In situ mass spectrometry imaging reveals heterogeneous glycogen stores in human normal and cancerous tissues (2022)
Figure 6

Figure 6. Glycogen accumulation is a therapeutic target in preclinical models of Ewing sarcoma.

A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A.

B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.

C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.

D. Schematic of targeting glycogen synthesis by CRISPR/Cas9 knockout or small molecule inhibitor, guaiacol, to prevent glycogen synthesis.

E. GCMS quantitation of glycogen abundance in A673 empty vector (EV) or glycogen synthase knockout (GYS1-KO) cells in vitro. Values are presented as mean +/- standard error (n=3 biological replicates per group). **0.001 < P < 0.01, analyzed by two-tailed t-test.

F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.

G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.

H. GCMS quantitation of glycogen abundance in A673 WT cells treated with PBS or Guaiacol (GYSi) (20µM). Values are presented as mean +/- standard error (n=3 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.

I. In vivo tumor growth of A673 cells injected in the flanks of athymic nude mice. Mice received daily injection of either PBS or GYSi (30mg/kg). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.

J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant).

K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.

L. Extracted phosphorylated CL5 abundance between normal human tibia, tibial ES tumor, and stroma. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001, analyzed by one-way ANOVA with Tukey's multiple comparison.

M. Relative mRNA abundance of Epm2a between normal muscle [GEOD-17674 (n=18) and GSE17679 (n=18) and Ewing Sarcoma tumors [GEOD-17674 (n=44), GSE17679 (n=65), and GEOD-37371 (n=20)] based on published RNAseq datasets. Values are presented as relative expression +/- standard error. ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image.

O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.

Q. Schematic of the role of laforin as a glycogen phosphatase. Loss of laforin leads to hyperphosphorylated glycogen in ES (bottom) while normal laforin function regulates glycogen phosphate content (top).