Biology subjects

Palma, P. V. B.

Publications and source records attributed to Palma, P. V. B..

3 recordsLinked to original sources

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↗

bioRxiv2024

A novel Mannan-specific chimeric antigen receptor M-CAR redirects T cells to interact with Candida spp. hyphae and Rhizopus oryzae spores

Invasive fungal infections (IFIs) are responsible for elevated rates of morbidity and mortality, causing around of 1.5 million deaths annually worldwide. One of the main causative agents of IFIs is Candida albicans, and non-albicans Candida species have emerged as a spreading global public health concernment. Furthermore, COVID-19 has contributed to a boost in the incidence of IFIs, such as mucormycosis, in which Rhizopus oryzae is the most prevalent causative agent. The effector host immune response against IFIs depends on the activity of T cells, which are susceptible to the regulatory effects triggered by fungal virulence factors. The fungal cell wall plays a crucial role as a virulence factor, and its remodeling compromises the development of a specific T-cell response. The redirection of Jurkat T cells to target Candida spp. by recognizing targets expressed on the fungal cell wall can be facilitated using chimeric antigen receptor (CAR) technology. This study generated an M-CAR that contains an scFv with specificity to -1,6 mannose backbone of fungal mannan, and the expression of M-CAR on the surface of modified Jurkat cells triggered a strong activation against Candida albicans (hyphae form), Candida tropicalis (hyphae form), Candida parapsilosis (pseudohyphal form), and Candida glabrata (yeast form). Moreover, M-CAR Jurkat cells recognized Rhizopus oryzae spores, which induced high expression of cell activation markers. Thus, a novel Mannan-specific CAR enabled strong signal transduction in modified Jurkat cells in the presence of Candida spp. or R. oryzae.

bioengineering↗