Abstract Of 1.6 million newly diagnosed breast cancer patients per year, about 10-16% will develop brain metastases. Among the different subtypes of breast cancer, HER2-overexpressing (HER2+) and triple-negative breast cancers (TNBC) have the highest incidence of brain metastasis. Recently, advances in targeted therapies for breast cancer (e.g., trastuzumab, T-DM1, and lapatinib) have prolonged patient survival through better control of the systemic disease. However, when patients have disease recurrence, their brain metastasis incidence doubles, which represents an imposing challenge. Current treatment options are limited and merely palliative for those patients with brain-metastatic breast cancer, and their one year survival is less than 20%. To develop effective treatments for brain metastasis, we strived to gain global understanding of the mechanisms of brain metastasis to guide rapid development of novel and clinically applicable targeted therapies. We recently found that the brain astrocytes secrete exosomes that contain PTEN-targeting miR-19a, among other biomolecules. After brain metastatic tumor cells uptake these astrocytes-derived exosomes, miR-19a reversibly downregulates PTEN expression in metastatic tumor cells, thereby increasing Akt and NF-kB signaling, leading to an increased secretion of CCL2 cytokine. CCL2 then recruits CCR2 positive and Iba1 positive myeloid cells that promote proliferation and inhibit apoptosis of brain metastatic tumor cells and facilitate metastatic tumor outgrowth to symptomatic brain metastasis. Importantly, inhibition of exosome secretion or stable ablation of CCL2 inhibits brain metastasis outgrowth in vivo, demonstrating the potential of exosome- and CCL2-targeting for therapeutic intervention of life-threatening brain metastases (Nature, 11/2015). Currently, we are exploring the clinical potential by systematic preclinical testing in animal models. Citation Format: Yu D. PTEN loss by exosomal microRNA primes brain metastasis outgrowth [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr BS2-2.

Radiolabeled tyrosine analogs enter cancer cells via upregulated amino acid transporter system and have been shown to be superior to18F-fluoro-2-deoxy-D-glucose (18F-FDG) in differential diagnosis in cancers. In this study, we synthesized O-[3-19F-fluoropropyl]-α-methyl tyrosine (19F-FPAMT) and used manual and automated methods to synthesize O-[3-18F-fluoropropyl]-α-methyl tyrosine (18F-FPAMT) in three steps: nucleophilic substitution, deprotection of butoxycarbonyl, and deesterification. Manual and automated synthesis methods produced18F-FPAMT with a radiochemical purity >96%. The decay-corrected yield of18F-FPAMT by manual synthesis was 34% at end-of-synthesis (88 min). The decay-corrected yield of18F-FPAMT by automated synthesis was 15% at end-of-synthesis (110 min).18F-FDG and18F-FPAMT were used forin vitroandin vivostudies to evaluate the feasibility of18F-FPAMT for imaging rat mesothelioma (IL-45).In vitrostudies comparing18F-FPAMT with18F-FDG revealed that18F-FDG had higher uptake than that of18F-FPAMT, and the uptake ratio of18F-FPAMT reached the plateau after being incubated for 60 min. Biodistribution studies revealed that the accumulation of18F-FPAMT in the heart, lungs, thyroid, spleen, and brain was significantly lower than that of18F-FDG. There was poor bone uptake in18F-FPAMT for up to 3 hrs suggesting itsin vivostability. The imaging studies showed good visualization of tumors with18F-FPAMT. Together, these results suggest that18F-FPAMT can be successfully synthesized and has great potential in mesothelioma imaging.

1173 Objectives Uptake of F-18 FDG in highly glycolytic areas could not differentiate infection and/or inflammation which cause false positive results. An alternative way is to target cell proliferation via nucleoside analogues. This study was aimed to synthesis a guanine analog for cell nucleus activity assessment. Methods N4-Guan is synthesized through a 9-step reaction. The hydroxyl groups of penciclovir was selectively protected. The unprotected hydroxyl group was converted to an amino group. The carboxyl acid group of the N4 mono-acid was reacted with amino-penciclovir to form amide linage. N4amG was obtained after deprotection and purification. Radiolabeling of N4amG with Tc-99m was performed in a standard manner. Cell uptake assay and cytoplasm/nucleus isolation was performed using breast cancer cell line. Biodistribution and planar imaging (n=3/time interval) of Tc-99m N4amG were conducted in breast tumor-bearing rats from 0.5-4hrs. Computer outlined region of interest (counts per pixel) was used to determine tumor-to-muscle (T/M) count density ratios. Dosimetry estimates were computed from respective biodistribution data using Olinda software. Results Mass spectronmetry, NMR and HPLC analysis for N4amG were performed to determine the structure. The purity was >97%. The total synthetic yield was 6%. Radiochemical purity of Tc-99m N4amG determined by ITLC was >95% (in saline). There was 2.3%ID uptake in cytoplasm and 1.5%ID in nucleus after 4 hours incubation indicated Tc-99m N4amG was involved in cell nucleus activity. In the breast tumor-bearing rat planar imaging from 0.5-4 hours, T/M ratio was 3.65+0.18 to 5.02+0.41. In the rat biodistribution from 0.5-4hrs, tumor, tumor/blood and tumor/muscle count density ratios were 0.32+0.06 to 0.43+0.04, 0.29+0.02 to 0.52+0.02 and 3.24+0.38to 4.30+0.59, respectively. Tc-99m N4amG showed total rad absorbed by each organ was below the proposed annual and total limits after dosimetry estimation. Conclusions Our data indicate that it is feasible to assess tumor nucleus activity with Tc-99m N4amG.

