The ability of C. elegans to sense attractive or repulsive odors is primarily facilitated by three pairs of olfactory neurons: AWA, AWB, and AWC. In the majority cases, worms detect attractive compounds using AWA and AWC olfactory neurons, while AWB neurons are responsible for sensing repulsive volatile compounds [24, 25]. Although chemotaxis is controlled by a complex set of neural and cellular mechanisms, it can be objectively quantified using chemotaxis assays with C. elegans nematodes [17, 23, 26, 27]. Some vertebrates have been shown to be able of detecting tumor growth [16, 28], however, the use of these animals in clinical applications is not feasible due to a number of limitations. To facilitate potential screening, it is essential to utilize a simple model organism that is suitable for high-throughput analysis. C. elegans meets these criteria because it has a highly developed chemosensory system, which allows it to detect a broad spectrum of volatile compounds. For example, Hirotsu et al. [19] conducted a study examining the chemotactic activity of C. elegans toward urine samples, serum, and tumor tissues of cancer patients. The highest level of attractive chemotaxis was observed in nematodes in response to urine samples, while the lowest level was detected in response to serum samples. According to these findings, the researchers postulated that the serum of cancer patients may also contain other odors that could repel C. elegans nematodes [19]. Furthermore, the researchers observed that olfactory neuron-ablated nematodes were not attracted to cancer patient urine, indicating that C. elegans is capable of detecting specific odors present in such samples. This finding provides a rationale for the N-NOSE (Nematode Nose) multi-cancer screening test, which has been shown to exhibit high sensitivity in the detection of early-stage cancers [20, 21, 29]. In addition to these findings, the objective of this study was to validate the documented results in the context of our institutional setting, with a particular focus on colorectal and mammary carcinomas. A significant difference in CI was observed between control and tumor samples (p < 0.001, Fig. 1). A subgroup analysis of urine samples from the patients with colon, rectosigmoid junction (C18/C19), and breast cancer (C50) also revealed significant differences compared to controls (p < 0.001, Figure S2). The test demonstrated high sensitivity, correctly identifying nearly all individuals with cancer (98% true positive results). However, the test demonstrated a lower specificity (62%), indicating that it correctly identified 62% of healthy individuals (i.e., true negative results). Conversely, 38% of individuals who tested positive in the control group did not have cancer, indicating a false positive result. This phenomenon can be attributed to the potential involvement of VOCs in non-cancerous pathologies such as tuberculosis [30], diabetes [31], or other as yet unidentified pathologies, which could compromise the assay's specificity. The positive predictive value (67%) indicates that in 67% of positive tests, the presence of cancer was confirmed. In contrast, the negative predictive value (97%) indicates a high degree of accuracy in the exclusion of cancer when the test result is negative. Taken together, the test demonstrated satisfactory performance, with a 78% detection rate (73% for breast cancer and 72% for colorectal cancer, respectively).

While mammography remains the primary method for breast cancer screening in women over the age of 40 (e. g., the European Commission recommends mammography screening starting at the age of 45), its use in younger cohorts is limited [32]. The limited use of mammography in younger cohorts is attributed to the presence of dense glandular tissue and the potential exposure to ionizing radiation associated with the procedure [33]. However, the increasing prevalence of breast cancer in younger women and the inherently aggressive nature of breast cancer in younger patients require the investigation of alternative approaches to detection [34, 35]. Since our results did not show a significant difference in CI between age groups (p > 0.05), it can be hypothesized that the aforementioned test could represent a potential benefit for the early detection of malignancies in all age categories. Furthermore, methods such as the C. elegans chemotaxis assay have the potential to serve as a valuable marker for breast cancer detection, even in younger women for whom mammography is often contraindicated. However, this assertion requires further validation through additional experimental studies and consideration of other important factors, such as cancer stage, tumor histology, patient age, and comorbidities that may influence laboratory results.

