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Pressure-tuning infrared spectroscopy: application to cancer research and diagnosis.

The last decade of the 20th century finds life sciences in a very exciting phase of development. The unprecedented ability for information processing and the study of the cell with the tools of molecular biology are rapidly altering the conceptual and practical framework of these. However, as knowledge is being rapidly translated into practical applications, there is an enormous strain placed on finite resources. On the other hand, it is now realized that while molecular biology has made revolutionary contributions towards our understanding of the life and regulation of the cell, additional approaches will be needed to study biological phenomena with enhanced resolution. In the last four years we systematically applied infrared spectroscopy combined with high pressure (pressure-tuning infrared spectroscopy) to the study of human cancer at the molecular level. Our early findings are encouraging and suggest that this physical method can potentially provide a versatile approach to the study of cell sand tissues. This method can also contribute to the rapid and inexpensive diagnosis of disease.

The Technology

Infrared spectroscopy is a powerful method to study not only the structure of chemical compounds, but also their relationship to surrounding molecules. [1] Its application to tissues has been hampered by problems in sample preparation for optimal spectra acquisition and, most importantly, the strong absorption of infrared light by water. By resolving technical and methodological problems [2,3,4] we are now able to apply infrared spectroscopy to the study of both animal and human tissues [4,5,6] as well as isolated cells.

Three important advances have contributed to out ability to obtain consistently high-quality IR spectra from tissues or cells. First, the development of novel sample holders, which decrease the amount of light scattering and interference fringes. [2,3] Second, the application of high pressure (pressuretuning) for the elucidation of the true nature of spectroscopic findings. And, third, the careful preparation and close monitoring of tissues under study in order to obtain meaningful data. The role of high pressure and sample monitoring will become evident as we summarize our work on cancer.

Cancer Findings

Our work in cancer started with colon cancer, the second most common malignancy in the western world. Colon cancer has three features that make it an attractive choice to investigate the role of infrared spectroscopy in the evaluation and diagnosis of cancer. First, it is both frequent enough and easily accessible, thanks to the availability of endoscopy of the colon (colonoscopy) by flexible fiberoptic instruments (colonoscopes). Second, it is preventable by removal of its precursor lesion (adenoma), which presents as a discrete anatomic entity (polyp). Third, its histology and biology are well studied;, it is considered the best studied human malignancy to date.

Initially, we studied paired histological sections of normal and malignant colon tissue from each patient. For each tissue section that we studied by infrared spectroscopy, we examined histologically an adjacent microtome section. We monitored the composition of the tissues studied very closely. This was particularly crucial in the case of colon cancer where histological heterogeneity is fairly frequent; benign and malignant elements coexist in up to 60% of the cases.

We demonstrated that the infrared spectra of microtome sections of malignant colon tissues were significantly different from those of corresponding normal tissues (see Figure 1). As shown in Figure 2, some spectroscopic parameters depend on the composition of the tissue section being studied. Atmospheric pressure spectra were consistently different between normal and cancer tissues.

Close study of our infrared spectroscopy data provided information encompassing several cell constituents, including nucleic acids, proteins, lipids and the degree of disorder and packing of cell membranes. The evaluation of the asymmetric phosphate stretching region is presented in Figures 3 and 4.

We have now studied in detail, besides colon cancer, hepatomas (liver cancer), cervical cancer and its premalignant lesion cervical dysplasia, as well as cancers of the esophagus, stomach, skin (basal cell carcinoma), ovary, vagina and breast. In addition, we have studied several cultured cell lines. The following features appear to be common to all these cancers: I. increased hydrogen-bonding of the phosphodiester groups of nucleic acids; 2. decreased hydrogen-bonding of the C-OH groups of proteins; 3. a shift of the band normally appearing at 1082 [cm.sup.-1] to 1086 [cm.sup.-1]. Cancers of glycogen-rich tissues demonstrate, in addition, reduced glycogen levels (see Figure 5). All cancers, except for basal cell carcinoma of the skin, how increase disorder of the methylene chains of the lipids of cell membranes. It was of great interest that the premalignant lesions of the human cervix (dysplasia) manifested spectroscopic changes intermediate between those of normal cervix and cervical cancer.

