Abstract: “Previous studies demonstrated that volatile organic compounds (VOCs) in urine are potential biomarkers of breast cancer. An unanswered question is how urinary VOCs change over the course of tumor progression. To explore this, urine samples were collected from female Balb/c mice injected with 4T1.2 murine tumor cells in the tibia prior to tumor injection (20 samples) and over the next three weeks (12, 15, and 18 samples collected during weeks 1, 2, and 3 respectively). Samples were analyzed by headspace solid phase microextraction coupled to GC-MS QTOF. Univariate analysis showed many VOCs dysregulated by cancer, with some varying significantly during cancer progression and others not. PCA using panels of VOCs could distinguish both Cancer Weeks 1-3 from Control and Cancer Week 1 from Cancer Week 3 with > 90% sensitivity and specificity. Forward feature selection and linear discriminant analysis identified a unique panel of five VOCs that could distinguish Cancer Weeks 1 and 3 from Control with a fivefold cross validated (CV) area under the receiver operator characteristic (AUROC) equal to 0.95. The same model could also distinguish Cancer Week 1 from Cancer Week 3 with a fivefold CV AUROC equal to 0.97. The results of this study show that VOCs can be used to monitor breast cancer progression in mice models.
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Important research! Nice talk!
VOC’s in urine depend on the diet as well. What was the diet of the mice and were they consistent through out the study? For example low protein diets generate a different set of VOCs when compared to a high protein diet.
I agree with the previous comments about setting more controls.
Thank you for the question. All mice were fed mouse-chow ad libitum throughout the entirety of the study.
Great talk! Can you explain further why denaturing of urinary proteins is a necessary sample preparation step? Why is having urinary proteins that bind to nonpolar analytes in hydrophobic pockets an issue for doing GC-MS analysis? Also, is there a reason why the number of urinary samples collected each week varies? Or is it just due to the number of samples that you were able to collect each week based upon the urinary output of the mice?
Thank you for listening and your questions. In general, VOCs are nonpolar in nature and bind tightly with major urinary proteins in urine. If these proteins are not denatured, the VOCs do not efficiently partition into the sample headspace and therefore would not be sampled by the SPME fiber. Even though the samples are heated, previous optimization experiments showed that GHCl increases the ability to detect VOCs by SPME GC-MS. Additionally, GHCl increases the ionic strength of the urine solution, which increases the ability of VOCs to partition into the headspace. The number of samples collected by week varies as not all mice gave urine samples, or did not give enough urine (at least 50 microliters) each time samples were collected.
Thank you for your thoughtful comments and response. The control samples were collected over the course of two days prior to tumor cell injection. There were no significant differences in urinary VOC concentration between the two days in which urine was collected from control. The authors agree that collecting urine samples from healthy mice over the course of the experiment would have enabled samples to be age matched.
No mice were injected with a vehicle control to elucidate the VOC changes due to injection injury. The collaborator has utilized this model in many previous studies, with no observable injury caused by the injection itself.
Nice poster, talk, and subject !! So two questions here – one, your control samples were all taken during the same time? I ask since one could expect variations in urine composition over time, and would have several temporally spaced controlled samples be better for establishing baseline? Also, was a second control done where the mice were injected with just the vehicle (minus tumor cells) to establish results due to cancer?