularly those resulting from the distinctive metabolic interactions between cancer cells and the surrounding stroma, offers a promising strategy for overcoming treatment challenges. Evidence demonstrating that stromal-driven metabolic pathways supply energy and building blocks and confer resistance to standard chemo- and immune-therapies is examined. Furthermore, innovative approaches to target these vulnerabilities in PDAC metabolic subtypes, including synthetic-lethal interactions and key transporters in metabolic pathways are explored. How functional precision medicine, which uses patient-derived models to identify metabolic vulnerabilities, may convert these insights into personalized therapies is examined. Moving from broad metabolic inhibition to precise targeting of the tumour-stroma metabolic ecosystem could substantially improve the prognosis for PDAC.

Adoptive T cell therapy holds great promise for the treatment of solid tumors but remains constrained by tumor heterogeneity, inefficient neoantigen targeting, and the complexity of T cell manufacturing. Here, we present a patient-specific, broadly applicable platform using physically inactivated tumor organoids (PIOs) to generate tumor-specific cytotoxic T cells ex vivo. Derived from droplet-engineered tumor organoids (DEOs), PIOs preserve the full antigenic repertoire of the patient's tumor without requiring synthetic peptides, antigen-presenting cells, or neoantigen prediction. Using matched tumor tissue and PBMCs from colorectal and liver cancer patients, we show that PIOs activate and expand tumor-specific T cells with enhanced infiltration, selective cytotoxicity, and robust secretion of IFN-γ and IL-2. Multi-round PIO stimulation achieves 80-400-fold expansion of CD8+CD137+ T cells within two weeks. Transcriptomic and epigenetic profiling suggest that PIOs modulate T cell programs linked to migration and persistence. This work redefines tumor organoids as immunotherapeutic materials and establishes a rapid, cost-effective platform for personalized T cell manufacturing. Our findings provide a new translational route for adoptive cell therapy in solid tumors using patient-derived materials.

Neutrophils are an ideal drug carrier because of their specific targeting and effective accumulation at sites of inflammation. However, the clinical application of neutrophils as drug carriers is constrained by the inability to sustain ex vivo culture and their short lifespan. Herein, we developed an in situ construction strategy to fabricate neutrophil-nanoparticle biohybrid systems (NE@NPs) in vivo by specifically hijacking the pro-inflammatory neutrophils in blood using thermoresponsive drug-loaded NPs. The pro-inflammatory neutrophils effectively transport these NPs to the tumor microenvironment across the vascular barriers, followed by the release of the NPs through neutrophil extracellular traps (NETs). Subsequent near-infrared (NIR) light-induced spatiotemporally drug-controlled release improved the therapeutic response. The therapeutic efficacy of in situ-constructed NE@NPs is successfully confirmed using multiple mouse models of cancer, pulmonary, and skin infections. This study illustrates that the in situ construction strategy of neutrophil-NP hybrid systems in vivo may be a promising approach for improving inflammation-associated disease treatment.

In this study, eleven 1,3,4-oxadiazolyl-thio-oxazines (4a-4k) were synthesized and characterized using H-1/C-1(3) NMR, IR spectroscopy, and mass spectrometry. Single-crystal XRD confirmed that 4c crystallized in the monoclinic P2(1)/n space group, revealing key intermolecular interactions through Hirshfeld surface analyses. DFT studies using the B3LYP/6-311++G(d,p) basis set optimized all the compounds without imaginary frequencies. Frontier molecular orbital analysis provided HOMO-LUMO gaps and global reactivity descriptors, whereas MEP surface analysis offered insights into the charge distribution and potential intermolecular interactions of the optimized structures. For all compounds, the key structural identity of the thiomethylene bond was confirmed by H-1 NMR doublets (3.5-4.5 ppm) from diastereotopic coupling near N&S and C-1(3) signals (25-40 ppm) indicating sulfur deshielding. The oxadiazole and oxazine rings were validated by C=N (1563-1613 cm(-)(1)) and C-O-C (1144-1201 cm(-)(1)) absorptions, with IR confirming C-S (696-713 cm(-)(1)) and S-CH2 (similar to 1400 cm(-)(1)) linkages. Mass spectra gave [M + H](+) ions as expected, with halogenated derivatives showing predicted isotopic patterns. Furthermore, the theoretical and experimental H-1 and C-1(3) NMR data were in agreement, as supported by the correlation coefficient and RMSD values. Alamar Blue assay on MCF-7 breast cancer cells identified 4c (IC50 = 33.06 mu M) and 4g (IC50 = 21.24 mu M) as active candidates. It was validated with molecular docking and 100 ns MD simulations, confirming 4g's strong binding affinity towards NF-kappa B p65 (-8.1 kcal/mol) via stable hydrogen bonds and hydrophobic contacts.

