Biology subjects

Orellana, M. D.

Publications and source records attributed to Orellana, M. D..

4 recordsLinked to original sources

bioRxiv2026

Coverslip Hypoxia High-Content Screening (CH-HCS): A 2D Imaging Platform for Spatially Resolved Analysis of CAR-T Function Under Oxygen Gradients

Hypoxia within the tumor microenvironment profoundly limits the efficacy of immune and cellular therapies, yet most in vitro cytotoxicity assays neglect spatial oxygen heterogeneity. We developed Coverslip Hypoxia High-Content Screening (CH-HCS), a simple, scalable 2D co-culture platform that enables quantitative, region-resolved evaluation of CAR-T cell activity across controlled oxygen gradients within a single well. In CH-HCS, a 5 mm glass coverslip placed over tumor-stroma co-cultures restricts oxygen diffusion, generating concentric hypoxia-normoxia zones in standard 96-well plates. Fluorescently labeled CD19+ Raji or CD19- K562 tumor cells, stromal cells, and anti-CD19 CAR-T cells were analyzed using multiparametric fluorescence imaging with an ImageXpress Micro XLS system coupled to custom CellProfiler-KNIME pipelines, enabling segmentation of spatial Regions of Interest--InnerCore, OuterCore, Periphery, and Outside--and single-cell quantification of tumor death (SYTOX Green) and T-cell morphodynamics. The platform reproducibly established oxygen gradients that strongly shaped cellular behavior: CAR-T cytotoxicity and motility were maximal in normoxic regions but markedly suppressed within hypoxic cores, whereas effector cell survival increased under low oxygen. Unlike bulk cytotoxicity assays, CH-HCS directly visualizes spatial functional heterogeneity within the same well, allowing simultaneous comparison of matched hypoxic and normoxic compartments. Together, CH-HCS provides a cost-effective, high-throughput, and physiologically relevant tool for preclinical screening of CAR-T products and therapeutic strategies aimed at overcoming hypoxia-driven immune resistance at the tumor-stroma interface. Key PointO_LISpatially resolved quantification: Simultaneous measurement of cellular behavior in matched hypoxic and normoxic compartments within the same well. C_LIO_LIPhysiologically relevant gradients: Coverslip geometry generates reproducible oxygen diffusion profiles that emulate the in vivo tumor-stroma interface. C_LIO_LIMultiparametric single-cell readouts: High-content imaging coupled with automated segmentation provides data on viability, cytotoxicity, and effector morphology. C_LIO_LIHigh-throughput and low-cost: Fully compatible with 96-well formats, enabling parallel pharmacological or genetic screening without specialized hypoxia chambers. C_LIO_LIGeneralizable design: Applicable beyond CAR-T assays to study stromal adaptation, drug resistance, or immune suppression across controlled oxygen gradients. C_LI

cancer biology↗

bioRxiv2025

Unraveling the spatial distribution of CAF subsets in PDAC spheroids through a novel spatial flow cytometry approach

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal compartment, predominantly composed of cancer-associated fibroblasts (CAFs), that contributes to immune exclusion and therapeutic resistance. Although the phenotypic and functional diversity of stromal cells within the TME is well characterized, their spatial distribution and the mechanisms driving this heterogeneity have not been thoroughly investigated. Here, we present SpheroMap Cytometry, an innovative spatial flow cytometry method that enables high-resolution analysis of spatially organized cellular phenotypes within spheroids. CAPAN-1 pancreatic tumor cells and either HS-5 bone marrow stromal or umbilical cord-derived mesenchymal stromal cells (UC-MSCs) were co-cultured in ultra-low-adhesion 96-well plates. After 48 hours aggregation, spheroids were incubated with Image-iT Green Hypoxia to mark hypoxic cells, and after 72 hours spheroids were dissociated and stained with antibodies against CD73 and CD140B. SpheroMap Cytometry revealed that the hypoxic core was significantly enriched for CD73+ and myCAF-like populations (CD140B+CD73high), indicating a functional link between hypoxia and this CAF subpopulation. Moreover, while hypoxia alone was sufficient to drive myCAF differentiation in heterotypic and monotypic spheroids, we found that normoxic induction of myCAF occurred only in the presence of tumor cells supporting the hypothesis that proximity to tumor cells synergizes with hypoxia to regulate CAF differentiation. Our findings demonstrate that hypoxia drives a distinct stromal architecture in PDAC. SpheroMap Cytometry provides a scalable, high-resolution method to dissect the spatial immunophenotype of 3D tumor models, overcoming limitations of static imaging and conventional flow cytometry, opening new avenues for preclinical assessment of stroma-targeting therapies and the development of immunotherapeutics that reprogram the TME.