99mTc-N4-guanine (99mTc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of 99mTc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using 99mTc-N4amG in tumor specific imaging.

We have developed ethylenedicysteine-glucosamine (ECG) as an alternative to 18 F-fluoro-2-deoxy-D-glucose ( 18 F-FDG) for cancer imaging. ECG localizes in the nuclear components of cells via the hexosamine biosynthetic pathway. This study was to evaluate the feasibility of imaging mesothelioma withT99mc-ECG andG68a-ECG. ECG was synthesized from thiazolidine-4-carboxylic acid and 1,3,4,6-tetra-O-acetyl-2-amino-D-glucopyranose, followed by reduction in sodium and liquid ammonia to yield ECG (52%). ECG was chelated withT99mc/tin (II) andG68a/ 69 Ga chloride for in vitro and in vivo studies in mesothelioma. The highest tumor uptake ofT99mc-ECG is 0.47 at 30 min post injection, and declined to 0.08 at 240 min post injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios forT99mc-ECG (30–240 min) were0.47±0.06to0.08±0.01;0.71±0.07to0.85±0.04;0.47±0.03to0.51±0.01, and3.49±0.24to5.06±0.25; forG68a-ECG (15–60 min) were0.70±0.06to0.92±0.08;0.64±0.05to1.15±0.08;0.42±0.03to0.67±0.07, and3.84±0.52to7.00±1.42; for 18 F-FDG (30–180 min) were1.86±0.22to1.38±0.35;3.18±0.44to2.92±0.34,4.19±0.44to19.41±2.05and5.75±2.55to3.33±0.65, respectively. Tumor could be clearly visualized withT99mc-ECG andG68a-ECG in mesothelioma-bearing rats.T99mc-ECG andG68a-ECG showed increased uptake in mesothelioma, suggesting they may be useful in diagnosing mesothelioma and also monitoring therapeutic response.

The purpose of this study was to develop an efficient way to synthesizeTc99m- O -[3-(1,4,8,11-tetraazabicyclohexadecane)-propyl]-tyrosine (Tc99m-N4-Tyrosine), a novel amino acid-based radiotracer, and evaluate its potential in breast cancer gamma imaging. Precursor N4-Tyrosine was synthesized using a 5-step procedure, and its total synthesis yield was 38%. It was successfully labeled withTc99mwith high radiochemical purity (>95%). Cellular uptake ofTc99m-N4-Tyrosine was much higher than that ofTc99m-N4 and the clinical gold standard 18 F-2-deoxy-2-fluoro-glucose ( 18 F-FDG) in rat breast tumor cells in vitro . Tissue uptake and dosimetry estimation in normal rats revealed thatTc99m-N4-Tyrosine could be safely administered to humans. Evaluation in breast tumor-bearing rats showed that althoughTc99m-N4-Tyrosine appeared to be inferior to 18 F-FDG in distinguishing breast tumor tissue from chemical-induced inflammatory tissue, it had high tumor-to-muscle uptake ratios and could detect breast tumors clearly by planar scintigraphic imaging.Tc99m-N4-Tyrosine could thus be a useful radiotracer for use in breast tumor diagnostic imaging.

Rationale and Objectives: Radio labeled tyrosine analogues that have been successfully used in tumor imaging accumulate in tumor cells via an upregulated L-type amino acid transporter system. The anticancer drug melphalan is an L-type amino acid transporter substrate. Therefore, radiolabeled tyrosine analogues may have great potential in evaluating treatment responses to melphalan. In this study, a Tc-99m-labeled tyrosine analogue, Tc-99m tyrosine using N,N'-ethylene-di-L-cysteine (EC) as a chelator, was developed and its potential for noninvasively assessing tumors' early response to melphalan determined.Materials and Methods: EC-tyrosine was synthesized in a three-step procedure and labeled with Tc-99m. To assess cellular uptake kinetics, the percentage uptake of Tc-99m-EC-tyrosine in the rat breast cancer cell line 13762 was measured. Planar imaging was performed in rats with 13762 cell derived tumors. To determine the transport mechanisms of Tc-99m-EC-tyrosine, a competitive inhibition study using L-tyrosine as an inhibitor was performed in vitro and in vivo. To assess tumors response to melphalan, tumor bearing rats were treated with different doses of melphalan, and planar imaging was performed 0 and 3 days after treatment Immunohistochemical analyses were conducted to determine expressions of L-type amino acid transporter 1 and cellular proliferation marker Ki-67.Results: L-tyrosine significantly inhibited Tc-99m-EC-tyrosine uptake in vitro and in vivo. Tumor volume decreased in a dose dependent manner with melphalan, and tumor/muscle ratios of Tc-99m-EC-tyrosine were significantly reduced in treated groups. Immunohistochemical data indicated that about 70% of tumor cells in the melphalan-treated groups underwent apoptosis, and the changes in tumor/muscle ratios reflected the decreased percentage of viable cells in treated tumors.Conclusions: These findings suggest that Tc-99m-EC-tyrosine has great potential for monitoring tumor response to melphalan in breast tumor-bearing rats.

