ABSTRACT: Neuroblastoma is a common type of pediatric cancer, accounting for approximately 7% of childhood cancers. Hypoxia tumor environment caused by fast growing cancer cell, hypoxic cancer cell would cause cell death and cell proliferation rate down.
Despite hypoxia causing tumor cell death and a decrease in cell proliferation rates, the hypoxic environment simultaneously activates various pathways, leading to the transformation of surviving hypoxic cells into malignant, proliferative tumors. Furthermore, these surviving cells may develop resistance to chemotherapy and radiotherapy.Tunneling nanotubes (TNTs) are intercellular conduits composed of F-actin that facilitate material transfer between cells.To understand the material transfer between normoxic and hypoxic cells via TNTs in the hypoxic tumor microenvironment, we conducted a TMT-based proteomic analysis of hypoxic and normoxic cells. TMT-Based proteomic analysis was used to investigate the changes in protein levels regulated via TNTs-mediated intercellular transfer between hypoxic and normoxic cells. We collected the conditional medium (CM) from the labeled normoxic (called normoxic CM) and hypoxic cells (called hypoxic CM), respectively. The hypoxic cells incubated with normoxic CM or normoxic cells incubated with hypoxic CM were used to exclude the influence of EVs and the soluble factors in the conditional medium. Following the FACS, six conditions of cells are all sorted and distinguished into two experimental batches, the normoxic to hypoxic batch (H batch: N-alone, H-alone, H (N CM), H-CO) and the hypoxic to normoxic batch (N batch: N-alone, N (H CM), N-CO). For TMT labeling, digested peptides from three biological replicates of the H batch and the N batch were labeled by TMTpro 16-plex kit and TMT 11-plex™ kit, respectively. The samples, sequentially analyzed by LC-MS/MS, were a total of 140159 MS/MS spectra, 46550 peptides, and 5835 proteins identified, and then we removed unreliable (potential contaminants) and low-quality (count ≤ 1) proteins. Finally, we got 4015 quantifiable proteins.
