Gasek, N. | Search bioRxiv

bioRxiv2026

High-Throughput, Multiplex Immunofluorescence for the Computer-automated Immunophenotyping of Mycosis Fungoides

The diagnosis of Mycosis Fungoides (MF) is difficult and often delayed, exacerbated by the constraint of conventional immunohistochemistry (IHC) to analyze only one antigen per tissue section, often necessitating repeat biopsies and extensive workups. We sought to validate a high-throughput Multiplex Immunofluorescence (MIF) method, coupled with computer-automated image analysis, to generate comprehensive immunophenotyping data from a single formalin-fixed, paraffin-embedded (FFPE) biopsy. We applied an 11-biomarker MIF panel across 18 archived skin specimens (9 MF/TCR clonality positive and 9 control/TCR clonality negative). Initial validation confirmed that MIF antigen expression and spatial localization were concordant with sequential IHC-stained sections. Whole slide image stacks were analyzed using both computer-assisted and fully computer-automated pipelines. Both methods successfully delineated immunophenotypic differences. MF specimens showed a significant expansion of hematopoietic cells and proliferative T-lymphocytes compared to controls. Crucially, MF tissues also exhibited a significant increase in the percentage of atypical T-lymphocytes. Our results validate the potential of MIF to obtain comprehensive, high-dimensional diagnostic information from a single tissue section. Integration with computer-automated analysis offers a scalable, high-throughput platform that can significantly aid in the timely and accurate diagnosis of cutaneous lymphomas.

pathology↗

bioRxiv2025

Senolytic-Resistant Senescent Cells Have a Distinct SASP Profile and Functional Impact: The Path to Developing Senosensitizers

The senescent cell (SC) fate is linked to aging, multiple disorders and diseases, and physical dysfunction. Senolytics, agents that selectively eliminate 30-70% of SCs, act by transiently disabling the senescent cell anti-apoptotic pathways (SCAPs), which defend those SCs that are pro-apoptotic and pro-inflammatory from their own senescence-associated secretory phenotype (SASP). Consistent with this, a JAK/STAT inhibitor, Ruxolitinib, which attenuates the pro-inflammatory SASP of senescent human preadipocytes, caused them to become "senolytic-resistant". Administering senolytics to obese mice selectively decreased abundance of the subset of SCs that is pro-inflammatory. In cell cultures, the 30-70% of human senescent preadipocytes or human umbilical vein endothelial cells (HUVECs) that are senolytic-resistant (to Dasatinib or Quercetin, respectively) had increased p16INK4a, p21CIP1, senescence-associated {beta}-galactosidase (SA{beta}gal), {gamma}H2AX, and proliferative arrest similarly to the total SC population (comprising senolytic-sensitive plus -resistant SCs). However, the SASP of senolytic-resistant SCs entailed less pro-inflammatory/ apoptotic factor production, induced less inflammation in non-senescent cells, and was equivalent or richer in growth/ fibrotic factors. Senolytic-resistant SCs released less mitochondrial DNA (mtDNA) and more highly expressed the anti-inflammatory immune evasion signal, glycoprotein non-melanoma-B (GPNMB). Transplanting senolytic-resistant SCs intraperitoneally into younger mice caused less physical dysfunction than transplanting the total SC population. Because Ruxolitinib attenuates SC release of pro-apoptotic SASP factors, while pathogen-associated molecular pattern factors (PAMPs) can amplify the release of these factors rapidly (acting as "senosensitizers"), senolytic-resistant and senolytic-sensitive SCs appear to be interconvertible.

cell biology↗