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pH Monitoring of Urine and Tumor Microenvironments in Rats

High Spatial Resolution Measurements Controlled with the Manual Micromanipulator

T. Kiskova1, Z. Steffekova2, M. Karasova2, N. Kokosova1
1P. J. Šafárik University in Košice, Faculty of Sciences, Košice, Slovakia
2University of Veterinary Medicine and Pharmacy in Košice, Slovakia

The aim of our study was to monitor pH of urine samples (< 100 µL) and of tumor microenvironments of anesthetized rats in a minimal invasive way. The small urine volumes of rats or mice make pH measurements difficult, as standard pH electrodes usually need a minimal volume of several milliliters to function. However, the amount of rat urine obtained is frequently < 100 µL, as in our study. Additionally, in vivo pH monitoring of tumor microenvironments is technically quite challenging. The PreSens Manual Micromanipulator (MM) together with  a needle-type (NTH) pH Microsensor offered a simple and effective way to do so. Our results show that pH of urine and tumor microenvironments was lower in tumor bearing rats compared to healthy individuals and tissues.

Solid tumors contain various cell types, such as cancer-, stromal-, or immune cells, but also proteins and various chemical messengers [1]. The interaction between these cells form the unique tumor microenvironment and influence both tumor growth and development [2]. As a result, pH differences have been observed in the tumor microenvironment. While the extracellular pH of malignant solid tumors is acidic, in the range of 6.5 to 6.9, the pH of normal tissues is significantly more alkaline around 7.2 to 7.5 [3]. Systemic acid-base balance is maintained in large parts by renal excretion of excess ions into the urine. Diet composition, body weight, as well as pathological state, such as cancer, may influence urine pH through the production of certain ions and organic acids [4]. We thus tested the hypothesis whether tumor formation in rats has an effect on urine pH. In our experiments a micro fiber optic pH meter with needle-type housed pH Microsensors (PreSens, Germany) was used. The pH Microsensors are miniaturized glass fiber pH sensors, with sensor tips below 150 µm, designed for measuring small sample volumes and with high spatial resolution. For measurements inside tumor tissue we additionally applied a Manual Micromanipulator (MM, PreSens, Germany) as it allows safe insertion of the microsensor and precise localization inside the sample.

Materials & Methods

Female Sprague-Dawley rats (n = 20) aged 31 days and weighing 100 - 130 g were obtained from Velaz (Únetice, Czech Republic) and adapted to standard vivarium conditions with temperature ranging from 21 - 24 °C, a relative humidity of 50 - 65 % and artificial 12:12 h light: dark regime. Rats were fed standard pellets (Peter Miško, Slovakia) and drank only tap water. Breast cancer was induced in 10 animals (NMU group) by 2 intraperitoneal doses (50 mg/kg body weight) of N-Methyl-N-Nitrosourea (NMU, Sigma, Germany) on the 43rd and the 50th postnatal day. The remaining 10 animals constituted the healthy control group (CON). The experiment was conducted according to the principles provided in Law No. 377 and 436/2012 of Slovak Republic for the Care and Use of Laboratory Animals. With the aim to monitor the pH of tumor development over time, 5 rats were randomly selected and anesthetized once a week over a 4 week period (10th to 13th postnatal week). Anesthesia was applied by isoflurane introduced below the false floor in an induction chamber of 1000 mL capacity. A 4 % concentration of isoflurane gas was adequate for short-term anesthesia (0.2 mL/L chamber volume). A schematic illustration of the experimental set-up for pH measurements in tumor tissue is shown in Figure 2. At the final week of the experiment (14th postnatal week), urine samples from each rat were obtained. During handling on a sterile underlay, the rats started to urinate spontaneously. The urine was immediately collected into sterile microtubes. pH was measured using pH Microsensors (PreSens, Germany) after urine collection. The measuring latency for one sample was shorter 10 minutes. The data were evaluated using GraphPad Prism 4.0 statistical software (GraphPad Software Inc., USA). The results were analyzed using a Mann-Whitney test. In order to see whether the pH value of urine correlated with the amount of tumor tissue in rats, an online Pearson Correlation coefficient calculator was used (www.socscistatistics.com). Significance levels are indicated in the legend of each figure.

pH Measurement Results

We discovered that the pH of the tumor microenvironment was only slightly decreased (pH 7.24 - 7.33), compared to the tissue of healthy individuals. Unfortunately, the uncontrolled growth and spread of the tumor tissue made a repeated monitoring over several weeks of a spatially defined tumor region difficult (see Fig. 3). That is why our data could not give conclusive results about how pH alters during the tumor formation. There are some studies indicating that urine pH changes during cancer development, despite the acid-base balance in the organism [4]. Our results clearly show pH changes in the urine of tumor bearing animals compared to healthy individuals (P < 0.001, Tab. 1). The urine pH only showed a very weak positive correlation (R = 0.21) with the amount of tumor tissue. This is probably caused by the low replication of the experiment (n = 10).

Conclusion

pH monitoring with micro-invasive, precise pH Microsensors was used for many experimental designs, including in vivo pH measurements of tumor tissue and low volumes of urine.The application of the Manual Micromanipulator with its µm reading accuracy allowed us safe insertion and precise localization of microsensors into the tissue. However, during in vivo pH monitoring of growing tumor tissue over several weeks it was difficult to determine a spatially defined measurement region. With a more precise protocol we hope to evaluate pH changes of tumor formation in the future.

References
[1] Hanahan D., Weinberg R. A., Cell (2011), 144(5), 646 - 74
[2] Yang L. V., Castellone R. D., Dong L., Cancer Prevention - From Mechanisms to Translational Benefits (2012), 3 - 40
[3] Obey I. F., Baggett B. K., Kirkpatrick N. D., Cancer Res. (2009), 69, 2260 - 68
[4] Wright M. E., Michaud D. S., Pietinen P., et al., Cancer Causes Control (2005), 16(9), 1117 - 23

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