KEYWORDS: Breast cancer, Receptors, Microscopes, Luminescence, Cancer, Light emitting diodes, Microscopy, Statistical analysis, Signal to noise ratio, Medicine
Significance: Stratification of malignancy is valuable for cancer treatment. Both optical redox imaging (ORI) indices and nuclear-to-cytoplasmic volume/area ratio (N:C ratio) have been investigated to differentiate between cancers with varying aggressiveness, but these two methods have not been directly compared. The redox status in the cell nucleus has not been studied by ORI, and it remains unknown whether nuclear ORI indices add new biological information.Aim: We sought to compare the capacity of whole-cell and subcellular ORI indices and N:C ratio to differentiate between breast cancer subtypes with varying aggressiveness and between mitotic and nonmitotic cells.Approach: ORI indices for whole cell, cytoplasm, and nucleus as well as the N:C area ratio were generated for two triple-negative (more aggressive) and two receptor-positive (less aggressive) breast cancer cell lines by fluorescence microscopy.Results: We found positive correlations between nuclear and cytoplasmic ORI indices within individual cells. On average, a nuclear redox status was found to be more oxidized than cytoplasm in triple-negative cells but not in receptor-positive cells. Whole-cell and subcellular ORI indices distinguished between the receptor statuses better than the N:C ratio. However, N:C ratio was a better differentiator between nonmitotic and mitotic triple-negative cells.Conclusions: Subcellular ORI analysis differentiates breast cancer subtypes with varying aggressiveness better than N:C area ratio.
KEYWORDS: Cancer, Endoscopy, Imaging systems, Breast cancer, Luminescence, Tissues, Fluorescence spectroscopy, Spectroscopy, In vivo imaging, In vitro testing
Despite various technological advancements in cancer diagnosis, the mortality rates were not decreased significantly. We aim to develop a novel optical imaging tool to assist cancer diagnosis effectively. Fluorescence spectroscopy/imaging is a fast, rapid, and minimally invasive technique which has been successfully applied to diagnosing cancerous cells/tissues. Recently, the ratiometric imaging of intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), as pioneered by Britton Chance and the co-workers in 1950-70’s, has gained much attention to quantify the physiological parameters of living cells/tissues. The redox ratio, i.e., FAD/(FAD+NADH) or FAD/NADH, has been shown to be sensitive to various metabolic changes in in vivo and in vitro cells/tissues. Optical redox imaging has also been investigated for providing potential imaging biomarkers for cancer transformation, aggressiveness, and treatment response. Towards this goal, we have designed and developed a novel fiberoptic-based needle redox imager (NRI) that can fit into an 11G clinical coaxial biopsy needle for real time imaging during clinical cancer surgery. In the present study, the device is calibrated with tissue mimicking phantoms of FAD and NADH along with various technical parameters such as sensitivity, dynamic range, linearity, and spatial resolution of the system. We also conducted preliminary imaging of tissues ex vivo for validation. We plan to test the NRI on clinical breast cancer patients. Once validated this device may provide an effective tool for clinical cancer diagnosis.
We previously showed that optical redox imaging (ORI) of snap-frozen breast biopsies by the Chance redox scanner readily discriminates cancer from normal tissue. Moreover, indices of redox heterogeneity differentiate among tumor xenografts with different metastatic potential. These observations suggest that ORI of fluorescence of NADH and oxidized flavoproteins (Fp) may provide diagnostic/prognostic value for clinical applications. In this work, we investigate whether ORI of formalin-fixed-paraffin-embedded (FFPE) unstained clinical tissue slides of breast tumors is feasible and comparable to ORI of snap-frozen tumors. If ORI of FFPE is validated, it will enhance the versatility of ORI as a novel diagnostic/prognostic assay as FFPE samples are readily available. ORI of fixed tissue slides was performed using a fluorescence microscope equipped with a precision automated stage and appropriate optical filters. We developed a vignette correction algorithm to remove the tiling effect of stitched-images. The preliminary data from imaging fixed slides of breast tumor xenografts showed intratumor redox heterogeneity patterns similar to that of the frozen tissues imaged by the Chance redox scanner. From ORI of human breast tissue slides we identified certain redox differences among normal, ductal carcinoma in situ, and invasive carcinoma. We found paraformaldehyde fixation causes no change in NADH signals but enhances Fp signals of fresh muscle fibers. We also investigated the stability of the fluorescence microscope and reproducibility of tissue slide fluorescence signals. We plan to validate the diagnostic/prognostic value of ORI using clinically annotated breast cancer sample set from patients with long-term follow-up data.