At this time we do not know the mechanism whereby these changes develop inside the cell. We have identified cultured cancer cells which manifest the spectroscopic changes that appear common to all cancers. They represent, therefore, an excellent model system to address mechanistic questions. Studies in this direction are already in progress.

Implications for the Study and

Diagnosis of Cancer

Our work was demonstrated that infrared spectroscopy can be used to study human tissues and cells in order to investigate a biological problem such as cancer. There are a number of advantages in using infrared spectroscopy in this type of study. First, only tiny amounts of unprocessed (ie. without staining or fixation) tissue sections are needed. Second, molecules are examined in their natural state in the intact cell and therefore their physical state and interactions with other molecules can be studied. For instance, the changes in hydrogen-bonding of nucleic acids in cancer could not be evaluated in isolated nucleic acids, since in the process they would be stripped of histones and be in an artificial solvent. Third, several molecules can be monitored simultaneously. This is also exemplified in our work, where we evaluated aspects of the amount, structure and interactions of proteins, lipids, nucleic acids, and carbohydrates in a single tissue section. Fourth, infrared spectroscopy is a relatively rapid and inexpensive method. Data acquisition can be accomplished by technical assistants following a brief period of training.

Our findings in cancer have prompted the consideration whether infrared spectroscopy has any value as a diagnostic tool in a cancer and, perhaps, other diseases. Given the results that we have obtained so far, one might be inclined to offer an affirmative answer. However this is a complex question. A clinically useful test requires sensitivity and specificity values close to or better than those of existing methodology, in this case histological evaluation of tissues. Hispathological techniques, developed over 100 years ago, are still the standard. The number of samples we have examined so far is limited for a definitive answer. We have calculated that, in order to establish the sensitivity and specificity of infrared spectroscopy in colon cancer, we need to study 397 patients. So, the answer to this question will have to await the results of additional studies, probably from several centers.

The Future

The development of precise and raid diagnostic methods for human diseases, and the elucidation of their pathogenesis are major challenges of biomedical research, as both can impact directly on their treatment. Rational therapeutic strategies are based upon an understanding of pathophysiology and accurate diagnosis. Infrared spectroscopy appears to be a method that can contribute in unique ways to the study of human tissues. We anticipate that in the future infrared spectroscopy will be more widely employed to the study of diseases.

The role of infrared spectroscopy in diganosis remains to be determined. There are obvious advantages in using infrared spectroscopy in diagnosis. It is rapid, inexpensive, autometable and requires minimal amounts of tissue. It should also compare most favorably with existing methods in terms of cost. A role as an adjunct for screening of large-volume histological examinations, such as cervical or bronchial smears is also possible. In these cases identifying only those samples that are definitely normal would greatly decrease the work load in clinical laboratories; infrared spectroscopy may be able to do that. Currently, large-scale evaluation of the use of infrared spectroscopy in the diagnosis of cervical smears has been initiated by a Cancer Diagnositic Technology, a Toronto-based company which licensed this technology from the National Research Council of Canada and Cornell University, the patent-holding institutions.

In conclusion, infrared spectroscopy emerges as a powerful research tool in molecular biology and possesses the potential to be utilized in clinical diagnosis. Additional work will clarify the full range of its applications to life sciences.

References

[1] P.T.T. Wong, in Vibrational Spectra and Structure, J.R. During, Ed. Elsevier, Amsterdam (16: 357-445 (1987).

[2] P.T.T. Wong, US Patent No. 4970396 (1990).

[3] P.T.T. Wong, US Patent No. 4980551 (1990).

[4] P.T.T. Wong and B. Rigas, Appl. Spectrosco. 44: 1715-1718 (1990).

[5] B. Rigas, S. Morgello, I.S. Goldman and P.T.T. Wong, Proc. Natl Acad. Sci. USA 87: 8140-8145 (1990).

[6] P.T.T. Wong, R.K. Wong, T.A. Caputo, T.A. Godwin and B. Rigas, Proc. Natl. Acad. Sci. USA (1991), in press.
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Author:Wong, Patrick T.T.; Rigas, Basil
Publication:Canadian Chemical News
Date:Nov 1, 1991
Words:1503
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