Biology subjects

Panepucci, R. A.

Publications and source records attributed to Panepucci, R. A..

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↗

bioRxiv2019

Focused screening reveals functional effects of microRNAs differentially expressed in colorectal cancer

BackgroundColorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs carcinogenesis. In fact, several microRNAs have been described as aberrantly expressed in CRC tissues and in the serum of patients. More specifically, microRNAs with dual roles in both cancer and stem cell survival represent a potential source of novel molecular targets in CRC due to their described functions in normal and deregulated proliferation. However, the functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs involved in the control of pluripotency of stem cells in the proliferation and cell death of a colorectal cancer cell line.\n\nMethodsWe performed transfection of 31 microRNA mimics in HCT116 CRC cells. Cell proliferation and cell death were measured after 4 days of treatment using fluorescence staining in a high content screening platform. Total number of live and dead cells were automatically counted and analyzed. To reveal mRNA targets, we used an oligonucleotide microarray. Functional classification of targets was done using DAVID tool. Gene expression of potential mRNA targets was performed by qPCR.\n\nResultsTwenty microRNAs altered the proliferation of HCT116 cells in comparison to control. Three microRNAs significantly repressed cell proliferation and induced cell death simultaneously (miR-22-3p, miR-24-3p, and miR-101-3p). Interestingly, all anti-proliferative microRNAs in our study had been previously described as poorly expressed in the CRC samples and were implicated in the disease. Microarray analysis of miR-101-3p targets revealed Wnt and cancer as pathways regulated by this microRNA. Specific repression of anti-apoptotic isoform of MCL-1, a member of the BCL-2 family, was also identified as a possible mechanism for miR-101-3p anti-proliferative/pro-apoptotic effect.\n\nConclusionsmicroRNAs described as upregulated in CRC tend to induce proliferation in vitro, whereas microRNAs described as poorly expressed in CRC halt proliferation and induce cell death in vitro. Selective inhibition of anti-apoptotic MCL-1 contributes to anti-tumoral activity of miR-101-3p.

cancer biology↗