Triple-negative breast cancer (TNBC), defined by the absence of ER, PR, and Her2, impacts over 46,000 U.S. women annually, disproportionately affecting minority ethnic groups and individuals with BRCA1 mutations. Despite advancements such as PARP inhibitors, TNBC remains highly aggressive, with frequent recurrences and a 50% mortality rate within four years, underscoring the urgent need for more effective targeted therapies. MicroRNAs (miRNAs) represent a novel therapeutic approach. In TNBC, overexpressed miR-21 drives tumor progression, immune evasion, treatment resistance, and metastasis. Targeted miR-21 inhibition could curb these effects while minimizing harm to normal cells. We developed a peptide-conjugated miR-21 inhibitor targeting TNBC cells via the overexpressed IGF1 receptor (IGF1R), associated with poor prognosis. Using aminomethyl-bridged nucleic acid (BNA) chemistry, a serum-stable, low-toxicity anti-miR-21 RNA analog was created and tested for its effects on TNBC cell proliferation, apoptosis, tumor suppressor expression, and immune checkpoint regulation. Conjugation to an IGF1 peptide analog improved delivery, demonstrating tumor-specific biodistribution, efficacy, and safety in TNBC-bearing mice. The miR-21 inhibitor-peptide conjugate reduced proliferation, induced apoptosis, elevated tumor suppressors, and suppressed immune checkpoints in TNBC cell lines. In vivo , it targeted tumors, halted growth, and showed no liver or kidney toxicity, supporting its potential as a targeted, low-toxicity TNBC therapy.

e13097 Background: Triple negative breast cancer (TNBC) is an orphan disease that attacks > 46,000 US women every year. TNBC recurs after standard-of-care surgery, chemotherapy and radiation, killing half its victims within 4 years. Poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates extend the survival of a fraction of TNBC patients for a few months. Thus, TNBC shows a critical need for molecularly-targeted therapy. Most TNBC cells show high microRNA 21 (miR-21), identical in humans and mice, which decreases tumor suppressor proteins that keep cell growth in check. Hypothesis: Short anti-miR-21 RNA analogs conjugated to a receptor ligand will direct TNBC cell uptake and slow the growth of TNBC orthotopic allografts with immune activation and minimal toxicity. Methods: We designed an anti-miR-21 RNA analog using aminomethyl bridged nucleic acid (BNA), exhibiting low toxicity, and serum stability. We lipofected a concentration ramp of the anti-miR-21 RNA analog into human TNBC cell lines (MDA-MB-231, MDA-MB-157, MDA-MB-436, MDA-MB-468, BT-20, BT-549, HCC1806, HCC1937, HCC1806) representing a spectrum of TNBC subtypes, then measured proliferation and biochemical markers. We also conjugated a peptide analog for endocytosis by the insulin-like growth factor 1 receptor (IGF1R), overexpressed in most TNBC lesions. The peptide ligand for IGF1R provides a unique strategy for delivering the anti-miR-21 RNA analog preferentially into TNBC cells. We administered 5 mg/kg of the anti-miR-21 RNA-peptide analog in sterile saline intraperitoneally twice a week for 14 days into immunocompetent female Balb/c mice bearing syngeneic EMT6 TNBC tumors in their mammary fat pads. Results: The anti-miR-21 RNA analog slowed proliferation, increased apoptosis, elevated tumor suppressor proteins, and suppressed immune checkpoint gene expression in multiple human TNBC lines. IC50 for inhibiting cell proliferation correlated with miR-21 copies/cell in 7 TNBC cell lines, but not in the non-tumorigenic breast epithelial cell line MCF-10A. The anti-miR-21 RNA-peptide analog stopped orthotopic TNBC allograft growth in syngeneic immunocompetent female Balb/c mice during 14 days of therapy, with apparent safety. Conclusions: This strategy optimizes cancer cell-specific delivery to block proliferation and immune checkpoints. The results support further preclinical proof-of-concept studies to enable an eIND submission for the anti-miR-21 RNA-peptide analog.

