Ultrasound-guided intramural inoculation of orthotopic bladder cancer xenografts: a novel high-precision approach

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice

Construction, expression and characterization of CD45-immunoglobulin fusion proteins

The aim of this work was to create, express, and characterize fusion proteins consisting of different alternatively spliced exons of murine CD45, a protein tyrosine phosphatase, linked to the heavy chain constant regions of murine immunoglobulin G. CD45-immunoglobulin fusion proteins were secreted as dimers in a relatively pure form using serum free media at an approximate yield of 1.5-4.5 ug / m l , depending on the isoform of CD45 and the cell line in which it was expressed. Fusion proteins secreted by Cos 7 cells had a higher apparent molecular weight by approximately 5-10 kDa than those expressed by X63-Ag8.653 or T28 cells. The interaction of CD45 with putative ligands may be mediated by specific carbohydrate residues on CD45, therefore, the carbohydrate residues expressed on CD45-immunoglobulin fusion proteins were characterized. O-glycosidase digestion and lectin analysis revealed that all fusion proteins were extensively O-glycosylated in a cell-specific manner. Neuraminidase digestion and analysis of subsequent Peanut agglutinin reactivity suggested that fusion proteins secreted by Cos 7 cells expressed more sialic acid when compared to that secreted by X63-Ag8.653 or T28 cells. Thrombin cleavage and PNGase F digestion revealed that the immunoglobulin portion was 34 kDa and the only site of N-linked carbohydrate addition. A l l fusion proteins reacted with anti-CD45 exon-specific antibodies as predicted with the exception of RA3 6B2, a B220-specific antibody that reacted with CD45RABCIg expressed by Cos 7 cells but not with that expressed by X63-Ag8.653 or T28 cells. RA3 6B2 reacted with fusion proteins containing exons A , B, and C inclusive in addition to fusion proteins containing only exon A. RA3 6B2 binding was not affected by neuraminidase treatment, but did correlate to the binding of wheat germ agglutinin. Once expressed and purified, CD45-immunoglobulin fusion proteins can be used as diagnostic tools in immunoadherence and adhesion assays in an attempt to further our understanding of T lymphocyte signalling via the identification an isoform-specific ligand(s) for murine CD45.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Harnessing Induced Essentiality: Targeting Carbonic Anhydrase IX and Angiogenesis Reduces Lung Metastasis of Triple Negative Breast Cancer Xenografts

Triple Negative Breast Cancer (TNBC) is aggressive, metastatic and drug-resistant, limiting the spectrum of effective therapeutic options for breast cancer patients. To date, anti-angiogenic agents have had limited success in the treatment of systemic breast cancer, possibly due to the exacerbation of tumor hypoxia and increased metastasis. Hypoxia drives increased expression of downstream effectors, including Carbonic Anhydrase IX (CAIX), a critical functional component of the pro-survival machinery required by hypoxic tumor cells. Here, we used the highly metastatic, CAIX-positive MDA-MB-231 LM2-4 orthotopic model of TNBC to investigate whether combinatorial targeting of CAIX and angiogenesis impacts tumor growth and metastasis in vivo to improve efficacy. The administration of a small molecule inhibitor of CAIX, SLC-0111, significantly reduced overall metastatic burden, whereas exposure to sunitinib increased hypoxia and CAIX expression in primary tumors, and failed to inhibit metastasis. The administration of SLC-0111 significantly decreased primary tumor vascular density and permeability, and reduced metastasis to the lung and liver. Furthermore, combining sunitinib and SLC-0111 significantly reduced both primary tumor growth and sunitinib-induced metastasis to the lung. Our findings suggest that targeting angiogenesis and hypoxia effectors in combination holds promise as a novel rational strategy for the effective treatment of patients with TNBC.Medicine, Faculty ofNon UBCBiochemistry and Molecular Biology, Department ofReviewedFacult

Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors

The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.Medicine, Faculty ofOther UBCNon UBCUrologic Sciences, Department ofReviewedFacultyResearcherPostdoctoralGraduat

Functional analysis of androgen receptor mutations that confer anti-androgen resistance identified in circulating cell-free DNA from prostate cancer patients

Background: The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors. Results: We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants. Conclusions: This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy.Other UBCNon UBCReviewedFacult

Ultrasound imaging.

<p>A. The mice were anesthetized with isoflurane and mounted on the heated imaging table with continuous monitoring of vital signs. After visualization of the bladder with the Vevo 700® small animal imaging platform the skin was perforated with a 30G needle. B. Ultrasound visualisation of normal mouse bladder in sagittal section with typical dimensions indicated (lumen dimensions 4.4×6.5 mm; wall thickness 0.25 mm).</p

Longitudinal imaging of xenograft growth.

<p>Tumor growth was measured at regular time intervals by: A. bioluminescence imaging, and B. ultrasound. C. Correlation of bioluminescence and xenograft volume for all three cell lines. D. H&E section of a representative UM-UC1 luc xenograft demonstrating invasive growth into the muscle (*) without invasion into adjacent organs. All tumors originated from the anterior bladder wall and often occupied most of the bladder lumen without infiltrating the posterior wall (**).</p