Bhalla, R. | bioRxiv Science

bioRxiv2026

Multi-omics and functional analysis of a bioengineered vascularized pancreatic cancer model reveal an immunosuppressive and therapy-resistant niche

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by therapy resistance and an immunosuppressive tumor microenvironment. To comprehensively characterize the complex stromal-immune cell interactions that drive PDAC aggressiveness, we applied an integrated multi-modal approach combining single-cell RNA sequencing, spatial transcriptomics, proteomics, immunofluorescence, and microfluidic-based functional assays to bioengineered spheroid models with increasing cellular complexity (up to four cell types) integrating human pancreatic cancer cells, pancreatic stellate cells, endothelial cells, and monocyte-derived macrophages. By incorporating vascularization within the OrganiX microfluidic platform, we enable studies of immune cell trafficking in vascularized tumors. Multi-omics phenotyping revealed coordinated molecular programs in our four-cell organotypic spheroid models, including enhanced hypoxic and glycolytic pathways, NF-{kappa}B activation, and ECM remodeling. Stromal and immune cells acquired tumor-associated phenotypes mirroring patient heterogeneity, including IL-1{beta}+ macrophages, inflammatory cancer-associated fibroblasts (iCAFs), and antigen-presenting CAFs (apCAFs). The four-cell model exhibited superior clinical relevance, with gene expression signatures that correlated more closely with poor-prognosis patient cohorts and cancer hallmarks that were functionally validated through microfluidic-based assays demonstrating enhanced tumor invasion, angiogenesis, and therapeutic resistance. Finally, live imaging combined with transcriptomic readouts captures dynamic interactions between neutrophils and cancer cells in the vascularized microtumor, enabling direct observation of intravascular events relevant to metastatic dissemination. This integrated analysis demonstrated the recreation of a human-relevant aggressive PDAC niche, establishing a framework that bridges in vitro cellular crosstalk studies with patient-relevant therapeutic responses, offering a powerful translational tool for therapy development in PDAC.

cancer biology↗

bioRxiv2025

Creatine synthesis is a tumor suppressor pathway hypostatic to one-carbon metabolism

Methylene tetrahydrofolate reductase 2 (MTHFD2), the rate-limiting enzyme of mitochondrial one-carbon metabolism, is one of the most highly expressed metabolic enzymes across diverse cancers and lymphoproliferative disorders. However, its exact roles in oncogenic metabolism remain poorly defined. We show that MTHFD2 is a key regulator of mitochondrial energetics in Epstein-Barr virus-transformed B lymphoblastoid cell lines (LCLs), an in vitro model of post-transplant lymphoproliferative disorder (PTLD). We also delineate a role for MTHFD2 in fueling de novo creatine synthesis; MTHFD2 mediates the production of glycine, a necessary substrate for creatine synthesis, through serine catabolism. Aminomethyltransferase (AMT) suppression short-circuits the glycine cleavage system (GCS) to augment LCL mitochondrial glycine levels. Creatine synthesis is hypostatic to mitochondrial one-carbon metabolism; inhibition of creatine synthesis improves LCL fitness only when MTHFD2 is lost. Our findings emplace MTHFD2 at the nexus of amino acid and energy metabolism pathways in LCLs, with potential clinical ramifications for PTLD. Highlights* Complete activation of creatine synthesis in an in vitro cellular model of PTLD * Creatine synthesis is a major sink for mitochondrial 1C-derived glycine * Reverse GCS activity due to AMT deficiency in lymphoblastoid cells * Epistasis between mitochondrial 1C metabolism and creatine synthesis eTOC BlurbLeung et al. demonstrate that MTHFD2 is crucial for creatine synthesis in lymphoproliferative disorders. MTHFD2 supports forward 1C flux through SHMT and drives reverse GCS activity to augment mitochondrial glycine, a substrate for creatine synthesis. Tumor-suppressive effects of creatine synthesis are unmasked with MTHFD2 loss, exhibiting metabolic epistasis.

molecular biology↗