Scintigraphic imaging of malignant glioblastoma (MG) continues to be challenging. We hypothesized that VPAC1 cell surface receptors can be targeted for positron emission tomography (PET) imaging of orthotopically implanted MG in a mouse model, using a VPAC1-specific peptide [64Cu]TP3805. The expression of VPAC1 in mouse GL261 and human U87 glioma cell lines was determined by western blot. The ability of [64Cu]TP3805 to bind to GL261 and U87 cells was studied by cell-binding. Receptor-blocking studies were performed to validate receptor specificity. GL261 tumors were implanted orthotopically in syngeneic T-bet knockout C57BL/6 mouse brain (N = 15) and allowed to grow for 2–3 weeks. Mice were injected i.v., first with ~ 150 μCi of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) then 24 h later with ~ 200 μCi of [64Cu]TP3805. In another set of tumor-bearing mice, (N = 5), ionic [64Cu]Cl2 was injected as a control. Mice were imaged at a 2-h post-injection using an Inveon micro-PET/CT, sacrificed and % ID/g of [64Cu]TP3805 and [64Cu]Cl2 were calculated in a tumor, normal brain, and other tissues. For histologic tissue examination, 3-μm thick sections of the tumors and normal brain were prepared, digital autoradiography (DAR) was performed, and then the sections were H&E stained for histologic examination. Western blots showed a strong signal for VPAC1 on both cell lines. [64Cu]TP3805 cell-binding was 87 ± 1.5 %. Receptor-blocking reduced cell-binding to 24.3 ± 1.5 % (P < 0.01). PET imaging revealed remarkable accumulation of [64Cu]TP3805 in GL261 MG with a negligible background in the normal brain, as compared to [18F]FDG. Micro-PET/CT image analyses and tissue distribution showed that the brain tumor uptake for [64Cu]TP3805 was 8.2 ± 1.7 % ID/g and for [64Cu]Cl2 2.1 ± 0.5 % ID/g as compared to 1.0 ± 0.3 % ID/g and 1.4 ± 0.3 % ID/g for normal mouse brains, respectively. The high tumor/normal brain ratio for [64Cu]TP3805 (8.1 ± 1.1) allowed tumors to be visualized unequivocally. Histology and [64Cu]TP3805 DAR differentiated malignant tumors from healthy brain and confirmed PET findings. Targeting VPAC1 receptors using [64Cu]TP3805 for PET imaging of MG is a promising novel approach and calls for further investigation.

