Trends in Biotechnology
Volume 24, Issue 10, October 2006, Pages 455-462
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Review
Chemical mapping of tumor progression by FT-IR imaging: towards molecular histopathology

https://doi.org/10.1016/j.tibtech.2006.08.005Get rights and content

Fourier-transform infrared (FT-IR) spectro-imaging enables global analysis of samples, with resolution close to the cellular level. Recent studies have shown that FT-IR imaging enables determination of the biodistribution of several molecules of interest (carbohydrates, lipids, proteins) for tissue analysis without pre-analytical modification of the sample such as staining. Molecular structure information is also available from the same analysis, notably for protein secondary structure and fatty acyl chain peroxidation level. Thus, several cancer markers can be identified from FT-IR tissue images, enabling accurate discrimination between healthy and tumor areas. FT-IR imaging applications are now able to provide unique chemical and morphological information about tissue status. With the fast image acquisition provided by modern mid-infrared imaging systems, it is now envisaged to analyze cerebral tumor exereses in delays compatible with neurosurgery. Accordingly, we propose to take FT-IR imaging into consideration for the development of new molecular histopathology tools.

Introduction

Cerebral cancer diagnosis for small-sized and diffuse solid tumors such as gliomas remains a major difficulty for histopathologists due to the unpredictable morphology of these tumors, which diffuse easily in the parenchyma [1]. Histopathology is precisely based upon the morphological appearance of tumors for classification and grading; thus, in many cases, neurosurgeons have no information about the efficiency of a tumor resection during surgery. Malignant gliomas have one of the worst prognoses in medicine, with median survival well below 18 months, despite technological advances in treatment [2]. Therefore, the development of histopathology-derived tools, based upon molecular information, for discriminating between tumor and healthy tissue is now viewed as a potent diagnostic tool [3].

In complex biological systems, the IR spectrum is the sum of the contributions of the biomolecules present (e.g. proteins, lipids, sugars and nucleic acids). Diagnostic methods based on Fourier-transform infrared (FT-IR) spectroscopy have emerged and developed rapidly during the past decade – currently, the main applications of FT-IR spectroscopy in the biomedical area are focused on bacterial phenotype identification [4], molecular structure determination [5], cancer DNA phenotype recognition 6, 7, clinical diagnosis [8], and molecular concentration determinations 9, 10, 11. One advantage of the FT-IR spectroscopy approach is that a spectrum from an intact cell can be recorded within a few seconds, and a recent technical advance has made imaging systems available that are able to provide fast FT-IR images of tissue sections, requiring only a few minutes to obtain a functional FT-IR image of a ∼ 1 mm2 area of tissue. Thus, useful diagnostic information can be extracted from IR spectra for different pathologies 12, 13, 14. However, until now, most cancer studies performed by FT-IR imaging have involved only statistical analyses of tissue spectral data [15]. This approach is a matter of controversy because tumor formation and growth is highly different among tissues and depends on a wide range of endogenous and exogenous stimuli. All these factors lie at the heart of the infinite mosaic of tumor forms.

Among the parameters underlying most occurrences of tissue carcinogenesis, oxygen delivery and glucose metabolism are interrelated and manifest in tumor cells as low intercellular oxygen tensions (pO2) and a high glycolytic metabolic profile [16]. Hypoxia influences the regulation and transcription of various genes involved in malignant growth and metastases, and promotes a more aggressive tumor phenotype. Tumor hypoxia essentially occurs when the growth of the tumor outstrips the accompanying angiogenesis. All cells must have a blood supply within a 1–2 mm3 volume for survival, and many parts of a developing tumor are hypoxic [17].

