Tumour-associated aldo-keto reductase activity targeted with exogenous volatile organic compound probes to detect lung cancer

Mariana Ferreira Leal 1, Alexandra Martin 1, Christiaan Labuschagne 1, Rob Smith 1, Connor Clarke 1, Ben Taylor 1, Matthew Hart 1, Max Allsworth 1, Billy Boyle 1 

1. Owlstone Medical Ltd., Cambridge, Cambridgeshire, UK

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Abstract: 

Introduction 

Early detection could improve cancer survival, particularly for lung cancer, but this requires accessible screening methods. Non-invasive breath tests targeting cancer metabolism could improve detection. Tumour cell metabolism is adapted for survival in the tumour microenvironment. Rapid growth, poor blood flow and persistent genetic errors in cancer cells contribute to a high level of oxidative stress characterized by an increase in reactive oxygen species (ROS). In turn, ROS promote destructive processes such as lipid peroxidation which produce aldehydes. Human lung cancers increase the expression of aldo-ketoreductase (AKRs) enzymes, including AKR1B10 and AKR1B15, to help process these excess aldehydes and reduce them to alcohols. These aldehydes and alcohols are volatile and could possibly be detected in breath to detect cancer. Here we measure AKR1B10 and AKR1B15 activity in lung cancer cells by administering aldehydes as exogenous volatile organic compound (EVOC) probes and monitoring alcohol production through in vitro headspace analysis.  

Material and Methods 

AKR activity was firstly assessed using a commercial colorimetric assay validated to AKR1B10, AKR1C1 and AKR1C3 activity analysis. Then, we measured AKR1B10/B15 activity in vitro in lung cancer cells by administering several aldehydes from different classes as EVOC® Probes and monitoring alcohol production. To assess relationships between volatiles and AKR metabolism we tested the effect of two AKR inhibitors (tolrestat and JF0064) in A549 and H460 cell lines, as well as in six A549 CRISPR-cas9 knockout mutants of AKR1B10/B15 (3 for each target) and mock-Cas9 control (wildtype cells). To assess evaporation, a parallel experiment analysed the same volatiles in cell culture medium (no cells). Supernatants was collected at several timepoints after the addition of aldehyde probes and analysed using an automated headspace HiSorb extraction platform (Centri) and GC-MS.  

Results and Discussions 

A dose-dependent effect of tolrestat in AKR activity in A549 and H460 was observed after 24h of treatment. However, JF0064 does not affect overall AKR activity after 24h of treatment in A549 and H460 cell lines. Using a commercial colorimetric AKR activity, a reduction in AKR activity of over 50% was detected in AKR1B10 knockouts and variable levels were observed in AKR1B15 knockout, with one clone showing AKR activity more similar to wild-type cells. Then, using our headspace analysis platform, we detected lower aldehyde and higher alcohol levels in wildtype and vehicle control samples compared to evaporation controls, confirming that AKRs are active in these cells. Moreover, as expected, activity was reduced by AKR inhibition. Overall, we observed no significant difference in aldehyde abundance but did see reduced production of alcohols in cells with inhibited AKR activity, including by the use of JF0064 compound. 

Conclusion 

Using in vitro study of human lung cancer cells, we have demonstrated the potential to monitor the metabolic conversion of administered EVOC Probe aldehydes into alcohols by AKRs, and have used AKR inhibition/silencing to show that this relationship is specific and sensitive to manipulation. Our data suggests the potential to use of these aldehydes as EVOC Probes for cancer early detection on breath by targeting upregulated AKR activity in lung cancer tissue.