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. 2022 Feb 2;3(2):100502. doi: 10.1016/j.xcrm.2021.100502

Figure 6.

Figure 6

Proteomic and metabolomic analyses upon treatment with SU086

(A) C4-2 and DU145 cells treated for 48 h with 1 μM SU086 or DMSO control were harvested, lysed, and digested with trypsin. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed. Cutoff values of 2-fold change with an FDR of <1% are included. Heatmap indicates −7 to +7 Z score of MS1 protein data. HSP90 is highlighted in red, and HSP90 interactors are indicated on the heatmap.

(B) A volcano plot of proteins showing Z score graphed as a function of p value for C4-2 and DU145 proteomic results. Blue and green indicate decreased and increased proteins (SU086 versus vehicle control) with 2-fold change or greater, respectively, with p < 0.05.

(C) Significantly decreased proteins were compared between cell lines, with 38 identified as overlapping.

(D) These common significantly downregulated proteins were graphed in String and show enrichment for glycolysis (red filled circles). Thickness of network edges indicate strength of data supporting the interaction. HSP90 interactors are highlighted in orange.

(E and F) Glycolytic challenge assay performed on Seahorse XF of C4-2 (E) and DU145 (F) cells pretreated for 24 h with 1 μM SU086 or DMSO. ECAR-extracellular acidification (mpH/min) indicates glycolysis. Flux cartridge injects glucose, Oligomycin (oligo), and 2-DG at the indicated points to measure metabolic initiation, peak glycolytic capacity, and depleted levels, respectively, and graphed ±SD. The sixth time point minus the third was quantified as glycolytic flux, while the ninth minus the third time point was quantified as glycolytic capacity. Scale bars represent 100 μm.

(G and H) DESI-MS analysis of OCT-embedded C4-2 (G) (n = 2 per condition) and DU145 (H) (n = 6 per condition) xenografts treated with SU086 or vehicle control from Figure 3, indicating localization of lactate and pyruvate levels in the tumors. DESI analyzed tissues were histologically analyzed and regions of non-necrotic tumor (outlined in black) were digitally pixelated and quantitatively analyzed using SAM analysis. Quantified lactate/pyruvate ratios across all pixels are graphed as violin plots with horizontal lines indicating median and quartiles. The median and average lactate/pyruvate ratios in C4-2 vehicle-treated tumors are 111.7 and 121.9, respectively; in C4-2 SU086-treated tumors are 101.8 and 112.3, respectively; DU145 vehicle-treated tumors are 80.6 and 94.7, respectively; in DU1145 SU086-treated tumors are 55.6 and 57.2, respectively. ∗p < 0.05, ∗∗p < 0.01. Scale bars for H&E represent 4 and 2 mm. Scale bars for DESI represnt 0.6 mm.