Christopher A. Cardi

Decorin Protein Core Inhibits in Vivo Cancer Growth and Metabolism by Hindering Epidermal Growth Factor Receptor Function and Triggering Apoptosis via Caspase-3 Activation

Decorin is not only a regulator of matrix assembly but also a key signaling molecule that modulates the activity of tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR). Decorin evokes protracted internalization of the EGFR via a caveolar-mediated endocytosis, which leads to EGFR degradation and attenuation of its signaling pathway. In this study, we tested if systemic delivery of decorin protein core would affect the biology of an orthotopic squamous carcinoma xenograft. After tumor engraftment, the animals were given intraperitoneal injections of either vehicle or decorin protein core (2.5-10 mg kg-1) every 2 days for 18-38 days. This regimen caused a significant and dose-dependent inhibition of the tumor xenograft growth, with a concurrent decrease in mitotic index and a significant increase in apoptosis. Positron emission tomography showed that the metabolic activity of the tumor xenografts was significantly reduced by decorin treatment. Decorin protein core specifically targeted the tumor cells enriched in EGFR and caused a significant down-regulation of EGFR and attenuation of its activity. In vitro studies showed that the uptake of decorin by the A431 cells was rapid and caused a protracted down-regulation of the EGFR to levels similar to those observed in the tumor xenografts. Furthermore, decorin induced apoptosis via activation of caspase-3. This could represent an additional mechanism whereby decorin might influence cell growth and survival.

Simplified quantification of small animal [18F]FDG PET studies using a standard arterial input function

PurposeArterial input function (AIF) measurement for quantification of small animal PET studies is technically challenging and limited by the small blood volume of small laboratory animals. The present study investigated the use of a standard arterial input function (SAIF) to simplify the experimental procedure.MethodsTwelve [18F]fluorodeoxyglucose ([18F]FDG) PET studies accompanied by serial arterial blood sampling were acquired in seven male Sprague-Dawley rats under isoflurane anaesthesia without (every rat) and with additional (five rats) vibrissae stimulation. A leave-one-out procedure was employed to validate the use of a SAIF with individual scaling by one (1S) or two (2S) arterial blood samples.ResultsAutomatic slow bolus infusion of [18F]FDG resulted in highly similar AIF in all rats. The average differences of the area under the curve of the measured AIF and the individually scaled SAIF were 0.11±4.26% and 0.04±2.61% for the 1S (6-min sample) and the 2S (4-min/43-min samples) approach, respectively. The average differences between the cerebral metabolic rates of glucose (CMRglc) calculated using the measured AIF and the scaled SAIF were 1.31±5.45% and 1.30±3.84% for the 1S and the 2S approach, respectively.ConclusionThe use of a SAIF scaled by one or (preferably) two arterial blood samples can serve as a valid substitute for individual AIF measurements to quantify [18F]FDG PET studies in rats. The SAIF approach minimises the loss of blood and should be ideally suited for longitudinal quantitative small animal [18F]FDG PET studies.

