Biology subjects

Queiroz, K.

Publications and source records attributed to Queiroz, K..

6 recordsLinked to original sources

bioRxiv2026

A scalable tumor-vasculature-on-chip for CAR T cell trafficking and efficacy studies

Most cellular therapies, like CAR T cells, remain ineffective in solid tumors. This is primarily due to a complex tumor microenvironment (TME), which creates biochemically hostile and often immunosuppressive conditions that limit efficacy of immunotherapies. Besides, cellular therapy efficacy is still often established in traditional 2D cultures that fail to simulate relevant aspects of solid tumor biology. Recent advances in three-dimensional (3D) and organ-on-chip culture systems have provided more physiologically relevant models for immunotherapy testing. These microphysiological systems (MPS) not only offer a 3D environment that alters tumor cell sensitivity to therapy but also enable inclusion of TME components and assessment of processes such as extravasation and infiltration, key steps in CAR T cell activity in vivo. This study focuses on applying an advanced culture technique and further building on the use of a scalable on-chip platform, the OrganoPlate, to grow EpCAM-positive and EpCAM-negative tumor cells in co-culture with an endothelial vessel to study EpCAM-targeting CAR T cell migration and killing kinetics. The CAR T cells specifically targeted and killed EpCAM-positive HT-29 tumor cells while EpCAM-negative A375 tumor cells were not affected. In addition, target cell killing was dependent on the ratio between CAR T and tumor cells (E:T ratio) and was enhanced by addition of IL-2. Inflammatory cytokines like INF-{gamma}, TNF and IL-6 increased overtime in cultures containing CAR T cells. Morphometric analyses of the endothelial compartment showed E:T ratio dependent disruption of endothelial vessels. Additionally, this system was able to distinguish EpCAM ScFv-CD28-CD3z and EpCAM ScFv-TM-4-1BB-CD3z CAR T cells killing abilities and was used for studying the effect of immune checkpoint inhibitors and Temozolomide, a DNA targeting drug, on CAR T cell performance. Altogether, this work adds to the available advanced culture techniques for immunotherapy developers by describing a model that is modular, scalable, and suitable for phenotypic and functional characterization of CAR T cells.

bioengineering↗

bioRxiv2025

Building the blood-brain barrier: a scalable self-assembling 3D model of the brain microvasculature under unidirectional flow

The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also poses a hurdle in the treatment of increasingly common neurological disorders. Additionally, BBB dysfunction is a hallmark of many neurological diseases, further emphasizing a need for a better understanding of BBB function in health and disease. We present a human self-assembling 3D model of the BBB in a microfluidic cell culture platform that allows culture of 48 models in parallel on one tissue culture plate. Human brain microvascular endothelial cells, pericytes, and astrocytes form highly reproducible BBB vascular networks under unidirectional perfusion and remain viable for a minimum of 14 days. Immunostaining reveals close cell-cell interactions with pericytes and astrocyte end-feet in direct contact with the brain microvasculature. Compared to endothelial monocultures, co-culture with astrocytes or pericytes results in improved barrier function, lower vessel diameters, increased branching, and alignment of the vessels in the direction of fluid flow. These results were most pronounced in tri-cultures containing all three cell types. Unlike similar models previously reported, this brain microvasculature model allows for unidirectional perfusion without the need for pumps and syringes. Combined with its high-throughput nature, this feature renders the model suitable for studies of BBB function in health and disease, and assessment of potential BBB restorative therapies.

