Paul J. Hauser

Restoring Barrier Function to Acid Damaged Bladder by Intravesical Chondroitin Sulfate

PURPOSE Chondroitin sulfate (Stellar Pharmaceuticals, London, Ontario, Canada), which is less expensive and more inert than heparinoids, hyaluronan or pentosan polysulfate, has been introduced to restore the barrier function lost due to epithelial dysfunction in interstitial cystitis cases. To our knowledge chondroitin sulfate binding to damaged bladder as a function of the urinary pH range, its efficacy in restoring the bladder permeability barrier and the capacity of the damaged bladder to bind chondroitin sulfate have not been determined previously. MATERIALS AND METHODS Chondroitin sulfate binding to bladder urothelium was investigated quantitatively using chondroitin sulfate highly labeled with Texas Red(R) and quantitative fluorescence microscopy in a mouse model of urothelial acid damage. The efficacy of restoring barrier function was determined using the passage of intravesically instilled (86)Rb, a potassium ion mimetic, through the urothelium into the bloodstream in a rat model of bladder damage. The binding capacity of acid damaged bladder was determined by fluorometry. RESULTS Chondroitin sulfate bound tightly and exclusively to the mouse bladder surface damaged by acid but showed only minimal binding to undamaged bladder. There was no systematic variation in pH. The model showed some variability in the degree of damage induced. In rats chondroitin sulfate instillation restored permeability to (86)Rb to control levels. Binding was saturable at a mean +/- SEM 0.67 +/- 0.13 mg/cm(2) of the bladder surface. CONCLUSIONS Chondroitin sulfate binds preferentially to damaged urothelium and restores the impermeability barrier. This suggests that the glycosaminoglycan layer is a major contributor to bladder urothelial impermeability. As determined by binding capacity, the dose applied in humans in Canada (400 mg per instillation) is sufficient to achieve maximum efficacy.

Urothelial expression of neuropilins and VEGF receptors in control and interstitial cystitis patients

Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.

Development and characterization of a preclinical model of breast cancer lung micrometastatic to macrometastatic progression.

Most cancer patients die with metastatic disease, thus, good models that recapitulate the natural process of metastasis including a dormancy period with micrometastatic cells would be beneficial in developing treatment strategies. Herein we report a model of natural metastasis that balances time to complete experiments with a reasonable dormancy period, which can be used to better study metastatic progression. The basis for the model is a 4T1 triple negative syngeneic breast cancer model without resection of the primary tumor. A cell titration from 500 to 15,000 GFP tagged 4T1 cells implanted into fat pad number four of immune proficient eight week female BALB/cJ mice optimized speed of the model while possessing metastatic processes including dormancy and beginning of reactivation. The frequency of primary tumors was less than 50% in animals implanted with 500–1500 cells. Although implantation with over 10,000 cells resulted in 100% primary tumor development, the tumors and macrometastases formed were highly aggressive, lacked dormancy, and offered no opportunity for treatment. Implantation of 7,500 cells resulted in >90% tumor take by 10 days; in 30–60 micrometastases in the lung (with many animals also having 2–30 brain micrometastases) two weeks post-implantation, with the first small macrometastases present at five weeks; many animals displaying macrometastases at five weeks and animals becoming moribund by six weeks post-implantation. Using the optimum of 7,500 cells the efficacy of a chemotherapeutic agent for breast cancer, doxorubicin, given at its maximal tolerated dose (MTD; 1 mg/kg weekly) was tested for an effect on metastasis. Doxorubicin treatment significantly reduced primary tumor growth and lung micrometastases but the number of macrometastases at experiment end was not significantly affected. This model should prove useful for development of drugs to target metastasis and to study the biology of metastasis.

Suppression and Activation of the Malignant Phenotype by Extracellular Matrix in Xenograft Models of Bladder Cancer: A Model for Tumor Cell “Dormancy”

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases.

Abnormalities in Expression of Structural, Barrier and Differentiation Related Proteins, and Chondroitin Sulfate in Feline and Human Interstitial Cystitis

