More Than Meets the Eye: Scientific Rationale behind Molecular Imaging and Therapeutic Targeting of Prostate-Specific Membrane Antigen (PSMA) in Metastatic Prostate Cancer and Beyond

Simple SummaryProstate-specific membrane antigen (PSMA) is a transmembrane protein that is overexpressed in prostate cancer and correlates with the aggressiveness of the disease. PSMA is a promising target for imaging and therapeutics in prostate cancer patients validated in prospective trials. However, the role of PSMA in prostate cancer progression is poorly understood. In this review, we discuss the biology and scientific rationale behind the use of PSMA and other targets in the detection and theranostics of metastatic prostate cancer.Prostate cancer is the second most common cancer type in men globally. Although the prognosis for localized prostate cancer is good, no curative treatments are available for metastatic disease. Better diagnostic methods could help target therapies and improve the outcome. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein that is overexpressed on malignant prostate tumor cells and correlates with the aggressiveness of the disease. PSMA is a clinically validated target for positron emission tomography (PET) imaging-based diagnostics in prostate cancer, and during recent years several therapeutics have been developed based on PSMA expression and activity. The expression of PSMA in prostate cancer can be very heterogeneous and some metastases are negative for PSMA. Determinants that dictate clinical responses to PSMA-targeting therapeutics are not well known. Moreover, it is not clear how to manipulate PSMA expression for therapeutic purposes and develop rational treatment combinations. A deeper understanding of the biology behind the use of PSMA would help the development of theranostics with radiolabeled compounds and other PSMA-based therapeutic approaches. Along with PSMA several other targets have also been evaluated or are currently under investigation in preclinical or clinical settings in prostate cancer. Here we critically elaborate the biology and scientific rationale behind the use of PSMA and other targets in the detection and therapeutic targeting of metastatic prostate cancer

Head and neck squamous cell carcinoma cell lines have an immunomodulatory effect on macrophages independent of hypoxia and toll-like receptor 9

Background: A low tissue oxygen level, Methods: Conditioned media (CMNOX or CMHOX) from cell lines UT-SCC-8, UT-SCC-74A, FaDu, MDA-MB-231 and HaCat cultured under normoxic (21% O-2) and hypoxic (1% O-2) conditions were used to polarize human monocyte-derived macrophages. Macrophage polarization was measured by flow cytometry and the production of cytokine mRNA using Taqman qPCR. To study the role of TLR9 in macrophage polarization, the lentiviral CRISPR/Cas9 method was used to establish a stable FaDu(TLR9def) clone.Results: Our results demonstrate that the soluble mediators produced by the cancer cells under normoxia polarize macrophages towards a hybridized M1/M2a/M2c phenotype. Furthermore, the results suggest that hypoxia has a limited role in altering the array of cancer-produced soluble factors affecting macrophage polarization and cytokine production. Our data also indicates that increased expression of TLR9 due to hypoxia in malignant cells does not markedly influence the polarization of macrophages. TLR9 transcriptional response to hypoxia is dissimilar to a HIF1-alpha-regulated LDH-A. This may indicate a context-dependent expression of TLR9 under hypoxia.Conclusions: HNSCC cell lines affect both macrophage activity (polarization) and functionality (cytokines), but with exception to iNOS expression, the effects appear independent of hypoxia and TLR9.</p

Dovitinib dilactic acid reduces tumor growth and tumor-induced bone changes in an experimental breast cancer bone growth model

Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[ 18 F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.</p

Synthesis of an Alkyne-Modified Bleomycin Disaccharide Precursor, Conversion to a F-18-Labeled Radiotracer, and Preliminary in vivo-PET Imaging Studies

