Hyperpolarized MRI Visualizes Warburg Effects and Predicts Treatment Response to mTOR Inhibitors in Patient-Derived ccRCC Xenograft Models

Abstract

The ever-changing tumor microenvironment constantly challenges individual cancer cells to balance supply and demand, presenting tumor vulnerabilities and therapeutic opportunities. Everolimus and temsirolimus are inhibitors of mTOR (mTORi) approved for treating metastatic renal cell carcinoma (mRCC). However, treatment outcome varies greatly among patients. Accordingly, administration of mTORi in mRCC is diminishing, which could potentially result in missing timely delivery of effective treatment for select patients. Here, we implemented a clinically applicable, integrated platform encompassing a single dose of [1-¹³C] pyruvate to visualize the in vivo effect of mTORi on the conversion of pyruvate to lactate using hyperpolarized MRI. A striking difference that predicts treatment benefit was demonstrated using two preclinical models derived from patients with clear cell RCC (ccRCC) who exhibited primary resistance to VEGFRi and quickly succumbed to their diseases within 6 months after the diagnosis of metastasis without receiving mTORi. Our findings suggest that hyperpolarized MRI could be further developed to personalize kidney cancer treatment. SIGNIFICANCE: These findings demonstrate hyperpolarized ^[1-13C]pyruvate MRI as a tool for accurately assessing the clinical success of mTOR inhibition in patients with ccRCC.

Figure 1.

In vitro characterization of metabolic flux with mTORi. A, CTs of patients from which each line was derived demonstrating widespread metastatic disease (red arrows indicate primary tumors and yellow arrow indicates metastasis). B, [1,6-¹³C₂] glucose metabolism through glycolysis and TCA cycle. EC 2.7.1.1 (hexokinase), EC 1.1.1.27 (L-lactate dehydrogenase), EC 1.2.4.1 (pyruvate dehydrogenase). C, Representative ¹HNMR spectra of intracellular JHRCC12 and JHRCC228 after treatment with vehicle (DMSO, black) and rapamycin (100nM, red). D, Total intracellular lactate shows almost no change in JHRCC12 (p<0.54) treatment with rapamycin comparing to DMSO (vehicle) and a significant drop in JHRCC228 treatment (p<0.027) (N=5) (¹HNMR studies). E, Labeled intracellular lactate from [1,6-¹³C₂] glucose shows no change in JHRCC12 (p<0.63) treatment with 100 nM rapamycin comparing to DMSO (vehicle) and a decrease in JHRCC228 (p<0.023) (N=5) (¹HNMR studies). F, Intracellular fractional enrichment exhibit similar levels of incorporation of ¹³C carbon in lactate pool. G, Extracellular total lactate has dropped in JHRCC12 (p<0.0008) and JHRCC228 (p<0.0027) (N=5). H, Extracellular fractional enrichment shows a slight change in JHRCC12 (p<0.04) and a greater decrease in JHRCC228 (p<0.0004) (N=5). I, Lactate production/ glucose consumption shows no changes in JHRCC12 (P<0.18) and decreases in JHRCC228 (P<0.006) (N=5). NS (not significant). Data are mean ± SD in D, E, F, G, H and I.

Figure 2.

Comparison of rapamycin response in two primary patient ccRCC cell lines. A, 4×10⁴ JHRCC12 and JHRCC228 cells were seeded, treated with rapamycin at the indicated concentrations, and counted at days 2, 4, 6, and 8 (n=3). Statistical significance was determined by Student’s t-test. B, Tables show genes with mutations in JHRCC12 (top) and JHRCC228 (bottom). Images show the cell morphology of each cell line. C, Immunoblots with the indicated antibodies in JHRCC12 and JHRCC228 cells treated with control and rapamycin (100nM) for 5 hours are shown. Data are mean ± SD in A and B.

Figure 3.

Comparison of in vivo rapamycin response. A-D, 2×10⁶ JHRCC12 or JHRCC228 cells were injected subcutaneously into NSG mice. When tumors reached 100–150mm, mice were randomly divided into two groups. One group was treated with vehicle (n=9) and the other group was treated with rapamycin (n=10). Tumor volume was measured twice per week for 37 days by caliper and tumor growth curve was generated using GraphPad Prism software. Statistical significance of tumor growth curves was analyzed using two-way ANOVA. Animal images were taken 19 days after treatment. E and F, Body weight was measured for 37 days and figures were generated using GraphPad Prism software for both models. G and H, Representative images of vehicle and rapamycin treated tumor grafts stained by H&E and p-S6K for JHRCC12 and JHRCC228. Scale bars indicate 200μm and 50μm, respectively. Data are mean ± SD in A, B, E and F.

Figure 4.

Metabolic imaging reveals changes in glycolytic flux within 24 hours of treatment. A and B, Anatomic T₂-weighted MRI and representative 2DCSI grid with accompanying HP ¹³C 2DCSI data demonstrating conversion of HP pyruvate to lactate in a tumor. C, Representative image overlays of normalized HP lactate demonstrating in vivo response to treatment. D, Quantitative comparison of models after 24 hours of treatment with a single dose of rapamycin (15 mg/Kg) in vivo (n=6 replicates per group). The ¹³C Lac/total 13C carbon represents the lactate signal divided by the sum of pyruvate and lactate as derived by eq. 1. E, Steady-state pool size measurements of lactate in each model derived from tumor extractions and ¹H NMR analysis (n=6 replicates per group). All data reported as mean ± SD with significance as a student’s t-test with p<0.05. F, Schematic for in vivo hyperpolarized [1-¹³C] pyruvate metabolism. NS (not significant)