Susan Evans-Axelsson

The STAT3 Inhibitor Galiellalactone Effectively Reduces Tumor Growth and Metastatic Spread in an Orthotopic Xenograft Mouse Model of Prostate Cancer

UNLABELLED Signal transducer and activator of transcription 3 (STAT3) is known to be involved in the progression of prostate cancer (PCa) and is a key factor in drug resistance and tumor immunoescape. As a result, it represents a promising target for PCa therapy. We studied the effects of the STAT3 inhibitor galiellalactone (GL) on tumor growth and metastatic spread in vitro and in vivo. The effect of GL on cell viability, apoptosis, and invasion was studied in vitro using androgen-independent DU145 and DU145-Luc cell lines. For in vivo studies, mice were injected orthotopically with DU145-Luc cells and treated with daily intraperitoneal injections of GL for 6 wk. GL significantly reduced the growth of the primary tumor and the metastatic spread of PCa cells to regional and distal lymph nodes in vivo. Treatment with GL also resulted in decreased cell proliferation and increased apoptosis compared with controls. In vitro, GL reduces the viability and invasive abilities of DU145-Luc cells and induces apoptosis. Our results showed that tumor growth and early metastatic dissemination of PCa can be significantly reduced by GL, indicating its potential use as a therapeutic compound in advanced metastatic PCa. PATIENT SUMMARY In this study, we tested the STAT3 inhibitor galiellalactone (GL) in an animal model of PCa. We found that mice treated with GL had smaller primary tumors and decreased lymph node metastases compared with mice treated with vehicle. GL has potential for treating advanced metastatic PCa.

Targeting Free Prostate-Specific Antigen for In Vivo Imaging of Prostate Cancer Using a Monoclonal Antibody Specific for Unique Epitopes Accessible on Free Prostate-Specific Antigen Alone.

This study investigated the feasibility of targeting the free, unbound forms of prostate-specific antigen (fPSA) for in vivo imaging of prostate adenocarcinomas (PCa), as PSA is produced and secreted at abundance during every clinical stage and grade of PCa, including castration-resistant disease. We injected (125)I-labeled monoclonal antibody PSA30 (specific for an epitope uniquely accessible on fPSA alone) intravenously in male nude mice carrying subcutaneous xenografts of LNCaP tumors (n=36). Mice were sacrificed over a time course from 4 hours to 13 days after injecting (125)I-labeled PSA30. Tissue uptake of (125)I-PSA30 at 48 and 168 hours after intravenous injection was compared with two clinically used positron emission tomography radiopharmaceuticals, (18)F-fluoro-deoxy-glucose ((18)F-FDG) or (18)F-choline, in cryosections using Digital AutoRadiography (DAR) and also compared with immunohistochemical staining of PSA and histopathology. On DAR, the areas with high (125)I-PSA30 uptake corresponded mainly to morphologically intact and PSA-producing LNCaP cells, but did not associate with the areas of high uptake of either (18)F-FDG or (18)F-choline. Biodistribution of (125)I-PSA30 measured in dissected organs ex vivo during 4 to 312 hours after intravenous injection demonstrated maximum selective tumor uptake 24-48 hours after antibody injection. Our data showed selective uptake in vivo of a monoclonal antibody highly specific for fPSA in LNCaP cells. Hence, in vivo imaging of fPSA may be feasible with putative usefulness in disseminated PCa.

Radioimmunotherapy for Prostate Cancer— Current Status and Future Possibilities

Prostate cancer (PCa) is one of the most common cancers in men and is the second leading cause of cancer-related deaths in the USA. In the United States, it is the second most frequently diagnosed cancer after skin cancer, and in Europe it is number one. According to the American Cancer Society, approximately 221,000 men in the United States would be diagnosed with PCa during 2015, and approximately 28,000 would die of the disease. According to the International Agency for Research on Cancer, approximately 345,000 men were diagnosed with PCa in Europe during 2012, and despite more emphasis placed on early detection through routine screening, 72,000 men died of the disease. Hence, the need for improved therapy modalities is of utmost importance. And targeted therapies based on radiolabeled specific antibodies or peptides are a very interesting and promising alternative to increase the therapeutic efficacy and overall chance of survival of these patients. There are currently several preclinical and some clinical studies that have been conducted, or are ongoing, to investigate the therapeutic efficacy and toxicity of radioimmunotherapy (RIT) against PCa. One thing that is lacking in a lot of these published studies is the dosimetry data, which are needed to compare results between the studies and the study locations. Given the complicated tumor microenvironment and overall complexity of RIT to PCa, old and new targets and targeting strategies like combination RIT and pretargeting RIT are being improved and assessed along with various therapeutic radionuclides candidates. Given alone or in combination with other therapies, these new and improved strategies and RIT tools further enhance the clinical response to RIT drugs in PCa, making RIT for PCa an increasingly practical clinical tool.