We have developed ethylenedicysteine-glucosamine (ECG) as an alternative to F-18-fluoro-2-deoxy-D-glucose (F-18-FDG) for cancer imaging. ECG localizes in the nuclear components of cells via the hexosamine biosynthetic pathway. This study was to evaluate the feasibility of imaging mesothelioma with (99)mTc-ECG and Ga-68-ECG. ECG was synthesized from thiazolidine-4-carboxylic acid and 1,3,4,6-tetra-O-acetyl-2-amino-D-glucopyranose, followed by reduction in sodium and liquid ammonia to yield ECG (52%). ECG was chelated with (99)mTc/tin (II) and Ga-68/Ga-69 chloride for in vitro and in vivo studies in mesothelioma. The highest tumor uptake of (99)mTc-ECG is 0.47 at 30 min post injection, and declined to 0.08 at 240 min post injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios for (99)mTc-ECG (30-240 min) were 0.47 +/- 0.06 to 0.08 +/- 0.01; 0.71 +/- 0.07 to 0.85 +/- 0.04; 0.47 +/- 0.03 to 0.51 +/- 0.01, and 3.49 +/- 0.24 to 5.06 +/- 0.25; for Ga-68-ECG (15-60 min) were 0.70 +/- 0.06 to 0.92 +/- 0.08; 0.64 +/- 0.05 to 1.15 +/- 0.08; 0.42 +/- 0.03 to 0.67 +/- 0.07, and 3.84 +/- 0.52 to 7.00 +/- 1.42; for F-18-FDG (30-180 min) were 1.86 +/- 0.22 to 1.38 +/- 0.35; 3.18 +/- 0.44 to 2.92 +/- 0.34, 4.19 +/- 0.44 to 19.41 +/- 2.05 and 5.75 +/- 2.55 to 3.33 +/- 0.65, respectively. Tumor could be clearly visualized with (99)mTc-ECG and Ga-68-ECG in mesothelioma-bearing rats. (99)mTc-ECG and Ga-68-ECG showed increased uptake in mesothelioma, suggesting they may be useful in diagnosing mesothelioma and also monitoring therapeutic response.

We have developed ethylenedicysteine-glucosamine (ECG) as an alternative to 18F-fluoro-2-deoxy-D-glucose (18F-FDG) for cancer imaging. ECG localizes in the nuclear components of cells via the hexosamine biosynthetic pathway. This study was to evaluate the feasibility of imaging mesothelioma with T99mc-ECG and G68a-ECG. ECG was synthesized from thiazolidine-4-carboxylic acid and 1,3,4,6-tetra-O-acetyl-2-amino-D-glucopyranose, followed by reduction in sodium and liquid ammonia to yield ECG (52%). ECG was chelated with T99mc/tin (II) and G68a/69Ga chloride for in vitro and in vivo studies in mesothelioma. The highest tumor uptake of T99mc-ECG is 0.47 at 30 min post injection, and declined to 0.08 at 240 min post injection. Tumor uptake (%ID/g), tumor/lung, tumor/blood, and tumor/muscle count density ratios for T99mc-ECG (30–240 min) were 0.47±0.06 to 0.08±0.01; 0.71±0.07 to 0.85±0.04; 0.47±0.03 to 0.51±0.01, and 3.49±0.24 to 5.06±0.25; for G68a-ECG (15–60 min) were 0.70±0.06 to 0.92±0.08; 0.64±0.05 to 1.15±0.08; 0.42±0.03 to 0.67±0.07, and 3.84±0.52 to 7.00±1.42; for 18F-FDG (30–180 min) were 1.86±0.22 to 1.38±0.35; 3.18±0.44 to 2.92±0.34, 4.19±0.44 to 19.41±2.05 and 5.75±2.55 to 3.33±0.65, respectively. Tumor could be clearly visualized with T99mc-ECG and G68a-ECG in mesothelioma-bearing rats. T99mc-ECG and G68a-ECG showed increased uptake in mesothelioma, suggesting they may be useful in diagnosing mesothelioma and also monitoring therapeutic response.