Rationale and Objectives
The clinical gold standard for breast cancer diagnosis relies on invasive biopsies followed by tissue fixation for subsequent histopathological examination. This process renders the specimens to be much less suitable for biochemical or metabolic analysis. Our previous metabolic imaging data in tumor xenograft models showed that the mitochondrial redox state is a sensitive indicator that can distinguish between normal and tumor tissue. In this study, we investigated whether the same redox imaging technique can be applied to core biopsy samples of human breast cancer and whether the mitochondrial redox state may serve as a novel metabolic biomarker that may be used to distinguish between normal and malignant breast tissue in the clinic. Our long-term objective was to identify novel metabolic imaging biomarkers for breast cancer diagnosis.
Materials and Methods
Both normal and cancerous tissue specimens were collected from the cancer-bearing breasts of three patients shortly after surgical resection. Core biopsies and tissue blocks were obtained from tumor and normal adjacent breast tissue, respectively. All specimens were snap-frozen with liquid nitrogen, embedded in chilled mounting medium with flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide reference standards adjacently placed, and scanned using the Chance redox scanner (ie, cryogenic nicotinamide adenine dinucleotide/oxidized flavoprotein fluorescence imager).
Results
Our preliminary data showed cancerous tissues had up to 10-fold higher oxidized flavoprotein signals and had elevated oxidized redox state compared to the normal tissues from the same patient. A high degree of tumor tissue heterogeneity in the redox indices was observed.
Conclusions
Our finding suggests that the identified redox imaging indices could differentiate between cancer and noncancer breast tissues without subjecting tissues to fixatives. We propose that this novel redox scanning procedure may assist in tissue diagnosis in freshly procured biopsy samples before tissue fixation.
Biochemical analysis of cancer core biopsies has little diagnostic value in the clinic so far. In this study, we demonstrated a novel approach using the intrinsic biochemical properties of breast tissues as measured by the Chance redox scanner (named after Dr. Britton Chance) to distinguish between normal and cancerous breast tissues using clinical biopsy samples. The Chance redox scanner is a low-temperature ex vivo fluorescence imaging system that uses a raster-scanning fiberoptic probe coupled with a pair of rotating filter wheels to select proper excitation and emission light wavelengths to measure the endogenous fluorescence signals of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoprotein (Fp) including flavin adenine dinucleotide (FAD) in tissue. With a tissue sample mounted in a liquid nitrogen-filled chamber and a miller to remove top tissues at different depth ( z axis), the scanner can image the three-dimensional distribution of NADH and Fp with a high spatial resolution down to 50 × 50 × 20 μm 3 . It requires only a small tissue sample (1 mm in two dimensions) for measurement of tissue mitochondrial redox states.
Redox scanning can become a diagnostic tool. NADH and reduced FAD are reducing equivalents used for the electron transport chain to generate ATP in mitochondria. It has been shown that the redox ratio [Fp/NADH or the normalized form, Fp/(Fp + NADH)] was a good representation of the tissue redox state and a sensitive indicator of mitochondrial metabolic state . It was discovered previously that the Fp redox ratio [ie, Fp/(Fp + NADH)] correlated with the degree of tumor invasiveness in mouse xenografts of human melanoma and breast cancer cell lines . In another study, the premalignant pancreatic tissue in the PTEN -null transgenic mouse model was shown to be more oxidized and heterogeneous in the mitochondrial redox state than the normal one . Redox scanning was also used to image the redox ratio variations in human dysplastic cervical tissues compared to normal controls .
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Methods
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Results and discussion
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Table 1
Values of the Redox Indices of the Tumor and Normal (Control) Breast Tissues from Three Patients Analyzed by the Global Averaging Method
Type Fp, μmol/L NADH, μmol/L Fp Redox Ratio Control ( n = 3) 118 ± 89 138 ± 45 0.40 ± 0.09 Tumor ( n = 3) 652 ± 93 382 ± 283 0.61 ± 0.10P (paired t test) .027 .25 .071
Fp, flavoproteins; NADH, reduced nicotinamide adenine dinucleotide.
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Table 2
Values of the Redox Indices of the Tumor and Normal (Control) Breast Tissues from Three Patients Analyzed by the General Linear Method Univariate Analysis
Type Fp, μmol/L NADH, μmol/L Fp Redox Ratio Control (6 sections) 118 ± 85 138 ± 42 0.40 ± 0.08 Tumor (14 sections) 620 ± 428 251 ± 210 0.67 ± 0.12P .015 .20 1.72E–04
Fp, flavoproteins; NADH, reduced nicotinamide adenine dinucleotide.
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Table 3
The Redox Indices of the Cancerous Core Biopsies Taken from Different Locations of Patient 1
Tissue Location Fp Redox Ratio NADH, μmol/L Fp, μmol/L a (2 sections) 0.54 ± 0.01 45 ± 13 57 ± 20 b (4 sections) 0.69 ± 0.03 139 ± 28 346 ± 38 d (5 sections) 0.82 ± 0.03 221 ± 38 1115 ± 344P ∗ .00001 .0004 .0017
Fp, flavoproteins; NADH, reduced nicotinamide adenine dinucleotide.
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Conclusions
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Acknowledgments
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References
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