PURPOSE We analyzed the urothelium of cats diagnosed with feline interstitial cystitis to determine whether abnormalities in protein expression patterns could be detected and whether the expression pattern was similar to that in patients with human interstitial cystitis/bladder pain syndrome. The proteins analyzed are involved in cell adhesion and barrier function, comprise the glycosaminoglycan layer or are differentiation markers. MATERIALS AND METHODS Formalin fixed biopsies from 8 cats with feline interstitial cystitis and from 7 healthy control cats were labeled by immunohistochemistry and scored with a modified version of a system previously used for human samples. Cluster analysis was performed to investigate relationships between markers and samples. RESULTS Of the feline interstitial cystitis bladders 89% showed abnormal protein expression and chondroitin sulfate patterns while only 27% of normal tissues showed slight abnormalities. Abnormalities were found in most feline interstitial cystitis samples, including biglycan in 87.5%, chondroitin sulfate, decorin, E-cadherin and keratin-20 in 100%, uroplakin in 50% and ZO-1 in 87.5%. In feline interstitial cystitis bladders about 75% of chondroitin sulfate, biglycan and decorin samples demonstrated absent luminal staining or no staining. Cluster analysis revealed that feline interstitial cystitis and normal samples could be clearly separated into 2 groups, showing that the urothelium of cats with feline interstitial cystitis is altered from normal urothelium. CONCLUSIONS Feline interstitial cystitis produces changes in luminal glycosaminoglycan and several proteins similar to that in patients, suggesting some commonality in mechanism. Results support the use of feline interstitial cystitis as a model of human interstitial cystitis.

Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA2 activation. Incubation with the membrane phospholipid-derived PLA2 hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA2 activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.

Phosphatidylserine targeted single-walled carbon nanotubes for photothermal ablation of bladder cancer

Bladder cancer has a 60%-70% recurrence rate most likely due to any residual tumour left behind after a transurethral resection (TUR). Failure to completely resect the cancer can lead to recurrence and progression into higher grade tumours with metastatic potential. We present here a novel therapy to treat superficial tumours with the potential to decrease recurrence. The therapy is a heat-based approach in which bladder tumour specific single-walled carbon nanotubes (SWCNTs) are delivered intravesically at a very low dose (0.1 mg SWCNT per kg body weight) followed 24 h later by a short 30 s treatment with a 360° near-infrared light that heats only the bound nanotubes. The energy density of the treatment was 50 J cm-2, and the power density that this treatment corresponds to is 1.7 W cm-2, which is relatively low. Nanotubes are specifically targeted to the tumour via the interaction of annexin V (AV) and phosphatidylserine, which is normally internalised on healthy tissue but externalised on tumours and the tumour vasculature. SWCNTs are conjugated to AV, which binds specifically to bladder cancer cells as confirmed in vitro and in vivo. Due to this specific localisation, NIR light can be used to heat the tumour while conserving the healthy bladder wall. In a short-term efficacy study in mice with orthotopic MB49 murine bladder tumours treated with the SWCNT-AV conjugate and NIR light, no tumours were visible on the bladder wall 24 h after NIR light treatment, and there was no damage to the bladder. In a separate survival study in mice with the same type of orthotopic tumours, there was a 50% cure rate at 116 days when the study was ended. At 116 days, no treatment toxicity was observed, and no nanotubes were detected in the clearance organs or bladder.

System level changes in gene expression in maturing bladder mucosa

PURPOSE Bladder problems clinically present early in life as birth defects that often lead to kidney failure and late in life as overactive bladder, incontinence and related disorders. We investigated the transcriptome of mouse bladder mucosa at juvenile and adult stages by microarray to identify the pathways associated with normal, healthy growth and maturation. We hypothesized that understanding these pathways could be key to achieving bladder regeneration or reawakening normal function in the elderly population. MATERIALS AND METHODS RNA was isolated from the mucosa at 3, 6, 20 and 30 weeks postnatally. Affymetrix® Mouse 430 v2 arrays were used to profile the expression of approximately 45,000 genes. The software program Statistical Analysis of Microarrays was used to identify genes that significantly changed during the time course. RESULTS No genes were significantly up-regulated during maturation. However, 66 well annotated genes demonstrated a statistically significant downward trend, of which 10 of 10 were confirmed by quantitative polymerase chain reaction. The main functions affected by age were transcription, regulation of cellular processes, neurogenesis, blood vessel development and cell differentiation. Notable genes included collagens, Mmp2, SPARC and several transcription factors, including Crebbp, Runx1, Klf9, Mef2c, Nrp1, Pex1 and Tcf4. These molecules were indirectly regulated by inferred Tgfb1 and Egf growth factors. Analysis of gene promoter regions for overrepresented upstream transcription factor binding sites identified specificity protein 1 and epidermal growth factor receptor-specific transcription factor as potentially major transcriptional regulators driving maturation related changes. CONCLUSIONS These findings identify a coherent set of genes that appear to be down-regulated during urothelial maturation. These genes may represent an attractive target for bladder regeneration or for treating age related loss of function.

