Highlights
- •Ants can rapidly be conditioned to associate the odor of cancer cells with a reward
- •Ants discriminate between cancerous and healthy cells and between two cancerous lines
- •Discrimination relies on volatile organic compounds that are specific of cell lines
Summary
Cancer is among the world’s leading causes of death. A critical challenge for public health is to develop a noninvasive, inexpensive, and efficient tool for early cancer detection. Cancer cells are characterized by an altered metabolism, producing unique patterns of volatile organic compounds (VOCs) that can be used as cancer biomarkers. Dogs can detect VOCs via olfactory associative learning, but training dogs is costly and time-consuming. Insects, such as ants, have a refined sense of smell and can be rapidly trained. We show that individual ants need only a few training trials to learn, memorize, and reliably detect the odor of human cancer cells. These performances rely on specific VOC patterns, as shown by gas chromatography/mass spectrometry. Our findings suggest that using ants as living tools to detect biomarkers of human cancer is feasible, fast, and less laborious than using other animals.
Introduction
Cancer cells possess specific features such as a deregulated cellular energetic metabolism, the ability to self-sustain themselves with proliferating signals or by exploiting tumor-promoting inflammation factors (Hanahan and Weinberg, 2000, Hanahan and Weinberg, 2011). Their metabolism produces volatile organic compounds (VOCs) that can act as biomarkers for cancer diagnosis using, for instance, gas chromatography or artificial olfactory systems (Krilaviciute et al., 2015; Lavra et al., 2015). However, the results of GC-MS analyses are extremely variable and most of the E-nose systems need to be optimized and are still at the prototype stage (Behera et al., 2019). Millions of years of evolution have shaped animals’ finely-tuned olfactory systems, which detect small odorant concentrations and have the computational power for discriminating among complex odorant blends. Dogs’ noses are well suited for medical diagnosis (Guest and Otto, 2020) and used for the detection of cancer-specific VOCs (Mazzola et al., 2020; Thuleau et al., 2019; Pirrone and Albertini, 2017; Schallschmidt et al., 2015), but training dogs in associative learning paradigms is expensive and time consuming (Pirrone and Albertini, 2017). The conditioning phase, in particular, takes several months and hundreds of trials are needed before the dog is operative. Consequently, studies report low sample sizes both in terms of individual dogs and numbers of tests performed. For instance, in one study, 90.3% of correct identification was achieved using two dogs, 5 months of training, and 1531 conditioning trials to perform 31 memory tests (Thuleau et al., 2019).Compared to dogs, insects can be easily reared in controlled conditions, they are inexpensive, they have a very well-developed olfactory system (Rössler and Stengl, 2013), and hundreds of individuals can be conditioned with very few trials (Guerrieri et al., 2005; Piqueret et al., 2019; Giurfa and Sandoz, 2012). There is evidence that insects can detect VOCs from cancer cell lines. In fruit flies, for instance, the odors from different cancer cell lines evoked specific olfactory receptor activity patterns in the antenna, suggesting that such insects could be used as cancer biodetectors (Strauch et al., 2014) by employing in vivo calcium imaging, a complex and expensive technique. Here, we combined the use of insects (the ant Formica fusca) with low-cost, easily transferable, behavioral analysis to provide a robust, yet affordable, bio detector tool for cancer VOCs. We previously demonstrated that individual worker ants of this species quickly learn to associate an olfactory stimulus with a food reward and retain this information for an extensive period of time (several days) (Piqueret et al., 2019). In the present study, individual ants were trained to associate the odor of a cell sample with food reward, and later had to discriminate this learned sample against a new one. The principle is that of classical conditioning, the association of an unconditioned stimulus (US, in our case a reward consisting of sucrose solution) with an initially neutral stimulus (the odor of cancer cells) that becomes a conditioned stimulus (CS) producing the response in the absence of the US during an unrewarded discrimination test.
Results
Ants can detect cells through olfaction
Individual ants (n = 36) were subjected to three training trials in a circular arena (Figure 1A), during which the odor of a human cancer cell sample (IGROV-1, ovarian cancer) cultured in medium (DMEM – Dulbecco modified Eagle’s minimal essential medium) was associated with a reward of sugar solution. The time the ants needed to find the reward decreased over the trials (Figure 1C and Table S1), indicating that they had learned to detect the presence of cells based on their emitted volatiles. This was confirmed by ants performing two consecutive memory tests in which no reward was present. In a similar circular arena (Figure 1B), we measured the time spent by the ants investigating two different odors: the odor of the cells (IGROV-1) (conditioned stimulus) and the odor of the culture medium alone (DMEM) (novel odor). Two empty tubes were also present as controls (Table S2). During these memory tests, ants spent significantly more time near the conditioned odor (cancer cells) than near the culture medium alone (Figure 1D), demonstrating that ants can recognize the presence of cells in a sample.