As a consequence of hypoxia, tumors consume more glucose than normal tissue: tumor studies show that there is always a net uptake of glucose, which is an essential substrate for cell proliferation. However, the limiting-factor for glucose and O2 uptake is the transport of these substances to the solid tumor as a result of poor blood flow, impaired microcirculation, greater diffusion distances between blood vessels and cancer cells [18] and cell dehydration [19]. Furthermore, higher glucose consumption leads to a several fold increase in lactic-acid production [20] but its removal is reduced because of the impaired surrounding microcirculation. Thus, acidic intracellular and extracellular pH is associated with tumor progression and ischemia. Carcinogenesis is also associated with an increased free-radical generation, which induces the formation of lipid, protein and DNA peroxidation products [21]. The consequences of an oxygen deficit within tumor tissues combined with the increased cellular acidosis are considered to be the main inducers of cellular apoptosis and necrosis [22]. Here, we review the most recent advances in cell and tissue analysis using FT-IR spectrometry and imaging, and we propose the use of the most omnipresent cancer markers accessible by FT-IR for making this technology a molecular histochemistry diagnostic tool for neurosurgeons.

Section snippets

FT-IR spectrometry for determining molecular concentrations

The FT-IR technique is based upon the absorption of infrared light by vibrational transitions in covalent bonds, where the intensities provide quantitative information and the frequencies relate to the nature of these bonds, their structure and their molecular environment (Figure 1). FT-IR data can be used as molecular signatures for physiological status once the spectral patterns are correlated with biological properties; therefore, FT-IR spectrometry is a unique resource to provide a global

FT-IR spectrometry for molecular structure analysis

FT-IR spectrometry can also provide unique information on molecular structure, mainly for determining the secondary structure of proteins [5], mutations of nucleic acids [30] and peroxidation of phospholipids [11]. Protein secondary structure can be determined in terms of percentages of α-helix, parallel and anti-parallel β-sheets, β-turn, and unordered structure (Figure 3). Pathologies induce changes in the extent of hydrogen bonding among the proteinous amide groups, conformational changes

FT-IR imaging for cancer diagnostic

A recent technological advance has been the development of FT-IR imaging systems. FT-IR imaging is a spatially resolved IR spectroscopy that involves the segmentation of radiation at the detection plane. Contributions from different sample areas are separated from the field of view by an array of IR-sensitive detection elements, with charge-coupled device instruments for optical microscopy. These IR multichannel detectors are termed focal plane array (FPA) detectors and consist of a series of

FT-IR imaging of tumor vasculature

The metabolic status of different areas of a tumor is heterogeneous, depending on the availability of oxygen and nutrients [47]. This is mainly because of the distance that exists between proliferating cells and tissue vasculature; it is believed that cells >200 μm from blood capillaries suffer from reduced oxygen diffusion and thus promote a higher glycolytic profile for survival [17]. Tumor angiogenic stress also induces a high proliferation of vascular endothelial cells, leading to abnormal

FT-IR imaging for the chemical mapping of metabolic parameters in tumors

Using FT-IR imaging, recent studies have shown that tumor metabolism can be evaluated from the characteristic absorptions of molecules, namely glucose, glycogen, lactic-acid and amino-acids, or from more global absorptions 57, 58. From the analysis of fluids or cells [11], spectral curve-fitting of the 1800–1500 and 1300–900 cm−1 spectral intervals revealed the characteristic IR absorptions of selected biomolecules for determining the metabolic status of tissues areas. This method has been

FT-IR imaging for the chemical mapping of phospholipids peroxidation

After FT-IR image acquisition of a tumor tissue, fatty acyl chain absorptions are easily determined by curve-fitting the 3100–2800 cm−1 spectral interval 28, 39, 43. These absorptions rely mainly on phospholipids, of which the peroxidation level can be evaluated [62]. Owing to the high morphological heterogeneity found in tumors, it has been proposed to use the changes in νdouble bond(CH):νas(CH3) and νas(CH2):νas(CH3) absorption ratios to avoid normalization of absorptions [28]. In this way, the data

FT-IR imaging for molecular histopathology

Various technologies have been developed for cellular and tissue tumor imaging [63], each one having advantages and limits that define their applicability to the investigation of cerebral pathology. The most useful or promising techniques include MRI [64], computed tomography scan [65], ultrasonography [66], bioluminescence [67], fluorescence [68], PET scan [69] and multi-dimensional power-Doppler imaging [70]. In most cases, the use of contrast chemicals is required for discriminating healthy

Acknowledgements

The authors are indebted to the Regional Council of Aquitaine, the National Center for Scientific Research (CNRS) and Perkin-Elmer for financial and/or technical supports.

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