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

Abstract

Hyperpolarized [1‐13C] pyruvate can be used to examine the metabolic state of cancer cells, highlighting a key metabolic characteristic of cancer: the upregulated metabolic flux to lactate, even in the presence of oxygen (Warburg effect). Thus, the rate constant of 13C exchange of pyruvate to lactate, kPL, can serve as a metabolic biomarker of cancer presence, aggressiveness and therapy response. Established in vitro hyperpolarized experiments dissolve the probe for each cell sample independently, an inefficient process that consumes excessive time and resources. Expanding on our previous development of a microcoil with greatly increased detection sensitivity (103‐fold) compared with traditional in vitro methods, we present a novel microcoil equipped with a 10‐μL vertical reservoir and an experimental protocol utilizing deuterated dissolution buffer to measure metabolic flux in multiple mass‐limited cell suspension samples using a single dissolution. This method increases efficiency and potentially reduces the methodological variability associated with hyperpolarized experiments. This technique was used to measure pyruvate‐to‐lactate flux in melanoma cells to assess BRAF‐inhibition treatment response. There was a significant reduction of kPL in BRAFV600E cells following 24 and 48 hours of treatment with 2 μM vemurafenib (P ≤ .05). This agrees with significant changes observed in the pool sizes of extracellular lactate (P ≤ .05) and glucose (P ≤ .001) following 6 and 48 hours of treatment, respectively, and a significant reduction in cell proliferation following 72 hours of treatment (P ≤ .01). BRAF inhibition had no significant effect on the metabolic flux of BRAFWT cells. These data demonstrate a 6‐8–fold increase in efficiency for the measurement of kPL in cell suspension samples compared with traditional hyperpolarized in vitro methods.

Real-Time Polarimetry of Hyperpolarized 13C Nuclear Spins Using an Atomic Magnetometer

“…The polarization, P (a dimensionless number ranging from 0, for unpolarized spins, to ±1, when spins are completely polarized), determines the effective concentration of the substrate seen by MRI and therefore is a crucial parameter in kinetic models of spin-label transfer along a metabolic pathway, especially in cases where hyperpolarized agents are administered to the subject repeatedly. 15 Additionally, any exogenous substance introduced in vivo is subject to strict quality control and dose requirements. Given that many hyperpolarization technologies are at an early development stage and not always consistent, P is currently a control variable; for instance, in dissolution dynamic nuclear polar-ization, polarization is monitored both before 16,17 and after 18,19 sample dissolution.…”

“…The procedure simulates a situation where fractions of a hyperpolarized sample are inserted into an MRI scanner one after the other. 15 The aliquots were then transferred sequentially by syringe into the magnetic shield located approximately 1 m from the polarizer and containing the OPM and sample chamber used for the 2-T-prepolarized water sample. A solenoid coil provided a guiding magnetic field 25 of 30 μT over the syringe during transport.…”

“…In these and other applications, such as noble gas MRI, there are several motives to quantify nuclear spin hyperpolarization. The polarization, P (a dimensionless number ranging from 0, for unpolarized spins, to ±1, when spins are completely polarized), determines the effective concentration of the substrate seen by MRI and therefore is a crucial parameter in kinetic models of spin-label transfer along a metabolic pathway, especially in cases where hyperpolarized agents are administered to the subject repeatedly . Additionally, any exogenous substance introduced in vivo is subject to strict quality control and dose requirements.…”

Real-time polarimetry of hyperpolarized $^{13}$C nuclear spins using an atomic magnetometer

“…In these and other applications, such as noble-gas MRI, there are several motives to quantify nuclear spin hyperpolarization. The magnitude or degree of polarization, P (a dimensionless number between zero and one), determines the effective concentration of the substrate seen by MRI and therefore is a crucial parameter in kinetic models of spin-label transfer along a metabolic pathway, especially in cases where hyperpolarized agents are administered to the subject repeatedly 15 . Additionally, any exogenous substance introduced in vivo is subject to strict quality control and dose requirements.…”

“…Immediately after the dissolution process, two 1.0 cm 3 aliquots of the polarized 13 C sample were collected and stored in the approximately 5 mT fringe magnetic field of the DNP polarizer. The procedure simulates a situation where fractions of a hyperpolarized sample are inserted into an MRI scanner one after the other 15 . The aliquots were then transferred sequentially by syringe into the magnetic shield located approximately 1 m from the po-larizer and containing the OPM and sample chamber used for the 2-T-prepolarized water sample.…”