Treatment with the WNT5A-mimicking peptide Foxy-5 effectively reduces the metastatic spread of WNT5A-low prostate cancer cells in an orthotopic mouse model

Prostate cancer patients with high WNT5A expression in their tumors have been shown to have more favorable prognosis than those with low WNT5A expression. This suggests that reconstitution of Wnt5a in low WNT5A-expressing tumors might be an attractive therapeutic approach. To explore this idea, we have in the present study used Foxy-5, a WNT5A mimicking peptide, to investigate its impact on primary tumor and metastasis in vivo and on prostate cancer cell viability, apoptosis and invasion in vitro. We used an in vivo orthotopic xenograft mouse model with metastatic luciferase-labeled WNT5A-low DU145 cells and metastatic luciferase-labeled WNT5A-high PC3prostate cancer cells. We provide here the first evidence that Foxy-5 significantly inhibits the initial metastatic dissemination of tumor cells to regional and distal lymph nodes by 90% and 75%, respectively. Importantly, this effect was seen only with the WNT5A-low DU145 cells and not with the WNT5A-high PC3 cells. The inhibiting effect in the DU145-based model occurred despite the fact that no effects were observed on primary tumor growth, apoptosis or proliferation. These findings are consistent with and supported by the in vitro data, where Foxy-5 specifically targets invasion without affecting apoptosis or viability of WNT5A-low prostate cancer cells. To conclude, our data indicate that the WNT5A-mimicking peptide Foxy-5, which has been recently used in a phase 1 clinical trial, is an attractive candidate for complimentary anti-metastatic treatment of prostate cancer patients with tumors exhibiting absent or low WNT5A expression.

Preclinical Characterization of 3β-(N-Acetyl l-cysteine methyl ester)-2aβ,3-dihydrogaliellalactone (GPA512), a Prodrug of a Direct STAT3 Inhibitor for the Treatment of Prostate Cancer

The transcription factor STAT3 is a potential target for the treatment of castration-resistant prostate cancer. Galiellalactone (1), a direct inhibitor of STAT3, prevents the transcription of STAT3 regulated genes. In this study we characterized 6 (GPA512, Johansson , M. ; Sterner , O. Patent WO 2015/132396 A1, 2015 ), a prodrug of 1. In vitro studies showed that 6 is rapidly converted to 1 in plasma and is stable in a buffer solution. The pharmacokinetics of 6 following a single oral dose indicated that the prodrug was rapidly absorbed and converted to 1 with a tmax of 15 min. Oral administration of 6 in mice increased the plasma exposure of the active parent compound 20-fold compared to when 1 was dosed orally. 6 treated mice bearing DU145 xenograft tumors had significantly reduced tumor growth compared to untreated mice. The favorable druglike properties and safety profile of 6 warrant further studies of 6 for the treatment of castration-resistant prostate cancer.

Establishing 177Lu-PSMA-617 radioligand therapy in a syngeneic model of murine prostate cancer

Clinical 177Lu-PSMA-617 radioligand therapy (RLT) is applied in advanced-stage prostate cancer. However, to the best of our knowledge murine models to study the biologic effects of various activity levels have not been established. The aim of this study was to optimize specific and total activity for 177Lu-PSMA-617 RLT in a syngeneic model of murine prostate cancer. Methods: Murine-reconstituted, oncogene-driven prostate cancer cells (0.1 × 106) (RM1), transduced to express human prostate-specific membrane antigen (PSMA), were injected into the left flank of C57Bl6 immunocompetent mice. RLT was performed by administering a single tail vein injection of 177Lu-PSMA-617 at different formulations for specific (60 MBq at high, 62 MBq/nmol; intermediate, 31 MBq/nmol; or low 15 MBq/nmol specific activity) or total activity (30, 60, or 120 MBq). Organ distribution was determined by ex vivo γ-counter measurement. DNA double-strand breaks were measured using anti–gamma-H2A.X (phospho S139) immunohistochemistry. Efficacy was assessed by serial CT tumor volumetry and 18F-FDG PET metabolic volume. Toxicity was evaluated 4 wk after the start of RLT. Results: Mean tumor-to-kidney ratios ± SEM were 19 ± 5, 10 ± 5, and 2 ± 0 for high, intermediate, and low (each n = 3) specific activity, respectively. Four of 6 (67%) mice treated with intermediate or high specific activity and none of 6 (0%) mice treated with low specific activity or formulation demonstrated significant DNA double-strand breaks (≥5% γ-H2A.X–positive cells). High when compared with intermediate or low specific activity resulted in a lower mean ± SEM tumor load by histopathology (vital tissue, 4 ± 2 vs. 8 ± 3 mm2; n = 3 vs. 6), day-4 18F-FDG PET (metabolic volume, 87 ± 23 vs. 118 ± 14 mm3; n = 6 vs. 12), and day-7 CT (volume, 323 ± 122 vs. 590 ± 46 mm3; n = 3 vs. 6; P = 0.039). 177Lu-PSMA-617 (120 MBq) with high specific activity induced superior tumor growth inhibition (P = 0.021, n = 5/group) without subacute hematologic toxicity (n = 3/group). Conclusion: 177Lu-PSMA-617 (120 MBq) and high specific activity resulted in the highest efficacy in a syngeneic model of murine prostate cancer. The model will be useful for studying the effects of PSMA-directed RLT combined with potentially synergistic pharmacologic approaches.