The bleomycins (BLMs) are known antitumor antibiotics composed of the tumoricidal and tumor seeking domains. The peptide structure of BLMs is responsible for the cytotoxicity by selective oxidative cleavage of DNA (and RNA), while the tumor cell selectivity and internalization resides in the disaccharide moiety (i.e. BLM disaccharide). This has prompted researchers to utilize BLM disaccharide and its derivatives as constituents for the selective recognition of tumor cells, which may find further applications as new tumor imaging tools or drug delivery vehicles. In the present study a high yielding synthesis of an alkyne modified BLM disaccharide precursor that may be used as a useful agent for the click conjugation, its conversion to a F-18-labeled radiotracer, and preliminary in vivo PET imaging studies of the tracer with breast cancer (MCF-7) xenograft mouse models are described

[18F]SPA-RQ/PET Study of NK1 receptors in the Whole Body of Guinea Pig and Rat

There is a substantial interest in the development of NK1 substance P antagonists as potential treatments for various neuropsychiatric and somatic disorders. The aim of this study was to determine whether [18F]SPA-RQ can be utilized as a tool for studying the whole body distribution and function of NK1 receptors in preclinical settings. The compound was injected into guinea pigs with or without premedication with a NK1 receptor antagonist (NK1A-2). For comparison, we included two rats in the study, as the affinity of antagonists for NK1 receptors is known to vary between species. The whole body biodistribution of the tracer was determined at several time points. The tracer showed specific binding in organs compatible with the known location of NK1-receptors. Premedication with a NK1 antagonist led to an inhibited uptake of [18F]SPA-RQ in several organs of guinea pigs, notably intestine, pancreas, urinary bladder, uterus, skin and lung. Specific binding was also seen in both cortex and striatum. In contrast, negligible specific binding was observed in the rat brain with [18F]SPA-RQ, whereas the tracer uptake in peripheral tissues was similar to that seen in guinea pigs. We conclude that [18F]SPA-RQ/PET is a useful tool to study the distribution and function of peripherally located NK1 receptors e.g. in different disease models.</div

Repeatability of tumour hypoxia imaging using [18F]EF5 PET/CT in head and neck cancer

</h3

Comparative Evaluation of Anti-HER2 Affibody Molecules Labeled with Cu-64 Using NOTA and NODAGA

Imaging using affi body molecules enables discrimination between breast cancer metastases with high and low expression of HER2, making appropriate therapy selection possible. This study aimed to evaluate if the longer half-life of Cu-64 (T-1/2 = 12.7h) would make Cu-64 a superior nuclide compared to Ga-68 for PET imaging of HER2 expression using affibody molecules. The synthetic ZHER2: S1 affibody molecule was conjugated with the chelators NOTA or NODAGA and labeled with Cu-64. The tumor-targeting properties of Cu-64-NOTA-ZHER2: S1 and Cu-64-NODAGA-ZHER2: S1 were evaluated and compared with the targeting properties of Ga-68-NODAGA-ZHER2: S1 in mice. Both 64 Cu-NOTA-ZHER2: S1 and Cu-64-NODAGA-ZHER2: S1 demonstrated specific targeting of HER2-expressing xenografts. At 2 h after injection of Cu-64-NOTA-ZHER2: S1, Cu-64-NODAGA-ZHER2: S1, and Ga-68-NODAGAZHER2: S1, tumor uptakes did not differ significantly. Renal uptake of Cu-64-labeled conjugateswas dramatically reduced at 6 and 24 h after injection. Notably, radioactivity uptake concomitantly increased in blood, lung, liver, spleen, and intestines, which resulted in decreased tumor-to-organ ratios compared to 2 h postinjection. Organ uptake was lower for Cu-64-NODAGA-ZHER2: S1. The most probable explanation for this biodistribution pattern was the release and redistribution of renal radiometabolites. In conclusion, monoamide derivatives of NOTA and NODAGA may be suboptimal chelators for radiocopper labeling of anti-HER2 affibody molecules and, possibly, other scaffold proteins with high renal uptake

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18-K20, 75-95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.</p

Evaluation of [F-18]F-DPA as a target for TSPO in head and neck cancer under normal conditions and after radiotherapy

