LNCaP Tumor Research Articles

Transcriptomic Analysis of LNCaP Tumor Xenograft to Elucidate the Components and Mechanisms Contributed by Tumor Environment as Targets for Dietary Prostate Cancer Prevention Studies.

LNCaP athymic xenograft model has been widely used to allow researchers to examine the effects and mechanisms of experimental treatments such as diet and diet-derived cancer preventive and therapeutic compounds on prostate cancer. However, the biological characteristics of human LNCaP cells before/after implanting in athymic mouse and its relevance to clinical human prostate outcomes remain unclear and may dictate interpretation of biological efficacies/mechanisms of diet/diet-derived experimental treatments. In this study, transcriptome profiles and pathways of human prostate LNCaP cells before (in vitro) and after (in vivo) implanting into xenograft mouse were compared using RNA-sequencing technology (RNA-seq) followed by bioinformatic analysis. A shift from androgen-responsive to androgen nonresponsive status was observed when comparing LNCaP xenograft tumor to culture cells. Androgen receptor and aryl-hydrocarbon pathway were found to be inhibited and interleukin-1 (IL-1) mediated pathways contributed to these changes. Coupled with in vitro experiments modeling for androgen exposure, cell-matrix interaction, inflammation, and hypoxia, we identified specific mechanisms that may contribute to the observed changes in genes and pathways. Our results provide critical baseline transcriptomic information for a tumor xenograft model and the tumor environments that might be associated with regulating the progression of the xenograft tumor, which may influence interpretation of diet/diet-derived experimental treatments.

Preclinical Evaluation of a Fluorine-18 Labeled Probe for the Detection of the Expression of PSMA Level in Cancer.

Prostate-specific membrane antigen (PSMA) is a prospect biomarker for the treatment of prostate cancer. Meanwhile, positron emission tomography (PET) is being developed as a significant imaging modality in cancer diagnosis. A new PET probe Glu-ureido-Lys-naphthylalanine-tranexamic acid-Gly(AMBF3)-triiodobenzoic acid (18F-GLNTGT) was radiosynthesized by a one-step 18F-labeled method. 18F-GLNTGT was obtained with a radioactivity yield (RCY) of 12.16 ± 6.4% and a good radiochemical purity (RCP > 96%). The cell uptakes of 18F-GLNTGT were determined to be 15.9 ± 0.43% ID and 9.47 ± 1.26% ID at 15 min in LNCaP cells and PC-3 cells, respectively. The cell internalization of 18F-GLNTGT was determined to be 12.89 ± 0.94% ID and 5.34 ± 0.15% ID at 15 min in LNCaP cells and PC-3 cells, respectively. It is suggested that the probe has good specificity targeting PSMA. From the results of 18F-GLNTGT binding affinity with PSMA, it has a higher affinity and a Ki value of 0.49 nM (95% confidence interval (CI): 0.35–0.67 nM). In PET imaging, 18F-GLNTGT showed the highest tumor uptake of 3.51 ± 0.15% ID/g at 45 min and the maximum tumor/muscle (T/Mmax) ratio of 3.68 ± 0.29 at 60 min post-injection (p.i.) in LNCaP tumors. The control probe 18F-AlF-NOTA-RGD2 presented the highest tumor uptake of 4.2 ± 0.54% ID/g at 7.5 min and the T/Mmax ratio of 2.72 ± 0.63 at 45 min p.i. in LNCaP tumors. The results showed that the probe has a higher tumor/muscle ratio compared with the control probe 18F-AlF-NOTA-RGD2. Although the probe 18F-GLNTGT has some limitations for CT signal detection both in cells and in vivo, it is still a promising PET probe for targeting PSMA membrane protein.

PSMA-D4 Radioligand for Targeted Therapy of Prostate Cancer: Synthesis, Characteristics and Preliminary Assessment of Biological Properties.

A new PSMA ligand (PSMA-D4) containing the Glu-CO-Lys pharmacophore connected with a new linker system (L-Trp-4-Amc) and chelator DOTA was developed for radiolabeling with therapeutic radionuclides. Herein we describe the synthesis, radiolabeling, and preliminary biological evaluation of the novel PSMA-D4 ligand. Synthesized PSMA-D4 was characterized using TOF-ESI-MS, NMR, and HPLC methods. The novel compound was subject to molecular modeling with GCP-II to compare its binding mode to analogous reference compounds. The radiolabeling efficiency of PSMA-D4 with 177Lu, 90Y, 47Sc, and 225Ac was chromatographically tested. In vitro studies were carried out in PSMA-positive LNCaP tumor cells membranes. The ex vivo tissue distribution profile of the radioligands and Cerenkov luminescence imaging (CLI) was studied in LNCaP tumor-bearing mice. PSMA-D4 was synthesized in 24% yield and purity >97%. The radio complexes were obtained with high yields (>97%) and molar activity ranging from 0.11 to 17.2 GBq mcmol−1, depending on the radionuclide. In vitro assays confirmed high specific binding and affinity for all radiocomplexes. Biodistribution and imaging studies revealed high accumulation in LNCaP tumor xenografts and rapid clearance of radiocomplexes from blood and non-target tissues. These render PSMA-D4 a promising ligand for targeted therapy of prostate cancer (PCa) metastases.

Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

AimTo investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies.MethodsBinding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice.ResultsPSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i.ConclusionsThe investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

18F]-JK-PSMA-7 and [18F]-FDG tumour PET uptake in treated xenograft human prostate cancer model in mice.

This preclinical study aims to evaluate the extent to which a change in prostate-specific membrane antigen (PSMA) expression of castration-resistant prostate cancer (CRPC) following standard treatment is reflected in [18F]JK-PSMA-7 PET/CT. Castrated mice supplemented with testosterone implant were xenografted with human LNCaP CRPC. After appropriate tumour growth, androgen deprivation therapy (ADT) was carried out by the removal of the implant followed by a single injection of docetaxel (400μg/20-g mouse) 2weeks later. [18F]JK-PSMA-7 PET/CT were performed before ADT, then before and at days 12, 26, 47 and 69 after docetaxel administration. The [18F]JK-PSMA-7 PET data were compared to corresponding unspecific metabolic [18F]FDG PET/CT and ex vivo quantification of PSMA expression estimated by flow cytometry on repeated tumour biopsies. ADT alone had no early effect on LNCaP tumours that pursued their progression. Until day 12 post-docetaxel, the [18F]JK-PSMA7 uptake was significantly higher than that of [18F]FDG, indicating the persistence of PSMA expression at those time points. From day 26 onwards when the tumours were rapidly expanding, both [18F]JK-PSMA7 and [18F]FDG uptake continuously decreased although the decrease in [18F]JK-PSMA uptake was markedly faster. The fraction of PSMA-positive cells in tumour biopsies decreased similarly over time to reach a non-specific level after the same time period. Applying PSMA-based imaging for therapy monitoring in patients with CRPC should be considered with caution since a reduction in [18F]JK-PSMA-7 PET uptake after successive ADT and chemotherapy may be related to downregulation of PSMA expression in dedifferentiated and rapidly proliferating tumour cells.

Role of HDL and Scavenger Receptor Class B Type I (SR-BI) Expression in LNCaP and PC-3 Prostate Cancer Cell Lines. Growth Patterns in the Chick Chorioallantoic Membrane Model

Cholesterol metabolism is a key pathway affecting androgen synthesis and prostate cancer cell proliferation. Major receptors and transporters involved in cellular cholesterol homeostasis and cholesterol fluxes are ATP-binding cassette protein A1 (ABCA1), LDL receptor (LDLR) and the HDL receptor scavenger receptor class B type I (SR-BI). Especially SR-BI deserves more and more attention because it has been shown to be overexpressed by the majority of malignant tumours, promoting their proliferation and metastasis. PC-3 and LNCaP are two of the commonly used cell lines for in vitro prostate cancer studies. A comparative overview of the expression of receptors involved in cholesterol uptake and their activities in these two cell lines was missing; therefore we reviewed the current knowledge on this topic. We investigated the expression of SR-BI in these two cell lines and the uptake of labelled HDL. In order to study and compare tumour development, we assessed the growth of both cell lines in the chick chorioallantoic membrane (CAM) model. Although microtumours successfully developed, size increase was limited as confirmed by the analysis of the proliferation marker Ki67. Interestingly prostate-specific antigen (PSA) secreted by LNCaP tumours could be determined in the chick embryo blood. SR-BI is a promising therapeutic target either by its direct inhibition or more importantly as an agent for drug delivery.

Synthesis and fluorine-18 radiolabeling of a phospholipid as a PET imaging agent for prostate cancer

IntroductionAltered lipid metabolism and subsequent changes in cellular lipid composition have been observed in prostate cancer cells, are associated with poor clinical outcome, and are promising targets for metabolic therapies. This study reports for the first time on the synthesis of a phospholipid radiotracer based on the phospholipid 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (PC44:12) to allow tracking of polyunsaturated lipid tumor uptake via PET imaging. This tracer may aid in the development of strategies to modulate response to therapies targeting lipid metabolism in prostate cancer. MethodsLipidomics analysis of prostate tumor explants and LNCaP tumor cells were used to identify PC44:12 as a potential phospholipid candidate for radiotracer development. Synthesis of phosphocholine precursor and non-radioactive standard were optimised using click chemistry. The biodistribution of a fluorine-18 labeled analogue, N-{[4-(2-[18F]fluoroethyl)-2,3,4-triazol-1-yl]methyl}-1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine ([18F]2) was determined in LNCaP prostate tumor-bearing NOD SCID gamma mice by ex vivo biodistribution and PET imaging studies and compared to biodistribution of [18F]fluoromethylcholine. Results[18F]2 was produced with a decay-corrected yield of 17.8 ± 3.7% and an average radiochemical purity of 97.00 ± 0.89% (n = 6). Molar activity was 85.1 ± 3.45 GBq/μmol (2300 ± 93 mCi/μmol) and the total synthesis time was 2 h. Ex vivo biodistribution data demonstrated high liver uptake (41.1 ± 9.2%ID/g) and high splenic uptake (10.9 ± 9.1%ID/g) 50 min post-injection. Ex vivo biodistribution showed low absolute tumor uptake of [18F]2 (0.8 ± 0.3%ID/g). However, dynamic PET imaging demonstrated an increase over time of the relative tumor-to-muscle ratio with a peak of 2.8 ± 0.5 reached 1 h post-injection. In contrast, dynamic PET of [18F]fluoromethylcholine demonstrated no increase in tumor-to-muscle ratios due to an increase in both tumor and muscle over time. Absolute uptake of [18F]fluoromethylcholine was higher and peaked at 60 min post injection (2.25 ± 0.29%ID/g) compared to [18F]2 (1.44 ± 0.06%ID/g) during the 1 h dynamic scan period. Conclusions and advances in knowledgeThis study demonstrates the ability to radiolabel phospholipids and indicates the potential to monitor the in vivo distribution of phospholipids using fluorine-18 based PET.