PET Imaging of VPAC1 Expression in Experimental and Spontaneous Prostate Cancer

Among U.S. men, prostate cancer (PC) accounts for 29% of all newly diagnosed cancers. A reliable scintigraphic agent to image PC and its metastatic or recurrent lesions and to determine the effectiveness of its treatment will contribute to the management of this disease. All PC overexpresses VPAC1 receptors. This investigation evaluated a probe specific for a 64Cu-labeled receptor for PET imaging of experimental human PC in athymic nude mice and spontaneously grown PC in transgenic mice. Methods: The probe, TP3939, was synthesized, purified, and labeled with 64Cu and 99mTc. Using a muscle relaxivity assay, biologic activity was assessed and inhibitory concentrations of 50% calculated. Receptor affinity (Kd) for human PC3 cells was determined using 99mTc-TP3939 and 64CuCl2. Blood clearance and in vivo stability were studied. After intravenous administration of either 64Cu-TP3939 or 64CuCl2 in PC3 xenografts and in transgenic mice, PET/CT images were acquired. Prostate histology served as the gold standard. Organ distribution studies (percentage injected dose per gram [%ID/g]) in normal prostate were performed. The ratios of tumor to muscle, tumor to blood, normal prostate to muscle, and tumor to normal prostate were determined. Results: Chemical and radiochemical purities of TP3939 were 96.8% and 98% ± 2%, respectively. Inhibitory concentrations of 50% and affinity constants were 4.4 × 10−8 M and 0.77 × 10−9 M, respectively, for TP3939 and 9.1 × 10−8 M and 15 × 10−9 M, respectively, for vasoactive intestinal peptide 28. Binding of 64CuCl2 to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of 64Cu-TP3939 to plasma proteins was less than 15%. 64Cu-TP3939 uptake in PC was 7.48 ± 3.63 %ID/g at 4 h and 5.78 ± 0.66 %ID/g at 24 h after injection and was significantly (P < 0.05) greater than with 64CuCl2 (4.79 ± 0.34 %ID/g and 4.03 ± 0.83 %ID/g at 4 and 24 h, respectively). The ratios of PC to normal prostate at 4 and 24 h were 4 and 2.7, respectively. 64Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but 18F-FDG and CT did not. Conclusion: Data indicate that TP3939, with its uncompromised biologic activity, delineated xenografts and cases of occult PC that were not detectable with 18F-FDG. 64Cu-TP3939 is a promising probe for PET imaging of PC. It may also be useful for localizing recurrent lesions and for determining the effectiveness of its treatment.

PET Imaging of CCND1 mRNA in Human MCF7 Estrogen Receptor–Positive Breast Cancer Xenografts with Oncogene-Specific [64Cu]Chelator-Peptide Nucleic Acid-IGF1 Analog Radiohybridization Probes

Treatment of breast cancer is hampered by a large unmet need for rapid, sensitive, specific staging and stratification of palpable and nonpalpable abnormalities. Mammography and physical examination miss many early breast cancers, yet detect many benign lesions. Cyclin D1, encoded by CCND1 messenger RNA (mRNA), and insulin-like growth factor 1 receptor (IGF1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. Therefore, we hypothesized that malignant breast masses could be imaged and quantitated externally by PET with a dual-specificity probe that targets both CCND1 mRNA and IGF1R. Methods: We designed a CCND1-specific peptide nucleic acid (PNA) hybridization sequence (CTGGTGTTCCAT), separated by a C-terminal spacer to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys), for IGF1R-mediated endocytosis. On the N-terminus we attached a chelator (1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl [DO3A]) for the positron-emitting nuclide 64Cu. We administered the [64Cu]CCND1-IGF1 analog radiohybridization probes, as well as sequence controls, by tail vein to immunocompromised female NCr mice bearing human MCF7 estrogen-dependent, receptor-positive xenografts. We imaged the mice by PET and CT 4 and 24 h later, and measured tissue distribution of the radiohybridization probes. Results: We observed 8 ± 2-fold higher PET intensity in the center of the breast cancer xenografts than in the contralateral tissues at 24 h after injection of the [64Cu]CCND1-IGF1 analog radiohybridization probe. IGF1 blocking yielded significantly weaker images (P < 0.05) relative to the tumor-free side at 24 h after injection, as did a PNA mismatch probe, a peptide mismatch probe, and free 64CuCl2. Conclusion: These results are consistent with our hypothesis for radiohybridization PET of overexpressed CCND1 mRNA, dependent on IGF1R-mediated endocytosis, in suspect masses. Early noninvasive detection of initial cancerous transformation, as well as invasive or recurrent breast cancer, with dual-specificity radiohybridization probes, might enable molecularly targeted staging, stratification, and choice of therapy.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) receptor specific peptide analogues for PET imaging of breast cancer: In vitro/in vivo evaluation.

