We have developed a high sensitivity near-infrared (NIR) optical imaging system for non-invasive cancer detection through molecular labeled fluorescent contrast agents. Near-infrared (NIR) imaging can probe tissue deeply thus possess the potential for non-invasively detection of breast or lymph node cancer. Recent developments in molecular beacons can selectively label various pre-cancer/cancer signatures and provide high tumor to background contrast. To increase the sensitivity in detecting fluorescent photons and the accuracy of localization, phase cancellation (in- and anti-phase) device is employed. This frequency-domain system utilizes the interference-like pattern of diffuse photon density wave to achieve high detection sensitivity and localization accuracy for the fluorescent heterogeneity embedded inside the scattering media. The opto-electronic system consists of the laser sources, fiber optics, interference filter to select the fluorescent photons and the high sensitivity photon detector (photomultiplier tube). The source-detector pair scans the tissue surface in multiple directions and the two-dimensional localization image can be obtained using goniometric reconstruction. In vivo measurements with tumor-bearing mouse model using the novel Cypate-mono-2-deoxy-glucose (Cypate-2-D-Glucosamide) fluorescent contrast agent, which targets the enhanced tumor glycolysis, demonstrated the feasibility on detection of 2 cm deep subsurface tumor in the tissue-like medium, with a localization accuracy within 2 ~ 3 mm. This instrument has the potential for tumor diagnosis and imaging, and the accuracy of the localization suggests that this system could help to guide the clinical fine-needle biopsy. This portable device would be complementary to X-ray mammogram and provide add-on information on early diagnosis and localization of early breast tumor.
The effect of photodynamic therapy (PDT) treatment on the metabolic state of tumor mitochondria is investigated by imaging of tumor redox status. PDT is performed using the photosensitizer pyropheophorbide-2-deoxyglucosamide (Pyro-2DG), which utilizes the glucose import pathway. It is found that Pyro-2DG-induced PDT resulting in a highly oxidized state of tumor mitochondria. This is determined from the redox ratio changes derived from the intrinsic oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) [i.e., reduced nicotinamide adenine dinucleotide (NADH)] fluorescence signals observed using a cryoimager. Thus, the redox ratio is a sensitive indicator for providing reliable and informative measurements of PDT-induced tissue damage. In the PDT treated region of the tumor, highly oxidized flavoprotein and diminishing NADH fluorescence is detected, suggesting that flavoprotein and NADH are oxidized by singlet oxygen produced in the photosensitization process.
In the previous study, we have proved that Pyro-2DG could be trapped and accumulated in the tumor. The fluorescent signal of Pyro-2DG has high correlation with the redox ratio. To further study the extrinsic probe Pyro-2DG, the characteristic of Pyro-2DG uptake by the liver tissue of rats and the distribution of Pyro-2DG in various tissues were detected using the Cryo-Imager. The result indicated that Pyro-2DG uptake by the liver resulted in the metabolic state shift towards an increased concentration of oxidized electron carriers and it completely recovered after 24hrs. Pyro-2DG with infusion injection could avoid the oxidized damage of the normal tissue induced by Pyro-2DG uptake. The distribution of Pyro-2DG in various normal tissues indicated that Pyro-2DG could be as a tumor special near infrared ray (NIR) probe besides the hepatic tumor and lymph system tumor.
We utilized the nude mouse model bearing the human hepatoblastoma G2 (HepG2) tumor and B-16 Murine Melanoma tumor to study the delivery and detection of the reconstituted Pyropheophorbide Cholesterol Oleate (r-pyroCE) molecular beacon. The delivery vehicle, low-density lipoprotein (LDL), labeled with the porphyrin derivative, was employed in response of the overexpression of LDL receptors in the HepG2 tumor. The B-16 melanoma tumor was also observed in this study for its overexpression of the LDL receptors. The tumors were imaged using the 3D low temperature scanner to produce images throughout several sliced sections of each tumor. The fluorescence signal of the pyropheophorbide was detected at 720nm when excited at 670nm in the tumor tissue. The uniform distribution of the signal in the HepG2 tumor shows extravasation of the beacon from the blood vessels. The B-16 tumor did not exhibit strong fluorescent signals and successful delivery as the HepG2 tumor outside the blood vessels and into the tumor tissue.
