Coregulation of glucose uptake and vascular endothelial growth factor (VEGF) in two small-cell lung cancer (SCLC) sublines in vivo and in vitro

Abstract

We examined the relationship between (18)F- labeled 2-fluro-2-deoxy-d-glucose (FDG) uptake, and expression of glucose transporters (GLUTs) in two human small-cell lung cancer (SCLC) lines CPH 54A and CPH 54B. Changes in the expression of GLUTs and vascular endothelial growth factor (VEGF) during 12-, 18-, and 24 hours of severe hypoxia in vivo (xenografts) and in vitro (cell cultures) were recorded for both tumor lines. The two SCLC lines are subpopulations of the same patient tumor. In spite of their common genomic origin they represent consistently different metabolic and microenvironmental phenotypes as well as treatment sensitivities. There were higher levels of Glut-1 protein in 54B and a correspondingly higher FDG uptake in this tumor line (P<.001). During hypoxia a significant upregulation of in VEGF mRNA, GLUT-1 mRNA, and Glut-1 and -3 protein occurred with a distinctly different time course in the two cell lines. A similar co-upregulation of GLUT and VEGF was seen in hypoxic tumors of both lines. There were no significant changes of HIF-1alpha mRNA during hypoxia in either of the cell lines. A more detailed understanding of such correlations between glucose metabolism, angiogenesis, and microenvironmental phenotype of tumors, by positron emission tomography (PET) and molecular techniques might further sophisticate our interpretation of glycolytic predominance in tumors as seen by 18FFDG PET.

Figure 1

In 20 mice a 54A tumor was grown at one flank and a 54B tumor at the other. The graph illustrates the distribution of differences in ¹⁸F-FDG uptake (54B minus 54A) in each individual pair of one 54B tumor and one 54A tumor in the same mouse. This difference was negative in only 3 of 20 pairs, documenting that the uptake was greatest in 54B tumors (P<.001, Extreme range test for paired differences, Lords test). The uptake was calculated as percent of injected dose per volume (p.i.d.). 54A median p.i.d.: 4.94%, range: (2.62% to 8.84%); 54B median p.i.d.: 7.12%, range: 2.64% to 13.7%.

Figure 2

PET scan of two tumor-bearing mice (Advance GE scanner, WI) following injection of 5 to 10 MBq of ¹⁸FFDG. Arrows indicate tumor locations. In the animal at the right side a deposit was accidentally left at the injection side.

Figure 3

Western blot of Glut-1 protein expression in 54A and 54B tumor xenografts. A higher Glut-1 expression in 54B tumors compared to 54A tumors. Lanes 1 and 2 were from the same animal as were 3 and 4, and 5 and 6. The quantitative, densitometric, ratios of Glut-1 to tubulin were significantly greater in 54B (P<.05), analysed with the extreme range test for paired differences, i.e., Lords test.

Figure 4

Northern blots of GLUT-1 (A), VEGF (B), and HIF-1α (C) mRNA expression in 54A and 54B cell cultures grown under hypoxic conditions for 12, 18, and 24 hours, compared with normoxic controls. The loading control is 28S ribosomal RNA.

Figure 5

VEGF and GLUT-1 mRNA in cell cultures. Relative expression of VEGF (A) and GLUT-1 (B) mRNA in 54A and 54B cells during hypoxia in vitro. A significant upregulation is seen in both cell lines. 54A levels off earlier than 54B. The GLUT-1 mRNA levels are constantly higher in 54B than in 54A. Each point represents cell cultures of between 5x10⁶ and 9x10⁶ cells. Bars represent standard error.

Figure 6

Glut-1 and Glut-3 protein expression in cell cultures. Relative expression of Glut-1 (A) and Glut-3 (B) protein in 54A and 54B cell cultures grown under hypoxic conditions for 12, 18, and 24 hours, compared with normoxic controls. 54A levels off earlier than 54B. Each point represent cell cultures of between 5x10⁶ and 9x10⁶ cells. Bars represent standard error.

Figure 7

VEGF and GLUT-1 MRNA from solid tumors. Relative expression of VEGF (A) and GLUT-1 (B) MRNA in 54A and 54B tumors during hypoxic incubation for 12 and 24 hours, compared with normoxic controls. Three tumors were examined for each of the time points. Bars indicate standard deviations.