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Preserving the phosphoproteome of clinical biopsies using a quick freeze collection device

ABSTRACT: The current gold standard for collecting and processing cancer biopsies is to flash freeze tumor samples in liquid nitrogen. However, in many clinical settings liquid nitrogen is not readily available, and neither the personnel nor the infrastructure is generally available to rapidly process the tumor samples. As a result, tumors are often subjected to prolonged ischemia and/or chemical fixatives, altering the phosphoproteome such that it may no longer reflect the true in vivo state of the tumor. There is a need for an economical, single-use device that can be stored at room temperature then activated at point of care to rapidly freeze the specimen. Our proof-of-concept quick-freeze prototype device focused on key requirements including cooling performance, device safety, minimizing use error, and the ability to ship via existing cold chain logistics. We have demonstrated that our device can cool a core sample below 0°C in less than 70 seconds, below -8°C in less than 150 seconds, and maintain that sample below 0°C for greater than 70 minutes. To demonstrate feasibility, the performance of our prototype was benchmarked against flash freezing in liquid nitrogen using melanoma-bearing PDX mice as a model system. After subjecting the mice to total body irradiation to elicit a phosphosignaling response in the DNA damage response pathway, tumors were harvested and quadrisected with two parts of the tumor snap frozen in liquid nitrogen, and the remaining two parts rapidly cooled in the prototype quick-freeze biospecimen containers for 1 hour. Phosphoproteins were profiled by LC-MS/MS. The prototype freeze device showed feasibility with bias within acceptable limits and slightly higher variability for a subset of phosphopeptides compared to flash freezing. The prototype device forms the framework for development of a commercial device that will improve tissue biopsy preservation for measurement of important phospho-signaling molecules.

ORGANISM(S): Mus Musculus

SUBMITTER: Jacob Kennedy

PROVIDER: PXD030367 | panorama | Mon Jul 18 00:00:00 BST 2022

REPOSITORIES: PanoramaPublic

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Project description:Endothelial inflammation contributes to the pathogenesis of numerous human diseases; however, the role of tumor endothelial inflammation in the growth of experimental tumors and its influence on the prognosis of human cancers is less understood. TNF-M-NM-1, an important mediator of tumor stromal inflammation, is known to target the tumor vasculature. In this study, we demonstrate that B16-F1 melanomas grew more rapidly in C57BL/6 wild-type (WT) mice than in syngeneic mice with germline deletions of both TNF-M-NM-1 receptors (KO). This enhanced tumor growth was associated with increased COX2 inflammatory expression in WT tumor endothelium compared to endothelium in KO mice. We purified endothelial cells from WT and KO tumors and characterized dysregulated gene expression, which ultimately formed the basis of a 6-gene Inflammation-Related Endothelial-derived Gene (IREG) signature. This inflammatory signature expressed in WT tumor endothelial cells was trained in human cancer datasets and predicted a poor clinical outcome in breast cancer, colon cancer, lung cancer and glioma. Consistent with this observation, conditioned media from human endothelial cells treated with pro-inflammatory cytokines (TNF-M-NM-1 and interferons) accelerated the growth of human colon and breast tumors in immune-deprived mice as compared with conditioned media from untreated endothelial cells. These findings demonstrate that activation of endothelial inflammatory pathways contributes to tumor growth and progression in diverse human cancers. To investigate the genes associated with TNF-M-NM-1 signaling in tumor endothelium, we performed expression profiling of tumor-associated endothelial cells isolated from B16F1 tumors grown in syngeneic TNFR 1, 2 -/- (KO) and C57BL/6 (WT) mice. Tumor endothelial cells were isolated from WT and KO tumors when tumor volumes were ~180 mm^3. This was based on a stepwise immunopurification of combined tumor tissue (Seaman, S. et al. Genes that distinguish physiological and pathological angiogenesis. Cancer Cell 11, 539-54 (2007)). Tumor endothelial cells were lysed to collect total RNA and analyzed in duplicates with Affymetrix GeneChipM-BM-. Mouse Genome 430 2.0 Arrays.

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Preserving the phosphoproteome of clinical biopsies using a quick freeze collection device

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