Characterization of Cancer Biopsy Tissues by Potentiometric Method

Transcription

1 M.S.R. Journal of Engineering and Technology Research Characterization of Cancer Biopsy Tissues by Potentiometric Method Chandraprabha M. N 1, Lokesh K. N 1*, Ahalya N 1, Rajeev A. G 2, Monika P 1 1 Department of Biotechnology, MSRIT, Bangalore. 2 Department of Radiation Oncology, MSRMC, Bangalore ABSTRACT Membrane composition changes, energy abnormalities, membrane charge distribution abnormalities, lower potassium and higher sodium concentrations of cancer cells results in a drop in the normal membrane potential and membrane capacitance of the cells. In the present work an attempt was made to characterize cancerous tissues based on electrical potential by potentiometric method. The clinical cancerous tissues of cervix, lung, buccal mucosa, base of tongue and rectum were collected from Radiation Oncology Department, M S Ramaiah Medical College and Hospital under the guidance of Dr. Rajeev. Samples from different patients were subjected to microscopic characterization to understand the cellular pattern of different cancer tissues and this information served as a basis to optimize disaggregation protocol. Tissue disaggregation protocol was standardized with respect to time and amplitude. The efficacy of disaggregation was assessed by total viability count using haemocytometer with aqueous toluidine blue as the stain. Electrical potential of the different cancerous tissues was determined by standard potentiometric method. Electrical potential was found to be unique for particular type of cancer tissue. Cervix tissue had maximum electric potential and lung tissue had the minimum electric potential. The mean electric potentials of different cancer samples were found to be Cervix = ± mv, Base of Tongue = ± mv, Lung = -38 ± mv, Buccal Mucosa = ± mv, Rectum= ± mv. These findings demonstrate the potential of potentiometric method as a feasible technique for characterization of cancer tissues. Keywords: Cancer tissue, characterization, membrane potential, potentiometric technique. 1. Introduction Cancer is the uncontrolled proliferation of cells in the body. Oncogenes are mutated forms of genes that cause normal cells to grow out of control. Different types of cancer can behave very differently. For example, Adenocarcinoma lung cancer and Adenocarcinoma colon cancer are very different diseases. They grow at different rates and respond to different treatments (Croce, 2008). Cancer is suspected based on a person s symptoms, the results of a physical examination, and sometimes the results of screening tests. Occasionally, X-rays obtained for other reasons such as injury show abnormalities that might be cancer. In men, PSA (Prostate specific antigen) levels in the blood may be used to screen for prostate cancer. The main drawback to its use as a screening test is the large number of false positive results, which generally lead to requirement for more invasive tests. The common screening test for colon cancer involves checking the stool for blood that cannot be seen by the naked eye. Some people with blood in the stool may have positive test results because they have consumed vitamin C or red meat (Greenwald, 2006). Because of the drawbacks and limitations of these conventional techniques, there is a need to find new diagnostic techniques * Corresponding author: knlokes@gmail.com