68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

BackgroundThe AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs.ResultsAAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay.ConclusionsThe AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

Investigating the performance of a novel pH and cathepsin B sensitive, stimulus-responsive nanoparticle for optimised sonodynamic therapy in prostate cancer

Nano-formulations that are responsive to tumour-related and externally-applied stimuli can offer improved, site-specific antitumor effects, and can improve the efficacy of conventional therapeutic agents. Here, we describe the performance of a novel stimulus-responsive nanoparticulate platform for the targeted treatment of prostate cancer using sonodynamic therapy (SDT). The nanoparticles were prepared by self-assembly of poly(L-glutamic acid-L-tyrosine) co-polymer with hematoporphyrin. The nanoparticulate formulation was characterized with respect to particle size, morphology, surface charge and singlet oxygen production during ultrasound exposure. The response of the formulation to the presence of cathepsin B, a proteolytic enzyme that is overexpressed and secreted in the tumour microenvironment of many solid tumours, was assessed. Our results showed that digestion with cathepsin B led to nanoparticle size reduction. In the absence of ultrasound, the formulation exhibited greater toxicity at acidic pH than at physiological pH, using the human prostate cells lines LNCaP and PC3 as targets. Nanoparticle cellular uptake was enhanced at acidic pH – a condition that was also associated with greater cathepsin B production. Nanoparticles exhibited enhanced ultrasound-induced cytotoxicity against both prostate cancer cell lines. Subsequent proof-of-concept in vivo studies demonstrated that, when ectopic human xenograft LNCaP tumours in SCID mice were treated with SDT using the systemically-administered nanoparticulate formulation at a single dose, tumour volumes decreased by up to 64% within 24 h. No adverse effects were observed in the nanoparticle-treated mice and their body weight remained stable. The potential of this novel formulation to deliver safe and effective treatment of prostate cancer is discussed.

Evaluation of a New 99mTc-labeled GnRH Analogue as a Possible Imaging Agent for Prostate Cancer Detection.

Prostate cancer is a serious threat to men's health so it is necessary to develop technics for early detection of this malignancy. The purpose of this research was the evaluation of a new99mTc-labeled GnRH analogue as an imaging probe for tumor targeting of prostate cancer. 99mTc-labeled-DLys6-GnRH analogue was prepared based on HYNIC as a chelating agent and tricine/ EDDA as coligands for labeling with 99mTc. HYNIC was coupled to epsilon amino group of DLys6 through aminobutyric acid (GABA) as a linker. Radiochemical purity and stability in normal saline and serum, were determined by TLC and HPLC methods. Furthermore, calculation of protein-binding and partition coefficient constant were carried out for 99mTc labeled peptide. The cellular experiments including receptor binding specificity and affinity were studied using three prostate cancer cell lines LN-CaP, DU-145 and PC-3. Finally, the animal assessment and SPECT imaging of radiolabeled GnRH analogue were evaluated on normal mice and nude mice bearing LN-CaP tumor. The GnRH conjugate was labeled with high radiochemical purity (~97%). The radiolabeled peptide showed efficient stability in the presence of normal saline and human serum. The in vitro cellular assays on three prostate cancer cell lines indicated that the radiotracer was bound to LN-CaP cells with higher affinity compared to DU-145 and PC-3 cells. The Kd values of 99mTc- HYNIC (tricine/ EDDA)-Gaba-D-Lys6GnRH were 89.39±26.71, 93.57±30.49 and107.3±18.82 in LN-CaP, PC-3 and DU-145 cells respectively. The biodistribution studies in normal mice and LN-CaP tumor-bearing nude mice showed similar results including rapid blood clearance and low radioactivity accumulation in non-target organs. High kidney uptake proved that the main excretion route of radiopeptide was through the urinary system. The tumor uptake was 1.72±0.45 %ID/g at 1h p.i. decreasing to 0.70±0.06%ID/g at 4h p.i. for 99mTc-HYNIC-Gaba-D-Lys6GnRH. The maximum tumor/ muscle ratio was 2.30 at 1h p.i. Pre-saturation of receptor using an excess of unlabeled peptide revealed that the tumor uptake was receptor mediated. The results of the SPECT image of LN-CaP tumor were in agreement with the biodistribution data. Based on this study, we suggest LN-CaP as a favorable cell line for in vivo studies on GnRH analogues. Moreover, this report shows that 99mTc-HYNIC (tricine/EDDA)-Gaba-D-Lys6GnRH may be a suitable candidate for further evaluation of prostate cancer.

