A new model of multi-visceral and bone metastatic prostate cancer with perivascular niche targeting by a novel endothelial specific adenoviral vector

Abstract

While modern therapies for metastatic prostate cancer (PCa) have improved survival they are associated with an increasingly prevalent entity, aggressive variant PCa (AVPCa), lacking androgen receptor (AR) expression, enriched for cancer stem cells (CSCs), and evidencing epithelial-mesenchymal plasticity with a varying extent of neuroendocrine transdifferentiation. Parallel work revealed that endothelial cells (ECs) create a perivascular CSC niche mediated by juxtacrine and membrane tethered signaling. There is increasing interest in pharmacological metastatic niche targeting, however, targeted access has been impossible. Here, we discovered that the Gleason 7 derived, androgen receptor negative, IGR-CaP1 cell line possessed some but not all of the molecular features of AVPCa. Intracardiac injection into NOD/SCID/IL2Rg -/− (NSG) mice produced a completely penetrant bone, liver, adrenal, and brain metastatic phenotype; noninvasively and histologically detectable at 2 weeks, and necessitating sacrifice 4-5 weeks post injection. Bone metastases were osteoblastic, and osteolytic. IGR-CaP1 cells expressed the neuroendocrine marker synaptophysin, near equivalent levels of vimentin and e-cadherin, all of the EMT transcription factors, and activation of NOTCH and WNT pathways. In parallel, we created a new triple-targeted adenoviral vector containing a fiber knob RGD peptide, a hexon mutation, and an EC specific ROBO4 promoter (Ad.RGD.H5/3.ROBO4). This vector was expressed in metastatic microvessels tightly juxtaposed to IGR-CaP1 cells in bone and visceral niches. Thus, the combination of IGR-CaP1 cells and NSG mice produces a completely penetrant metastatic PCa model emulating end-stage human disease. In addition, the metastatic niche access provided by our novel Ad vector could be therapeutically leveraged for future disease control or cure.

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice

“…We noticed that 3-kb upstream promoter of ROBO4 contains information for endothelial cell typespecific expression in the intact endothelium; 46 this observation was confirmed by our published proteome data, which suggest that LSECs have highest ROBO4 abundance in murine liver, 47 and endothelial cell-specific adenovirus was also created based on this demonstration. 39 Furthermore, RGD, which is considered an endothelial cellspecific polypeptide, 48 was used to modify these adenoviruses and PEG particles carried with honokiol, ensuring the endothelial cell specificity of adenovirus and nanoparticle. Moreover, adenovirus had higher gene transfer efficiency than lentivirus and more space to assemble LSEC-specific elements than recombinant adeno-associated virus (rAAV) to ensure the high LSEC targeting, which may act as a more effective vector involved in anti-fibrotic treatment.…”

“…To avoid any nonspecific effects of chemical Erk1/2 and Akt agonists and inhibitors, we used arginylglycylaspartic acid-roundabout guidance receptor 4 (RGD-ROBO4) modified adenovirus to separately express constitutively active mitogen-activated protein kinase (MEK)1 (Ad-CA MEK1) and dominant-negative Akt (Ad-DN Akt), which are specific for endothelial cells, 39,40 to investigate the role of Erk1/2 and Akt activity in LSECs during chronic liver injury. Most of the Ad-RGD-ROBO4 vectors were located in the liver sinusoidal endothelium (Figure S5A).…”

Wnt Signaling Pathway Is among the Drivers of Liver Metastasis

“…For example, a study has shown that DKK1 expression increases early in prostate cancer development and decreases during progression from primary tumor to metastasis, which would indicate the activation of the Wnt pathway [190]. Additionally, a recent study presented a new model of multi-visceral and bone metastatic prostate cancer with activation of Notch and Wnt pathways [191] adding to the complexity of Wnt signaling in contribution to a metastatic process in prostate cancer.…”

Targeting Tumor Neoangiogenesis via Targeted Adenoviral Vector to Achieve Effective Cancer Gene Therapy for Disseminated Neoplastic Disease

“…Sectioning of frozen organs was carried out using the CryoJane Taping System (Leica Biosystems Inc). All frozen section slides were subject to immunofluorescence staining analysis in accordance with the standard techniques (8). Primary antibodies used in this study included hamster anti-CD31 (EMD Millipore), rat anti-endomucin (eBioscience), rat anti-PDGFRb (eBioscience), rabbit anti-HSVtk (Dr. Summers's laboratory), and Alexa Fluor 488 or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).…”

“…Triple-immunodeficient NSG mice allow for modeling of all metastatic sites as published previously (8,9). For systemic metastases, mice were injected intracardially with 5 Â 10 5 786-0 renal carcinoma cells (Luciferase gene expressed).…”