iBeacon Indoor Positioning Method Combined with Real-Time Anomaly Rate to Determine Weight Matrix.

This paper proposes an indoor positioning method based on iBeacon technology that combines anomaly detection and a weighted Levenberg-Marquadt (LM) algorithm. The proposed solution uses the isolation forest algorithm for anomaly detection on the collected Received Signal Strength Indicator (RSSI) data from different iBeacon base stations, and calculates the anomaly rate of each signal source while eliminating abnormal signals. Then, a weight matrix is set by using each anomaly ratio and the RSSI value after eliminating the abnormal signal. Finally, the constructed weight matrix and the weighted LM algorithm are combined to solve the positioning coordinates. An Android smartphone was used to verify the positioning method proposed in this paper in an indoor scene. This experimental scenario revealed an average positioning error of 1.540 m and a root mean square error (RMSE) of 1.748 m. A large majority (85.71%) of the positioning point errors were less than 3 m. Furthermore, the RMSE of the method proposed in this paper was, respectively, 38.69%, 36.60%, and 29.52% lower than the RMSE of three other methods used for comparison. The experimental results show that the iBeacon-based indoor positioning method proposed in this paper can improve the precision of indoor positioning and has strong practicability.

EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low.

PURPOSE: The potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes. RESULTS: Cancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB. MATERIALS AND METHODS: Ten cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB. CONCLUSIONS: EpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.

Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)(-)/CD44(+) hormone-independent breast cancer cells, but not of the ER(+)/CD44(-/low) hormone-dependent breast cancer cells. Coincidentally, CD44(+) breast cancer cells were abundant in metastatic lung and brain lesions in ER(-) breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER(-)/CD44(+) breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44(+) cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER(-)/CD44(+) breast cancer.

Characterization of a novel radiation-induced sarcoma cell line.

BACKGROUND: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. METHODS: We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. RESULTS: Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. CONCLUSION: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS.

Expression profiling of circulating tumor cells in metastatic breast cancer.

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients' blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.

Trans-resveratrol alters mammary promoter hypermethylation in women at increased risk for breast cancer.

Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. Based upon in vitro studies demonstrating that trans-resveratrol downregulates the expression of 1) DNA methyltransferases and 2) the cancer promoting prostaglandin (PG)E(2), we determined if trans-resveratrol had a dose-related effect on DNA methylation and prostaglandin expression in humans. Thirty-nine adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 wk. Methylation assessment of 4 cancer-related genes (p16, RASSF-1α, APC, CCND2) was performed on mammary ductoscopy specimens. The predominant resveratrol species in serum was the glucuronide metabolite. Total trans-resveratrol and glucuronide metabolite serum levels increased after consuming both trans-resveratrol doses (P < .001 for both). RASSF-1α methylation decreased with increasing levels of serum trans-resveratrol (P = .047). The change in RASSF-1α methylation was directly related to the change in PGE(2) (P = .045). This work provides novel insights into the effects of trans-resveratrol on the breast of women at increased breast cancer risk, including a decrease in methylation of the tumor suppressor gene RASSF-1α. Because of the limited sample size, our findings should be validated in a larger study.

Quantitative evaluation of DNA hypermethylation in malignant and benign breast tissue and fluids.

The assessment of DNA had demonstrated altered methylation in malignant compared to benign breast tissue. The purpose of our study was to (i) confirm the predictive ability of methylation assessment in breast tissue, and (ii) use the genes found to be cancer predictive in tissue to evaluate the diagnostic potential of hypermethylation assessment in nipple aspirate fluid (NAF) and mammary ductoscopic (MD) samples. Quantitative methylation specific (qMS)-PCR was conducted on three specimen sets: 44 malignant (CA) and 34 normal (NL) tissue specimens, 18 matched CA, adjacent normal (ANL) tissue and NAF specimens, and 119 MD specimens. Training and validation tissue sets were analyzed to determine the optimal group of cancer predictive genes for NAF and MD analysis. NAF and MD cytologic review were also performed. Methylation of CCND-2, p16, RAR-beta and RASSF-1a was significantly more prevalent in tumor than in normal tissue specimens. Receiver operating characteristic curve analysis demonstrated an area under the curve of 0.96. For the 18 matched CA, ANL and NAF specimens, the four predictive genes identified in cancer tissue contained increased methylation in CA vs. ANL tissue; NAF samples had higher methylation than ANL specimens. Methylation frequency was higher in MD specimens from breasts with cancer than benign samples for p16 and RASSF-1a. In summary, i) routine quantitative DNA methylation assessment in NAF and MD samples is possible, and ii) genes hypermethylated in malignant breast tissue are also altered in matched NAF and in MD samples, and may be useful to assist in early breast cancer detection.

