Metabolic imaging using hyperpolarized ¹³C‐pyruvate to assess sensitivity to the B‐Raf inhibitor vemurafenib in melanoma cells and xenografts

Abstract

Nearly all melanoma patients with a BRAF‐activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) 13C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP 13C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate‐to‐lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT‐PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time‐points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate‐to‐lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi‐mediated impairment of glycolysis. The paradoxical increase of pyruvate‐to‐lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.

Multi‐sample measurement of hyperpolarized pyruvate‐to‐lactate flux in melanoma cells

“…Altered cell metabolism is a key component of the downstream consequence of activating BRAF mutations in melanoma, 38 with evidence of reduced oxidative phosphorylation activity and increased glycolysis 39 . The data presented here are in agreement with the current hypotheses that indicate treatment with vemurafenib leads to a reduction of in vitro aerobic glycolysis and glucose consumption in BRAF‐mutant melanoma cells 22,23 …”

“…In the experimental protocol described here, deuterated solvation of HP [1‐ 13 C] pyruvate combined with a highly sensitive microcoil system with a micro‐reservoir in the vertical plane, provided a means of increasing the throughput of in vitro DNP‐MRS experiments. Traditional HP experiments using cell suspensions require more than 10 7 cells, 22–25 which utilizes excessive resources and incubator space. The microcoil system allows for reproducible measurements of k PL with ~10 5 cells.…”

Enhancing Cancer Diagnosis with Real-Time Feedback: Tumor Metabolism through Hyperpolarized 1-13C Pyruvate MRSI

“…BRAF is one of the genes upregulated in melanoma cells, and inhibition of BRAF signaling has been a top target for therapy. Metabolic consequences as a result of the inhibition of BRAF by vemurafenib have been investigated using HP [1- 13 C] pyruvate MRSI in melanoma cell lines [67][68][69]. One of the studies showed a reduction in pyruvate-to-lactate conversion in the BRAF mutant melanoma cell line compared to the vehicle control, whereas an increased pyruvate-to-lactate conversion was recorded in the BRAF wildtype melanoma cell line compared to vehicle control [67].…”

Tumor Metabolism Is Affected by Obesity in Preclinical Models of Triple-Negative Breast Cancer

“…Hyperpolarized 13 C-NMR data were acquired, as previously described [26]. 1-13 C pyruvic acid (Sigma-Aldrich, Saint Louis, MO, USA) was mixed with 15 mM trityl radical OXO63 and doped with 2 mM gadolinium (Guerbet, Villepinte, France).…”