1282 Objectives: Our previous studies in which we used Copper-64 labeled VPAC1 receptor specific PACAP analogue (TP-3805) to image breast cancer (BC) in humans, showed that BC can be imaged at 15 minute post injection with 100% sensitivity and specificity. No triple negative BC (TNBC) were included. The objective here was to determine if 64Cu-TP3805 could be used to image TNBC in nude female mice bearing MDA-MB-231xenografts. Methods: 30 µL of 64CuCl2 in 0.1M HCl, was added to peptide-conjugate (20 µg) in 200 µL of 0.2M glycine buffer (pH=9.27), and incubated at 70°C for 90 min. The radiochemical purity was determined by radio-HPLC. Expression of VPAC1 receptors on MDA-MB-231, along with three other human BC cells lines namely MD-MBA-231, MD-MBA-157, MD-MBA-436 and MD-MBA-468, was determined by western blots. Cell binding assay was performed by incubating 64Cu-TP3805 with 1.5 x 106 MDA-MB-231, human TNBC cells. 107 MDA-MB-231 cells grown in the tissue culture were administered subsequently in the right thigh of the mice (N=5) and tumors were allowed to grow to no more than 6mm in diameter. PET/CT imaging, receptor blocking, and tissue distribution were performed. Results: radio-HPLC showed retention time for 64Cu-peptide at 5.3 min as compared to free 64CuCl2 at 3.4. The radiochemical purity of 64Cu-TP3805 was 96.3 ± 0.5 %. We found strong signal for VPAC1 receptor on western blot (Fig-1). The cell binding for 64Cu-peptide was 83.96 ± 3.5 %. Micro-PET/CT images were analyzed and tissue distribution showed tumor to muscle (T/M) ratio of 5 ± 0.5 post 2h injection. Receptor blocked imaging studies with 64Cu-TP3805 showed ~60% decrease in T/M (1.9 ± 0.2). The tissue distribution showed a similar pattern as observed in PET imaging. Conclusions: Western blot studies confirmed high VPAC1 expression on the TNBC cells lines examined. TP3805 showed excellent labeling efficiency, and high uptake in TNBC cells. Receptor blocking confirmed the specificity of the 64Cu-TP3805 for the VPAC1 receptors in TNBC cells, which makes it a highly suitable biomarker to image TNBC. Acknowledgments: The research, in part, was supported by NIH/NCI RO1CA157372 (MLT), NIH/NCI 1S10OD012406 (MLT) and NIH/NCI S10RR23709 (MLT). References 1- VPAC1 Receptors for Imaging Breast Cancer: A Feasibility Study. J Nucl Med. 2013 Jul;54(7):1019-25, Mathew L. Thakur, Kaijun Zhang, Adam Berger, Barbara Cavanaugh, Sung Kim, Chaitra Channappa, Andrea J. Frangos, Eric Wickstrom and Charles M. Intenzo.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative genetic disorder caused by CAG repeat expansion in exon 1 of the HTT gene. Expression of the mutant gene results in the production of a neurotoxic polyglutamine (polyQ)-expanded huntingtin (Htt) protein. Clinical trials of knockdown therapy of mutant polyglutamine-encoding HTT mRNA in Huntington's disease (HD) have begun. To measure HTT mRNA knockdown effectiveness in human cells, we utilized a fluorescent hybridization imaging agent specific to the region encompassing the human HTT mRNA initiation codon. We designed, synthesized, purified, and characterized Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides. The human HTT PNA 12mer complement was CATGGCGGTCTC, while the rat htt equivalent 12mer contained the sequence CATGaCGGcCTC, with two bases differing from the human sequence. The cyclized IGF1 tetrapeptide fragment d(CSKC) that promotes IGF1 receptor-mediated endocytosis was bonded to the C-terminus. We tested the reliability of HTT mRNA imaging with Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides in human embryonic kidney (HEK) 293T cells that express endogenous HTT and IGF1 receptor. By qPCR, we quantitated HTT mRNA in HEK293T cells with and without HTT mRNA knockdown by three different siRNAs. By confocal fluorescence imaging, we quantitated the accumulation of fluorescent HTT hybridization agent in the same cells. A rat homologue differing from the human sequence by two bases showed negligible fluorescence. qPCR indicated 86 ± 5% knockdown of HTT mRNA by the most effective siRNA. Similarly, Cal560- HTT PNA-peptide fluorescence intensity indicated 69 ± 6% reduction in HTT mRNA. We concluded that the fluorescence hybridization method correlates with the established qPCR method for quantitating HTT mRNA knockdown by siRNA in HEK293T cells, with a Pearson correlation coefficient of 0.865 for all three siRNA sequences. These results will enable real time imaging and quantitation of HTT mRNA in animal models of HD.

In recent years, considerable progress has been made in the use of gallium-68 labeled receptor-specific peptides for imaging oncologic diseases. The objective was to examine the stability and pharmacokinetics of [68Ga]NODAGA and DOTA-peptide conjugate targeting VPAC [combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)] receptors on tumor cells.

