Sniffer mice discriminate urine odours of patients with bladder cancer: A proof-of-principle study for non-invasive diagnosis of cancer-induced odours

Similar to fingerprints, humans have unique, genetically determined body odours. In case of urine, the odour can change due to variations in diet as well as upon infection or tumour formation. We investigated the use of mice in a manner similar to “sniffer dogs” to detect changes in urine odour in patients with bladder cancer. We measured the odour discrimination thresholds of mice in a Y-maze, using urine mixtures from patients with bladder cancer (Stage I) and healthy volunteers (dietary variations) as well as occult blood- or antibiotic drug metabolite-modulated samples. Threshold difference indicated that intensities of urinary olfactory cues increase in the following order: dietary variation < bladder cancer < occult blood < antibiotic drug metabolites. After training with patient urine mixtures, sniffer mice discriminated between urine odours of pre- and post-transurethral resection in individual patients with bladder cancer in an equal-occult blood diluted condition below the detection level of dietary variations, achieving a success rate of 100% (11/11). Furthermore, genetic ablation of all dorsal olfactory receptors elevated the discrimination thresholds of mice by ≥ 105-fold. The marked reduction in discrimination sensitivity indicates an essential role of the dorsal olfactory receptors in the recognition of urinary body odours in mice.

. Initial training of wild-type (WT) and ΔD mice for odour discrimination. (A), %Correct of WT mice (black closed circles). (B), %Correct of ΔD mice (red open squares) that genetically lost all dorsal olfactory receptors. Two alternative forced choice assays with target vs. non-target odorus were performed in a Y-maze. %Correct ± standard error of the mean (SE; 6-24 trials × 4-7 mice) is shown. Broken lines indicate the %Correct significantly above chance performance (P = 0.05 for 24 to 144 trials, 6-24 trials/mouse). Black arrowheads indicate chance levels (50%). Table ST1. %Correct of wild-type (WT) and ΔD mice in initial training for odour discrimination in a Y-maze.
Each WT mouse was performed twelve to twenty four trials of odour discrimination per day with an award of a drop of water for the concordance odour choice. The %Correct for P = 0.05 level was calculated for 36-126 trials (indicated by the chain line in Supplementary Fig. S1). SE, standard error. Table ST1. %Correct of WT and ΔD mice in initial training for odour discrimination in a Y-maze (continued).
Each ΔD mouse was performed six to twenty four trials of odour discrimination per day with an award of a drop of water for the concordance odour choice. The %Correct for P = 0.05 level were calculated for 24-144 trials (indicated by the chain line in Supplementary   Fig. S1). SE, standard error. Table ST2. %Correct of wild-type (WT) and ΔD mice for serial 10-or 100-fold diluted equi-occult blood urine samples in a Y-maze.
Ui, individual patient urine mixture (Ui) of equal volumes of five urine samples from each patient. N:Um, N-series urine mixture (Um) of equal volumes of 25 urine samples from five patient N6-N10 on five different days for each pre-and post-transurethral resection (post-TUR) after antibiotic exposure. Abbreviation: blad., bladder. Ranges of patient 1st-5th sampling days are shown, when day 0 is the ablative operation day of the patient. Table ST4. Blood, proteins, glucose testing in K-and A-series patient and healthy volunteer urine samples using urine test strips.
Ui, individual patient urine mixture (Ui) of equal volumes of five urine samples from each patient. K:Um, K-series urine mixture (Um) of equal volumes of 25 urine samples from five patient K3-K5, A1 and T2 on five different days for each pre-transurethral resection (pre-TUR) and antibiotic-exposure post-TUR except for a patient (four patients* + one patient**); Extra-dilution rates were determined by using linear regression of Hb = (0.15, 0.03) † for diluted urine = (10 -1 , 10 -2 ). H1-3, an equal-volume mixture of 18 urine samples from six healthy volunteer on three (n = 5) or two (n = 1, including one-day AM and PM sampling) different days; H4-6, an equal-volume mixture of 18 urine samples from the identical six healthy volunteers on three (n = 6) different days. Ranges of patient 1st-5th, healthy volunteer 1st-3rd ‡ and 4th-6th ¶ sampling days are shown, when day 0 is the ablative operation day of the patient or the fourth sampling day of the healthy volunteer.  