Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Lipidomic Profiling Reveals Biological Differences between Tumors of Self-Identified African Americans and Non-Hispanic Whites with Cancer

“…De-identified human lung tissues cryopreserved in optimal cutting temperature compound (OCT), obtained during standard-of-care procedures and banked when judged to be in excess of that required for patient diagnosis and treatment, were procured from the Virginia Commonwealth University (VCU) Tissue and Data Acquisition and Analysis Core (TDAAC) under a VCU IRB-approved protocol (#HM2471) as described in [ 15 , 16 ]. Samples were provided under an honest broker system, and biorepository participants who signed the TDAAC informed consent documentation agreed to have their residual tissues utilized for any research question, including lipidomic analysis and health information for translational research.…”

“…Prior to mass spectrometry analysis, human specimens were processed with three rounds of the “sOCTrP” method to remove OCT cryoprotectant as described with few modifications [ 15 , 16 ]. Briefly, tissues were resuspended in 10 mL of ice-cold (4 °C) ultrapure Milli-Q water, vortexed for 20 s, centrifuged (5000× g , 4 °C) in a swinging bucket centrifuge equipped with ClickSeal biocontainment lids (Thermo Fisher, Whaltam, MA, USA, #75007309), and the supernatant aspirated without disturbing the pellets containing the tissues.…”

“…Briefly, tissues were resuspended in 10 mL of ice-cold (4 °C) ultrapure Milli-Q water, vortexed for 20 s, centrifuged (5000× g , 4 °C) in a swinging bucket centrifuge equipped with ClickSeal biocontainment lids (Thermo Fisher, Whaltam, MA, USA, #75007309), and the supernatant aspirated without disturbing the pellets containing the tissues. After the third round of washing/vortexing/centrifugation, tissues were resuspended in ice-cold phosphate-buffered saline (PBS; Gibco, New York, NY, USA, #20012027), transferred to a preweighed 1.5 mL tube (USA Scientific, Ocala, FL, USA, #1615-5500), centrifuged (7500× g , 4 °C), the supernatant removed, and excess liquid removed using a wick made out of laboratory grade tissue paper (Kimtech, Roswell, GA, USA, #34120) (for detailed instructions on wicking please see [ 15 , 16 ]). The tube and tissues were then weighed using an analytical balance and then placed on ice.…”

“…In cancers, it is well established that dysregulation of sphingolipid metabolism and of their signaling pathways contributes to disease development, progression, and responses to therapy. Tumors of human patients with various types of cancer have significant alterations of sphingolipid levels as compared to normal adjacent tissues [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. There is also broad consensus that key sphingolipid-metabolism-regulating enzymes impact malignancy, responses to therapy, and prognosis, and regulators of sphingolipid levels are considered important therapeutic targets in cancer, with several being tested in clinical trials [ 8 , 21 , 22 , 23 ].…”

“…In addition, given the recognized importance of sphingolipid alterations in the etiology of cancer and their therapeutic potential, precisely establishing how sphingolipid metabolism is altered may lead to the development of targeted therapies. Therefore, to decrease the gap in our understanding of the reprogramming of lipid metabolism in the tumors of individuals self-identifying as AA, we used methodology we previously developed [ 15 , 16 ] to conduct a modern sphingolipidomic analysis of the normal adjacent uninvolved (abbreviated “unin.” henceforth) tissues and tumors of self-identified AA and NHW males with lung adenocarcinoma (LUAD), colorectal adenocarcinoma (COAD), liver adenocarcinoma (HCC), and head and neck squamous cell carcinoma (HNSCC) and of AA and NHW females with endometroid endometrial carcinoma (EEC).…”