Circulating microRNAs in breast cancer and healthy subjects.

BACKGROUND: It has been demonstrated that extracellular mRNA can be detected in the circulation. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of breast cancer patients compared to controls. FINDINGS: We measured miRNA in the serum of samples with and without the addition of miRNA prior to analysis. To test our RNA extraction efficiency, we spiked-in serial dilutions of single-strand C elegens miR-39 (cel-miR-39) and human miR-145 (has-miR-145) into goat serum and a 10 year old human serum specimen. We next analyzed miR-16, -145, and -155 in archived serum specimens from 21 participants, 13 of whom did and 8 of whom did not have breast cancer. We were able to detect the miRNAs from all the serum samples to which the miRNAs had been added. We were also able to detect endogenous miR-16, -145, and -155 in all serum samples. While the expression of all three miRNAs was similar in samples from healthy women compared to those with breast cancer, women with progesterone receptor (PR, p = 0.016) positive tumors had higher miR-155 expression than tumors that were negative for these receptors. CONCLUSION: 1) RNA species can be detected in archived serum; 2) miR-155 may be differentially expressed in the serum of women with hormone sensitive compared to women with hormone insensitive breast cancer. Screening serum for miRNAs that predict the presence of breast cancer is feasible, and may be useful for breast cancer detection.

Soy isoflavones have an antiestrogenic effect and alter mammary promoter hypermethylation in healthy premenopausal women.

We determined if soy isoflavones have dose-related estrogenic and methylation effects. Thirty-four healthy premenopausal women were randomized to 40 mg or 140 mg isoflavones daily through one menstrual cycle. Breast specific and systemic estrogenic effects were assessed measuring the estrogenic marker complement (C)3 and changes in cytology, whereas methylation assessment of 5 cancer related genes (p16, RASSF1A, RARbeta2, ER, and CCND2) was performed on intraductal specimens. Serum genistein significantly increased after consuming both isoflavone doses. Cytology did not significantly change at either isoflavone dose. Serum C3 levels posttreatment were inversely related to change in serum genistein (r =-0.76, P = 0.0045) in women consuming low but not high dose isoflavones. The RAR beta 2 hypermethylation increase posttreatment correlated with the posttreatment genistein level considering the entire group (r = 0.67, P = 0.0017) and those receiving high-dose isoflavones (r = 0.68, P = 0.021). At the low but not the high isoflavone dose, CCND2 hypermethylation increase correlated with posttreatment genistein levels (r = 0.79, P = 0.011). In summary, the inverse correlation between C3 and genistein suggests an antiestrogenic effect. Isoflavones induced dose-specific changes in RARbeta2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.

uPA is upregulated by high dose celecoxib in women at increased risk of developing breast cancer.

BACKGROUND: While increased urokinase-type plasminogen activator (uPA) expression in breast cancer tissue is directly associated with poor prognosis, recent evidence suggests that uPA overexpression may suppress tumor growth and prolong survival. Celecoxib has been shown to have antiangiogenic and antiproliferative properties. We sought to determine if uPA, PA inhibitor (PAI)-1 and prostaglandin (PG)E2 expression in nipple aspirate fluid (NAF) and uPA and PGE2 expression in plasma were altered by celecoxib dose and concentration in women at increased breast cancer risk. METHODS: NAF and plasma samples were collected in women at increased breast cancer risk before and 2 weeks after taking celecoxib 200 or 400 mg twice daily (bid). uPA, PAI-1 and PGE2 were measured before and after intervention. RESULTS: Celecoxib concentrations trended higher in women taking 400 mg (median 1025.0 ng/mL) compared to 200 mg bid (median 227.3 ng/mL), and in post- (534.6 ng/mL) compared to premenopausal (227.3 ng/mL) women. In postmenopausal women treated with the higher (400 mg bid) celecoxib dose, uPA concentrations increased, while PAI-1 and PGE2 decreased. In women taking the higher dose, both PAI-1 (r = -.97, p = .0048) and PGE2 (r = -.69, p = .019) in NAF and uPA in plasma (r = .45, p = .023) were correlated with celecoxib concentrations. CONCLUSION: Celecoxib concentrations after treatment correlate inversely with the change in PAI-1 and PGE2 in the breast and directly with the change in uPA in the circulation. uPA upregulation, in concert with PAI-1 and PGE2 downregulation, may have a cancer preventive effect.

Can mitochondrial DNA mutations in circulating white blood cells and serum be used to detect breast cancer?

