He N. Xu

Quantitative magnetic resonance and optical imaging biomarkers of melanoma metastatic potential

Noninvasive or minimally invasive prediction of tumor metastatic potential would facilitate individualized cancer management. Studies were performed on a panel of human melanoma xenografts that spanned the full range of metastatic potential measured by an in vivo lung colony assay and an in vitro membrane invasion culture system. Three imaging methods potentially transferable to the clinic [dynamic contrast-enhanced (DCE) MRI, T1ρ-MRI, and low-temperature fluorescence imaging (measurable on biopsy specimens)] distinguished between relatively less metastatic and more metastatic human melanoma xenografts in nude mice. DCE-MRI, analyzed with the shutter-speed relaxometric algorithm and using an arterial input function simultaneously measured in the left ventricle of the mouse heart, yielded a blood transfer rate constant, Ktrans, that measures vascular perfusion/permeability. Ktrans was significantly higher in the core of the least metastatic melanoma (A375P) than in the core of the most metastatic melanoma (C8161). C8161 melanoma had more blood vascular structures but fewer functional blood vessels than A375P melanoma. The A375P melanoma exhibited mean T1ρ values that were significantly higher than those of C8161 melanoma. Measurements of T1 and T2 relaxation times did not differ significantly between these 2 melanomas. The mitochondrial redox ratio, Fp/(Fp + NADH), where Fp and NADH are the fluorescences of oxidized flavoproteins and reduced pyridine nucleotides, respectively, varied linearly with the in vitro invasive potential of the 5 melanoma cell lines (A375P, A375M, A375P10, A375P5, and C8161). This study shows that a harsh microenvironment may promote melanoma metastasis and provides potential biomarkers of metastatic potential.

Quantitative mitochondrial redox imaging of breast cancer metastatic potential

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.

Monitoring response to chemotherapy of non‐Hodgkin's lymphoma xenografts by T2‐weighted and diffusion‐weighted MRI

An effective method for in vivo detection of early therapeutic response of patients with non‐Hodgkins lymphoma would enable personalized clinical management of cancer therapy and facilitate the design of optimal treatment regimens. This study evaluates the feasibility of T2‐weighted MRI (T2WI) and diffusion‐weighted MRI (DWI) for in vivo detection of response of human diffuse large B‐cell lymphoma xenografts in severe combined immunodeficient mice to chemotherapy. Each cycle of combination chemotherapy with cyclophosphamide, hydroxydoxorubicin, Oncovin, prednisone, and bryostatin 1 (CHOPB) was administered to tumor‐carrying mice weekly for up to four cycles. T2WI and DWI were performed before the initiation of CHOPB and after each cycle of CHOPB. In order to corroborate the MRI results, histological analyses were carried out on control tumors and treated tumors after completion of all MRI studies. DWI revealed a significant (P < 0.03) increase in the mean apparent diffusion coefficient in CHOPB‐treated tumors as early as 1 week after initiation of CHOPB. However, a significant (P < 0.03) decrease in mean T2 was observed only after two cycles of CHOPB. Both MRI methods produced high‐resolution (0.1 × 0.1 × 1.0 mm3) maps of regional therapeutic response in the treated tumors based on local apparent diffusion coefficient and T2. Only a specific region of the tumors (in 3 of the 5 tumors) corresponding to about one third of the tumor volume exhibited a response‐associate increase in ADC and decrease in T2. An adjacent region exhibited an increase in T2 and no change in ADC. The rest of the tumor was indistinguishable from sham‐treated controls by MRI criteria. The therapeutic response of the treated tumors detected by MRI was accompanied by changes in tumor cell density, proliferation and apoptosis revealed by histological studies performed upon completion of the longitudinal study. The mechanism producing the regional response of the tumor remains to be elucidated. Copyright

MITOCHONDRIAL REDOX IMAGING FOR CANCER DIAGNOSTIC AND THERAPEUTIC STUDIES.