177Lu-Labeled Albumin-Binder-Conjugated PSMA-Targeting Agents with Extremely High Tumor Uptake and Enhanced Tumor-to-Kidney Absorbed Dose Ratio.

The use of an albumin binder has been shown to improve tumor uptake of prostate-specific membrane antigen (PSMA)-targeting radiotherapeutic agents. The aim of this study was to develop improved radiotherapeutic agents that combine an optimized affinity-modifying group and optimized albumin binders to maximize the tumor-to-kidney absorbed dose ratio. Methods:68Ga-labeled DOTA-conjugated lysine-ureido-glutamate-based PSMA-targeting agents bearing various affinity-modifying groups or albumin binders were synthesized and evaluated by PET/CT imaging and biodistribution studies in LNCaP tumor-bearing mice. The optimized affinity-modifying group and albumin binders were combined, and the resulting derivatives were radiolabeled with 177Lu and evaluated by SPECT/CT imaging and biodistribution studies in LNCaP tumor-bearing mice. Radiation dosimetry was calculated using the OLINDA/EXM software. Results: Affinity-modifying group optimization revealed that 68Ga-HTK03041 bearing a tranexamic acid-9-anthrylalanine affinity-modifying group had the highest tumor uptake (23.1 ± 6.11 percentage injected dose [%ID]/g at 1 h after injection). Albumin binder optimization showed that 68Ga-HTK03055 and 68Ga-HTK03086 bearing the N-(4-(p-chlorophenyl)butanoyl)-Gly and N-(4-(p-methoxyphenyl)butanoyl)-Gly motifs, respectively, had relatively faster tumor accumulation (∼30 %ID/g at 3 h after injection) and lower average kidney uptake (<55 %ID/g at both 1 and 3 h after injection). Combining the tranexamic acid-9-anthrylalanine affinity-modifying group with N-(4-(p-chlorophenyl)butanoyl)-Gly and N-(4-(p-methoxyphenyl)butanoyl)-Gly albumin-binding motifs generated HTK03121 and HTK03123, respectively. 177Lu-HTK03121 and 177Lu-HTK03123 had extremely high peak uptake (104 ± 20.3 and 70.8 ± 23.7 %ID/g, respectively) in LNCaP tumor xenografts, and this peak was sustained up to 120 h after injection. Dosimetry calculation showed that compared with 177Lu-PSMA-617, 177Lu-HTK03121 and 177Lu-HTK03123 delivered 18.7- and 12.7-fold higher absorbed dose to tumor but only 6.4- and 6.3-fold higher absorbed dose to kidneys, leading to 2.9- and 2.0-fold improvement in the tumor-to-kidney absorbed dose ratios. Conclusion: With greatly enhanced tumor uptake and tumor-to-kidney absorbed dose ratio, 177Lu-HTK03121 and 177Lu-HTK03123 have the potential to improve treatment efficacy using significantly lower quantities of 177Lu and are promising candidates for clinical translation to treat metastatic castration-resistant prostate cancer.

Development of 177Lu-scFvD2B as a Potential Immunotheranostic Agent for Tumors Overexpressing the Prostate Specific Membrane Antigen

The clinical translation of theranostic 177Lu-radiopharmaceuticals based on inhibitors of the prostate-specific membrane antigen (PSMA) has demonstrated positive clinical responses in patients with advanced prostate cancer (PCa). However, challenges still remain, particularly regarding their pharmacokinetic and dosimetric properties. We developed a potential PSMA-immunotheranostic agent by conjugation of a single-chain variable fragment of the IgGD2B antibody (scFvD2B) to DOTA, to obtain a 177Lu-labelled agent with a better pharmacokinetic profile than those previously reported. The labelled conjugated 177Lu-scFvD2B was obtained in high yield and stability. In vitro, 177Lu-scFvD2B disclosed a higher binding and internalization in LNCaP (PSMA-positive) compared to PC3 (negative control) human PCa cells. In vivo studies in healthy nude mice revealed that 177Lu-scFvD2B present a favorable biokinetic profile, characterized by a rapid clearance from non-target tissues and minimal liver accumulation, but a slow wash-out from kidneys. Micro-SPECT/CT imaging of mice bearing pulmonary microtumors evidenced a slow uptake by LNCaP tumors, which steadily rose up to a maximum value of 3.6 SUV at 192 h. This high and prolonged tumor uptake suggests that 177Lu-scFvD2B has great potential in delivering ablative radiation doses to PSMA-expressing tumors, and warrants further studies to evaluate its preclinical therapeutic efficacy.

Targeted Nanobubbles Carrying Indocyanine Green for Ultrasound, Photoacoustic and Fluorescence Imaging of Prostate Cancer.

