DOI: 10.20350/digitalcsic/15674
title: Uncovering Cellular Senescence as a Therapeutic Target in Human Vestibular Schwannoma

description: [Description of methods used for collection/generation of data] Freshly harvested samples of sporadic VS were obtained from indicated surgeries and transported to the laboratory on ice. All protocols involving patient samples were approved by the local Ethics Review Board of La Paz University Hospital and the CSIC, and were conducted in accordance with the Helsinki Declaration. Written informed consent was obtained from all participants. Freshly harvested VS tissue was rinsed several times and minced under sterile conditions into sub-fragments, which were then dissociated with and enzymatic digestion. Cells were cultured i for 2–3 weeks. In addition, we used an establish cell line called HEI-193 cell line, derived from a VS of a patient with NF2.
description: [Methods for processing the data] To generate the data we performed different experimental techniques: cell viability assays using crystal violet staining; analysis of B-galactosidase enzyme activity associated with senescence; cell cycle analysis using PI staining in the flow cytometer; study of protein and mRNA expression by Western blotting and RT-qPCR, respectively; analysis of the numer of apoptotic cells using Annexin V-FITC and propidium iodide dual staining.
description: Cellular senescence is a crucial response against cancer development. It has been demonstrated that this cellular state is a defining feature of premalignant tumors that could be important in cancer diagnosis. This tumor-supressor role of cellular senescence has not been studied so far in human vestibular schwannomas (VS). VS are benign tumors originating from the Schwann cells that form the myelin sheath of the cochleovestibular nerve. VS grow slowly and cause hearing loss both due to compression of the auditory nerve and the release of ototoxic substances. Currently, patients undergo periodic MRI, surgery, gamma-radiosurgery or radiotherapy as tumor treatment but there are no pharmacological FDA-approved therapies to treat these tumors. In this study, we used HEI-193 cell cultures and patient derived VS primary cell cultures to address whether conventional chemotherapy, mainly DNA damage drugs, may induce a senescent response in these cellular systems that makes cells susceptible to be targeted by senolytic agents. Our data show that bleomycin can induce several senescent markers in VS primary cell culture as well as in an established cell line derived from a patient with neurofibromatosis type II, HEI-193 cell line. We analyzed the SASP-related secretory profile and observed that VS expressed higher levels of cytokines, metalloproteases and other SASP components than those obtained in bleomycin-treated HEI-193 cells. Our results also show that treatment with navitoclax, a senolytic agent, decreases the viability of the bleomycin- induced senescent cells without affecting the viability of the proliferative ones. This two-punch strategy based in the combination of senogenic and senolytic agents could constitute a potential alternative for the treatment of VS.
description: In the Data Acumulated_03112023 folder you will find two files: a folder called "figures" and an excel file entitled "statistics and figure details". The figures folder contains the original files and the excell files of our work, separated by figure number as designed for the scientific article we have just submitted to the journal Molecular Oncology and which I will detail later. The excel file called statistics and figure details contains as many sheets as figures we have designed for this article. The details of the brightfield and fluorescence microscopy photographs and the statistics used in each panel of each figure are described. Going back to the "figures" folder, you will find 11 subfolders where each of the panels of the figure appears with raw data and excel files.