neuroscience↗

bioRxiv2025

Modeling Hepatocellular Carcinoma and its microenvironment on a chip

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Its incidence is increasing and is closely related to advanced liver disease. Interactions in the HCC microenvironment between tumor cells and the associated stroma actively regulate tumor initiation, progression, metastasis, and therapy response. Effective drug development increasingly requires advanced models that can be utilized in the earliest stages of compound and target discovery. Here we report a phenotypic screen on an advanced HCC patient-derived chip (PDChip) model. The vascularized HCC PDChip models include relevant cellular players of the HCC microenvironment. We assessed the effect of 28 treatment conditions on a panel of 8 primary HCC tumors and 2 cell lines. Approximately 1200 HCC PDchips were grown under perfusion flow, exposed to treatments, and subsequently assessed for viability, tumor-associated vasculature responses and chemokine and cytokine changes. Although the SoC therapeutics sorafenib and lenvatinib reduced culture viability and produced profound changes in the organization of the vascular beds, they did not affect the tumor cell population in these cultures. Atorvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, reduced tumor viability but did not affect vascular bed organization. Sorafenib, lenvatinib and atorvastatin also affected chemokine and cytokine release. Tocilizumab, galunisertib, and vactosertib decreased the level of IL6, a relevant prognostic marker for HCC, while IL6 was increased by halofuginone. In conclusion, HCC PDChip models enabled a detailed evaluation of drug-induced responses in the tumor and associated microenvironment, highlighting their importance in preclinical research for understanding diseases and developing new drugs.

bioengineering↗

bioRxiv2025

Scalable Image-Based Quantification of Cell Permeability and Actin Remodeling: An Example in a Gut-on-Chip Platform

Evaluating cellular responses to toxic compounds is essential for assessing the safety and potential hazards of drugs, environmental pollutants, and food contaminants. Traditional in vitro methods often lack the precision and scalability required for comprehensive toxicological assessments. This protocol presents an efficient and scalable approach for quantifying cellular damage and toxicity using organ-on-chip technology, specifically the OrganoPlate(R) platform. By combining fluorescent probes--DRAQ7 for cell membrane integrity, ActinGreen for cytoskeletal changes, and NucBlue for nuclear counting--with high-throughput image analysis via CellProfiler, this method provides detailed and quantitative insights into cellular damage induced by toxic compounds. The protocol includes step-by-step instructions for staining, image acquisition, and data analysis, as well as troubleshooting guidance. CellProfilers open-source nature, flexibility, and automation capabilities enable reproducible, high-throughput workflows, offering significant advantages over traditional manual image analysis. Its ability to assess cytotoxicity in human tissue models makes this protocol a valuable tool for safety testing in drug discovery and environmental toxicology. Furthermore, the approach is highly adaptable, accommodating a variety of cell types and toxic compounds, and is well-suited for rapid screening and risk assessment across diverse research and industrial applications.

cell biology↗

bioRxiv2024

Mycotoxin candidalysin impairs epithelial barrier integrity and initiates inflammation in a gut-on-chip model

Candida albicans is an opportunistic pathogenic yeast commonly found in the gastrointestinal tract, vagina, and mouth of healthy humans. Under certain conditions, it can become invasive, causing mucosal or life-threatening systemic infections. One mechanism used by C.albicans to breach the epithelial barrier is the secretion of candidalysin, a cytolytic peptide toxin. Candidalysin damages epithelial membranes and activates the innate epithelial immune response, making it key to C.albicans pathogenicity and a promising therapeutic target. Although candidalysin mediates C. albicans translocation through intestinal layers, its impact on epithelial responses is not fully understood. This study aims to characterize this response and develop scalable, quantitative methodologies to assess candidalysins toxicological effects using gut-on-chip models. We used the OrganoPlate(R), a microfluidic platform to culture up to 64 perfused, membrane-free intestinal epithelial tubes. We exposed Caco-2 tubes to candidalysin and evaluated their response with trans-epithelial electrical resistance (TEER), protein detection, and immunostaining. We then validated our findings in a proof-of-concept experiment using human intestinal organoid tubules. Candidalysin impaired barrier integrity, as indicated by decreased TEER and increased permeability in a fluorescent dye assay. It also induced actin remodeling and DRAQ7 dye uptake, a marker of cell permeability. This disruption was associated with the release of LDH, cytokines, and the antimicrobial peptide LL37, suggesting cellular damage, inflammation, and antimicrobial activity. This study strengthens our understanding of candidalysins role in C. albicans pathogenesis and suggests new therapeutic strategies targeting this toxin. Moreover, the use of patient-derived organoids shows promise for capturing patient heterogeneity and developing personalized treatments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/621017v4_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@dd8e35org.highwire.dtl.DTLVardef@1a33d18org.highwire.dtl.DTLVardef@113eorg.highwire.dtl.DTLVardef@6c2b9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in GBM

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

cancer biology↗