Circulating mitochondrial DNA (mtDNA) affected by mutations have been detected in melanoma, prostate cancer, and digestive neoplasms involving the pancreas, liver, and the colon. We sought to detect such mutations in women with breast cancer to assess if the method could be used to aid in the diagnosis of breast cancer. Blood was collected and mtDNA extracted; 27 samples included 14 patients who had breast cancer and 13 healthy controls. White blood cells and serum were separated. The mitochondrial D-loop region was amplified using PCR followed by automated DNA sequencing. The collected data was analyzed with computer software to detect both polymorphisms and mutations. mtDNA sequencing was successful in 93% of the samples (n=23). No mutations were found in any of the study groups. Polymorphisms were detected in all specimens, three of which had not been previously reported. The method used did not detect mtDNA mutations in the blood of women with breast cancer, but was extremely sensitive in polymorphism detection.

Nipple aspirate fluids from women with breast cancer contain increased levels of group IIa secretory phospholipase A2.

Arachidonic acid, a bioactive molecule metabolized into prostaglandins and leukotrienes, contributes to cellular proliferation and tumor progression. Group IIa secretory phospholipase A2 (sPLA2-IIa) can facilitate arachidonate release from cellular phospholipids, suggesting its involvement in tumor evolution. Analysis of breast nipple aspirate fluid (NAF), a noninvasive research tool, allows the identification of biomarkers of breast cancer. We sought to determine whether sPLA2-IIa expression might be related to breast cancer development or progression. sPLA2-IIa expression was evaluated in NAF samples from 110 women (57 women with and 53 without breast cancer) using ELISA and western blotting; ultrastructural immunolocalization was performed in epithelial cells floating in NAF. Immunocytochemistry revealed that sPLA2-IIa is a constitutive intracellular protein suggesting that breast ductal cells synthesize and secrete the 14 kDa protein in NAF. Among all 110 subjects, sPLA2-IIa expression was significantly increased both in NAF and within ductal epithelial cells from cancer containing breasts. While in healthy women menopausal status did not influence sPLA2-IIa expression (P = 0.457), among patients with breast cancer there was a significant down-regulation in postmenopausal subjects (P < 0.0001). Moreover, sPLA2-IIa concentration in NAF from breast cancer patients was positively correlated with tumor stage (r (2) = 0.979, P = 0.0012), suggesting an active secretion/accumulation of the enzyme in NAF based on tumor burden. sPLA2-IIa activity may serve a dual role in breast carcinogenesis, beneficial in its release of arachidonate and detrimental in the metabolic conversion of arachadonic acid into prostaglandins and leukotrienes.

Black cohosh does not exert an estrogenic effect on the breast.

Women's Health Initiative findings indicate that hormone replacement therapy may increase breast cancer and cardiovascular disease risk. Black cohosh extract (BCE) is a popular alternative that reduced menopausal symptoms in several clinical trials. Preclinical studies have addressed the estrogenic properties of BCE, with conflicting results. The estrogenic influence of BCE on the breast has not been investigated. Black cohosh is standardized to triterpenes, but the activity and mechanism of action of these compounds are unknown. The study goals were to determine 1) triterpene content of 2 commercially available BCE preparations and 2) the effect of BCE on circulating and breast-specific estrogenic markers. Two black cohosh preparations were analyzed for triterpene content. Postmenopausal women took BCE for 12 wk followed by a 12-wk washout. One BCE preparation contained trace amounts and another contained 2.5% triterpenes. Women taking BCE with 2.5% triterpenes experienced relief of menopausal symptoms, with reversion toward baseline after washout. BCE had no effect on estrogenic markers in serum and no effect on pS2 or cellular morphology in nipple aspirate fluid. Triterpene content in commercially available black cohosh preparations varies. BCE standardized to 2.5% triterpenes relieved menopausal symptoms without systemic or breast-specific estrogenic effects.

Increased expression of the inflammatory protein YKL-40 in precancers of the breast.