Abstract Triple-negative breast cancer (TNBC) attacks >40,000 young women annually in the US, and has no molecular targeted therapies. MicroRNAs drive many forms of cancer, and they modulate multiple gene pathways simultaneously. Highly expressed microRNA miR-17-5p is distinctive in TNBC and contributes to cancer cell survival. We hypothesized that reduction of miR-17-5p activity by delivering oligonucleotide-based antagomiRs specifically to breast cancer cells via receptor-mediated endocytosis would inhibit metastatic behavior of TNBC cells. Using a luciferase reporter system harboring a miR-17-5p binding site in the 3′ untranslated region of the luciferase gene, we assessed the efficacy of various antagomiRs. Contrary to conventional wisdom, that only one of the two strands in a pre-miRNA duplex is active, such as miR-17-5p, we previously observed that a full-length miR-17-5p DNA-LNA blocker created additional off-target effects by mimicking miR-17-3p, implying that therapeutic microRNA blockers should be designed to avoid resemblance to the opposing strands. We designed guide strand specific blockers of miR-17-5p composed of gapmers with anionic backbone derivatives 2′-fluoro-arabino nucleic acid (FANA), and 2′-aminomethyl-bridged nucleic acid (NC-BNA). Since most breast cancer cells overexpress insulin-like growth factor receptor (IGF1R), a peptide derivative of the IGF1 was conjugated to anti-miR-17-5p antagomiRs to achieve breast cancer cell-specific delivery. Our results showed that in multiple TNBC cell lines, the NC-BNA 15mers displayed high efficacy with sub-nM activity for miR-17-5p blockade and significant inhibition of cell growth. Anti-miR-17-5p-IGF1 peptide conjugate successfully inhibited miR-17-5p activity in the luciferase reporter system. We are further examining the effects of NC-BNA-peptides in multiple TNBC cell lines. Our study provides a good basis for the development of a new TNBC targeted therapy. Supported by Bound Therapeutics LLC. Conflict of interest: pending TJU PCT/US2015/015681 patent application licensed to Bound Therapeutics LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Antisense oligonucleotide therapeutics with receptor-targeted delivery in triple-negative breast cancer cells via microRNA blockade without passenger strand side effects [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A57.

ObjectiveTo validate a hypothesis that prostate cancer can be detected non‐invasively by a simple and reliable assay by targeting genomic VPAC receptors expressed on malignant prostate cancer cells shed in voided urine.Patients/Subjects and MethodsVPAC receptors were targeted with a specific biomolecule, TP4303, developed in our laboratory. With an Institutional Review Board exempt approval of use of de‐identified discarded samples, an aliquot of urine collected as a standard of care, from patients presenting to the urology clinic (207 patients, 176 men and 31 women, aged ≥21 years) was cytospun. The cells were fixed and treated with TP4303 and 4,6‐diamidino‐2‐phenylindole (DAPI). The cells were then observed under a microscope and cells with TP4303 orange fluorescence around the blue (DAPI) nucleus were considered ‘malignant’ and those only with a blue nucleus were regarded as ‘normal’. VPAC presence was validated using receptor blocking assay and cell malignancy was confirmed by prostate cancer gene profile examination.ResultsThe urine specimens were labelled only with gender and presenting diagnosis, with no personal health identifiers or other clinical data. The assay detected VPAC positive cells in 98.6% of the men with a prostate cancer diagnosis (141), and none of the 10 men with benign prostatic hyperplasia. Of the 56 ‘normal’ patients, 62.5% (35 patients, 10 men and 25 women) were negative for VPAC cells; 19.6% (11, 11 men and no women) had VPAC positive cells; and 17.8% (10, four men and six women) were uninterpretable due to excessive crystals in the urine. Although data are limited, the sensitivity of the assay was 99.3% with a confidence interval (CI) of 96.1–100% and the specificity was 100% with a CI of 69.2–100%. Receptor blocking assay and fluorescence‐activated cell sorting (FACS) analyses demonstrated the presence of VPAC receptors and gene profiling examinations confirmed that the cells expressing VPAC receptors were malignant prostate cancer cells.ConclusionThese preliminary data are highly encouraging and warrant further evaluation of the assay to serve as a simple and reliable tool to detect prostate cancer non‐invasively.

Infections are a devastating complication of titanium alloy orthopedic implants. Current therapies include antibiotic-impregnated bone cement and antibiotic-containing coatings. Daptomycin (DAP) (1) is a novel peptide antibiotic that penetrates the cell membranes of Gram-positive bacteria. Few DAP-resistant strains have appeared so far. We hypothesized that when DAP covalently bonded via a flexible, hydrophilic spacer it could prevent bacterial colonization of titanium alloy surfaces. We designed and synthesized a series of DAP conjugates for bonding to the surface of Ti6Al4V foils through tetra(ethylene glycol) spacers via thioether linkages. The stability and antimicrobial activity of the attached conjugates were evaluated using Staphylococcus aureus ATCC 25923. Colonization of the Ti6Al4V foils was inhibited by 72% at 8 h and 54% at 24 h. The strategy described in this report provides a new, more facile way to prepare bactericidal Ti6Al4V implants.

