Mutant KRAS Exosomes Alter the Metabolic State of Recipient Colonic Epithelial Cells.

See editorial on page 647.

In colorectal cancer (CRC) cells, mutant Kirsten rat sarcoma (KRAS) cell-autonomously imparts Warburg-like metabolic changes through induction of Glucose transporter 1 (GLUT-1) (SLC2A1).2, 3 We previously reported that mutant KRAS has marked effects on the constituents of CRC exosomes, including proteins and enzymes involved in metabolism and glycolysis.4, 5 The present studies were designed to test whether mutant KRAS exosomes can alter the metabolic state cell-nonautonomously in recipient colonic epithelial cells.

We isolated exosomes purified from Daniel L. Dexter derived 1 (DLD-1) cells, which contain 1 wild-type (WT) and 1 mutant KRAS allele, and those from DLD-1 isogenic cell variants genetically engineered to express only the WT KRAS allele (DKs-8) or only the mutant KRAS allele (DKO-1). Adding DKO-1 exosomes to DKs-8 cells significantly reduced glucose concentrations in the medium, suggesting increased cellular glucose uptake in these WT KRAS cells (Figure 1A). After 48-hour exposure to DKO-1 exosomes, a significantly higher percentage of recipient DKs-8 cells were in S and G2/M phases of the cell cycle (Supplementary Figure 1A and B), and cell number was increased at 120 hours (Supplementary Figure 1C). We next used the endogenous fluorescent characteristics of Nicotinamide adenine dinucleotide reduced (NADH) and Flavin adenine dinucleotide (FAD) to determine the relative balance between glycolysis and oxidative phosphorylation, as previously reported.7, 8, 9 Addition of mutant KRAS exosomes selectively and significantly increased the redox ratio in recipient normal mouse colonic cells cultured in Matrigel or on plastic (Supplementary Figure 2A–D) and WT KRAS DKs-8 cells (Supplementary Figure 2E).

Figure 1

Mutant KRAS exosomes alter metabolism in vitro and in vivo. (A) Glucose consumption of exosome-treated DKs-8 cells (N = 3 in triplicate). Data are plotted as means ± SD. (B) Normalized redox ratio of exosome-treated Apc colonic tumors. (C) 18FSPG tumor-to-muscle uptake ratio in WT or Apc mice injected with exosomes analyzed by 1-way analysis of variance followed by a post hoc Tukey test. *P < .05.

Supplementary Figure 1

Mutant KRAS exosomes enhance cell-cycle progression and proliferation of DKs-8 cells cultured in low-glucose medium. DKs-8 cells were incubated with DKs-8 or DKO-1 exosomes or mock-treated (A) for 48 hours or (B) for times shown. Cell-cycle state was assayed by Hoechst staining followed by flow cytometric analysis (n = 3 in triplicate). (C) Growth of exosome-treated DKs-8 cells after 120 hours as determined by relative fluorescence intensity (RFU) of stained nuclei. (A and C) *P < .05 for comparison indicated. (B) *P < .05 for pairwise comparisons between DKO-1 exosomes vs DKs-8 exosomes or mock treatment.

Supplementary Figure 2

Characterization of exosome effects in vitro. (A) Normal mouse colonoid cultures were characterized by antibody staining as indicated. Most epithelial cells express non–cell-surface E-cadherin (E-Cad; green); smooth muscle actin (SMA; red) marks pericryptal fibroblasts. T-antigen (green) indicates the presence of young adult mouse colon (YAMC) cells. Na/K adenosine triphosphatase (ATPase) (green) marks epithelial cells separate from Pan-Cadherin–expressing cells. Most epithelial cells express cytokeratin (CYT) 8/18 (green). (B) Representative images for panel C. Redox ratios for (C) normal mouse colonoids cultured in Matrigel, (D) normal mouse colonic cells cultured on plastic, and (E) DKs-8 cells exposed for 48 hours to the treatments indicated. Data are mean normalized redox ratio ± SEM. *P < .05.

To test whether mutant KRAS CRC exosomes function in vivo, we used the Adenomatous polyposis coli multiple intestinal neoplasia (Apc) mouse model in which adenomas develop throughout the gastrointestinal tract. Apc mice received intraperitoneal injections of DKs-8 or DKO-1 exosomes over 4 successive days. The redox ratio of tumors treated with DKO-1 exosomes was increased significantly (Figure 1B and Supplementary Figure 3A), suggesting that treatment with these mutant KRAS exosomes increases aerobic glycolysis in recipient tumor cells. We also performed (S)-4-(3-[18F]-fluoropropyl)-L-glutamic acid (18F-FSPG) positron emission tomography imaging 2 hours after the last injection of exosomes. 18F-FSPG is a novel positron emission tomography tracer that follows the import of cystine by the glutamate/cysteine antiporter SLC7A11, which is overexpressed in CRC. We found that Apc mice injected with DKO-1 exosomes had a significant increase in 18F-FSPG uptake in the tumor region (Figure 1C and Supplementary Figure 3B and C), suggesting that mutant KRAS CRC exosomes can alter tumor cell metabolism in vivo.

