Using exogenous volatile organic compound (EVOC) probes to target tumour-associated aldo-keto reductase activity: a potential tool to detect lung cancer

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

Owlstone Medical, Analytical Science, Cambridge, United Kingdom, Owlstone Medical, Clinical and Translational Science, Cambridge, United Kingdom, Owlstone Medical, Leadership team, Cambridge, United Kingdom.

Email: breathbiopsy@owlstone.co.uk

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Introduction
Detection and treatment of early-stage lung cancer confer the best chance of long-term cure. Non-invasive breath tests targeting cancer metabolism could improve detection. Aldo-keto reductase (AKR) 
enzymes including AKR1B10 and AKR1B15 are upregulated in lung cancers to reduce aldehydes, 
produced by lipid peroxidation, into corresponding alcohols. These aldehydes and alcohols are volatile 
and could be detected in breath to detect cancer. Here, we measure AKR1B10/B15 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
We modulated AKR activity using small compounds (tolrestat and JF0064) in A549 and H460 lung 
cancer cell lines, as well as by developing A549 CRISPR-cas9 AKR1B10/B15 knockouts. Vehicle groups 
and mock-Cas9 (wildtype) were used as controls. AKR activity was measured using a colorimetry assay 
and eVOC probe. For this last approach, we treated cells with aldehydes (multiple classes) and 
collected supernatants at multiple timepoints for the analysis of volatiles. Substrate and bioproduct 
levels were analyzed using headspace HiSorb extraction and GC-MS. To assess evaporation, a parallel 
plate was set up with the same aldehydes spiked in the cell culture medium (no cells).

Results and Discussions
Using a colorimetric assay, a dose-dependent effect of tolrestat in AKR activity in A549 and H460 was 
observed. However, JF0064 does not affect overall AKR activity in both cell lines. In addition, 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, cells with a knockout or inhibited AKR activity, including using JF0064 
AKR inhibitor compound, show reduced production of alcohol bioproducts.

Conclusion
Using in vitro study of lung cancer cells, we showed the potential to monitor the metabolic conversion 
of administered EVOC Probe aldehydes into alcohols by AKRs. Our data suggest the potential to use 
these aldehydes as EVOC Probes for cancer early detection on the breath by targeting upregulated AKR 
activity in lung cancer tissue.