It has been shown that a malignant tumor is akin to a complex organ comprising of various cell populations
including tumor cells that are genetically, metabolically and functionally different. Our redox imaging data
have demonstrated intra-tumor redox heterogeneity in all mouse xenografts derived from human melanomas,
breast, prostate, and colon cancers. Based on the signals of NADH and oxidized flavoproteins (Fp, including
flavin adenine dinucleotide (FAD)) and their ratio, i.e., the redox ratio, which is an indicator of mitochondrial
metabolic status, we have discovered several distinct redox subpopulations in xenografts of breast tumors
potentially recapitulating functional/metabolic heterogeneity within the tumor. Furthermore, xenografts of
breast tumors with higher metastatic potential tend to have a redox subpopulation whose redox ratio is
significantly different from that of tumors with lower metastatic potential and usually have a bi-modal
distribution of the redox ratio. The redox subpopulations from human breast cancer samples can also be very
complex with multiple subpopulations as determined by fitting the redox ratio histograms with multi-
Gaussian functions. In this report, we present a new method for identifying the redox subpopulations within
individual breast tumor xenografts and human breast tissues, which may be used to differentiate between
breast cancer and normal tissue and among breast cancer with different risks of progression.
Abnormal metabolism can be a hallmark of cancer occurring early before detectable histological changes and may serve
as an early detection biomarker. The current gold standard to establish breast cancer (BC) diagnosis is histological
examination of biopsy. Previously we have found that pre-cancer and cancer tissues in animal models displayed
abnormal mitochondrial redox state. Our technique of quantitatively measuring the mitochondrial redox state has the
potential to be implemented as an early detection tool for cancer and may provide prognostic value. We therefore in this
present study, investigated the feasibility of quantifying the redox state of tumor samples from 16 BC patients. Tumor
tissue aliquots were collected from both normal and cancerous tissue from the affected cancer-bearing breasts of 16
female patients (5 TNBC, 9 ER+, 2 ER+/Her2+) shortly after surgical resection. All specimens were snap-frozen with
liquid nitrogen on site and scanned later with the Chance redox scanner, i.e., the 3D cryogenic NADH/oxidized
flavoprotein (Fp) fluorescence imager. Our preliminary results showed that both NADH and Fp (including FAD, i.e.,
flavin adenine dinucleotide) signals in the cancerous tissues roughly tripled to quadrupled those in the normal tissues
(p<0.05); and the redox ratio Fp/(NADH+Fp) was about 27% higher in the cancerous tissues than in the normal ones
(p<0.05). Our findings suggest that the redox state could differentiate between cancer and non-cancer breast tissues in
human patients and this novel redox scanning procedure may assist in tissue diagnosis in freshly procured biopsy
samples prior to tissue fixation. We are in the process of evaluating the prognostic value of the redox imaging indices for
BC.
Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp/(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.
Clinically-translatable redox imaging methods developed in the Chance laboratory have been used for imaging
mitochondrial metabolic states in tissues. The fluorescence of reduced pyridine nucleotide (PN or NADH) and oxidized
flavoproteins (Fp) in the respiratory chain is sensitive to intracellular redox states. The redox ratios, i.e., Fp/(Fp+NADH)
and NADH/(Fp+NADH) provide important metabolic information in living tissues. Usually the higher the metabolic
flux, the less NADH, the more oxidized Fp, and the higher Fp redox ratio. Snap-freezing tissue samples under the liquid
nitrogen condition preserves the tissue metabolic state in vivo. Here we report our work on the calibration of a homebuilt
Charged Coupled Device (CCD) cryogenic redox imager using a series of snap-frozen solution standards of NADH
and Fp. The NADH concentration ranged from 0-1318 μM and Fp from 0-719 μM. The sensitivity ratio of NADH and
Fp channels was determined from the slope ratio of the two calibration curves and was used to correct the redox ratio of
a human melanoma mouse xenograft. The NADH and Fp reference standards were placed adjacent to the tissue samples
and their emission intensities were used to quantitatively determine the concentrations of NADH and Fp in a mouse
xenograft of a human breast cancer line. Our method of imaging tissue samples along with reference NADH and Fp
standards should facilitate the comparison of redox images obtained at different times or with different instrument
parameters.
The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) such
as flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular redox states and
have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a threedimensional
(3D) redox cryo-imager previously developed by Chance et al., can quantitatively determine the metabolic
properties of tissue samples by acquiring the fluorescence images of NADH and Fp. The redox ratios, i.e.,
Fp/(Fp+NADH) and NADH/(Fp+NADH), obtained on the basis of relative signal intensity ratios, provide a sensitive
index of steady-state of the mitochondrial metabolism that has been determined for a variety of biological tissues. This
paper presents the further development of the instrument by establishing a calibration method to quantify the
concentration of the fluorophores and facilitate the comparison of redox images obtained at different time or with
different instrument functions. Calibration curves of both NADH and Fp have been obtained using snap-frozen standard
references with NADH concentration ranging from 150-1400 μM and Fp from 80-720 μM. Snap-freeze tissue samples
such as human breast tumors xenografted in mice, normal mouse pancreases and spleens were imaged. The NADH and
Fp concentrations as well as the redox ratios in the tissue samples were quantified based on the adjacent solution
standards of NADH and Fp. The obtained multi-slice redox images revealed high heterogeneity of the tissue samples
which can be quantitatively interpreted.
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