Biology subjects

Cuenca, M.

Publications and source records attributed to Cuenca, M..

4 recordsLinked to original sources

bioRxiv2024

Basement membrane hydrogels dampen CAR-T cell activation: nanofibrillar cellulose gels as alternative to preserve T cell function in 3D cell cultures.

BackgroundHydrogel-based 3D culture systems are emerging as a valuable tool for preclinical screening of cell-based immunotherapies against solid and hematological malignancies, such as chimeric antigen receptor T (CAR-T) cells. Hydrogels can influence T cell function in a non-desired manner due to their mechanical properties and chemical composition, potentially skewing results in preclinical testing of novel immunotherapeutic compounds. MethodsIn this study, we assess CD4+ T and CAR-T cell activation and proliferation in chemically-undefined matrices (Matrigel and basement membrane extract, BME) and compare them to a synthetic nanofibrillar cellulose (NFC) hydrogel. ResultsRheometric analyses show that NFC is more rigid than Matrigel and BME. Murine CD4+ T cells acquire a regulatory T cell (Treg) phenotype in Matrigel and BME, while this is not observed in NFC. Proliferation and activation of human T cells are higher in NFC than in Matrigel or BME. Similarly, we show that CAR-T cell activation and proliferation is significantly impaired in Matrigel and BME, in contrast to NFC. ConclusionsOur findings highlight the impact of hydrogel choice on (CAR-)T cell behavior, with direct implications for preclinical immunotherapy testing. In contrast to Matrigel and BME, NFC offers a chemically-defined 3D environment where T cell function is preserved. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSIn 3D (preclinical) tumor-killing assays for evaluating engineered T cell cytotoxicity, the surrounding matrix can influence immune cell phenotype and function, potentially skewing T cell activity. Basement membrane hydrogels such as Matrigel and basement membrane extract (BME), widely used as scaffolds for 3D culture, are inherently heterogeneous and contain extracellular matrix components that can influence lymphocyte function. What this study addsHere, we show that (CAR-)T cell function is significantly reduced in Matrigel and BME as compared to standard (2D) culture conditions. In contrast, (CAR-)T cell activity is preserved in synthetic nanofibrillar cellulose (NFC) gels. Importantly, murine T cells spontaneously acquire a Treg phenotype in Matrigel and BME. T cell proliferation and cytokine secretion are >10-fold lower in Matrigel than in NFC. Similarly, CAR-T cell survival and expansion are 10-fold higher in NFC than in Matrigel or BME. How this study might affect research, practice or policyWe report that the intrinsic cytotoxic and proliferative potential of (CAR-)T cells can be underestimated when performing assays in 3D cultures based on Matrigel or BME. As an alternative, we suggest the use of chemically defined synthetic gels, and we show that nanofibrillar cellulose hydrogels are suitable 3D matrices for preserving T cell phenotype and activation.

immunology↗

bioRxiv2024

Engineering BCMA CAR T cells for myeloma-targeted cargo delivery

Clinical responses with chimeric antigen receptor (CAR) T cells are encouraging, however, primary resistance and relapse after therapy prevent durable remission in a large fraction of cancer patients. One of the underlying causes comprises apoptosis resistance mechanisms in cancer cells that limit killing by CAR T cells. Therefore, we developed a technology that boosts tumor cell apoptosis induced by CAR T cells. We reveal that B cell maturation antigen (BCMA) CAR T cells equipped with a granzyme B-NOXA fusion construct improves killing of multiple myeloma (MM) cells, both in vitro and in a xenograft mouse model, by localizing NOXA to cytotoxic granules that are released into cancer cells upon contact. Since MM cells critically depend on MCL-1 expression, inhibition by its natural ligand NOXA effectively induces apoptosis. Overall, this strategy allows specific delivery of cargo into cancer cells and improves killing efficacy of CAR T cells in a tailor-made manner.

immunology↗

bioRxiv2021

Genetic regulation of RNA splicing in human pancreatic islets

Genetic variants that influence transcriptional regulation in pancreatic islets play a major role in the susceptibility to type 2 diabetes (T2D). For many susceptibility loci, however, the mechanisms are unknown. We examined splicing QTLs (sQTLs) in islets from 399 donors and observed that genetic variation has a widespread influence on splicing of genes with important functions in islet biology. In parallel, we profiled expression QTLs, and used transcriptome-wide association and co-localization studies to assign islet sQTLs or eQTLs to T2D susceptibility signals that lacked candidate effector genes. We found novel T2D associations, including an sQTL that creates a nonsense isoform in ERO1B, a regulator of ER-stress and proinsulin biosynthesis. The expanded list of T2D risk effectors revealed overrepresented pathways, including regulators of G-protein-mediated cAMP production. This data exposes an underappreciated layer of genetic regulation in pancreatic islets, and nominates molecular mediators of T2D susceptibility.

genetics↗

bioRxiv2020

STAG2 loss-of-function affects short-range genomic contacts and modulates urothelial differentiation in bladder cancer cells

Cohesin exists in two variants, containing either STAG1 or STAG2. STAG2 is one of the most commonly mutated genes in human cancer, and a major bladder cancer tumor suppressor. Little is known about how its inactivation contributes to tumor development. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the resulting genomic effects by integrating gene expression and chromatin interaction data. Cohesin-STAG2 is required for DNA contacts within topological domains, but not for compartment maintenance of domain boundary integrity. Cohesin-STAG2-mediated interactions are short-ranged and engage promoters and gene bodies with higher frequency than those mediated by cohesin-STAG1. STAG2 knockdown resulted in a modest but consistent down-regulation of the luminal urothelial differentiation signature, mirroring differences between STAG2-high and STAG2-low bladder tumors. Both lost and gained contacts were enriched among STAG1/STAG2 common sites as well as STAG2-enriched sites. Contacts lost upon depletion of STAG2 were significantly assortative, indicating their proximity at the 3D level, and were associated with changes in gene expression. Overall, our findings indicate that, in urothelial cells, STAG2 is required for the establishment and/or maintenance of DNA looping that, in turn, sustains the luminal differentiation program. This mechanism may contribute to the tumor suppressor function of STAG2 in bladder cancer.

cancer biology↗