Another prevalent malignant neoplasm among the Western population is colorectal cancer. It is the second most common diagnosis in women, following only breast cancer, and the third most common in men, surpassed only prostate and lung cancer [36]. Currently, fecal occult blood testing (FOBT) is the primary screening modality for colorectal cancer [37]. However, the limitations associated with FOBT have prompted researchers to explore alternative methods. These initiatives are guided by two key objectives: first, to foster patient participation in screening programs, and second, to minimize the burden of unnecessary colonoscopies resulting from false positive FOBT outcomes. Given the potential for increased patient compliance, urine-based assays hold significant promise. Consequently, scientific research is currently being conducted in order to develop new colorectal cancer screening tests that utilize metabolome and volatilome analysis of urine samples [38]. In view of the increasing prevalence of colorectal cancer in South Korea, Lee et al. [39] conducted an RNA sequencing analysis of tumor and adjacent normal tissue samples from 214 patients with this disease, with the aim of obtaining their transcriptomic profiles. The study yielded valuable insights into the molecular mechanisms involved, and may serve as a platform for identifying potential target genes for this disease [39]. The combination of volatilome analysis with other -omics data, including genomics, transcriptomics, and proteomics, has been proposed as a means of enhancing diagnostic accuracy and deepening the understanding of the molecular mechanisms underlying cancer [38, 40].

In a study by Kusumoto et al. [20], the feasibility of using a cancer screening method based on the chemotaxis of C. elegans (N-NOSE tests) was evaluated in post-surgical patients with colon or stomach cancer. The findings demonstrated that N-NOSE exhibited superior performance compared to conventional tests (based on the detection of carcinoembryonic and carbohydrate antigen) in evaluating the completeness of cancer resection. Researchers concluded that the use of C. elegans chemosensory responses facilitated the detection of gastrointestinal malignancies in urine samples with a high degree of sensitivity [20] Similarly, Lanza et al. [41] observed that C. elegans exhibited a significant preference for samples collected from women with breast cancer, while urinary controls demonstrated the capacity to act as avoidance-promoting chemorepellents. The sensitivity of the test in the experiment was 75%, while the specificity was 97.22%, resulting in an accuracy of 86.11% [41].

The findings of our research and numerous other studies regarding the exceptional sensitivity of C. elegans chemotaxis assays indicate their potential utility in the early identification of cancer, a critical stage for therapeutic intervention. For example, C. elegans-based detection tests demonstrated 100% sensitivity for breast cancer and 88.9% sensitivity for colorectal cancer [42]. However, it is important to note, that the methodology used in the study mentioned above differed slightly from that used in the present study. It should be noted that the current study used a single dilution of the urine sample, whereas Inaba et al. [42] used data from two dilutions (tenfold and 100-fold). However, our results were consistent with those of previous studies, indicating that C. elegans hermaphrodites can accurately identify urine samples from colorectal and breast cancers with a high degree of sensitivity compared to control samples. Although our study focused on the identification of only two distinct cancers, breast and colorectal, a novel assay that makes use of C. elegans' behavioral responses shows promise in the identification of a wide range of tumor types from urine samples [21, 43, 44]. Compared to current screening techniques, which are typically cancer-specific and require distinct tests for each form of malignancy, this could represent a paradigm shift. Furthermore, di Luccio et al. [12] propose that C. elegans-based cancer screening assays offer several advantages over the "one-test-one-cancer" approach, including protein tumor markers and cfDNA/ctDNA technologies. Such non-invasive tests may prove to be more cost-effective and easier to implement on a large scale within cancer screening programs. Additionally, the nematode C. elegans is highly sensitive to environmental changes and is capable of detecting even very low concentrations of specific substances, which could facilitate the identification of early-stage cancer. Despite the promising potential of chemotactic assays with C. elegans for cancer screening represent a promising method, several challenges remain to be addressed to ensure the accuracy and clinical utility of this method. A biological sample, such as urine, contains a variety of substances other than those associated with cancer. These substances can mask or modify chemotactic signals, which can lead to false positive or negative results.

Future research aimed at identifying the urinary chemoattractants responsible for the response of the helminth may provide new insights into the mechanisms of carcinogenesis. This is of particular importance in light of the increasing global prevalence of cancer, with an alarming tendency for early-stage malignancies. Early detection of cancer can be an effective tool, as it allows the administration of the most appropriate treatment before patient´s present symptoms. This indicates the potential significance of C. elegans-based screening in this context.

In conclusion, the early diagnosis of cancer is a key factor in the successful treatment of the disease. The findings of our study confirm the remarkable sensitivity of C. elegans nematodes to discriminating volatile organic compounds associated with cancer in urine samples. It appears that this tiny nematode may help in the development of a rapid, painless, and cost-effective diagnostic screening test for cancer detection with high sensitivity based on the simple collection of a readily available biological sample (urine). This method demonstrates potential, yet further investigation is required to ascertain its suitability for widespread clinical application, particularly in differentiating between malignant and benign conditions. Therefore, further research is necessary to develop more effective diagnostic tools.