Vasoactive intestinal peptide and pituitary adenylate cyclase activating peptide have high affinity for VPAC1, VPAC2 and PAC1 receptors overexpressed on human cancer cells. Four potent analogues of these peptides, TP3939, TP3982, TP4200 and TP3805 were labeled with (64)Cu and evaluated ex vivo and in vivo to asses their biological activity and receptor specificity. The ultimate goal is to utilize (64)Cu analogues for positron emission tomography (PET) imaging of breast cancers in humans. Radiochemical purity of each analogue was >92%. The muscle relaxivity assay revealed IC(50) to be 5.3x10(-8) M, 4.4x10(-8) M, 8.1x10(-8) M, 8.1x10(-9) M and Kd values determined by receptor specific cell binding assays were 3.3 nM, 0.33 nM, 0.2 nM and 0.72 nM for TP3805, TP3939, TP3982, and TP4200 respectively. The receptor affinity, using human breast cancer tissues, was 10.93 times greater than normal breast tissues. RT-PCR confirmed increased VPAC1 receptor expression on human breast tumor cells over normal cells and corroborated with autoradiography data. The blood clearance was rapid and in vivo translocation of (64)Cu to plasma protein was <15%. Data demonstrate that these analogues are potent, have uncompromised biological activity and are worthy of further evaluation for accurate PET imaging of human breast cancers and in determining malignant and benign lesions.

External imaging of CCND1, MYC, and KRAS oncogene mRNAs with tumor-targeted radionuclide-PNA-peptide chimeras.

Abstract: In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one‐quarter of cancers in women aged 40‐49 years, and one‐fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5‐year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor‐positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin‐like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C‐terminus, and a Tc‐99m‐chelator peptide on the N‐terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R‐overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc‐99m‐chelator PNA‐peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu‐64‐chelator PNA‐peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4‐24 h after tail‐vein administration of MYC or CCND1 Tc‐99m‐chelator PNA‐peptides, but not after administration of mismatch controls. Similarly, human Panc‐1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail‐vein administration of a KRAS Tc‐99m‐chelator PNA‐peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail‐vein adminstration of a KRAS Cu‐64‐chelator PNA‐peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs in solid tumors with radiolabel‐PNA‐peptide chimeras might in the future provide additional genetic characterization of pre‐invasive and invasive breast cancers.

First results of a dedicated breast PET imager, BPET, using NaI(Tl) curve plate detectors

We present the first imaging results from phantom measurements of a dedicated, breast-only positron emission imager, BPET, using NaI(Tl) Curve Plate detectors. The scanner uses 19 mm thick NaI(Tl) detectors in a split-ring design which surrounds the breast as the woman lies prone and the breast hangs down from the body. Because the detectors are close to the breast and the scanner detects photons that do not pass through the body, system sensitivity and spatial resolution are both optimized. The split ring design provides for flexibility for needle aspirations of masses or alternate viewing orientations. We have measured energy resolution, spatial resolution, scatter fraction, and system sensitivity. We have compared the BPET scanners performance to our clinical whole-body scanner using a breast phantom with hot spheres simulating lesions. The results show that for activity concentrations that correspond to clinical FDG doses, the dedicated scanner has better lesion detectability than the whole-body scanner for the 20 cm detector separation used.

Design of (Gd-DO3A)n-Polydiamidopropanoyl-Peptide Nucleic Acid-d(Cys-Ser-Lys-Cys) Magnetic Resonance Contrast Agents