Background Many malignant tumours have increased TSPO expression, which has been related to a poor prognosis. TSPO-PET tracers have not comprehensively been evaluated in peripherally located tumours. This study aimed to evaluate whether N,N-diethyl-2-(2-(4-([F-18]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([F-18]F-DPA) can reflect radiotherapy (RT)-induced changes in TSPO activity in head and neck squamous cell carcinoma (HNSCC). Methods RT was used to induce inflammatory responses in HNSCC xenografts and cells. [F-18]F-DPA uptake was measured in vivo in non-irradiated and irradiated tumours, followed by ex vivo biodistribution, autoradiography, and radiometabolite analysis. In vitro studies were performed in parental and TSPO-silenced (TSPO siRNA) cells. TSPO protein and mRNA expression, as well as tumour-associated macrophages (TAMs), were also assessed. Results In vivo imaging and ex vivo measurement revealed significantly higher [F-18]F-DPA uptake in irradiated, compared to non-irradiated tumours. In vitro labelling studies with cells confirmed this finding, whereas no effect of RT on [F-18]F-DPA uptake was detected in TSPO siRNA cells. Radiometabolite analysis showed that the amount of unchanged [F-18]F-DPA in tumours was 95%, also after irradiation. PK11195 pre-treatment reduced the tumour-to-blood ratio of [F-18]F-DPA by 73% in xenografts and by 88% in cells. TSPO protein and mRNA levels increased after RT, but were highly variable. The proportion of M1/M2 TAMs decreased after RT, whereas the proportion of monocytes and migratory monocytes/macrophages increased. Conclusions [F-18]F-DPA can detect changes in TSPO expression levels after RT in HNSCC, which does not seem to reflect inflammation. Further studies are however needed to clarify the physiological mechanisms regulated by TSPO after RT

Evaluation of [F-18]F-DPA PET for Detecting Microglial Activation in the Spinal Cord of a Rat Model of Neuropathic Pain

Purpose Recent studies have linked activated spinal glia to neuropathic pain. Here, using a positron emission tomography (PET) scanner with high spatial resolution and sensitivity, we evaluated the feasibility and sensitivity of N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin3-yl)acetamide -([F-18]F-DPA) imaging for detecting spinal cord microglial activation after partial sciatic nerve ligation (PSNL) in rats.Procedures Neuropathic pain was induced in rats (n = 20) by PSNL, and pain sensation tests were conducted before surgery and 3 and 7 days post- injury. On day 7, in vivo PET imaging and ex vivo autoradiography were performed using -[F-18]F-DPA or -[C-11]PK11195. Ex vivo biodistribution and PET imaging of the removed spinal cord were carried out with -[F-18]F-DPA. Sham-operated and PK11195pretreated animals were also examined.Results Mechanical allodynia was confirmed in the PSNL rats from day 3 through day 7. Ex vivo autoradiography showed a higher lesion-to-background uptake with -[F-18]F-DPA compared with -[C-11]PK11195. Ex vivo PET imaging of the removed spinal cord showed -[F-18]F-DPA accumulation in the inflammation site, which was immunohistochemically confirmed to coincide with microglia activation. Pretreatment with PK11195 eliminated the uptake. The SUV values of in vivo -[F-18]F-DPA and -[C-11]PK11195 PET were not significantly increased in the lesion compared with the reference region, and were fivefold higher than the values obtained from the ex vivo data. Ex vivo biodistribution revealed a twofold higher -[F-18] F-DPA uptake in the vertebral body compared to that seen in the bone from the skull.Conclusions[F-18]F-DPA aided visualization of the spinal cord inflammation site in PSNL rats on ex vivo autoradiography and was superior to -[C-11]PK11195. In vivo -[F-18]F-DPA PET did not allow for visualization of tracer accumulation even using a high-spatial-resolution PET scanner. The main reason for this result was due to insufficient SUVs in the spinal cord region as compared with the background noise, in addition to a spillover from the vertebral body.</p