cancer biology↗

bioRxiv2025

SpheroMap Cytometry: a novel spatial flow cytometry approach to evaluate immune response in PDAC spheroids

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stroma that contributes, along with hypoxia, to immune exclusion and therapeutic resistance. Here, we introduce SpheroMap Cytometry, an innovative spatial flow cytometry platform designed to preserve and quantify the relative localization of immune cells from 3D spheroid models. CAPAN-1 pancreatic tumor cells and either HS-5 bone marrow stromal cells or umbilical cord-derived mesenchymal stromal cells (UC-MSCs) co-cultured in ultra-low-adhesion 96-well plates. PBMC cells (activated or non-activated) were added to infiltrate pre-formed tumor-stromal/mesenchymal spheroids. Then after 48 hours post-seeding and 24 hours post-PBMC incubation, spheroids were incubated with Image-iT Green Hypoxia to mark hypoxic cores, and after 72 hours spheroids were dissociated and stained with CD3, CD4, CD8, CD25 and CD127 antibodies. SpheroMap Cytometry revealed that non-activated immune cells infiltrated spheroids with a distinct pattern between normoxic and hypoxic regions, with an enrichment of CD4+ over CD8+ cells in the hypoxic core and a higher proportion of CD4+CD25+CD127- Treg-like cells. Pre-activation of PBMCs enhanced CD8+ cell infiltration into the hypoxic region and increased CD25+/CD25high/CD127- Tregs in both compartments, indicating that T-cell activation, while facilitating CD8+ cell entry into hypoxic zones, may also promote immunosuppressive populations that impair cytotoxic function. Notably, the effects we observed from the infiltration of pre-activated lymphocytes into tumor-stromal spheroids were not seen in spheroids formed with tumor-mesenchymal cells, which may reflect the immunosuppressive role of umbilical-cord mesenchymal cells. Our findings demonstrate that hypoxia modulates T-cell infiltration and activation, underscoring the critical interplay between oxygen gradients and immune evasion in PDAC. SpheroMap Cytometry provides a scalable, high-resolution method to dissect the spatial immunophenotype of lymphocytes in PDAC models, overcoming limitations of static imaging and conventional flow cytometry, making it an ideal platform for testing therapies aimed at overcoming immune exclusion.

cancer biology↗

bioRxiv2021

5-Azacytidine: Effects on the Expression of alpha-Cardiac Actin in Pericytes from Human Adipose Tissue

DNA methylation patterns are closely related to the chromatin structure, and its remodeling is considered an important mechanism in the control of gene transcription during cell differentiation. In rodent, several studies have related the possibility that multipotent mesenchymal stromal cells (MSCs) undergo cardiomyogenesis. However, it has not been completely elucidated if human adult stem cell exhibits true differentiation potential for a cardiac lineage. In this study, the action of the DNA methylation inhibitor 5-azacytidine (5-aza) was examined in human adipose tissue pericytes (hATPCs: 3G5+) regarding their possible capacity to induce myocytes in vitro. Real-Time PCR revealed that cells treated with 5-aza presented time-dependent decrease in the mRNA expression of -cardiac actin (-CA). At 24 h, this diminution was statistically significant; however, there was not a correlation with the highest level of DNA demethylation at the same period using Methylation-Sensitive High Resolution Melting-PCR (MS-HRM-PCR). An evident increase in the -CA protein expression was observed by Western blotting in hATPCs treated with 5-aza at 24 h. The mRNA expression of -SMA (-smooth actin) also showed a time-dependent decrease after the treatment, however, it was not significant. The ultrastructural analysis showed similar structures such as like-cell junctions, caveolae, and actin myofilaments, which aligned in parallel. These phenotypic alterations were found only after the treatment; however, the hTAPCs after 5-aza treatment were not able to form thick myofilaments and consequently sarcomeres. These results indicated that a terminal cardiac differentiation of hTAPCs was not achieved and that the cardiomyogenesis failure could be related to the non-muscle origin of the adipose tissue.

cell biology↗