2 Chandraprabha M. N, Lokesh K. N, Ahalya N, Rajeev A. G, Monika P which are reliable and accurate. Surface potential of the cells can be considered as a distinct property to characterize different tissues. Present studies were aimed at potentiometric characterization of biopsied cancerous tissues, evaluation of viability profile of cancerous tissues with respect to disaggregation protocol, determinination of the electric potential of different cancer cells by electrochemical method (potentiometer) and to determine the difference in the electric potential of clinical samples of cancer. 2. Review of Related Literature Cancerous cells are characterised by various features that affect their electrical activity. Cancer cells are less efficient in their production of cellular energy (ATP). Cell membranes have different lipid and sterol content than normal cells that exhibit different electrochemical properties and a different distribution of electrical charges than normal tissues (Cure, 1991;1995). Altered membrane composition and membrane permeability in them results in the movement of potassium, magnesium and calcium out of the cell and the accumulation of sodium and water into the cell (Seeger and Wolz, 1990). Since the membrane potential in a cancer cell is consistently weaker than the membrane potential of a healthy cell. The electrical field across the membrane of a cancer cell will be reduced. The reduction in membrane electrical field strength will in turn cause alterations in the metabolic functions of the cell. Two of the most outstanding electrical features of cancer cells are that they constantly maintain their membrane potential at a low value and their intracellular concentration of sodium at a high concentration (Cone, 1975). Also a sustained elevation of intracellular sodium may act as a mitotic trigger causing cells to go into cell division (Cone, 1985). The accumulation of an excessive amount of negative charges on the exterior surface of cancer cells will depolarize cancer cell membranes. The depolarization (fall in membrane potential) of the cancer cell membrane due to the accumulation of excess negative surface charges may precede and create the reduction in intracellular potassium and the rise in the intracellular sodium launching the cell into a carcinogenic state (Cure, 1991). Andrew et al., (1994), reported the cell membrane potential in breast tissue and breast epithelial cell. The breast tissue obtained was from 9 women with infiltrating ductal carcinoma and women with benign breast disease. Cell membrane potential was also measured in MCF 10A cells (ATCC, Rockville, Md USA) a non-transformed cell line from a patient with fibrocystic breast disease, MCF 7 cells(atcc), an estrogen receptor-protein (ER+) cell line from patient with breast adenocarcinoma and MDA 435L2, an estrogen negative (ER-) cell line from a patient with breast ductal carcinoma. The cell lines were implanted in the breast of female nude mouse to obtain solid tumours. The role of K + and Na + was studied by adding tetra ethyl ammonium, 4- amino pyridine (4-AP) or tetrodoxin (TTX). Membrane potential was measured for isolated cells, using a silver chloride electrode in micropipette filled with 3 M KCl connected to an electrometer. The reference electrode was silver chloride in bath solution. They reported a significant depolarisization in patients diagnosed with infiltrating ductal carcinoma, compared to patients with benign breast disease with the mean V m values of ± 2.2 mv and ± 2.8 mv, respectively. The transformed cell line was significantly depolarised compared to the normal cells. In the transformed cell line there was a significant decrease of V m on addition of 4-AP, K + channel blocker. There was no decrease in V m on addition of Na + blocker, TTX. They suggested an association between V m and oncogenesis as a result of increased mitosis in cancer cell compared to normal cell. 3. Research Methodology 3.1 Collection of Collection and Storage of Biopsy Tissues The clinical cancerous tissues of cervix, lung, buccal mucosa, base of tongue and rectum were collected from Radiation Oncology Department, M S Ramaiah Medical College and Hospital under the guidance of Dr. Rajeev. They were transferred and stored in 10 % formalin. Table 1 gives the location of different cancer biopsy tissues. Table 1. List of biopsy Samples Sl.No. Sample code Location 1 B2 Cervix 2 BT02002 Cervix 3 BT02003 Base of tongue 4 BT02004 Cervix 5 BT02005 Lung 6 BT02006 Cervix 7 BT02007 Buccal mucosa 8 BT02008 Rectum 9 BT02009 Cervix 10 BT02010 Cervix 33

3 Characterization of Cancer Biopsy Tissues by Potentiometric Method 3.2 Microscopic characterization of cancerous tissues The cancer cells and other associated cells and structures with the cancer tissue were microscopically examined by making permanent slides. The method used for making permanent slides is Touch/Imprint method. This method is useful to optimize time and amplitude for ultrasonication by relating the structures associated with the cancer tissue. H & E staining method was used for staining of the permanent slides. 3.3 Disaggregation of cancerous tissues and Viability determination The cancerous tissues were subjected for disaggregation by sonicating in tissues in Ultrasound at frequency of 20 khz for time periods of 2-10 min at amplitude of 50-80%. The ultrasound subjected samples were viability counting by using with 1 % aqueous toluidine blue indicator. 3.4 Characterization of Electrical Potentials of Cancerous Tissues by Potentiometer The disaggregated test samples were characterized using a digital potentiometer. The electric potential or emf of the cancer cells was measured in triplicates of each sample using the standard protocol of potentiometer. Platinum and calomel (reference electrode). 4. Results and Discussion The biopsies punched after clinical examination from the edge of the suspected cancer tissue were transferred and stored in 10 % formalin. The reason for the storage of the living tissues in formalin is, it fixes the cell membrane and preserves viability of the cells. Formalin is an isotonic solution which maintains the osmolarity of the cancer cells. The cancer samples were obtained from Dr. Rajeev A.G., Department of Radiation Therapy, M S Ramaiah Medical College and hospital. Ten samples were collected from different origins and from different patients for the study of their electric potentials by potentiometric method. The permanent slides which were prepared by Haematoxylin and Eosin method (H & E staining method) were microscopically examined. The cells and structures associated with the cancer tissues were stoma as connective tissue, red blood cells, lymphocytes and neutrophils. The white blood cells were found in very few numbers. The permanent slides were prepared in order to find the cells and structures associated along with cancer cells that helped in optimising time and amplitude for disaggregation. Some tissues were soft and were found to have less connective tissue associated with it and the ones that were hard had dense connective tissue associated with it. The permanent slides of cervix cancer tissue ( hard) compared to other cancer tissues) and buccal mucosa (soft) were made by Touch/ Imprint method and permanently stained using standard protocol of H & E staining. Optimization of time and amplitude was done for different time periods and amplitude as shown in Table no 2. This study optimized the disaggregation parameters for ultrasonication. Optimization study with different time periods and amplitude showed that disaggregation of cancer tissue by ultrasonicator for 2 min, 50 % amplitude released maximum number of free cancer cells, the cell count of per ml was observed. Whereas other time periods and amplitudes showed reduced number of cells per ml when counted in haemocytometer. It was observed that with 2 min and varying amplitudes there was no significant difference in the cell count. Increase in time from 2 min to 4 min showed reduction in the cells per ml. This is because the free cells in disaggregated cell suspension were getting damaged by ultrasonic waves that were produced during ultrasonication. At 8 min and varying amplitudes most of the cells died and there was a drastic reduction in the number of cells per ml. This proved the fact that, ultrasonic waves damaged the viable cancer cells (cell lysis) and hence it can be concluded that cancer cells gets destructed with Table 2. Standardization of time and amplitude for disaggregation Sl no. Time (min) Amplitude (%) Total viable cell count per square (avg) No of viable cells per ml