KEYWORDS: Tumors, Luminescence, Animal model studies, Scattering, Near infrared, Phased arrays, In vivo imaging, Breast, Signal detection, Imaging systems
We have developed a novel imaging system for determining the localization of tumors labeled by fluorescent contrast agents and embedded several centimeters inside the highly scattered medium. This frequency-domain system utilizes the phased cancellation configuration with a goniometric probe. The instrumentation performance on the phantom test can detect 3 mm diameter sphere filled with 1 nM fluorescent dye, Indocyanine Green (ICG), and 3 cm deep inside the scattering medium with similar optical properties as human breast tissue within a 1 mm localization confidence. Mouse tumor model immersed in appropriate scattering/absorbing medium is used for animal test. Intra-tumor injection of ICG demonstrates the localization of the tumor (5 mm in diameter) submerged 3 cm deep inside the highly scattered medium with 2 mm position error. Results with NIR804-D-Glucosamide on the AR42J tumor bearing nude mouse are also presented with 3 mm localization error. The accuracy of the localization suggest that this system would be helpful to guide the clinical fine-needle biopsy for early breast cancer detection.
This study imaged the metabolic state of a growing tumor and the relationship between energy metabolism and the ability of glucose uptake in whole tumor tissue with cryo-imaging at 77° K. A MTB/TOM mouse model, bearing c-MYC-induced mammary tumor, was very rapidly freeze-trapped 2 hrs post Pyro-2DG injection. The fluorescence signals of oxidized flavoprotein (Fp), reduced pyridine nucleotide (PN), pyro-2DG, and the reflection signal of deoxy-hemoglobin were imaged every 100 μm from the top surface to the bottom of the tumor sequentially, 9 sections in total. Each of the four signals was constructed into 3D images with Amira software. Both Fp and PN signals could be detected in the growing tumor regions, and a higher reduction state where was shown in the ratio images. The necrotic tumor regions displayed a very strong Fp signal and weak PN signal. In the bloody extravasation regions, Fp and PN signals were observably diminished. Therefore, the regions of high growth and necrosis in the tumor could be determined according to the Fp and PN signals. The content of deoxy-hemoglobin (Hb) in the tumor was positively correlated with the reduced PN signal. Pyro-2DG signal was only evident in the growing condition region in the tumor. Normalized 3D cross-correlation showed that Pyro-2DG signal was similar to the redox ratio. The results indicated that glucose uptake in the tumor was consistent with the redox state of the tumor. And both Pyro-2DG and mitochondrial NADH fluorescence showed bimodal histograms suggesting that the two population of c-MYC induced mammary tumor, one of which could be controlled by c-MYC transgene.
Proteases are involved in the invasion and metastasis of tumor cells. Cathepsin B overexpression has been shown in some neoplastic tissues. This study assesses the expression of Cathepsin B in the human fibrosarcoma (HT1080) in the mouse model by near-infrared (NIR) imaging. The nude mice were intravenously injected "a stealth probe" - an activable Cathepsin B sensing near-infrared fluorescence (NIRF) probe (24 hours before sacrifice) and the dye Cy5.5 (30 seconds before sacrifice). The animals were freeze-trapped and NIR images were obtained by the low temperature NIR scanner at the following excitation-emission wavelength pairs: 366, 450nm (NADH), 436, 520nm (FAD), and 670, 695nm (Cathepsin B probe). After imaging, the samples were submitted for histopathological evaluation. The tumor redox ratio NADH/(NADH+FAD) increased significantly because of the hypoxic state of tumor tissue with respect to normal tissue. The Cathepsin B probe was uniformly distributed throughout the tumor. This study indicated the efficient usage of the Cathepsin B probe in the molecular imaging for the detection of the early stage tumors.
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