Triple-negative breast cancer (TNBC) remains resistant to immunotherapy because of its profoundly immunosuppressive tumor microenvironment. Here, we establish a droplet-engineered organoid (DEO) platform that preserves endogenous TILs and supports rapid, immunocompetent drug evaluation. Optimizing ROCK pathway modulation reveals that early withdrawal of Y-27632 maintains TIL viability, whereas ROCK activation by pentanoic acid (PA) substantially enhances CD8+ T cell infiltration and cytotoxicity within DEOs. In 4T1 tumor-bearing mice, PA monotherapy or alternating PA/Y-27632 treatment significantly reduces tumor volume without detectable systemic toxicity. To validate translational relevance, we applied PA to patient-derived organoids, which exhibited increased T cell activation and abundance, along with a higher proportion of apoptotic cells within the organoid. Integrated transcriptomic and protein analyses reveal that PA induces a cytolytic program coupled to ROCK-dependent effector pathways. Clinical dataset analyses further associate ROCK activation signatures with improved overall survival in basal-like immune-suppressed (BLIS) subtype TNBC. Collectively, our findings highlight that alternating ROCK pathway modulation, in particular ROCK activation, is a promising strategy to convert TNBC into an immune-responsive state.

The mechanisms by which circRNAs regulate estrogen receptor (ER)-positive breast progression and therapeutic resistance remain poorly defined. By screening circRNAs involved in ER signaling, circESR1 was identified as a novel circRNA exhibiting high specificity of expression in ER+ breast cancer. CircESR1 interacted with HNRNPAB, which was transcriptionally activated by ER/SP1 signaling. HNRNPAB promoted the back-splicing and expression of circESR1 by binding to the Alu elements of cognate pre-mRNA; and circESR1 transcripts increased the stability and expression of HNRNPAB, ensuring an efficient positive feedback loop as reflected in antiestrogen-resistant breast cancer cells. Furthermore, HNRNPAB interacted and stabilized CDK1 and CDK6 mRNA, which was facilitated by its asymmetrical binding of circESR1, to promote cell cycle progression. Patients whose cancer exhibited high levels of circESR1 and/or HNRNPAB exhibited advanced prognostic stage and poor survival. Combined use of circESR1 ASO and CDK4/6 inhibitors were shown to be an effective therapeutic approach overcoming antiestrogen resistance in breast cancer xenograft models. Hence, these findings elucidated a novel signaling complex centered around circESR1 and HNRNPAB in ER+ breast cancer, and suggested that circESR1 might represent a potential therapeutic target for this disease.

Advanced algorithms have significantly improved the efficiency of in vitro screening for protein-interactive compounds. However, target antigen (TAA/TSA)-based drug discovery remains challenging, as predictions of compound-protein interaction (CPI) based solely on molecular structure fail to fully elucidate the underlying mechanisms. In this study, we utilized deep learning, specifically TransformerCPI to screen active molecules from a Chinese herb compound library based on protein sequences. Two natural products, Polyphyllin V and Polyphyllin H, were identified as targeting the pan-cancer marker CD133. Their anti-tumor efficacy and safety were confirmed across validation in cancer cell lines, tumor patient-derived organoids, and animal models. Despite their analogous structures and binding affinity to CD133, Polyphyllin V suppresses the PI3K-AKT pathway, inducing pyroptosis and blockage of mitophagy, whereas Polyphyllin H inhibits the Wnt/β-catenin pathway and triggers apoptosis. These distinct mechanisms underscore the potential of combining AI-driven screening with biological validation. This AI-to-patient pipeline identifies Polyphyllin V and Polyphyllin H as CD133-targeted drugs for pan-cancer therapy, and reveals the limitations of virtual screening alone and emphasizes the necessity of live model evaluation in AI-based therapeutic discovery.