Mitochondrial redox states provide important information about energy-linked biological processes and signaling events in tissues for various disease phenotypes including cancer. The redox scanning method developed at the Chance laboratory about 30 years ago has allowed 3D high-resolution (~ 50 × 50 × 10 μm3) imaging of mitochondrial redox state in tissue on the basis of the fluorescence of NADH (reduced nicotinamide adenine dinucleotide) and Fp (oxidized flavoproteins including flavin adenine dinucleotide, i.e., FAD). In this review, we illustrate its basic principles, recent technical developments, and biomedical applications to cancer diagnostic and therapeutic studies in small animal models. Recently developed calibration procedures for the redox imaging using reference standards allow quantification of nominal NADH and Fp concentrations, and the concentration-based redox ratios, e.g., Fp/(Fp+NADH) and NADH/(Fp+NADH) in tissues. This calibration facilitates the comparison of redox imaging results acquired for different metabolic states at different times and/or with different instrumental settings. A redox imager using a CCD detector has been developed to acquire 3D images faster and with a higher in-plane resolution down to 10 μm. Ex vivo imaging and in vivo imaging of tissue mitochondrial redox status have been demonstrated with the CCD imager. Applications of tissue redox imaging in small animal cancer models include metabolic imaging of glioma and myc-induced mouse mammary tumors, predicting the metastatic potentials of human melanoma and breast cancer mouse xenografts, differentiating precancerous and normal tissues, and monitoring the tumor treatment response to photodynamic therapy. Possible future directions for the development of redox imaging are also discussed.

QUANTITATIVE REDOX SCANNING OF TISSUE SAMPLES USING A CALIBRATION PROCEDURE

The fluorescence properties of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) including flavin adenine dinucleotide (FAD) in the respiratory chain are sensitive indicators of intracellular metabolic states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox scanner, a three-dimensional (3D) low temperature imager previously developed by Chance et al., measures the in vivo metabolic properties of tissue samples by acquiring fluorescence images of NADH and Fp. The redox ratios, i.e. Fp/(Fp+NADH) and NADH/(Fp+NADH), provided a sensitive index of the mitochondrial redox state and were determined based on relative signal intensity ratios. Here we report the further development of the redox scanning technique by using a calibration method to quantify the nominal concentration of the fluorophores in tissues. The redox scanner exhibited very good linear response in the range of NADH concentration between 165-1318μM and Fp between 90-720 μM using snap-frozen solution standards. Tissue samples such as human tumor mouse xenografts and various mouse organs were redox-scanned together with adjacent NADH and Fp standards of known concentration at liquid nitrogen temperature. The nominal NADH and Fp concentrations as well as the redox ratios in the tissue samples were quantified by normalizing the tissue NADH and Fp fluorescence signal to that of the snap-frozen solution standards. This calibration procedure allows comparing redox images obtained at different time, independent of instrument settings. The quantitative multi-slice redox images revealed heterogeneity in mitochondrial redox state in the tissues.

Heterogeneity of mitochondrial redox state in premalignant pancreas in a PTEN null transgenic mouse model.

Pancreas-specific deletion of PTEN in mice revealed progressive premalignant lesions such as ductal metaplasia with infrequent malignant transformation. In this study, we aimed at evaluating the mitochondrial redox state of the metaplastic pancreas in a pancreas-specific PTEN null transgenic mouse model. The two intrinsic fluorophores, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) such as flavin adenine dinucleotide (FAD), in the respiratory chain in mitochondria are sensitive indicators of mitochondrial redox states and have been applied to the studies of mitochondrial function with energy-linked processes. The redox ratio, Fp/(Fp+NADH) provides a sensitive index of mitochondrial redox state. We have obtained optical images of the in vivo mitochondrial redox states of the snap frozen pancreases from pancreas-specific PTEN null mice (Pdx1-Cre;PTEN(lox/lox), N=3) and the controls (PTEN(lox/lox), N=3) using the redox scanner at low temperature. The results showed high spatial heterogeneity of mitochondrial redox state in the mutated pancreases with hot spots of much higher Fp redox ratios whereas the normal ones, were relatively homogenous. The cystic dilation regions in the metaplastic pancreases showed little to no NADH or Fp signal. Histological analysis confirmed no cells existed in these regions. It is the first time that the in vivo mitochondrial redox states of the metaplastic mouse pancreas were optically imaged. Our previous results on human melanoma and breast cancer mouse xenografts have shown that mitochondrial redox state quantitatively correlates with cancer metastatic potential. The more oxidative mitochondrial redox state (higher Fp redox ratio) corresponded to the higher metastatic potential of the tumors. As mitochondrial redox state imbalance is associated with abnormal mitochondrial function, and redox state mediates the generation of reactive oxygen species and many signal transduction pathways, this research may provide insights for studying basic biology and developing early diagnostic imaging biomarkers for pancreatic cancer.

Imaging the redox states of human breast cancer core biopsies.

