Biology subjects

Rodriguez-Garcia, A.

Publications and source records attributed to Rodriguez-Garcia, A..

6 recordsLinked to original sources

bioRxiv2026

SCITRAM: a Single-cell Integrated transcription regulation modeler for predicting master transcription factors

Single-cell omics technologies have provided unprecedented access to molecular cell heterogeneity, revolutionizing our understanding of complex living systems. Although generating such type of data has become a streamlined process, data processing still relies on command-line driven tools, or commercial solutions. Furthermore, single-cell transcriptomics analysis is usually reduced to stratifying cells on the grounds of gene expression signatures, while deconvolving gene regulatory programs (and their associated master transcription factors) responsible for cell heterogeneity are not classically addressed with the available tools, or performed under different data processing platforms. Herein, we present SCITRAM (Single-Cell Integrated TRAnscription regulation Modeler), a user-friendly stand-alone computational solution for predicting master transcription factors from single-cell transcriptomics data. SCITRAM first reconstructs a primary gene regulatory network and subsequently uses this network as template to model transcriptional cascades driven by the in-silico activation of transcription factors identified within the system. We validated SCITRAM performance across a variety of cell fate transition events including T cell exhaustion during solid tumor treatment, lineage stratification during pancreatic endocrinogenesis, as well as nervous tissue formation in cerebral organoid models. Overall, SCITRAM provides an accessible and integrated platform for processing (large amounts of) single-cell transcriptomics data, enabling the investigation of transcriptional regulatory programs and their master transcription factors without requiring a command-line driven environment.

bioinformatics↗

bioRxiv2026

Context-dependent tonic signaling shapes the performance and manufacturability of a 4-1BB- based HER2 CAR-T cell therapy

The development of clinically effective CAR-T cell therapies for solid tumors requires careful optimization of receptor design, functional fitness, and manufacturability. While advancing low-affinity HER2-targeting CAR-T cells toward clinical application, we found that the candidate with the strongest in vivo antitumor activity--comprising a CD8 hinge and transmembrane region and a 4-1BB co-stimulatory domain--exhibited measurable tonic signaling. This basal antigen-independent signaling, likely driven by high CAR surface expression, was associated with increased apoptosis and reduced ex vivo expansion under research-grade manufacturing conditions. Modification of the transmembrane domain reduced CAR surface expression but did not alleviate tonic signaling and instead impaired antitumor activity. By contrast, transient pharmacologic inhibition of CAR signaling with dasatinib rescued expansion and reduced apoptosis in small-scale research cultures. Notably, these tonic-signaling-associated defects were largely absent during large-scale, GMP-compliant manufacturing, which enabled robust CAR-T cell expansion without additional benefit from dasatinib supplementation. Together, these findings show that tonic signaling is not inherently detrimental to CAR-T cell performance and that its functional consequences are highly dependent on manufacturing context. Our study underscores the importance of evaluating CAR candidates within clinically relevant production platforms and supports the advancement of this 4-1BB-based HER2-specific CAR-T cell product toward clinical testing.

immunology↗

bioRxiv2026

In vivo CRISPR-based screen identifies ZC3H12C as a mediator of CAR-T cell dysfunction in solid tumors

CAR-T cell therapy has shown limited efficacy in solid tumors, largely due to T cell dysfunction driven by chronic antigen exposure. To uncover mediators of this dysfunction, we developed an in vivo screening platform using an ovarian xenograft tumor model in which CD28-based CAR-T cells undergo exhaustion leading to tumor escape. Transcriptomic profiling of tumor-infiltrating CAR-T cells at different stages revealed dynamic upregulation of exhaustion-associated genes. We used this data to design a focused CRISPR/Cas9 library and performed an in vivo screen. We identified 14 significantly enriched candidate genes, among which ZC3H12C emerged as the top hit. Single-cell RNA and ATAC-seq confirmed ZC3H12C expression in CAR-T cells undergoing early exhaustion in vivo. ZC3H12C disruption enhanced CAR-T cell persistence and antitumor efficacy while reducing exhaustion, across both CD28- and 4-1BB-based CARs targeting distinct antigens. These results highlight ZC3H12C as a promising target to improve CAR-T therapy in solid tumors.

immunology↗

bioRxiv2022

Modulation of multiple gene clusters expression by a single PAS-LuxR transcriptional regulator