ObjectiveTo construct prostate-specific membrane antigen (PSMA)-targeting, indocyanine green (ICG)-loaded nanobubbles (NBs) for multimodal (ultrasound, photoacoustic and fluorescence) imaging of prostate cancer.MethodsThe mechanical oscillation method was used to prepare ICG-loaded photoacoustic NBs (ICG NBs). Then, PSMA-binding peptides were connected to the surface of ICG NBs using the biotin–avidin method to make targeted photoacoustic NBs, namely, PSMAP/ICG NBs. Their particle sizes, zeta potentials, and in vitro ultrasound, photoacoustic and fluorescence imaging were examined. Confocal laser scanning microscopy and flow cytometry were used to detect the binding ability of the PSMAP/ICG NBs to PSMA-positive LNCaP cells, C4-2 cells, and PSMA-negative PC-3 cells. The multimodal imaging effects of PSMAP/ICG NBs and ICG NBs were compared in nude mouse tumor xenografts.ResultsThe particle size of the PSMAP/ICG NBs was approximately 457.7 nm, and the zeta potential was approximately −23.5 mV. Both confocal laser scanning microscopy and flow cytometry confirmed that the PSMAP/ICG NBs could specifically bind to both LNCaP and C4-2 cells, but they rarely bound to PC-3 cells. The ultrasound, photoacoustic and fluorescence imaging intensities of the PSMAP/ICG NBs in vitro positively correlated with their concentrations. The ultrasound and photoacoustic imaging effects of the PSMAP/ICG NBs in LNCaP and C4-2 tumor xenografts were significantly enhanced compared with those in PC-3 tumor xenografts, which were characterized by a significantly increased duration of ultrasound enhancement and heightened photoacoustic signal intensity (P < 0.05). Fluorescence imaging showed that PSMAP/ICG NBs could accumulate in LNCaP and C4-2 tumor xenografts for a relatively long period.ConclusionThe targeted photoacoustic nanobubbles prepared in this study can specifically bind to PSMA-positive prostate cancer cells and have the ability to enhance ultrasound, photoacoustic and fluorescence imaging of PSMA-positive tumor xenografts. Photoacoustic imaging could visually display the intensity of the red photoacoustic signal in the tumor region, providing a more intuitive imaging modality for targeted molecular imaging. This study presents a potential multimodal contrast agent for the accurate diagnosis and assessment of prostate cancer.

Preclinical Evaluation of a High-Affinity Sarcophagine-Containing PSMA Ligand for 64Cu/67Cu-Based Theranostics in Prostate Cancer.

The application of small molecules targeting prostate-specific membrane antigen (PSMA) has emerged as a highly promising clinical strategy for visualization and treatment of prostate cancer. Ligands that integrate the ability to both quantify the distribution of radioactivity and treat disease through the use of a matched pair of radionuclides have particular value in clinical and regulatory settings. In this study, we describe the development and preclinical evaluation of RPS-085, a ligand that binds PSMA and serum albumin and exploits the 64/67Cu radionuclide pair for prostate cancer theranostics. RPS-085 was synthesized by conjugation of a PSMA-targeting moiety, an Nε-(2-(4-iodophenyl)acetyl)lysine albumin binding group, and a bifunctionalized MeCOSar chelator. The IC50 of the metal-free RPS-085 was determined in a competitive binding assay. The affinity for human serum albumin of the radiolabeled compound was determined by high-performance affinity chromatography. Radiolabeling was performed in NH4OAc buffer at 25 °C. The stability of the radiolabeled compounds was assessed in vitro and in vivo. The biodistribution of [64/67Cu]Cu-RPS-085 was determined following intravenous administration to male BALB/c mice bearing LNCaP tumor xenografts. The radiochemical yields of [64/67Cu]Cu-RPS-085 were nearly quantitative after 20 min. The metal-free complex is a potent inhibitor of PSMA (IC50 = 29 ± 2 nM), and the radiolabeled compound has moderate affinity for human serum albumin (Kd = 9.9 ± 1.7 μM). Accumulation of the tracer in mice was primarily evident in tumor and kidneys. Activity in all other tissues, including blood, was negligible, and the radiolabeled compounds demonstrated high stability in vitro and in vivo. Tumor activity reached a maximum at 4 h post injection (p.i.) and cleared gradually over a period of 96 h. By contrast, activity in the kidney cleared rapidly from 4 to 24 h p.i. As a consequence, by 24 h p.i., the tumor-to-kidney ratio exceeds 2, and the predicted dose to tumors is significantly greater than the dose to kidneys. [64Cu]Cu-RPS-085 combines rapid tissue distribution and clearance with prolonged retention in LNCaP tumor xenografts. The pharmacokinetics should enable radioligand therapy using [67Cu]Cu-RPS-085. By virtue of its rapid kidney clearance, the therapeutic index of [67Cu]Cu-RPS-085 likely compares favorably to its parent structure, [177Lu]Lu-RPS-063, a highly avid PSMA-targeting compound. On this basis, [64/67Cu]Cu-RPS-085 show great promise as PSMA-targeting theranostic ligands for prostate cancer imaging and therapy.