Antisense and antigene DNA therapy of aberrant genes represents an exciting possibility. A variety of synthetic DNA derivatives have been applied to control many different pathogenic genes in cell culture, and a few in whole organisms, such as mice. The array of offshoots from the original phosphodiester backbone include methylphosphonate, phosphorothioate, phosphoramidate, α-phosphodiester, phosphorodithioate, boranophosphate, formacetal, and polyamide, to name only a few. In one example of an animal trial, transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (Εμ-myc) have been treated with antisense DNA methylphosphonates targeted against c-myc mRNA. A single intravenous dose of 300 nmol inhibited production of c-MYC protein in peripheral and splenic lymphocytes. In addition, DNA methylphosphonates did not induce acute toxicity following i.v. administration of a 300-nmol dose. An identically administered scrambled sequence oligomer did not decrease c-MYC protein or induce toxicity. Finally, recovery of DNA methylphosphonates from the blood plasma of treated mice indicated that the oligomers remained intact up to 3 h, while their concentrations decreased rapidly for the first hour, then slowly decreased over the 86next 2 h. Beyond this simple demonstration of antisense DNA therapy in an animal model, realistic design of DNA-based human therapeutic strategies requires many aspects of a candidate disease to be considered: disease prevalence, the number and nature of genes and mutations involved, and the tissues which must be targeted. For each DNA derivative intended for therapy, methods of targeting, mode of administration, pharmacokinetics, tissue distribution cellular uptake, toxicity, degradation, and excretion must be considered.

OBJECTIVE:The authors have conjugated chelating agents (DOTA and NODAGA) with a peptide (pituitary adenylate cyclase-activating peptide [PACAP] analogue) that has a high affinity for VPAC1 receptors expressed on cancer cells. To determine a suitable chelating agent for labeling with (68)Ga, they have compared the labeling kinetics and stability of these peptide conjugates.METHODS:For labeling, (68)GaCl3 was eluted in 0.1 M HCl from a [(68)Ge-(68)Ga] generator. The influences of peptide concentration, pH, and temperature on the radiolabeling efficiency were studied. The stability was evaluated in saline, human serum, DTPA, transferrin, and metallic ions (FeCl3, CaCl2, and ZnCl2). Cell binding assay was performed using human breast cancer cells (T47D). Tissue biodistribution was studied in normal athymic nude mice.RESULTS:Optimal radiolabeling (>95.0%) of the DOTA-peptide conjugates required a higher (50°C-90°C) temperature and 10 minutes of incubation at pH 2-5. The NODAGA-peptide conjugate needed incubation only at 25°C for 10 minutes. Both radiocomplexes were stable in saline, serum, as well as against transchelation and transmetallation. Cell binding at 37°C for 15 minutes of incubation with (68)Ga-NODAGA-peptide was 34.0% compared to 24.5% for (68)Ga-DOTA-peptide. Tissue biodistribution at 1 hour postinjection of both (68)Ga-labeled peptide conjugates showed clearance through the kidneys.CONCLUSIONS:NODAGA-peptide showed more convenient radiolabeling features than that of DOTA-peptide.

