Imaging human pancreatic cancer xenografts by targeting mutant KRAS2 mRNA with [(111)In]DOTA(n)-poly(diamidopropanoyl)(m)-KRAS2 PNA-D(Cys-Ser-Lys-Cys) nanoparticles.

95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, (64)Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([(111)In]DOTA-AEEA)(n)-PDAP(m)-AEEA(2)-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [(111)In](n)-PDAP(m)-KRAS2 G12D probes increased from 3.1 +/- 0.2 at n = 2, m = 1, to 4.1 +/- 0.3 at n = 8, m = 3, to 6.2 +/- 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [(111)In](n)-PDAP(m)-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 +/- 0.6 at n = 2, m = 1, 2.6 +/- 0.9 at n = 8, m = 3, and 3.7 +/- 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [(111)In]DOTA control results. Simultaneous administration of nonradioactive Gd(n)-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [(111)In](8)KRAS2 G12 V probes 3-6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of (111)In uptake increased from 3.1 +/- 0.5 to 6.5 +/- 1.0, and the kidney/tumor ratio of (111)In uptake decreased by more than 5-fold from 174.8 +/- 17.5 to 30.8 +/- 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd(n)-KRAS2 G12 V probes, enhanced tumor uptake of [(111)In](n)KRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd(n)-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.

PET imaging of VPAC1 expression in experimental and spontaneous prostate cancer.

UNLABELLED: 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 (64)Cu-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 (64)Cu and (99m)Tc. 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 (99m)Tc-TP3939 and (64)CuCl(2.) Blood clearance and in vivo stability were studied. After intravenous administration of either (64)Cu-TP3939 or (64)CuCl(2) 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 x 10(-8) M and 0.77 x 10(-9) M, respectively, for TP3939 and 9.1 x 10(-8) M and 15 x 10(-9) M, respectively, for vasoactive intestinal peptide 28. Binding of (64)CuCl(2) to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of (64)Cu-TP3939 to plasma proteins was less than 15%. (64)Cu-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 (64)CuCl(2) (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. (64)Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but (18)F-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 (18)F-FDG. (64)Cu-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.

UNLABELLED: 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 (64)Cu. We administered the [(64)Cu]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 [(64)Cu]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 (64)CuCl(2). 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.

Radionuclide imaging of small-cell lung cancer (SCLC) using 99mTc-labeled neurotensin peptide 8-13.

OBJECTIVES: To prepare 99m technetium (99mTc)-labeled neurotensin (NT) peptide and to evaluate the feasibility of imaging oncogene NT receptors overexpressed in human small-cell lung cancer (SCLC) cells. METHODS: The NT analogue (Nalpha-His)Ac-NT(8-13) was synthesized such that histidine was attached at the N-terminus. The analogue was labeled with [99mTc(H2O)3(CO)3] at pH 7. 99mTc-(Nalpha-His)Ac-NT(8-13) in vitro stability was determined by challenging it with 100 times the molar excess of DTPA, human serum albumin (HSA) and cysteine. The affinity, 99mTc-(Nalpha-His)Ac-NT(8-13) binding to SCLC cell line NCI-H446, was studied in vitro. Biodistribution and imaging with 99mTc-(Nalpha-His)Ac-NT(8-13) were performed at 4 and 12 h postinjection, and tissue distribution and imaging after receptor blocking were carried out at 4 h in nude mice bearing human SCLC tumor. Blood clearance was determined in normal mice. RESULTS: The affinity constant (Kd) of 99mTc-(Nalpha-His)Ac-NT(8-13) to SCLC cells was 0.56 nmol/L. When challenged with 100 times the molar excess of DTPA, HSA or cysteine, more than 97+/-1.8% radioactivity remained as 99mTc-(Nalpha-His)Ac-NT(8-13). Tumor-to-muscle ratio was 3.35+/-1.01 at 4 h and 4.20+/-1.35 at 12 h postinjection. The excretory route of 99mTc-(Nalpha-His)Ac-NT(8-13) was chiefly through the renal pathway. In the receptor-blocking group treated with unlabeled (Nalpha-His)Ac-NT(8-13), tumor-to-muscle ratio at 4 h was 1.25+/-0.55. CONCLUSION: The results suggest that 99mTc-(Nalpha-His)Ac-NT(8-13) specifically binds to the SCLC cells and made 99mTc-(Nalpha-His)Ac-NT(8-13) a desirable compound for further studies in planar or SPECT imaging of oncogene receptors overexpressed in SCLC cells.

Imaging thromboembolism with fibrin-avid 99mTc-peptide: evaluation in swine.