Achieving high maturity and functionality in in vitro skeletal muscle models is essential for advancing our understanding of muscle biology, disease mechanisms, and drug discovery. However, current models struggle to fully recapitulate key features such as sarcomere structure, muscle fiber composition, and contractile function while also ensuring consistency and rapid production. Adult stem cells residing in muscle tissue are known for their powerful regenerative potential, yet tissue-derived skeletal muscle organoids have not been established. In this study, we introduce droplet-engineered skeletal muscle organoids derived from primary tissue using cascade-tubing microfluidics. These droplet-engineered organoids (DEOs) exhibit high maturity, including well-developed striated sarcomeres, spontaneous and stimulated contractions, and recapitulation of parental muscle fiber types. Notably, DEOs are produced in just 8 d without the need for primary cell culture—substantially accelerating the 50- to 60-d process required by classical organoid models. Additionally, the cascade-tubing microfluidics platform enables high-throughput production of hundreds of uniform DEO replicates from a small tissue sample, providing a scalable and reproducible solution for skeletal muscle research and drug screening.

Tumor dormancy is a substantial clinical obstacle in treatment of estrogen receptor positive mammary carcinoma (ER+MC), contributing to drug resistance, metastatic outgrowth, relapse, and consequent mortality. Preclinical models mimicking clinical anti-estrogen-induced ER+MC dormancy were generated in vivo. Function and a mechanism-based combination treatment were determined in the generated dormancy-like models in vitro, ex vivo, and in vivo. The dormancy models display molecular features of dormancy and tumor mass and cellular dormancy with associated clinical dormancy behavior. Both serum and cancer tissue expression of Trefoil factor 3 (TFF3) are identified as prognostic indicators of dormant ER+MC with TFF3 functioning as an epigenetically regulated driver of dormancy-associated behaviors. BCL2-dependent pro-survival functions of TFF3 coupled with enhanced attributes of stemness designates TFF3 as an actionable target. Moreover, combination screening of a TFF3 small-molecule-inhibitor (AMPC) with compounds used clinically to treat anti-estrogen-resistant ER+MC identifies strong synergism between AMPC and CDK4/6 inhibitors in the dormancy-like models. The combination results in concomitant suppression of CCND1 expression and CDK4/6 kinase activity to decrease RB phosphorylation, with reduced BCL2 expression, leading to both ER + MC cell cycle arrest and apoptosis. The combined TFF3-CDK4/6 inhibition impedes metastatic outgrowth and ameliorates host animal survival in the dormancy-like models, producing a complete response in a percentage of animals. Hence, in vivo models of anti-estrogen induced dormancy of ER+MC generated herein, identify TFF3 as a driver of this process. The combined inhibition of TFF3 and CDK4/6 may potentially alleviate the clinical challenges posed by anti-estrogen-induced dormancy in ER+MC. Estrogen receptor positive mammary carcinoma is a common type of breast cancer. It is difficult to cure as the cancer cells stop dividing but survive upon treatment leaving the cancer dormant. It then recurs years or even decades later. To address this challenge, mouse models of dormant estrogen receptor positive mammary carcinoma were developed. It was observed that a protein, TFF3, reduced the chance of survival. A combination treatment was found to be able to treat the dormant cancer in the mice models. The combination approach uses an experimental drug to inhibit TFF3 with clinically used drugs. These results suggest a possible treatment for people with this type of cancer. Chen et al. generate mimicking dormant clinical anti-estrogen-induced estrogen receptor positive mammary carcinoma (ER+MC) models. TFF3 is identified as a prognostic indicator and epigenetically regulated oncogenic driver of anti-estrogen-induced dormancy in ER+MC with combined inhibition of TFF3 and CDK4/6 exhibiting potential to ameliorate outcomes in anti-estrogen-induced ER+MC.

Brain organoids have been proposed as suitable human brain model candidates for a variety of applications. However, the lack of appropriate maturation limits the transferability of such functional tools. Here, we present a method to facilitate neuronal maturation by integrating astrocyte-secreted factors into hPSC-derived 2D and 3D neural culture systems. We demonstrate that protein- and nutrient-enriched astrocyte-conditioned medium (ACM) accelerates neuronal differentiation with enlarged neuronal layer and the overproduction of deep-layer cortical neurons. We captured the elevated changes in the functional activity of neuronal networks within ACM-treated organoids using comprehensive electrophysiological recordings. Furthermore, astrocyte-secreted cues can induce lipid droplet accumulation in neural cultures, offering protective effects in neural differentiation to withstand cellular stress. Together, these data indicate the potential of astrocyte secretions to promote neural maturation.