We hypothesized that chelating Gd(III) to 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would provide a magnetic resonance genetic imaging agent capable of hybridization to a specific mRNA. Because of the low sensitivity of Gd(III) as an magnetic resonance imaging (MRI) contrast agent, a single Gd-DO3A complex per PNA hybridization agent could not provide enough contrast for detection of cancer gene mRNAs, even at thousands of mRNA copies per cell. To increase the Gd(III) shift intensity of MRI genetic imaging agents, we extended a novel DO3An-polydiamidopropanoyl (PDAPm) dendrimer, up to n = 16, from the N-terminus of KRAS PNA hybridization agents by solid phase synthesis. A C-terminal D(Cys-Ser-Lys-Cys) cyclized peptide analog of insulin-like growth factor 1 (IGF1) was included to enable receptor-mediated cellular uptake. Molecular dynamic simulation of the (Gd-DO3A-AEEA)16-PDAP4-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) genetic imaging nanoparticles in explicit water yielded a pair correlation function similar to that of PAMAM dendrimers, and a predicted structure in which the PDAP dendron did not sequester the PNA. Thermal melting measurements indicated that the size of the PDAP dendron included in the (DO3A-AEEA)n-PDAPm-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) probes (up to 16 Gd(III) cations per PNA) did not depress the melting temperatures (Tm) of the complementary PNA/RNA hybrid duplexes. The Gd(III) dendrimer PNA genetic imaging agents in phantom solutions displayed significantly greater T1 relaxivity per probe (r1 = 30.64 +/- 2.68 mM(-1) s(-1) for n = 2, r1 = 153.84 +/- 11.28 mM(-1) s(-1) for n = 8) than Gd-DTPA (r1 = 10.35 +/- 0.37 mM(-1) s(-1)), but less than that of (Gd-DO3A)32-PAMAM dendrimer (r1 = 771.84 +/- 20.48 mM(-1) s(-1)) (P < 0.05). Higher generations of PDAP dendrimers with 32 or more Gd-DO3A residues attached to PNA-D(Cys-Ser-Lys-Cys) genetic imaging agents might provide greater contrast for more sensitive detection.

Performance of a GSO brain PET camera

A high sensitivity, high resolution brain PET scanner has been developed. The scanner comprises 58 rows of 320 4/spl times/4/spl times/10 mm/sup 3/ gadolinium orthosilicate (GSO) crystals, coupled to a continuous light guide that is sampled by 288 39-mm photomultiplier tubes in a hexagonal grid. A distortion removal algorithm has been developed to remove position non-linearities and to identify individual crystal regions in a flood image with physical crystal locations on the scanner. A central profile through a sinogram of a point source in the center of the scanner shows that the spatial resolution (fwhm) is 3.5 mm (fwtm=8.4 mm). The Hoffman brain phantom has been successfully imaged to demonstrate the capability of the scanner.

Receptor-mediated internalization of chelator-PNA-peptide hybridization probes for radioimaging or magnetic resonance imaging of oncogene mRNAs in tumours

Early external detection of cancer gene activity might enable early treatment of cancer and might reduce cancer mortality. We hypothesized that oncogene mRNA overexpressed at thousands of copies per malignant cell in a zone of transformed cells could be imaged externally by scintigraphic imaging, PET (positron emission tomography) or MRI (magnetic resonance imaging) with PNA (peptide nucleic acid) hybridization probes that include chelators for metal cations and a cyclized peptide analogue of IGF-1 (insulin-like growth factor 1), D(Cys-Ser-Lys-Cys), to mediate internalization by IGF1R (IGF-1 receptor) overexpressed on cancer cells. We observed that human MCF7 breast cancer cells that overexpress IGF1R efficiently internalized fluorescein-chelator-PNA-D(Cys-Ser-Lys-Cys) to the cytoplasm, but not with D(Cys-Ala-Ala-Cys). Scintigraphic imaging of MCF7 xenografts in immunocompromised mice revealed that CCND1 and MYC [(99m)Tc]chelator-PNA-D(Cys-Ser-Lys-Cys) probes yielded xenograft. PET imaging with [(64)Cu]chelator-PNA-D(Cys-Ser-Lys-Cys) yielded stronger signals. Scintigraphic imaging of human AsPC1 pancreas cancer xenografts with [(99m)Tc]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys) yielded strong xenograft signals. Stronger xenograft image intensities were obtained by PET imaging of [(64)Cu]chelator-KRAS PNA-D(Cys-Ser-Lys-Cys). MRI required extension of chelator-polydiamidopropanoate dendrimers from the N-termini of the PNA probes to increase the number of contrast paramagnetic gadolinium (III) cations per probe. These results provide a basis for detection of oncogene activity in tissues from outside the body by hybridization with metal-chelator-PNA-peptides that are selectively internalized by cancer cells.