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. Author manuscript; available in PMC: 2023 Oct 9.
Published in final edited form as: J Thorac Oncol. 2023 Jul 16;18(10):1362–1385. doi: 10.1016/j.jtho.2023.07.012

Figure 5.

Figure 5.

Proteomic analysis of network-forming GEMM cells. (A) LC-MS/MS experimental outline. NE (HES1−/GFP−) and non-NE (HES1+/GFP+) cells were generated from RBL2 GEMM tumors by flow cytometry on the basis of Hes1-GFP reporter expression and seeded onto plastic and Matrigel with a blank Matrigel control. Protein lysates were harvested, processed, and analyzed by LC-MS-MS in biological triplicate and technical duplicate. (B) Volcano plot of proteins in GEMM non-NE cells forming tubules on Matrigel versus growth on plastic. Red circles significantly differentially expressed proteins (fold change >1, −log(pvalue) >1). There were two independent tumor replicates analyzed in triplicate per condition. (C) Venn diagram showing 49 VM candidates overlapping between the 322 up-regulated proteins in the network-forming non-NE GEMM cells and the 2836 up-regulated genes in the CDX non-NE cells. (D) Transcript fold change in CDX non-NE versus NE cells of the top 10 overlapping protein and RNA hits identified in (C). (E) Representative immunoblots of CDX NE and non-NE cell lysates. There are 2 to 3 independent replicate tumors per CDX. Tubulin loading control was run subsequently for each marker illustrated on the same blot. (F) Representative images of CD31/PAS immunohistochemistry (top left) and intravenous tomato lectin/CD31/VCAM1/DAPI multiplex IF (bottom left) in serial tissue sections of a CDX17P tumor harvested after mice received intravenous tomato lectin injection. Individual perfused VCAM1+ EC vessels (images a and b; intravenous lectin+/CD31+/VCAM1+) and perfused VCAM1+ VM vessels (images c and d; intravenous lectin+/CD31−/VCAM1+) are illustrated on the right with single channel IF for CD31 (yellow), intravenous tomato lectin (pink), VCAM1 (green) and nuclei (DAPI, blue). Scale bars 50 μm (left panels) and 10 μm (right panels). (G) Gene Ontology Biological Processes representing the 49 overlapping protein and RNA hits identified in the GEMM and CDX. CDX, circulating tumor cell-derived explant; DAPI, 4’,6-diamidino-2-phenylindole; EC, endothelial cell; GEMM, genetically engineered mouse model; IF, immunofluorescence; LC-MS/MS, liquid chromatography - tandem mass spectrometry; NE, neuroendocrine; PAS, Periodic Acid Schiff stain; VM, vasculogenic mimicry.