Figure 2.

Gene Specific Expression Analysis Reveals a Strongly Glycolytic Signature in Mono-S-Treated Tumors

(A) Gene set overlapping analysis on the top 2,500 upregulated genes in sunitinib treatment compared to vehicle treated controls, revealing that glycolysis and hypoxia were among the most enriched pathway signatures (left). The glycolysis enrichment plot for sunitinib-treated tumors, including the profile of running ES Score are shown (right).

(B) Top five ranked genes from the hallmark glycolysis gene set, adopted from the Molecular Signatures Database of the Broad Institute, for the sunitinib-treated cohort.

(C) MCT4 expression was assessed using IHC on 15 week control tumors (Con, left) and tumors treated with rapamycin monotherapy (Mono-R, middle left), axitinib (Mono-A, middle right), or sunitinib (Mono-S, right). The MCT4 expression is high in 4/4 larger and 5/6 smaller Mono-A- and 4/4 larger Mono-S-treated tumors and absent in Con and Mono-R-treated tumors. The scale bars represent 2 mm in top image and 200 μm in the bottom image.

(D) Pimonidazole (pimo, green) staining was performed to assess tumor hypoxia (center left); the glucose transporter, GLUT1, is shown in red (center right), and the leftmost image depicts the merged images. A merged image of R+A-treated tumors for MCT4 and pimonidazole is depicted in the rightmost image. The GLUT1 and MCT4 staining is highest in the most pimonidazole+/hypoxic regions, but can also be found in the peri-hypoxic areas. The scale bars represent 100 μm in the three left images and 50 μm in the right image.

(E) IHC using anti-GLUT1 (middle) and anti-MCT4 (right) indicates that their expression is highly reduced/absent in tumors containing a cell-type-specific (β-cell) gene knock out of HIF1α that were treated with sunitinib. The top row shows a representative Rip1Tag2_Rip1Cre_Hif1αWT tumor, while the bottom row shows a Rip1Tag2_Rip1Cre_Hif1αfl/fl littermate whose tumors do not express Hif1α; this result is representative of all tumors from 4/4 wild-type (WT) versus 6/6 HIF1α KO mice, all similarly treated with sunitinib. The scale bar represents 100 μm.

(F) Monotherapy with sunitinib (middle row) or axitinib (bottom row) elicits upregulation of MCT4 (green, first, second, and fourth) versus control untreated tumors (top row); in addition, MCT1 (in red) is upregulated in both AI-treated arms (middle and bottom rows, first, third, and fourth) versus controls (top row, first, third, and fourth). The scale bar represents 25 μm.