MP11-15 DEVELOPMENT OF A TARGETED GADOLINIUM CONTRAST FOR NONINVASIVE OF MAGNETIC RESONANCE (MR) IMAGING OF NON-MUSCLE INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Development of methods to noninvasively detect bladder cancer and eliminate cystoscopy remains the golden ring of urology research. While muscle invasive cancer is relatively easily imaged with standard CT and MRI technology, the ability to image non-muscle invasive bladder cancer (NMIBC) in a noninvasive manner remains a challenge. We report our experience with a tumor-targeted gadolinium contrast agent for MR imaging NMIBC. METHODS: The peptide ligand (CSNRDARRC) has previously been reported as having strong specificity for urothelial cancer. The peptide linked to a FITC was tested against a panel of human and rodent malignant and benign urothelial cell lines and imaged fluorescently. The peptide ligand was then conjugated to Gadolinium-DOTA (Gd). The linked Gd-ligand was studied in vitro against urothelial cell lines and compared to a scrambled peptide linked to Gd and visualized with MRI. The linked Gd-ligandide ligand was delivered intravesically into a murine model of non-muscle invasive bladder cancer for 90 minutes and then washed clear with saline and then imaged with MR. This approach was repeated using a intravenous (IV)delivery and renal filtering into the bladder to image the surface cancer. Intravenous delivery was also used to image the same tumor growing in the subcutis of the mouse. RESULTS: The FITC-ligand reproducibly bound to the surface of the various cancer lines but no to the benign immortalized urothelial and nonurothelial cell lines increasing fluorescence in cells 2.5-fold. The ligand-linked Gd was bound to the cancer cell growing in vitro increasing “MR signal” by 300% compared to scramble peptide-linked Gd. Intravesically delivered ligand-linked Gd successfully bound to the surface tumor and allowed for easy imaging for up to 6 hours after infusion. Similar results were found when the ligand-linked Gd was delivered intravenously and imaged 12e24 hour later. CONCLUSIONS: Cancer targeted Gd can be delivered intravesically and intravenously to allow for enhanced MR imaging of superficial bladder cancer. This approach may be first step toward MRbased noninvasive surveillance of the bladder for recurrent non-muscle invasive cancer.

Abstract 4023: Identification of novel compounds to target dormant micrometastatic cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In spite of decades of research and billions of dollars, cancer-specific death rates have changed little, mainly because 90% of cancer patients who die succumb to metastatic disease. Our hypothesis is that the most vulnerable target for cancer therapy is the micrometastatic cell that leaves the primary tumor early in the disease history, lies dormant or suppressed by the normal extracellular matrix (ECM) and then emerges even decades later. We here describe a novel approach to identifying compounds that target micrometastatic cells suppressed by normal ECM. We first developed an in vitro 96-well format screen based on an earlier observation in our lab that the normal ECM suppresses the malignant phenotype. Using paired wells, one of which contains a normal ECM gel (small intestine submucosa, SISgel) and the other is just bare plastic, known cancer therapeutics show lower activity (more resistance) in cells grown on SISgel than on plastic. Our endpoint was the opposite pattern, namely more resistant on plastic. We screened 3,000 compounds in an NCI diversity set and identified 3 with the requisite activity. All had low toxicity in mice (<45 mg/kg). EC50 values were in the range of 3-30 µM, depending on the cell line. Screening of an additional 10K compounds identified 2 additional compounds, but they were too toxic to mice to be useful. We evaluated the activities of these compounds in vivo using a novel flank xenograft method we developed in which GFP-labeled cancer cells are co-injected with SISgel into the flank. The SISgel reprograms the cells and substantially normalizes them, even after the SISgel is resorbed such that a flat, glowing green spot marks the suppressed cells. Histopathology confirmed a substantial normalization. Cancer cells co-injected with Matrigel show rapid tumor growth. The lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model modified for physiological metastatic progression; surprisingly, 2 of the 3 lead compounds given intraperitoneally or in osmotic pumps decreased primary tumor growth more effectively than did docetaxel and resulted in small decreases in small lung micrometastases. However, as predicted, the main effect was to significantly (P<0.01, one-way ANOVA/Tukey post test) reduce the formation of lung macrometastases almost to zero. Given this result, we tested the activity of these compounds against ALDH+ CD44v3+ stem cells (SC) isolated from the parental line and found that one of the compounds specifically targeted the SC (EC50 = 50 µM vs 630 µM for doxorubicin). The SC also retained their stemness on SISgel. Mechanistic studies showed a weak, late apoptotic response but depending on the compound, blocking either at G1/S or G2/M in the cell cycle. In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their differentiation into cancer cells that form tumors. Citation Format: Robert E. Hurst, Michael A. Ihnat, Paul J. Hauser, Lora C. Bailey-Downs, Lilly Y. Bourgignon. Identification of novel compounds to target dormant micrometastatic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4023. doi:10.1158/1538-7445.AM2014-4023