Modeling hyperpolarized lactate signal dynamics in cells, patient‐derived tissue slice cultures and murine models

Abstract

Determining the aggressiveness of renal cell carcinoma (RCC) noninvasively is a critical part of the diagnostic workup for treating this disease that kills more than 15,000 people annually in the USA. Recently, we have shown that not only the amount of lactate produced, as a consequence of the Warburg effect, but also its efflux out of the cell, is a critical marker of RCC aggressiveness and differentiating RCCs from benign renal tumors. Enzymatic conversions can now be measured in situ with hyperpolarized (HP) 13C magnetic resonance (MR) on a sub‐minute time scale. Using RCC models, we have shown that this technology can interrogate in real time both lactate production and compartmentalization, which are associated with tumor aggressiveness. The dynamic HP MR data have enabled us to robustly characterize parameters that have been elusive to measure directly in intact living cells and murine tumors thus far. Specifically, we were able to measure the same intracellular lactate longitudinal relaxation time in three RCC cell lines of 16.42 s, and lactate efflux rate ranging from 0.14 to 0.8 s−1 in the least to the most aggressive RCC cell lines and correlate it to monocarboxylate transporter isoform 4 expression. We also analyzed dynamic HP lactate and pyruvate data from orthotopic murine RCC tumors using a simplified one‐compartment model, and showed comparable apparent pyruvate to lactate conversion rate (kPL) values with those measured in vitro. This kinetic modeling was then extended to characterize the lactate dynamics in patient‐derived living RCC tissue slices; and even without direct measurement of the extracellular lactate signal the efflux parameter was still assessed and was distinct between the benign renal tumors and RCCs. Across all these preclinical models, the rate parameters of kPL and lactate efflux correlated to cancer aggressiveness, demonstrating the validity of our modeling approach for noninvasive assessment of RCC aggressiveness.

A continuous flow bioreactor system for high‐throughput hyperpolarized metabolic flux analysis

“…Revisiting the reported rate constant CV of this system, a comparison with other literature investigating cells in various bioreactor and NMR systems would provide valuable insights for evaluating the current performance. The mean CV of control groups (n ≥ 3) in six studies 18,23,25,26,37,40 along with our own findings (Figure S2) equaled C mean V ¼ 23% AE 15% (range 3%-46%). Details of the pooled data can be found in Table S2.…”

“…We were able to detect downstream products of pyruvate in leukemic cells with all three wells independently. Reproducibility experiments revealed a sufficient robustness, and longitudinal experiments showed preservability in the system for at least 6 h. Additionally, tissue preservation was also shown to be viable for at least 1 h. Although both high-throughput HP setups and HP bioreactor setups previously have been demonstrated, 3,4,[18][19][20][21][22][24][25][26] to the best of our knowledge, this is the first demonstration of a combined high-throughput bioreactor for HP experimentation.…”

“…Data from these studies suggest that the CV varies largely between both cell line and setup used for acquiring HP data. As an example, Ahamed et al 18 showed that the HK-2 cell line had a CV of 6% and that of the UOK262 cell line had a CV of 46% on the same bioreactor and NMR system.…”

“…Combining a bioreactor system with hyperpolarized spectroscopy has proven very useful in metabolic studies of ex vivo and in vitro systems; however, these are limited by low throughput. 4,[18][19][20][21][22][23] Different high-throughput hyperpolarization approaches have recently been demonstrated, but these lack the active biological environments that are necessary for longitudinal and therapeutic response monitoring studies. 3,[24][25][26] An additional advantage of the ex vivo bioreactor application with the hyperpolarization technology is that it provides an opportunity for examinations of multiple hyperpolarized biomarkers simultaneously, one that is not applicable in vivo in patients.…”

“…Bioreactor systems can play an important role because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. Combining a bioreactor system with hyperpolarized spectroscopy has proven very useful in metabolic studies of ex vivo and in vitro systems; however, these are limited by low throughput 4,18–23 . Different high‐throughput hyperpolarization approaches have recently been demonstrated, but these lack the active biological environments that are necessary for longitudinal and therapeutic response monitoring studies.…”