Radiolabeled antibodies in prostate cancer: A case study showing the effect of host immunity on antibody bio-distribution

OBJECTIVES Human tumors xenografted in immunodeficient mice are crucial models in nuclear medicine to evaluate the effectiveness of candidate diagnostic and therapeutic compounds. However, little attention has been focused on the biological profile of the host model and its potential effects on the bio-distribution and tumor targeting of the tracer compound under study. We specifically investigated the dissimilarity in bio-distribution of (111)In-DTPA-5A10, which targets free prostate specific antigen (fPSA), in two animal models. METHODS In vivo bio-distribution studies of (111)In-DTPA-5A10 were performed in immunodeficient BALB/c-nu or NMRI-nu mice with subcutaneous (s.c.) LNCaP tumors. Targeting-specificity of the tracer was assessed by quantifying the uptake in (a) mice with s.c. xenografts of PSA-negative DU145 cells as well as (b) BALB/c-nu or NMRI-nu mice co-injected with an excess of non-labeled 5A10. Finally, the effect of neonatal Fc-receptor (FcRn) inhibition on the bio-distribution of the conjugate was studied by saturating FcRn-binding capacity with nonspecific IgG1. RESULTS The inherent biological attributes of the mouse model substantially influenced the bio-distribution and pharmacokinetics of (111)In-DTPA-5A10. With LNCaP xenografts in BALB/c-nu mice (with intact B and NK cells but with deficient T cells) versus NMRI-nu mice (with intact B cells, increased NK cells and absent T cells), we observed a significantly higher hepatic accumulation (26 ± 3.9 versus 3.5 ± 0.4%IA/g respectively), and concomitantly lower tumor uptake (25 ± 11 versus 52 ± 10%IA/g respectively) in BALB/c-nu mice. Inhibiting FcRn by administration of nonspecific IgG1 just prior to (111)In-DTPA-5A10 did not change tumor accumulation significantly. CONCLUSIONS We demonstrated that the choice of immunodeficient mouse model importantly influence the bio-distribution of (111)In-DTPA-5A10. This study further highlighted important considerations in the evaluation of preclinical tracers, with respect to gaining information on their performance in the translational setting. Investigators utilizing xenograft models need to assess not only radiolabeling strategies, but also the host immunological status.

Microscale radiosynthesis, preclinical imaging and dosimetry study of [ 18 F]AMBF 3 -TATE: A potential PET tracer for clinical imaging of somatostatin receptors

BACKGROUND Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced. METHODS Both [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE. RESULTS The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50 ± 6% (n = 15), overall decay-corrected radiochemical yield of 16 ± 1% (n = 5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26 × 10-2 mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds. CONCLUSIONS The combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.