S2. Urine odour discrimination performance of wild-type (WT) mice for individual patient urine mixtures (Ui) and antibiotic drug metabolite-containing urine mixtures (Um) in the Y-maze. (A), Odour discrimination between equi-occult blood individual patient pre-transurethral resection (TUR) Ui vs. post-TUR Um after re-training with the equioccult blood pre-vs. post-TUR Um. Extra-dilution rates for equi-occult blood Ums were 1/6* v/v, 1/9 § v/v, 1/10 & v/v, and 1/13** v/v. P value of paired difference in %Correct is indicated by # (P ≥0.05, not significant) and **(0.001 ≤P <0.01). (B), Odour discrimination between equally-diluted pre-vs. (during antibiotic exposure) post-TUR Um of other five patients with bladder cancer. Post-assay, 10 -3 pre-vs. post-TUR Um and identical Um pair: 10 -3 pre-vs. pre-TUR Um. Tasks performed at thresholds are marked by the star. Chain lines indicate the %Correct significantly above chance performance (P = 0.05 for 90 to 126 trials, 18 trials/mouse). Black arrowheads indicate chance levels (50%). Table ST5. %Correct of wild-type (WT) for serial diluted urine samples in a Y-maze. Extra-dilution rates for equi-occult blood urine samples were 1/6* v/v, 1/9 § v/v, 1/10 & v/v, 1/13** v/v, and 1/15 † v/v. (+)-car, (S)-(+)-carvone (w/w). An outlier of %Correct = 38.9 # (statistic T = 2.384 > T0 = 2.285, P = 0.05, one-way) among 12 data ※ for K5:Ui was excluded from analysis. P values of paired difference in %Correct was calculated by using the Student's t-test. ‡ P value is for the N:Um pair of the first and last %Correct, and ¶ P value is for the pair of the K5:Ui and last identical Um %Correct. SE, standard error. Table ST6. %Correct of wild-type (WT) mice for re-training and individual patient preand post-transurethral resection (post-TUR) urine mixture (Ui) discrimination.
Extra-dilution rates for equi-occult blood urine samples were 1/3 ¶ v/v, 1/6* v/v, 1/9 § v/v, 1/10 & v/v, and 1/13** v/v. P value is for the pair of successive %Correct. SE, standard error. Table ST7. %Correct of wild-type (WT) mice for serial 10-fold diluted mixtures of urine samples collected during antibiotic exposure after transurethral resection (post-TUR). SE, standard error.  The %Correct of ∆D mice (red open squares) that genetically lost all dorsal olfactory receptors. Two alternative forced choice assays with target vs. non-target odours were performed in a Y-maze. %Correct ± standard error of the mean (SE; 6-24 trials x 4-7 mice) is shown. Broken lines indicate the %Correct significantly above chance performance (P = 0.05 for 24 to 144 trials, 6-24 trials/mouse). Black arrowheads indicate chance levels (50%). 3.5 58.7 Each WT mouse was performed twelve to twenty four trials of odour discrimination per day with an award of a drop of water for the concordance odour choice. The %Correct for P = 0.05 level were calculated for 36-126 trials (indicated by the chain line in Supplementary Fig. S1). SE, standard error. Table ST1. %Correct of wild-type (WT) and ΔD mice in initial training for odour discrimination in a Y-maze. 58.5 Each ΔD mouse was performed six to twenty four trials of odour discrimination per day with an award of a drop of water for the concordance odour choice. The %Correct for P = 0.05 level were calculated for 24-144 trials (indicated by the chain line in Supplementary Fig. S1). SE, standard error.    59.4 Extra-dilution rates for equi-occult blood urine samples were 1/6* v/v and 1/13** v/v. (-)car, (R)-(-)-carvone (w/w); solvent, di(propylene) glycol. SE, standard error.      (5 days and T2 on five different days for each pre-TUR and antibiotic-exposure post-TUR except for a patient (four patients* + one patient**). Abbreviation: blad., bladder. Extra-dilution rates of urine samples were determined by using linear regression of Hb = (0.15, 0.03) † for diluted urine = (10 -1 , 10 -2 ). H1-3 , an equal-volume mixture of 18 urine samples from six healthy volunteer on three (n = 5) or two (n = 1, including one-day AM and PM sampling) different days; H4-6 , an equal-volume mixture of 18 urine samples from the identical six healthy volunteers on three different days. Ranges of patient 1st-5th, healthy volunteer 1st-3rd ‡ and 4th-6th ¶ sampling days are shown, when day 0 is the ablative operation day of the patient or the fourth sampling day of the healthy volunteer. Table ST4. Blood, proteins, glucose testing in K -, and A -series patient and healthy volunteer urine samples using urine test strips.   Table ST5. %Correct of wild-type (WT) mice for serial diluted urine samples and urine discrimination in a Y-maze.   Table ST6. %Correct of wild-type (WT) mice for re-training and individual pre-and post-TUR urine mixture (U i ) odor discrimination in a Y-maze. Extra-dilution rates for equi-occult blood urine samples were 1/3 ¶ v/v, 1/6* v/v, 1/9 § v/v, 1/10 & v/v, and 1/13** v/v. P value is for the pair of successive %Correct or the pair of A3 :U i and identical N :U m # . SE, standard error.