1171 Objectives Our laboratory has successfully designed and characterized a peptide which has high affinity for VPAC1 receptors expressed in high density on breast (BC) and prostate cancers (PC). The purpose was to conjugate the peptide with two different chelating agents- NODAGA (1,4,7-triazacyclononane-1-glutamic acid-4,7-diacetic acid) and N2S2 [bis(thiosemicarbazone)], to label the conjugates with gallium-68 (Ga-68) and evaluate them for stability, ability to micro-PET image human breast cancer in a mouse model and to determine their biodistribution. Methods The NODAGA-peptide (TP-3718) was synthesized with C-terminal NODAGA chelator on a Wang resin using a peptide synthesizer (Protein Technologies, Tucson, Arizona, USA) while N2S2-peptide (TP-3805) was purchased from American Peptide Co, Sunniville, CA. Both peptide-conjugates were characterized by electrospray mass spectroscopy. Ga-68 was eluted from [Ge-68/Ga-68] generator using 0.1 N HCl (5.0 mL). The eluate was then passed through a cation exchange column and the column was washed with 80% acetone (in 0.1 N HCl). Ga-68 retained on column was eluted with 98% acetone (in 0.05 N HCl) and the excess of acetone was evaporated by heating at 70° C for 5 min. For labeling TP-3718, 20 µg of peptide-conjugate in 200 µL of deionized water, and 20 µL of Ga-68-Cl3 were incubated at 90°C for 30 min. The final pH was adjusted to 7.2 ± 0.2 with 0.1 N NaOH. For radiolabeling TP-3805, 200 µL of glycine buffer (pH=9.2), 20 µL SnCl2 (10 mg/mL in 50 mg/mL glucoheptonate), 20 µg TP-3805 and 20 µL of Ga-68-Cl3 were mixed (pH- 7.2 ± 0.2) and incubated at 90°C for 30 min. The radiochemical purity and in vitro stability of the radiolabeled peptides were determined by Radio-HPLC. Ga-68-labeled peptides (~ 100 µCi in 200 µL) each was injected intravenously to a separate group of immunocompromised female nude mice (n = 5) bearing BT474 human BC. Micro-PET/CT imaging was performed at 1 hr post-injection and data were analyzed by calculating regional standardized uptake values. After imaging, the animals were sacrificed and % injected dose per gram was calculated for each organ. Results As determined by mass spectrometry, the purity of the peptide was 99.9%. Radio-HPLC showed retention time for Ga-68-TP-3718 at 9.9 ± 0.3 min and Ga-68-TP-3805 at 4.9 ± 0.4 min as compare to free Ga-68-Cl3 at 3.4 ± 0.4 min. The radiolabeling efficiency for each peptide conjugate was > 95.0 % and their stability was greater than 95.0% at 4 h. Micro-PET images showed tumor to muscle ratio of 3.2 ± 0.2 for Ga-68-TP-3718 and 3.4 ± 0.2 for Ga-68-TP-3805 at 1 hr post injection. The biodistribution data at 1 hr post injection showed that Ga-68-TP-3718 had maximum uptake in the kidneys (30.4 ± 5.0 %ID/g) associated with high renal clearance whereas Ga-68-TP-3805 had lesser uptake in kidneys (4.1 ± 2.5 %ID/g). Conclusions The post processing of Ga-68 ensured removal of cationic impurities and yielded higher specific activity. Data demonstrated that either Ga-68-TP-3718 or Ga-68-TP-3805 could be used as a potential agent for PET imaging for breast cancer. Research Support: The research, in part, was supported by NIH/NCI RO1CA157372 (MLT), NIH/NCI 1S10OD012406 (MLT) and NIH/NCI S10RR23709 (MLT).

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1–17 cells per 5 μl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 μl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the ‘liquid biopsy’ was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ∼90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%–100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (∼12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube–CTC chip study.

Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we previously reported that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins in human MDA-MB-231 TNBC cells, instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of the miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by the miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and the miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3′UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knockdown miR-17-5p reduced PDCD4 and PTEN protein expression, instead of raising them. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells also reduced PDCD4 and PTEN protein levels. To test the hypothesis that miR-17-3p passenger strand can also modulate the same mRNA targets as miR-17-5p, we performed luciferase assays using reporter constructs harboring predicted miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs. We found that the miR-17-5p mimic lowered the expression of luciferase vectors containing one predicted binding site from the PDCD4 or PTEN 3′UTR domains. However, the miR-17-3p mimic lowered the expression of luciferase vectors containing all four 3′UTR target sites of PDCD4, and two of the six predicted PTEN 3′UTR target sites. In addition, we used luciferase assays to test the hypothesis that antisense DNA-LNA against miR-17-5p mimicked passenger strand miR-17-3p. We found that anti-miR-17-5p DNA-LNA lowered the expression of luciferase reporter vectors containing two of the four predicted PDCD4 3′UTR target sites for miR-17-3p, and one of the six predicted PTEN 3′UTR target sites for miR-17-3p. From these results, we concluded that the miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs, just like the miR-17-5p guide strand. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked the miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. Supported by the Department of Biochemistry and Molecular Biology. Conflict of interest: pending PCT/US2015/015681 patent application licensed to Nikita RNA LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B41.