Study on the cellular internalization mechanisms and in vivo anti-bone metastasis prostate cancer efficiency of the peptide T7-modified polypeptide nanoparticles

Bone-metastasis prostate cancer (BMPCa)-targeting gene therapy is gaining increasing concern in recent years. The peptide T7-modified polypeptide nanoparticles for delivery DNA (CRD-PEG-T7/pPMEPA1) was prepared as our previous study. However, the feasibility of CRD-PEG-T7/pPMEPA1 for BMPCa treatment, the mechanisms underlying cellular uptake, anti-BMPCa effect, and administration safety requires further research. LNCaP cells treated with endocytosis inhibitors and excessive T7 under different culture condition were carried out to investigate the mechanisms of cellular uptake of the CRD-PEG-T7-pPMEPA1. A transwell assay was applied to evaluate the cell migration ability. Besides, the tumor volume and survival rates of the PCa xenograft mice model were recorded to estimate the anti-tumor effect. In addition, the weight profiles of the PCa tumor-bearing mice, the blood chemistry, and the HE analysis of visceral organs and tumor was conducted to investigate the administration safety of CRD-PEG-T7/pPMEPA1. The results showed that PCa cellular uptake was decreased after treating with excessive free T7, endocytosis inhibitors and lower incubation temperature. Besides, CRD-PEG-T7/pPMEPA1 could inhibit the LNCaP cells chemotaxis and tumor growth. In addition, the survival duration of the PCa tumor-bearing mice treating with CRD-PEG-T7/pPMEPA1 was significantly prolonged with any systemic toxicity or damage to the organs. In conclusion, this research proposes a promising stratagem for treatment BMPCa by providing the biocompatible and effective carrier for delivery DNA therapeutic agents.

Radiohybrid Ligands: A Novel Tracer Concept Exemplified by 18F- or 68Ga-Labeled rhPSMA Inhibitors.

When we critically assess the reason for the current dominance of 68Ga-labeled peptides and peptide-like ligands in radiopharmacy and nuclear medicine, we have to conclude that the major advantage of such radiopharmaceuticals is the apparent lack of suitable 18F-labeling technologies with proven clinical relevance. To prepare and to subsequently perform a clinical proof-of-concept study on the general suitability of silicon-fluoride-acceptor (SiFA)–conjugated radiopharmaceuticals, we developed inhibitors of the prostate-specific membrane antigen (PSMA) that are labeled by isotopic exchange (IE). To compensate for the pronounced lipophilicity of the SiFA unit, we used metal chelates, conjugated in close proximity to SiFA. Six different radiohybrid PSMA ligands (rhPSMA ligands) were evaluated and compared with the commonly used 18F-PSMA inhibitors 18F-DCFPyL and 18F-PSMA-1007. Methods: All inhibitors were synthesized by solid-phase peptide synthesis. Human serum albumin binding was measured by affinity high-performance liquid chromatography, whereas the lipophilicity of each tracer was determined by the n-octanol/buffer method. In vitro studies (IC50, internalization) were performed on LNCaP cells. Biodistribution studies were conducted on LNCaP tumor–bearing male CB-17 SCID mice. Results: On the laboratory scale (starting activities, 0.2–9.0 GBq), labeling of 18F-rhPSMA-5 to -10 by IE was completed in < 20 min (radiochemical yields, 58% ± 9%; radiochemical purity, >97%) with molar activities of 12–60 GBq/μmol. All rhPSMAs showed low nanomolar affinity and high internalization by PSMA-expressing cells when compared with the reference radiopharmaceuticals, medium-to-low lipophilicity, and high human serum albumin binding. Biodistribution studies in LNCaP tumor–bearing mice revealed high tumor uptake, sufficiently fast clearance kinetics from blood, low hepatobiliary excretion, fast renal excretion, and very low uptake of 18F activity in bone. Conclusion: The novel 18F-rhPSMA radiopharmaceuticals developed under the radiohybrid concept show equal or better targeting characteristics than the established 18F-PSMA tracers 18F-DCFPyL and 18F-PSMA-1007. The unparalleled simplicity of production, the possibility to produce the identical 68Ga-labeled 19F-68Ga-rhPSMA tracers, and the possibility to extend this concept to true theranostic radiohybrid radiopharmaceuticals, such as F-Lu-rhPSMA, are unique features of these radiopharmaceuticals.