UNLABELLED: A pentapeptide, Gly-Pro-Arg-Pro-Pro, with high affinity for alpha-chain-fibrin was labeled with (99m)Tc ((99m)Tc-TP850) and evaluated in swine to image experimental venous thromboembolism (deep vein thrombosis [DVT]) and pulmonary embolism (PE). METHODS: Scatchard analysis was performed to determine fibrin affinity for TP850 and the number of binding sites (receptors) per milligram of fibrin. DVT was induced in the left jugular vein and PE was induced by introducing a preformed autologous blood clot into the right atrium using a 7-French introducer sheath inserted into the right jugular vein. (99m)Tc-TP850 was injected at 4, 24, 48, 72, 96, or 120 h later. Animals were imaged for up to 4 h after injection, heparinized, and sacrificed. Lungs were extirpated, radiographed, and imaged, and the PE was removed. Other tissues, including blood and normal lungs, were harvested and, concomitantly, (99m)Tc was counted for determination of target-to-tissue ratios and the percentage injected dose per gram of tissue. RESULTS: The affinity for human fibrin was 10(-9) mol/L and there were >10(15) receptors per milligram of fibrin. DVT and PE were visualized for up to 4 h after injection with high DVT/blood (7.9-22.6), DVT/muscle (31.1-89.4), PE/blood (1-155), and PE/lung (0.8-245) ratios. Thereafter, the PEs fragmented spontaneously below the spatial resolution of the gamma-camera and, despite the high associated radioactivity, could not be localized in vivo. The fragmented clots were detectable by scintigraphy on excised lungs and provided excellent concordance with radiograms. CONCLUSION: (99m)Tc-TP850 with its modest affinity (10(-9) mol/L), rapid blood clearance, and high DVT and PE uptake is a promising agent for imaging vascular thrombosis.

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

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.

Noninvasive molecular imaging of MYC mRNA expression in human breast cancer xenografts with a [99mTc]peptide-peptide nucleic acid-peptide chimera.

Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.

External imaging of CCND1 cancer gene activity in experimental human breast cancer xenografts with 99mTc-peptide-peptide nucleic acid-peptide chimeras.

UNLABELLED: Detection of a new or recurrent breast cancer lesion relies on physical examination and imaging studies, primarily mammography, followed by histopathologic evaluation of biopsy tissue for morphologic confirmation. Approximately 66%-85% of detected lesions are not malignant. Therefore, biopsies are unnecessary for at least two thirds of patients. Human estrogen receptor-positive breast cancer cells typically display an elevated level of cyclin D1 protein because of the overexpression of CCND1 messenger RNA (mRNA) and an elevated level of insulin-like growth factor 1 (IGF1) receptor (IGF1R) because of the overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of CCND1 peptide nucleic acid (PNA) hybridization probes with a (99m)Tc-chelating peptide on the N terminus and an IGF1 peptide loop on the C terminus could detect CCND1 mRNA in human MCF7 breast cancer xenografts in nude mice from outside the body. METHODS: We prepared the CCND1 probes as well as mismatched controls by solid-phase synthesis. We used fluorescence microscopy to detect the cellular uptake of fluoresceinyl probes and quantitative reverse transcription-polymerase chain reaction to detect the hybridization of probes to mRNA. We imaged (99m)Tc-probes in MCF7 xenografts scintigraphically and measured distribution by scintillation counting of dissected tissues. RESULTS: IGF1R-overexpressing MCF7 cells internalized the fluorescein-chelator-CCND1 PNA-IGF1 peptide but not the mismatched control peptide. The chelator-CCND1 PNA-IGF1 peptide but not the control peptide lowered the level of cyclin D1 protein in IGF1R-overexpressing MCF7 xenografts in nude mice after intratumoral injection. IGF1R-overexpressing MCF7 xenografts in nude mice were visualized at 4, 12, and 24 h after tail vein administration of the (99m)Tc-CCND1 antisense probe but not the control probe. (99m)Tc-chimeras were distributed normally in the kidneys, liver, tumors, and other tissues. CONCLUSION: Cancer gene activity can be detected from outside the body by probing with radionuclide-chelator-PNA-peptide chimeras.

PET imaging of oncogene overexpression using 64Cu-vasoactive intestinal peptide (VIP) analog: comparison with 99mTc-VIP analog.