MSC infiltration of diabetic vasculature.

STAT3 has emerged as a validated target in cancer, being functionally associated with breast cancer (BC) development, growth, resistance to chemotherapy, metastasis, and evasion of immune surveillance. Previously, a series of compounds consisting of imidazo[1,2-a]pyridine tethered 2-pyrazolines (referred to as ITPs) were developed that inhibit STAT3 phosphorylation in estrogen receptor-positive (ER+) BC cells. Herein, a new library of derivatives consisting of imidazo[1,2-a]pyridine clubbed 2-pyrazolines 2(a-o) and its amide derivatives 3(a-af) have been synthesized. Among these derivatives, 3n and 3p displayed efficacy to reduce ER+ BC cell viability, with IC50 values of 55 and 15 nM, respectively. Molecular docking simulations predicted that compound 3p bound to STAT3 protein, with a binding energy of -9.56 kcal/mol. Using Western blot analysis, it was demonstrated that treatment of ER+ BC cells with compound 3p decreased the levels of phosphorylated STAT3 at the Tyr705 residue. In conclusion, this investigation presents the synthesis of imidazopyridine clubbed 2-pyrazolines that exhibit significant efficacy in reducing viability of ER+ BC cells. In silico docking and Western blot analyses together support compound 3p as a promising novel inhibitor of STAT3 phosphorylation, suggesting its potential as a valuable candidate for further therapeutic development.

Estrogen signaling dysregulation plays a critical role in the development of anti-estrogen resistance and bone metastasis of ER+ mammary carcinoma. Using quantitative proteomic screening, we identified FXR1 as an estrogen-regulated RNA-binding protein associated with anti-estrogen resistance. Mechanistically, estrogen and IGF1 facilitate FXR1 protein translation via the PI3K/AKT/mTOR/EIF4E pathway. FXR1 enhances cellular resistance to apoptosis and ferroptosis by facilitating the maturation of BCL2 pre-mRNA and stabilizing GPX4 mRNA, respectively. Anti-estrogen resistant cells exhibit elevated FXR1 expression, and FXR1 depletion restores their sensitivity to tamoxifen. Moreover, combining FXR1 depletion with a ferroptosis inducer induces synergistic lethal in anti-estrogen resistant cells. Finally, we provide proof-of-concept evidence supporting FXR1 antagonism as a potential treatment for bone metastases in ER+ breast cancer. Our findings highlight FXR1 as a promising therapeutic target to improve existing therapeutic regimes for ER+ breast cancer patients.

Epidermal growth factor receptor (EGFR) is a key target in breast cancer (BC) treatment due to its significant role in disease progression. This study aims to synthesize novel heterocyclic compounds by tailoring adamantane with pyrazoline followed by piperazines to target EGFR in breast cancer cells. Synthesis of the heterocyclic compounds was performed by Claisen-Schmidt reaction followed by cyclization and concludes with substitution by secondary amines. Structure of the lead compound 1-(3-((3r,5r,7r)-adamantan-1-yl)-5-(4-nitrophenyl)-4,5-dihydro1H-pyrazol-1-yl)-2-(4-(2-nitrophenyl)piperazin-1-yl)ethenone (6c) is confirmed by HRMS, 1H & 13C NMR, IR and Single crystal XRD. In silico docking studies were performed to investigate the molecular interactions of the compound 3, 4 & 6c with the EGFR binding site and to calculate their binding energies. Molecular dynamics simulations were conducted to explore stability of protein-ligand complex for compound 6c within the active site groove of EGFR protein in comparison with its precursors. Additionally, cytotoxic effects of these compounds against MCF-7 breast cancer cells were evaluated using IC-50 assays. Among the synthesized compounds, 6c exhibited significant cytotoxic effects against MCF-7 cells, with IC-50 values of 1.22 mu M. Compound 6c had binding energy of-8.6 kcal/mol, indicating strong interactions within the EGFR binding site indicating their anti- breast cancer potential.