Abstract LB-A01: Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab

Abstract Background: Programmed cell death 1 (PD-1) and programmed death ligand 1 (PD-L1) expression is typically low in primary prostate cancer but increases at advanced stages. However, targeting PD-1 or PD-L1 has not proven efficacy in prostate cancer patients, and recent clinical trials are directed to combination therapies. About 85% of advanced prostate cancer patients develop skeletal metastases, and the focus in this context should be addressed to the treatment of these metastases. These efforts have been hindered by the lack of relevant preclinical bone metastasis models in immunocompetent mice. We aimed to establish a prostate cancer bone metastasis model in humanized mice and to assess pembrolizumab efficacy in the established model. Materials and methods: Two million LNCaP human prostate cancer cells were inoculated into tibia bone marrow of male CIEA NOG® mice engrafted with human CD34+ hematopoietic stem cells to generate humanized mice. Serum prostate-specific antigen (PSA) levels were measured at 4 weeks, and the mice were allocated to receive either pembrolizumab (anti-PD-1, Keytruda®, 5 mg/kg, Q5D) or human IgG4 isotype control for 6 weeks. Tumor growth was monitored by measuring serum PSA levels. Tumor-induced bone changes were monitored by measuring serum levels of the bone formation marker N-terminal propeptide of type I procollagen (PINP), and by X-ray imaging of tibia. Changes in quantity of circulating T cells was monitored by flow cytometry. At study termination, tissue samples were collected for histological and immunohistochemical evaluation of tumor-infiltrating lymphocytes (TILs). Results: A tumor take of 90% was observed in the humanized mice evaluated by serum PSA levels at endpoint. No significant differences were observed in serum PSA levels, but in a few mice treated with pembrolizumab, only modest tumor growth was observed. Intratibial LNCaP tumors induced osteoblastic-mixed lesions as evaluated by X-ray imaging. No differences were observed in the bone lesion area or in serum PINP levels between the isotype control and pembrolizumab groups. No changes in circulating levels of CD4+, CD8+, or CD4+CD8+ cells were observed. Histology will be performed to confirm tumor morphology and overall tumor viability, and immunohistochemical analysis will reveal the quantity and location of TILs in the tumor. Conclusions: A novel preclinical model of bone metastasis in humanized mice was established. Intratibial prostate cancer tumors induced osteoblastic mixed bone lesions and increased serum PSA levels, mimicking the clinical situation in patients. Resembling recent clinical findings, no or modest responses with pembrolizumab as monotherapy were observed. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Azusa Tanaka, Philip Dube, Jussi M Halleen, Jenni Bernoulli. Establishment of a novel prostate cancer bone metastasis model in humanized mice and early efficacy results of pembrolizumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A01. doi:10.1158/1535-7163.TARG-19-LB-A01

Abstract 4850: The relevance of using proper preclinical models when developing therapeutics for localized or bone metastatic prostate cancer

Abstract Early stages of prostate cancer are sensitive to androgen deprivation therapy, but upon progression the disease develops to metastatic castration-resistant prostate cancer, where bone is the dominant metastatic site. Despite of recent advances in drug development, the advanced stage is still incurable. Tumor microenvironment in metastatic locations differs from the primary site and may cause resistance to used therapy and modulate tumor properties. Therefore, it is crucial to use proper models in preclinical drug development. Aim of this study was to establish predictive preclinical in vitro and in vivo prostate cancer models that can be used in drug development when targeting cancer cells or tumor microenvironment at different stages. Androgen receptor (AR) positive human prostate cancer cell line LNCaP was used in all studies. In the in vitro assay, the effects of the antiandrogen enzalutamide on viability of LNCaP cells was determined using CellTiter Glo assay in the presence and absence of a synthetic androgen R1881. In two in vivo studies, male NMRI nude mice were used. In a subcutaneous model, part of the mice received dihydrotestosterone (DHT) supplement prior to cancer cell inoculation. In a bone metastasis model, LNCaP cells were inoculated into tibia bone marrow. The mice were randomized to treatment groups based on similar serum PSA levels and cancer-induced changes in bone determined by X-ray imaging at 6 weeks after inoculation of the cancer cells. The mice were treated with 300 kBq/kg of Radium-223 dichloride or vehicle at 6 and 10 weeks. Bone lesions were followed by X-ray imaging during the study. The study was terminated at 12 weeks after inoculation of the cancer cells and the tibias were removed for histological analysis and AR staining. In the in vitro assay, 0.1 and 0.01 nM R1881 increased LNCaP cell viability, and enzalutamide reduced cell viability in the presence of R1881 compared to the group with only R1881. In the subcutaneous model, tumors formed within 2-3 weeks after inoculation and the maximum tumor volume was reached within 10-12 weeks. LNCaP tumors grew in NMRI nude mice without DHT supplement, but DHT supplement supported tumor growth. In the bone metastasis model, LNCaP tumors induced osteoblastic-mixed bone lesions without additional androgen supplement, and immunohistochemical staining demonstrated strong AR expression. Radium-223 dichloride reduced the progression of tumor-induced bone lesions, decreased serum PSA levels, and decreased tumor area analyzed by histology. This study showcases relevant models that can be used at different phases of preclinical drug development when evaluating efficacy or safety of new therapeutics as mono- or combination therapies. The choice of a preclinical model and key readouts should be carefully selected based on whether developing therapeutics for localized or metastatic prostate cancer. Citation Format: Tiina E. Kähkönen, Jenni H.E. Mäki-Jouppila, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli. The relevance of using proper preclinical models when developing therapeutics for localized or bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4850.