UNLABELLED: The purpose of this study was to assess the feasibility of PET imaging of oncogene VPAC1 receptors overexpressed in human breast cancer cells. METHODS: Vasoactive intestinal peptide (VIP) analog (TP3982) was synthesized to harbor a carboxy-terminus lysine (Lys) residue separated from VIP-asparagine (Asn(28)) by 4-aminobutyric acid (Aba) as a spacer. Lys was derivatized with diaminopropionic acid coupled to a pair of dibenzoylthioglycolic acid residues as protecting groups. The analog was labeled with (64)Cu at pH 9 ((64)Cu-TP3982) and (99m)Tc at pH 12 ((99m)Tc-TP3982). (99m)Tc-TP3982 and VIP derivatized with Aba-GAGG and labeled with (99m)Tc ((99m)Tc-TP3654) were used as reference agents. Smooth muscle relaxivity assays performed with each derivative and compared with unaltered VIP(28) demonstrated functional integrity. In vitro stability of (64)Cu-TP3982 was determined by challenging the complex with 100-mol excess of diethylenetriaminepentaacetic acid (DTPA), human serum albumin (HSA), and cysteine. In vivo stability was determined in urine and serum for up to 24 h. The mass of the Cu-TP3982 complex was determined by mass spectrometry. Human T47D breast tumor xenografts were grown in athymic nude mice. Planar scintigraphic imaging was performed at 4 and 24 h after the intravenous administration of (99m)Tc-TP3982 and (99m)Tc-TP3654 and PET imaging was performed using a small animal MOSAIC PET scanner, also at 4 and 24 h after injection of (64)Cu-TP3982. Tissue-distribution studies were also performed. In a separate experiment, receptors were blocked by intravenous injection of authentic VIP(28) 30 min before the administration of (64)Cu-TP3982 and tissue distribution was examined. RESULTS: (64)Cu-TP3982 labeling yields were 98% +/- 1.2% and those for (99m)Tc-TP3982 and (99m)Tc-TP3654 were 98.2% +/- 1.1% and 97% +/- 1.6%, respectively. The biologic activity of both VIP analogs was uncompromised. When (64)Cu-TP3982 was challenged with 100-mol excess of DTPA, HSA, or cysteine, >98% radioactivity remained as (64)Cu-TP3982. In vivo, >98% of (64)Cu circulating in plasma remained as (64)Cu-TP3982. Of the (64)Cu excreted in urine 4, 20, and 24 h after injection, >98%, 89.9% +/- 0.9%, and 85% +/- 3%, respectively, were bound to TP3982. The mass of Cu-TP3982 as determined by surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) was 4,049.7 Da. Four hours after receptor blocking with VIP(28), there was a significant reduction in uptake of all tissues except in the liver. With (64)Cu-TP3982, the 4-h postinjection tumor uptake was 10.8 +/- 2.1 %ID/g versus 0.5 +/- 0.02 %ID/g and 0.24 +/- 0.08 %ID/g for (99m)Tc-TP3982 and (99m)Tc-TP3654, respectively. Twenty-four hours after injection, the corresponding numbers were 17 +/- 0.7 %ID/g, 0.77 +/- 0.1 %ID/g, and 0.23 +/- 0.1 %ID/g. The severalfold greater uptake (21.2-74) of (64)Cu-TP3982 is attributable to the in vivo stability of the agent. CONCLUSION: The results suggest that the uncompromised biologic activity and the significantly greater tumor uptake of (64)Cu-TP3982, combined with the high sensitivity and enhanced resolution of PET imaging, make (64)Cu-TP3982 highly desirable for further studies in PET imaging of oncogene receptors overexpressed in breast and other types of cancers.

Imaging oncogene expression.

In 2003, approximately 39,800 women in the US will die from breast cancer. Mammography and physical examination miss up to 40% of early breast cancers. Moreover, if an abnormality is found, an invasive diagnostic procedure must still be performed to determine if the breast contains atypia or cancer, even though approximately 85% of abnormalities are benign. Scintigraphic imaging of gene expression in vivo by noninvasive means could direct physicians to appropriate targets for intervention at the onset of disease and thereby significantly impact patient management. Until now, no method has been available to image specific overexpressed oncogene mRNAs in vivo by scintigraphic imaging. We hypothesize that gamma-emitting Tc-99m-PNA-peptides can be taken up by human ER+ and ER- breast cancer xenografts, hybridize to complementary mRNA targets in those cells, and concentrate sufficiently in tumor tissue to allow noninvasive imaging of oncogene overexpression. To prepare the probes, peptide analogs of insulin-like growth factor 1 (IGF1) were extended from a solid support by Fmoc coupling. Peptide nucleic acid (PNA) dodecamers antisense to CCND1 and MYC mRNAs were then extended from the N-terminus of IGF1, followed by a chelator peptide, using Fmoc coupling for all residues. The cysteine thiols were cyclized on the solid support, either before or after PNA extension. This simplified synthetic approach allows preparation of a variety of multipeptide disulfide-bridged PNA chimeras. A chelating peptide-PNA chimera antisense to MYC mRNA was then labeled efficiently with Tc-99m, yielding a single product. Tissue distribution studies of antisense and mismatch chimeras at 4 h and 24 h after administration displayed modest accumulation in the liver and kidneys, with appreciable levels in tumors. This result enables testing of Tc-99m-peptide-PNA probes to image gene expression in tumors.