Abstract Reprogramming of energy metabolism exerts pivotal functions in cancer progression and immune surveillance. Identification of the mechanisms mediating metabolic changes in cancer may lead to improved strategies to suppress tumor growth and stimulate antitumor immunity. Here, it was observed that the secretomes of hypoxic breast cancer cells and breast cancer stem cells (BCSC) induced reprogramming of metabolic pathways, particularly glycolysis, in normoxic breast cancer cells. Screening of the BCSC secretome identified MIF as a pivotal factor potentiating glycolysis. Mechanistically, MIF increased c-MYC–mediated transcriptional upregulation of the glycolytic enzyme aldolase C by activating WNT/β-catenin signaling. Targeting MIF attenuated glycolysis and impaired xenograft growth and metastasis. MIF depletion in breast cancer cells also augmented intratumoral cytolytic CD8+ T cells and proinflammatory macrophages while decreasing regulatory T cells and tumor-associated neutrophils in the tumor microenvironment. Consequently, targeting MIF improved the therapeutic efficacy of immune checkpoint blockade in triple-negative breast cancer. Collectively, this study proposes MIF as an attractive therapeutic target to circumvent metabolic reprogramming and immunosuppression in breast cancer. Significance: MIF secreted by breast cancer stem cells induces metabolic reprogramming in bulk tumor cells and engenders an immunosuppressive microenvironment, identifying MIF targeting as a strategy to improve immunotherapy efficacy in breast cancer.

Intrinsic and acquired resistance represent major obstacles to optimize outcomes in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeted therapy in lung adenocarcinoma (LUAD). Hence, a deeper understanding of EGFR-TKI resistance mechanisms in LUAD will potentially assist in formulating strategies to delay or overcome such resistance. Herein, it was observed that trefoil factor 3 (TFF3) is a crucial mediator of the LUAD EGFR-TKI response. TFF3 conferred intrinsic resistance to EGFR inhibition in LUAD by promotion of EGFR activation. TFF3 expression was also increased in acquired EGFR-TKI resistant LUAD, accompanied by reduced EGFR activation. YAP, a key mediator of the Hippo signaling, was positively regulated by TFF3 by post-transcriptional mechanisms and was responsible for acquired EGFR-TKI resistance mediated by TFF3. Inhibition of TFF3 by a small molecule inhibitor not only enhanced EGFR-TKI sensitivity in LUAD cells but also restored the sensitivity of acquired EGFR-TKI resistant LUAD cells to EGFR-TKIs in vitro and in vivo. These findings demonstrate a pivotal function of TFF3 in mediating both intrinsic and acquired EGFR-TKI resistance in LUAD and may offer a potential therapeutic mechanism for delaying or overcoming resistance to EGFR-TKIs.

Objective Mitomycin C (MMC), a DNA-damaging chemotherapeutic, is commonly used clinically for recurrent cervical carcinoma (CC), either alone or in combination. MMC generates DNA damage resulting in CC cell death yet also induces increased AKT-BAD phosphorylation associated with drug resistance and reduced clinical benefit. The present study evaluates the efficacy of combined MMC and a BAD phosphorylation inhibitor in CC. Methods The association and function of phosphorylation of BAD on serine 99 (pBADS99) for cell survival of both MMC-resistant or sensitive-CC cells was explored. BAD was mutated to BADS99A to examine the requirement of BADS99 for CC cell survival and a novel small-molecule inhibitor of pBADS99 was utilized. Cell proliferation, survival, foci formation, and patient-derived organoids (PDOs) assays were utilized to determine efficacy, synergy and related mechanisms. Results MMC IC50 was positively correlated to the cell line pBADS99/BAD ratio. Increased BADS99 phosphorylation was observed in both MMC-sensitive or -resistant CC cells after MMC treatment. Inhibition of pBADS99 in CC cell lines produced synergistic apoptosis through BAD-mediated apoptotic pathways and enhanced DNA damage in response to MMC. The concurrent use of pharmacological inhibition of pBADS99 and MMC was synergistic, resulting in diminished cell viability and inducing apoptotic cell death in MMC-sensitive and -resistant CC cell lines or patient-derived organoids. Conclusion A combination of MMC with inhibition of BAD phosphorylation potentiated efficacy compared to single agent treatment. The potential further development of such strategies may provide outcome benefits to patients with CC.

Supplementary Figure S7 shows that MIF is associated with immunosuppressive Tumor microenvironment.

Supplementary Figure S2 shows that BCSC CM enhances the aerobic glycolysis not oxidative phosphorylation.