Cancer

It is possible to conduct in-vivo pO2 measurements in conscious mouse? It is a well-known fact that the oxygenation level is a significant determinant when it comes to identifying and monitoring neoplastic changes. Another fact is that anesthetics affect the physiology of the oxygen inside the animal’s body, which could cause inaccuracies during in vivo experiments. The question is whether it is possible to monitor conscious animal, avoiding the anesthesia’s impact and, simultaneously, collect high-quality data. To answer this question, we begin by taking a closer look at the study conducted by Matsumoto et al. “Dynamic Monitoring of Localized Tumor Oxygenation Changes Using RF Pulsed Electron Paramagnetic Resonance in Conscious Mice”. The authors used an electron paramagnetic resonance (EPR) spectroscopy at frequency of 750 MHz to assess the pO2 level in tumor bearing mice. Crystals of lithium phthalocyanine (LiPc) was implanted into squamous cell carcinoma (SCC) tumor and femoral muscle on opposing legs of mice. It has been examined whether there is a correlation between oxygenation and factors such as anesthesia, body temperature and carbogen inhalation. As a consequence of the conducted experiment, this correlation has been confirmed by Matsumoto and colleagues. In conclusion, EPR spectroscopy, due to a rapid acquisition of spectral data, enables the pO2 measurements in conscious mice and minimizing the influence of anesthetics. This ability makes the EPR with LiPc a powerful tool in oncology researches, especially. in studies on tissues oxygenation. References: Matsumoto S. et al.; Dynamic monitoring of localized tumor oxygenation changes using RF pulsed electron paramagnetic resonance in conscious mice; Magn Reson Med. 2008 Mar;59(3):619-25; DOI: 10.1002/mrm.21500.

Biological differences between cancerous tissues and healthy ones. Nowadays, a fight against cancer seems to be one of the most important issue for scientists. Research groups all around the world are working on new cancer therapies which target tumour cells. One of the approaches to this problem consists in learning the physiological differences between normal and neoplastic tissue. The differences could appeared among physiological parameters such as redox state, oxygenation and pH value. The fact is that Electron Paramagnetic Resonance is a non-invasive and rapid method that enables detection of insignificant changes of above-mentioned characteristics and thus it is a great tool for preclinical cancer research and has a great potential to become a method used in clinical trials. A hypothesis that the redox state and pO2 value differ in normal and neoplastic tissue, have been presented in a paper “Noninvasive Imaging of Tumor Redox Status and Its Modification by Tissue Glutathione Levels” by Kuppusamy et al. The main goal of the research was:  i) develop a non-invasive technique that enables measurement and imaging of the tissue’s redox state ii)  acquire images of redox state from normal and malignant tissue iii) analyse the role of glutathione (GSH) in changes of the redox status. In examinations conducted by the research group, in vivo EPR spectroscopy and imaging (EPRI) with a nitroxide spin probe were used. Buthionine sulfoximine (BSO), as a GSH synthesis inhibitor, was applied to decrease the level of GSH in a tissue. The subject was tumour-bearing mice with implanted radiation-induced fibrosarcoma (RIF-1) tumour growing in the right leg. The normal tissue from the left leg was used as a control. The results of experiments have confirmed the hypothesis posed by the research group. It has been shown that physiological parameters such as redox status, in the normal tissue and in the neoplastic tissue significantly differ from each other. Obtained data showed that it was a higher concentration of GSH in malignant tissue. It was also noted that BSO treatment causes differences in reduction of GSH level. Tumour tissue showed higher reduction rate in comparison to healthy tissue. Physiological differences between malignant tissue and normal tissue are a significant factor involved in improving cancer treatments, and what is important, EPR technique is a very promising and helpful tool in such researches.  References: Kuppusamy P. et al.; Noninvasive imaging of tumor redox status and its modification by tissue glutathione levels; Cancer Res. 2002 Jan 1;62(1):307-12.

EPR is making its way to clinical studies There are  subtle physiological differences between normal tissue and tumour tissue. These differences mainly concern characteristics such as redox status and pO2 levels. Thus, imaging methods that enable detection of these biological variations may be valuable in developing treatment strategies. Electron Paramagnetic Resonance Imaging is capable of non-invasive measurements of redox state and pO2 level, involving proper nitroxide probes. The study of the distribution and lifetime of nitroxides in normal and tumour tissue was the aim of Kuppusamy’s research paper “In vivo Electron Paramagnetic Resonance Imaging of Tumour Heterogeneity and Oxygenation in a Murine Model”. As has been pointed out, nitroxides occur in biological systems in two forms: as a free radical and/or as a diamagnetic hydroxylamine which is a product of one-electron reduction of the nitroxide free radical. The reduction rate of nitroxides is increasing during  hypoxia conditions. A specificity of these compounds may indicate some of the physiological differences in tissues, which exist under various oxygenation levels. In order to detect tissue heterogeneity, three nitroxides were used: TPL (4-OH, 2,2,6,6-tetramethylpiperidine-1-oxyl ), 3-CP (3-carbamoyl-proxyl) and PDT (4-oxo-L,2,6,6-tetramethylpiperadine-1-oxyl). TPL has been specified as an in vitro radioprotector, the 3-CP, due to greater biostability in tissues, was used in in vivo EPRI experiments and PDT was used as a probe for pO2 levels assessment.  Mice bearing tumour in size approximately 10 – 15 mm in diameter were examined. Electron Paramagnetic Resonance Imaging was used to perform measurements of reduction rates, distribution and levels of nitroxides in normal and tumour tissues. The obtained results have shown that the peak level of nitroxides was significantly lower in the tumour tissue. Moreover, the reduction rate of nitroxides was much higher in tumours, compared to normal tissues. 3D imaging has confirmed a significant heterogeneity in the distribution of spin probes and redox state in tumour tissue, whereas normal tissues has remained homogenous. With the use of PDT, a significant difference in pO2 level between normal tissue and tumour tissue have been shown. An average level of oxygenation in tumours was three times lower, then in normal tissue. A state of hypoxia in tumours is linked to lower nitroxides content and its greater reduction rate. The study conducted by Kuppusamy’s group has confirmed, that EPRI is a technique that enables performing both the spatial bio-distribution and functional imaging as a result of spectral properties of nitroxides. EPRI may be a breakthrough approach to plan  treatment of neoplastic diseases. References:  Kuppusamy P et al.;In vivo electron paramagnetic resonance imaging of tumor heterogeneity and oxygenation in a murine model; Cancer Res. 1998 Apr 1;58(7):1562-8.