4 Chandraprabha M. N, Lokesh K. N, Ahalya N, Rajeev A. G, Monika P the increase in time and amplitude. This study showed that 2 min 50% amplitude was optimal time and amplitude parameters for disaggregation of cancer tissue. It was found that 1 min disaggregation also showed similar results as 2 min, but at 1 min 50 % amplitude most of the cancer tissues that were rigid and hard (example cervix) did not get disaggregated completely and yielded less viable cells ( per ml) compared to 2 min 50 %. Softer tissues got disaggregated completely at 2 min 50 % amplitude and not less than that range justifying the point that 2 min 50 % amplitude is the optimal time and amplitude for disaggregation of all types of cancer tissues. In order to characterize the cancerous tissues by potentiometric method by effect of balanced salt solution i.e. normal saline was blanked out, in order to avoid electrical gradients developed by the balanced salts. All clinical samples showed characteristic electric potential which was found unique for particular type. The cervical cancer sample exhibited variation in their electrical potential (standard deviation/ standard error) within the trials. The consolidated electrical potential was found to unique with respect to the type of cancerous tissues AS listed in Table 1), which is summarized as Table No 3. Table 3. Variation in electrical potential (standard deviation/ standard error) within the trials. Sl No Trial 1 Trial 2 Trial Mean ± SD (mv) ± ± ± ± ± ± ± ± ± ± Standard error From Figure 1 it can be concluded that electrical potential of cervical cancer varies within different patients. Since there are six samples for cervix cancers each from different patients, and each sample showed slightly different electric potential readings, a range for electric potential of cervical cancer was obtained. The mean electrical potential of cervical cancer samples was found to be mv. Electric potential range for cervical cancer is from -105 ± to ± Above investigations reveals the following important findings Electrical potential is unique for particular type of cancer. Electrical potentials do vary with respect to the different patients having same cancer origins (example cervix). Further investigations with multiple samples are necessary to minimise error within the sample. A particular electric potential value corresponds to a particular type of cancer. Hence, the property of electrical potential was used as a basis for the detection of particular type of cancer. 5. Future Recommendations The following are recommended to enhance efficiency of eletropotentiometric characterization of cancerous tissues Developments of Ion patch clamp technique for normal and different cancerous tissues. Micro eletropotentiometric method Primary or secondary cell line based experiments. 6. Conclusion The electrical potential was found to be unique and used as a basis for fabrication and design of instrument for Figure 1. Electrical potential of cancer cells. 35

5 Characterization of Cancer Biopsy Tissues by Potentiometric Method diagnosis. It was found that cervix had maximum electric potential and lung had minimum electric potential. As we received multiple samples of cervical cancer, the electrical potential range was obtained. Significant readings were obtained for lung, buccal mucosa, base of tongue and rectum as well. Further investigation will be carried out with multiple clinical samples of different categories of cancer and focus will be on minimizing the error within samples and comparative interpretation with advanced potentiometer. By conducting further research with multiple samples, potentiometric method can be one of the most reliable, fast and cost effective method for cancer detection. References Andrew A.M., Ilko G.I., Michael A.S., Enrique G., Kevin C.M. and Carol A.F., (1994) Association between cell membrane potential and breast cancer. Tumour Biol, 15: Cone, C.D., (1975). The role of surface electrical transmembrane potential in normal and malignant mitogenesis. Ann NY AcadSci, 238: Cone, C.D., (1985). Transmembrane Potentials and Characteristics of Immune and Tumor Cells. CRC Press: Boca Raton, Florida. pp Croce, C.M., (2008). Oncogenes and cancer. The New England journal of medicine, 358(5): Cure, J.C., (1991. Cancer an electrical phenomenon. Resonant, 1(1): Cure, J.C., (1991). Cancer an electrical phenomenon. Resonant, 1(1): Cure, J.C., (1995). On the electrical characteristics of Cancer: Electrochemical Treatment of Cancer. Jupiter, Florida. Greenwald, B., (2006). The DNA Stool Test - An emerging technology in colorectal cancer screening. Gastroenterology Nursing, 28(1): Seeger,P.G. and Wolz, S., (1990). Successful Biological Control of Cancer: By Combat Against the Causes. Neuwieder Verlagsgesellschaft Mbh, Neuweid, Germany. 36