Real‐time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor

Abstract

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) 13C MR and interrogated HP [1-13C] lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP 13C MR studies were performed on 3 immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC and UOK262, an aggressive and metastatic RCC. The intra- (Lacin) and extracellular (Lacex) HP lactate signals were robustly resolved in dynamic 13C spectra of the cell lines due to a very small but reproducible chemical shift difference(0.031 ± 0.0005 ppm). Following HP [1-13C] pyruvate delivery, the ratio of HP Lacin/Lacex was significantly lower for UOK262 cells compared to both UMRC6 as well as the HK2 cells due to a significant (p<0.05) increase in the Lacex pool size. The Lacin/Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient derived RCC tissue slices using HP [1,2-13C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-13C]glutamate consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export.

Separation of extra- and intracellular metabolites using hyperpolarized 13C diffusion weighted MR

“…UOK262 cells were used for this experiment because they are known to transport relatively large amounts of lactate out into the extracellular environment [7,8]. Fig.…”

“…This finding is consistent with a prior published MR compatible cell culture bioreactor study of UOK262 cells, in which flow-rate modulations in the bioreactor demonstrated that hyperpolarized 13 C lactate was transported out of the cell and washed-away during the time frame of the hyperpolarized MR study [7]. Furthermore, a recent publication studying UOK262 cells in a 5 mm MR compatible bioreactor, which enabled a spectral line-width narrow enough for the separation of the intra- and extracellular hyperpolarized 13 C lactate peaks, showed that when subjected to DIDS inhibition, the ratio of intra- to extracellular lactate increased by 1.64-fold [8]. This study showed a 2.35-fold increase in the intra- to extracellular lactate ratio with DIDS inhibition (Fig.…”

“…After several hyperpolarized 13 C acquisitions, the same batch of cells were treated with 1 mM 4,4′-Di-isothiocyanostilbene-2,2′-dis ulphonic acid (DIDS), an inhibitor of monocarboxylate transporters (MCTs) [8], within the bioreactor for 40 min before injection of the hyperpolarized solution to modulate the extra- and intracellular distribution of lactate pools. To clearly see the relative change in the ratios before and after treatment, the pre-treatment data was normalized to 1.…”

“…In that ex vivo study, metastatic RCC UOK262 cells rapidly transported hyperpolarized 13 C lactate out of the cells during the course of the hyperpolarized 13 C MR experiment [7]. Additionally, it was recently shown that the extra- and intracellular hyperpolarized 13 C lactate signal can be directly measured in RCC cells in the same bioreactor system, and confirmed that the metastatic RCC cells have higher extracellular lactate pool [8]. By correlating the hyperpolarized lactate signals to the relative expression levels of MCT4 between the indolent and metastatic RCC cells, these studies demonstrated that the localization of hyperpolarized 13 C lactate between the extra- and intracellular environment could provide valuable information concerning tumor aggressiveness and metastatic potential.…”

The use of hyperpolarized carbon-13 magnetic resonance for molecular imaging

“…Renal cell carcinomas (RCCs) are a broad group of tumors affecting the kidney that have been studied in some detail using hyperpolarized 13 C. In vitro HP [1- 13 C]pyruvate studies of two different RCC cell lines (UOK262 and UMRC6) and healthy proximal tubule epithelial (HK-2) cells have shown that the observed pyruvate to lactate ratio and the ratio of HP intracellular lactate to extracellular lactate (efflux ratio) can be used to distinguish the three different cell lines from each other [262,263]. Furthermore, the HP lactate efflux ratio strongly correlates with MCT4 mRNA expression, suggesting its potential use as a biomarker for differential diagnosis of RCCs.…”