Currently, the gold standard to establish benign vs. malignant breast tissue diagnosis requires an invasive biopsy followed by tissue fixation for subsequent histopathological examination. This process takes at least 24 h resulting in tissues that are less suitable for molecular, functional, or metabolic analysis. We have recently conducted redox scanning (cryogenic NADH/flavoprotein fluorescence imaging) on snap-frozen breast tissue biopsy samples obtained from human breast cancer patients at the time of their breast cancer surgery. The redox state was readily determined by the redox scanner at liquid nitrogen temperature with extraordinary sensitivity, giving oxidized flavoproteins (Fp) an up to tenfold discrimination of cancer to non-cancer of breast in our preliminary data. Our finding suggests that the identified metabolic parameters could discriminate between cancer and non-cancer breast tissues without subjecting tissues to fixatives. The remainder of the frozen tissue is available for additional analysis such as molecular analysis and conventional histopathology. We propose that this novel redox scanning procedure may assist in tissue diagnosis in ex vivo tissues.

Ratiometric analysis in hyperpolarized NMR (I): Test of the two-site exchange model and the quantification of reaction rate constants

Conventional methods for the analysis of in vivo hyperpolarized 13C NMR data from the lactate dehydrogenase (LDH) reaction usually make assumptions on the stability of rate constants and/or the validity of the two‐site exchange model. In this study, we developed a framework to test the validity of the assumption of stable reaction rate constants and the two‐site exchange model in vivo via ratiometric fitting of the time courses of the signal ratio L(t)/P(t). Our analysis provided evidence that the LDH enzymatic kinetics observed by hyperpolarized NMR are in near‐equilibrium and satisfy the two‐site exchange model for only a specific time window. In addition, we quantified both the forward and reverse exchange rate constants of the LDH reaction for the transgenic and mouse xenograft models of breast cancer using the ratio fitting method developed, which includes only two modeling parameters and is less sensitive to the influence of instrument settings/protocols, such as flip angles, degree of polarization and tracer dosage. We further compared the ratio fitting method with a conventional two‐site exchange modeling method, i.e. the differential equation fitting method, using both the experimental and simulated hyperpolarized NMR data. The ratio fitting method appeared to fit better than the differential equation fitting method for the reverse rate constant on the mouse tumor data, with less relative errors on average, whereas the differential equation fitting method also resulted in a negative reverse rate constant for one tumor. The simulation results indicated that the accuracy of both methods depends on the width of the transport function, noise level and rate constant ratio; one method may be more accurate than the other based on the experimental/biological conditions aforementioned. We were able to categorize our tumor models into specific conditions of the computer simulation and to estimate the errors of rate quantification. We also discussed possible approaches to the development of more accurate rate quantification methods for hyperpolarized NMR. Copyright

Imaging heterogeneity in the mitochondrial redox state of premalignant pancreas in the pancreas-specific PTEN-null transgenic mouse model

BackgroundMetabolic alteration is one of the hallmarks of carcinogenesis. We aimed to identify certain metabolic biomarkers for the early detection of pancreatic cancer (PC) using the transgenic PTEN-null mouse model. Pancreas-specific deletion of PTEN in mouse caused progressive premalignant lesions such as highly proliferative ductal metaplasia. We imaged the mitochondrial redox state of the pancreases of the transgenic mice approximately eight months old using the redox scanner, i.e., the nicotinamide adenine dinucleotide/oxidized flavoproteins (NADH/Fp) fluorescence imager at low temperature. Two different approaches, the global averaging of the redox indices without considering tissue heterogeneity along tissue depth and the univariate analysis of multi-section data using tissue depth as a covariate were adopted for the statistical analysis of the multi-section imaging data. The standard deviations of the redox indices and the histogram analysis with Gaussian fit were used to determine the tissue heterogeneity.ResultsAll methods show consistently that the PTEN deficient pancreases (Pdx1-Cre;PTENlox/lox) were significantly more heterogeneous in their mitochondrial redox state compared to the controls (PTENlox/lox). Statistical analysis taking into account the variations of the redox state with tissue depth further shows that PTEN deletion significantly shifted the pancreatic tissue to an overall more oxidized state. Oxidization of the PTEN-null group was not seen when the imaging data were analyzed by global averaging without considering the variation of the redox indices along tissue depth, indicating the importance of taking tissue heterogeneity into account for the statistical analysis of the multi-section imaging data.ConclusionsThis study reveals a possible link between the mitochondrial redox state alteration of the pancreas and its malignant transformation and may be further developed for establishing potential metabolic biomarkers for the early diagnosis of pancreatic cancer.

Calibration of redox scanning for tissue samples

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.