PAS-LuxR transcriptional regulators are highly conserved enzymes governing polyene macrolide antifungal biosynthesis. PteF is one of such regulators, situated in the polyene macrolide filipin gene cluster from Streptomyces avermitilis. Its mutation leads to a drastic filipin production decline, but also to a severe loss of oligomycin production, an ATP-synthase inhibitor of macrolide structure, and a delay in sporulation, thus it has been considered as a transcriptional activator. Transcriptomic analyses were carried out in S. avermitilis {Delta}pteF and its parental strain S. avermitilis NRRL 8165 as control. Both strains were grown in YEME medium without sucrose, and samples were taken in the exponential and stationary growth phases. 257 genes showed altered expression in the PteF-deleted mutant, most of them in the exponential phase of growth. Surprisingly, despite PteF being an activator of filipin biosynthesis, a majority of the genes affected upon mutation showed overexpression thus suggesting a negative modulation of those genes. Genes affected were related to various metabolic processes, including genetic information processing; DNA, energy, carbohydrate, and lipid metabolism; morphological differentiation; and transcriptional regulation; among others, but particularly to secondary metabolite biosynthesis. Notably, ten secondary metabolite gene clusters out of 38 encoded by the genome, some of them encoding cryptic compounds, showed altered expression profiles in the mutant, suggesting a regulatory role for PteF wider than expected. Transcriptomic results were validated by quantitative reverse transcription polymerase chain reaction. These findings provide important clues to understand the intertwined regulatory machinery that modulates antibiotic biosynthesis in Streptomyces.

microbiology↗

bioRxiv2022

Targeted nanocarriers coopting pulmonary leukocytes for drug delivery to the injured brain

Selective drug delivery to injured regions of the brain is an elusive, but biomedically important, goal. It is tempting to co-opt migrating white blood cells (WBC) to carry drugs to the injured brain, using natural WBC tropism. Current approaches to load cargoes to WBC have limited utility, particularly in acute conditions, due to the need for time consuming ex vivo manipulation and loading of cells. Physiological, in vivo loading of WBC may be advantageous in this scenario. Here we devised such a strategy, capitalizing on the unique features of the direct blood exchange between brain and lungs. Mediators emanating from the injured brain directly travel to the pulmonary vasculature via venous flow. In response to these mediators, WBCs, transiently residing in the pulmonary microvascular lumen, disembark and flow with arterial blood to the brain microvasculature, where they adhere and transmigrate to the brain parenchyma via the local chemoattractant gradient. We posited that direct in vivo targeting of cargoes to the pulmonary WBC pool may provide drug transfer to brain via this natural mechanism. To test this, we intravenously injected agents targeted to intercellular adhesion molecule 1 (ICAM) in mice with acute brain inflammation caused by direct injection of tumor necrosis factor alpha (TNF-). We found that: A) At 2 hours, >20% of ICAM/NP accumulated in lungs, predominantly in WBCs; B) At 6 and 22 hours, ICAM/NP pulmonary uptake markedly decreased; C) In contrast, ICAM/NP uptake in brain increased ~5-fold in this time interval, concomitantly with migration of WBCs to the brain. Cranial window fluorescent microscopy confirmed WBC transport of ICAM/NP to the brain in TNF--challenged mice beyond the BBB. Importantly, demonstrating the pharmacologic relevance of this strategy, dexamethasone-loaded ICAM/liposomes abrogated brain edema in this model. In sum, coopting the natural homing of WBC from the lungs via ICAM-targeting to injured brain is an attractive strategy for precise interventions for treatment of acute brain injuries. VISUAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

pharmacology and toxicology↗

bioRxiv2021

Epigenetic state determines inflammatory sensing in neuroblastoma

Immunotherapy has revolutionized cancer treatment, but many cancers are not impacted by currently available immunotherapeutic strategies. Here, we investigated inflammatory signaling pathways in neuroblastoma, a classically "cold" pediatric cancer. By testing the functional response of a panel of 20 diverse neuroblastoma cell lines to three different inflammatory stimuli, we found that all cell lines have intact interferon signaling and all but one lack functional cGAS-STING signaling. However, toll-like receptor (TLR) signaling, particularly through TLR3, was heterogeneous. Six cell lines showed robust response, five of which are in the mesenchymal epigenetic state, while all 14 unresponsive cell lines are in the adrenergic state. Genetically switching the adrenergic BE2(c) cell line towards the mesenchymal state fully restored TLR responsiveness. In responsive cells, TLR3 activation results in the secretion of pro-inflammatory cytokines, enrichment of inflammatory transcriptomic signatures, and increased tumor killing by T-cells in vitro. Using single cell RNA sequencing data, we show that human neuroblastoma cells with stronger mesenchymal signatures have a higher basal inflammatory state, demonstrating intra-tumoral heterogeneity in inflammatory signaling that has significant implications for immunotherapeutic strategies in this aggressive childhood cancer.

cancer biology↗