Supplementary Figure 3

Metabolic imaging in vivo. WT or Apc mice were injected with DKs-8 or DKO-1 exosomes or mock-treated. (A) Colonic tumors were removed and the normalized redox ratio was calculated. Y-axis plots redox ratios of individual cells showing distribution of ratios for pair-wise comparisons. Mutant KRAS DKO-1 exosomes show a shift toward higher redox ratios. (B) 18F-FSPG uptake was monitored by positron emission tomography imaging; representative positron emission tomography images are shown with brackets highlighting colonic regions of interest with corresponding magnetic resonance image in bottom panel. (C) Whole-mount and colonoscopic images from mice in each treatment group. *Distal colonic tumors.

Levels of GLUT-1 were increased in DKO-1 exosomes, as well as in cell lysates (Figure 2A). To test whether these mutant KRAS exosomes contained functional GLUT-1, we measured 18F-fluorodeoxyglucose incorporation. After 1 hour, 18F-fluorodeoxyglucose uptake was significantly higher in DKO-1 exosomes (Figure 2B). The purified exosomes showed the characteristic cup-shaped morphology and size reported for exosomes (40–100 nm) (Supplementary Figure 4A and B). Further purification of exosomes on an iodixanol density gradient showed that GLUT-1 was present in the fractions that contain established exosomal markers (Supplementary Figure 4C). Thus, mutant KRAS exosomes contain increased levels of functional GLUT-1.

Figure 2

Exosomal GLUT-1 partially drives metabolic changes in recipient cells. (A) Immunoblot analysis of cells and exosomes. After normalization to syntenin-1, levels of GLUT-1 were increased 2.5- and 3.1-fold in cell lysates and 3.1- and 5.2-fold in exosomes of DLD-1 and DKO-1 cells, respectively. (B) Percent 18F-fluorodeoxyglucose (18FDG) uptake in exosomes isolated from DKs-8 and DKO-1 cells (N = 3 in triplicate). (C) Fold-change of 18FDG uptake in exosomes isolated from parental and GLUT-1 KO DLD-1 cells. (D) nuclear magnetic resonance (NMR) determination of glutamate and lactate secretion in recipient DLD-1 GLUT-1 KO cells 43 hours after treatment with exosomes from parental DLD-1 or GLUT-1 KO cells (N = 2 in triplicate). Data are plotted as the means ± SD. *P < .05.

Supplementary Figure 4

Morphologic and biochemical analysis of exosomes. (A) DKO-1 exosomes show characteristic appearance by transmission electron microscopy (Materials and Methods). (B) DKs-8 and DKO-1 exosomes have a similar mean particle diameter. Four independent preparations of DKs-8 and DKO-1 exosomes were subjected to nanoparticle tracking analysis (Materials and Methods). Data are plotted as average diameter ± SD. (C) Immunoblot analysis of DKO-1 cell–derived exosomes fractionated by iodixanol density gradient centrifugation. (D) Immunoblot analysis of DLD-1 parental and GLUT-1 KO cells and exosomes.

Targeted disruptions of both alleles of GLUT1 in DLD-1 cells (GLUT-1 knockout [KO]) led to loss of detectable GLUT-1 protein (Supplementary Figure 4D) and significantly reduced uptake of radiolabeled glucose in these exosomes (Figure 2C). None of the other glucose transporters tested (GLUT-2, GLUT-3, and GLUT-4) were detected in these exosomes (data not shown). We treated recipient DLD-1 GLUT-1 KO cells with exosomes isolated from DLD-1 parental or GLUT-1 KO cells for 43 hours and measured metabolite uptake and secretion using 1H-MRS. Compared with GLUT-1 KO exosomes, parental DLD-1 exosomes significantly increased secretion of lactate and glutamate in recipient cells (Figure 2D), suggesting that exosomal GLUT-1 contributes to the altered metabolic state of recipient cells.

In summary, we show that mutant KRAS exosomes are able to confer a Warburg-like effect on recipient colonic epithelial cells in vitro and in vivo. Increased functional exosomal GLUT-1 contributes to metabolic changes in recipient cells. These preliminary observations should prompt further study of exosomes in tumor metabolism.