An attenuation method for reducing count rate losses in preclinical PET during intratherapeutic imaging

In pre-clinical imaging, tumor response to radionuclide therapy can be monitored with PET imaging. Radionuclides used for therapy such as 177Lu emit a significant amount of low energy photons. These photons may have an energy high enough to penetrate the imaged object and are the prone to be detected. Although these phtons are likely to be rejected electronically, they add dead-time to the system since they need to be processed by the electronics. This is a problem in high-sensitivity pre-clinical PET system with a low number of readout channels, such as the Genisys G8 investigated in this work. The low energy gammas may also affect image quality due to increased probability of pulse pile-up. The use of high-attenuating shields designed to absorb most of the low energy photons emitted from the therapeutic radionuclide were investigated. Cylindrical led shields were constructed with thicknesses between 1 and 3 mm. A 3mm thick cylindrical shield was also constructed out of Rose metal (50% Bi, 28% Pb, and 22% Sn). The diameter of the shield was wide enough to accommodate a NEMA IQ phantom and a mouse. The attenuation of the shields for annihilation radiation was measured with a 22Na point source placed at the center of the FOV. Measurements of the coincidence rate were performed with the lead shields in place. At a of thicknesses of 1 and 3mm, the coincidence rate was reduced by a factor or 0.70 and 0.40, respectively. To study the effect of the presence of a background of low energy gammas on the coincidence count rate and the efficacy of the lead shields, 177Lu was added to a 1 cm diameter hollow sphere. In the presence of 100 MBq of 177Lu, the coincidence count rate was reduced by a factor 0.20 due to the detector dead-time. Although the count rate was reduced by a factor of 0.40 with the 3mm shield around the source, the dead-time effects due to the 177Lu background were less than 7%. 18F imaging of a NEMA phantom and a tumor bearing mouse showed dramatic image distortions in the presence of the 177Lu background. When imaging of with the 3mm shield in place, the image distortions were eliminated and were comparable in to the images acquired without the background activity.

Abstract 4476: Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer

Background and Aims Wnt5a protein levels are upregulated in prostate cancer (PCa) compared to benign tissue and patients with high Wnt5a protein levels have a better outcome after radical prostatectomy compared to patients with low Wnt5a levels (Syed Khaja AS, 2011; 2012). Thus, the reconstitution of the Wnt5a signaling pathway could be a promising therapeutic approach in PCa, as already shown in breast cancer (Safholm A, 2008). Constitutively active STAT3 (pSTAT3) has been correlated to PCa progression and disruption of its signaling pathway could represent a promising strategy for the treatment of patients with advanced PCa (Hellsten R, 2008). We have analyzed the expression of Wnt5a and pSTAT3 in a cohort of patients with metastatic Castration Resistant Prostate Cancer (mCRPC). Using in vitro and in vivo techniques we have explored the effect of the Wnt5a-mimicking peptide FOXY5 for the reconstitution of the Wnt5a pathway in PCa, and we have studied the effect of the STAT3 inhibitor Galiellalactone (GL) on PCa cell viability, apoptosis, invasion, tumor growth and metastases. Methods Wnt5a and pSTAT3 protein expression was analyzed by IHC on a TMA construct with duplicate cores of malignant tissues from 38 patients with mCRPC. Statistical and correlation analyses were performed using the SPSS version 21. Cell viability was determined by MTT assay; apoptotic cells were visualized with M30 CytoDeath antibody 24h after treatment with FOXY5 or GL. Cell invasion was analyzed using matrigel pre-coated cell culture inserts in the presence or absence of FOXY5 or GL. For the animal study, NMRI nude mice were injected orthotopically with 1×10 6 DU145-Luc cells, and treated IP with GL every day or with FOXY5 every second day. Primary tumor growth and metastatic spread were evaluated weekly by in vivo luminescence using the IVIS Lumina II system. 9 weeks after injection animals were sacrificed and organs were analyzed for the presence of metastases. Results IHC analyses of TMA showed high pSTAT3 and low Wnt5a expression in CRPC patients, with a small but significant correlation (r = 0.4646; p = 0.0038). Our results show that GL inhibits viability and induces apoptosis of DU145-Luc cells in vitro, and that it reduces tumor growth and metastatic spread to lymph nodes in vivo. FOXY5 treatment has no effects on cell viability or on primary tumor growth, but it significantly reduces cell invasion in vitro and metastatic spread to lymph nodes in vivo. Conclusions Our results confirm that the reconstitution of the Wnt5a pathway and the inhibition of the STAT3 pathway are a promising therapeutic approach for the treatment of mCRPC. We found that GL and FOXY5 are good candidates for the treatment of mCRPC, as they inhibit tumor growth and metastatic spread to lymph nodes. Since these two compounds act through different mechanisms, our future studies will explore the possibility of a combination of GL and FOXY5 for the treatment of PCa. Citation Format: Giacomo Canesin, Susan Evans-Axelsson, Rebecka Hellsten, Nicholas Don-Doncow, Tommy Andersson, Anders Bjartell. Targeting Wnt5a and STAT3 pathways for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4476. doi:10.1158/1538-7445.AM2015-4476