Abstract 1292: A new generation of N-terminal domain androgen receptor inhibitors, with improved pharmaceutical properties, in castration-resistant prostate cancer models

Abstract Introduction: The androgen receptor (AR) pathway continues to drive castration-resistant prostate cancer (CRPC) even in late stages of the disease, and ligand binding domain (LBD)-linked resistance inevitably emerges. Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its’ transcription even in the presence of anti-androgen resistance. A Phase I clinical trial of the first-generation AR NTD inhibitor, EPI-506 (EPI-002 pro-drug), demonstrated PSA declines in anti-androgen resistant metastatic CRPC patients. However, these declines were minor and of short duration, revealing the need for more potent and metabolically stable NTD inhibitors. A new generation of NTD transcriptional inhibitors (Anitens) has been generated. Examples of this new class, EPI-7170 and EPI-7245, demonstrate improved potency, metabolic stability and pharmaceutical properties, and are potent against anti-androgen resistant prostate cancers in in vitro and in preclinical models. Methods: Chemical structure activity relationships were developed to identify more potent molecules as measured by both cellular and in vivo assays, while metabolic stability improvements were assessed in in vitro ADME assays and in animal pharmacokinetic studies. In addition, the on-target activity and selectivity were also optimized using a variety of cellular experiments. Results: These next generation Anitens demonstrated a 10-20-fold improvement on AR-driven cellular potency, with IC50’s of 0.5-1 uM compared to 10 uM for EPI-002. Aniten AR inhibition was specific to the NTD, as demonstrated by the absence of LBD binding and the inhibition of the AR-V7-driven transcription inLNCaP95 model. In vitro proliferation assays demonstrated AR-dependent activity, with an IC50 ~ 1 uM in LNCaP and &amp;gt;10 uM in the AR-independent cell model PC-3. The antiproliferative effect aligned with the inhibitory effect on a subset of AR-driven genes. In vivo, next generation Anitens demonstrated PSA serum decreases along with significant tumor growth inhibition in mice bearing LNCaP tumors. While EPI-7170 represents a major advance, subsequent chemistry efforts led to the generation of EPI-7245 and other next generation Anitens which exhibit IC50’s &amp;lt;500 nM. These new compounds exhibited favorable ADME and PK profiles, with half-life ~ 8 hrs in mice, and predicted low human clearance. Conclusions: Promising next-generation Aniten compounds have been identified which retain the NTD specificity of first-generation agents. These new molecules demonstrate major improvements in potency and metabolic stability in comparison to the first-generation clinical compound EPI-002. These next generation Anitens, by selectively inhibiting AR transcription in both hormone sensitive and castrated resistant prostate cancer, represent an important next step in AR targeted therapeutics. Citation Format: Ronan Le Moigne, Nasrin R. Mawji, C. Adriana Banuelos, Jun Wang, Kunzhong Jian, Raymond J. Andersen, Marianne D. Sadar, Han-Jie Zhou, Peter Virsik. A new generation of N-terminal domain androgen receptor inhibitors, with improved pharmaceutical properties, in castration-resistant prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1292.

Abstract 1141: Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate

Abstract The PSMA targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) is a targeted alpha therapy approach for metastatic castration resistant prostate cancer that has shown strong anti-tumor activity in PSMA-positive prostate cancer models (Hammer et al. AACR 2017/2018/2019). PSMA-TTC consists of a fully human IgG1 PSMA-targeted antibody covalently linked to a 3,2-HOPO chelator moiety, stably complexing the alpha emitter thorium-227. As this chelator can also complex zirconium-89, positron emission tomography (PET) imaging using the zirconium-89 labeled variant of the PSMA antibody-chelator conjugate could support clinical development of PSMA-TTC by enabling monitoring of organ distribution. Herein, we describe radiolabeling of the PSMA antibody-chelator conjugate with zirconium-89 and its quality control using bioanalytical assays. In addition, the zirconium-89 labeled PSMA antibody-chelator conjugate was injected into mice bearing LNCaP xenograft tumors and biodistribution was assessed by measurement of radioactivity in tumor and organs as well as PET imaging at different time points after injection. The PSMA antibody-chelator conjugate was successfully labeled with zirconium-89 at high yield, purity and specific activity. Target binding was confirmed by determination of the immunoreactive fraction. Biodistribution experiments revealed high, long lasting uptake of zirconium-89 in PSMA-positive LNCaP tumors after single injection of the zirconium-89 labeled conjugate. An uptake of 40-50% of the injected dose per gram of tumor was measured at 24, 48, 72, 168 and 240h after injection. The PET images showed that the zirconium-89 labeled PSMA antibody-chelator conjugate provided an excellent image contrast for the delineation of LNCaP xenografts between 48 and 168 h after administration. Other organs showed only very little background and tumor-to-heart ratio was greater than 10 when PET imaging was performed between 48 and 168 h after injection. These results indicate that the zirconium-89 labeled PSMA antibody-chelator conjugate shows promise for PET imaging studies. These properties may allow its use to support clinical development of PSMA-TTC. Citation Format: Stefanie Hammer, Sabine Zitzmann-Kolbe, Felix Oden, Joerg Jannsen, Eva Bickel, Andre Mueller, Lothar Everz, Urs B. Hagemann, Jenny Karlsson, Olav B. Ryan, Hartwig Hennekes, Patricia E. Cole, Dominik Mumberg. Preclinical analysis of biodistribution and PET imaging of a zirconium-89 labeled PSMA-targeted antibody-chelator conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1141.