Serum levels of YKL-40 have been associated with inflammatory diseases and breast cancer. Our purpose was to determine if YKL-40 in breast tissue, nipple aspirate fluid (NAF) and serum is (i) concentrated in NAF compared to matched serum, (ii) increased in the NAF, serum or tissue of women with biopsy proven precancer or cancer compared to healthy women and (iii) influenced by menopausal status. 118 women (61 healthy subjects, 10 with precancer and 47 with breast cancer) aged 17-95 years provided NAF with or without serum samples for analysis. Matched tissue was analyzed from a subset of subjects who underwent breast biopsy. All NAF and serum samples had detectable levels of YKL-40. Median YKL-40 levels for the entire cohort were 683 fold higher in NAF than serum. Premenopausal subjects had higher NAF and lower serum levels of YKL-40 than postmenopausal subjects. YKL-40 levels in NAF but not serum were higher in women with precancer (atypical hyperplasia and lobular carcinoma in situ) than in either healthy subjects (p = 0.025) or subjects with breast cancer (p = 0.015). In women with precancer, YKL-40 distribution in tissue correlated with YKL serum level (p = 0.043). YKL-40 is concentrated in NAF, with the highest concentrations in premenopausal women. NAF levels of YKL-40 are significantly higher in women with precancers than healthy subjects, suggesting that measuring YKL-40 in NAF may improve the identification of women at increased breast cancer risk.

The 8-epimer of prostaglandin F(2alpha), a marker of lipid peroxidation and oxidative stress, is decreased in the nipple aspirate fluid of women with breast cancer.

Breast cancer (BC), a worldwide disease with increasing incidence, develops from ductal/lobular epithelium. Nipple aspirate fluid (NAF), secreted from the breast ducts and lobules, can be analyzed to assess breast metabolic activity. Whether lipid peroxidation in the mammary gland promotes or prevents tumorigenesis is unclear. Malondialdehyde (MDA) and the 8-epimer of Prostaglandin F(2alpha) (8-iso-PGF(2alpha)), two lipid peroxidation markers, were studied in milk (n = 10), NAF (n = 140) and plasma (n = 35) samples. MDA was detected in all plasma, in 80% of milk samples and in 95% of NAF samples. MDA levels in NAF and plasma were significantly higher than in milk (p = 0.016 and p = 0.029, respectively). We found no significant difference between levels of MDA in NAF samples from BC patients compared to healthy controls. 8-iso-PGF(2alpha) was detectable in all samples. 8-iso-PGF(2alpha) median levels in NAF were significantly higher than in both milk and plasma (p < 0.0001). The highest 8-iso-PGF(2alpha) levels were found in NAF from healthy women, significantly higher than in women with BC (p < 0.0001). No significant differences were found in both markers after the age-adjustment. High levels of lipid peroxidation products in NAF suggest their in situ production in the nonlactating breast. Active lipid peroxidation may have a physiologic role in the normal mammary gland. Lower levels of 8-iso-PGF(2alpha) in NAF from BC patients suggest altered production of arachidonic acid metabolites during breast carcinogenesis.

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.

Mitochondrial DNA mutations in breast cancer tissue and in matched nipple aspirate fluid.

Unlike nuclear (n)DNA, of which there is one paired copy per cell, there are many copies of mitochondrial (mt)DNA per cell, making PCR amplification of mtDNA easier in samples of limited cellularity. The aims of this study were to (i) determine the mutation patterns of breast cancers through a comprehensive screen of mtDNA mutations, and (ii) assess if mutations in the cancers are also detectable in breast nipple aspirate fluid (NAF), a physiologic fluid which contains shed ductal epithelial cells. Fifteen breast cancers, matched benign tissues and NAF were collected. Nine overlapping primer sets were used to sequence the entire mitochondrial genome from tissue samples. For NAF samples, we focused on the 19 nucleotide positions (np) where mutations were found in a 3701 bp region (np 15331 to 2463), which includes the displacement (D)-loop, a mtDNA mutation hot spot. Fourteen of the fifteen (93%) cancer samples had > or =1 somatic mtDNA mutation for a total of 45 at 35 np (9 np reported previously, 26 new). Nine of fifteen tumors had > or =2 mutations. The D-loop contained 17 of 45 (38%) and non-D-loop (coding) regions contained 28 (62%) mutations. Of the 28 mutations in the coding loci, 11 led to an amino acid change. The frequency of mtDNA mutations was higher in the D-loop region (1.5 versus 0.18% of loci). 155 polymorphisms were identified (98 reported previously, 57 new). Sixteen of forty-five (36%) mutations were located at polymorphism sites. Four of nineteen mtDNA mutations in 10 cancers located between np 15331 and 2463 were found in matched NAF (two of eleven mutations in the D-loop and two of eight in non-D-loop regions). No mutations were found in five matched NAF samples from women whose cancers lacked a mutation in the same region. In conclusion, mtDNA mutations in breast cancer occur both within and outside of the D-loop, though the mutation rate in the D-loop is over 7-fold higher than in coding areas. We identified 26 new mutation loci (25 in regions sequenced by others, one in an area not). The high frequency of mtDNA mutations at polymorphic loci requires further investigation. Specific mtDNA mutations can be detected in a subset of NAF samples from women with breast cancer.

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.