Detection of carcinogen-induced bladder cancer by fluorocoxib A

Bladder cancer is the 6th most common type of cancer with an estimated 80,000 newly diagnosed cases and 17,000 deaths per year in the United States [1]. Bladder cancer incidence is four times higher in men than in women. The most common type of bladder cancer is urothelial carcinoma, also known as transitional cell carcinoma, which accounts for over 90% of all bladder cancer cases in the United States. The extent of the bladder cancer spread through the body is determined by a staging based on physical exams, biopsies, surgery, and imaging tests. The staging system in TNM system is the most often used for the bladder cancer. In that staging system T indicates the spread of tumor through the bladder wall and nearby tissues, N indicates any cancer has spread to lymph nodes near the bladder, and M indicates any cancer has spread (metastasized) to distal sites and organs. There are five stages of bladder cancer, with stage IV being the most advanced metastatic disease stage. The cancers at Stage 0a (Ta, N0, M0) found on the surface of the inner lining of the bladder, Stage 0is (Tis, N0, M0) classified as a flat tumor or carcinoma-in-situ (CIS) and Stage I (T1, N0, M0) belong to a group of non-muscle invasive bladder carcinomas (NMIBC). The cancers at Stages II, (T2a or T2b, N0, M0), Stage IIIA (T3a, T3b or T4a, N0, M0; or T1-4a, N1, M0), Stage IIIb (T1-4a, N2 or N3, M0), Stage IVA (T4b, Any N, M0 or Any T, Any N, M1a) and Stage IVB (Any T, Any N, M1b) are more advanced stages, as the cancer has progressed through the muscle layer of the bladder wall to surrounding local pelvic and later to distal organs, such as bones, liver or lungs (M1b) belong to muscle-invasive bladder cancers (MIBC) [2].

Treatment management depends on whether the bladder cancer is diagnosed as NMIBC or MIBC. Currently, the gold-standard treatment for MIBC is neoadjuvant platinum-based chemotherapy followed by radical cystectomy [3, 4]. In an attempt to reduce the morbidity associated with open radical cystectomy, less invasive approaches, such as laparoscopic/robotic cystectomy have been explored [5, 6]. Standard treatments for NMIBC are a transurethral resection of bladder tumor (TURBT) or opened radical cystectomy depending on patient preferences and anatomy and location of cancer. Risk stratification based on accurate pathologic staging is then employed to determine the need for adjuvant intravesical treatment with chemotherapy (mitomycin C or gemcitabine) or immunotherapy (Bacille Calmette-Guérin) [7‐9]. The detection of bladder cancer at the early stages and more accurate detection of cancer during TURBT procedures is needed to improve patient treatment outcomes.

White light cystoscopy (WLC) is the current standard of care for the detection of papillary or larger cancerous lesions in the bladder. WLC has been used for several decades to detect bladder tumors, but there are several limitations associated with WLC, including difficulties in detecting early non-invasive stages of bladder cancer (Ta, T1, CIS), as well as the inability to detect tumor margins during resection procedures leading to the potential for incomplete resection of the tumor [10]. Newer technologies, including fluorescence cystoscopy/photodynamic diagnosis (PDD), narrow band imaging (NBI), confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT) [11, 12] have been developed to improve the quality of detection of the non-invasive disease from MIBC lesions during diagnostic and resection procedures [13, 14]. Fluorescence cystoscopy/PDD and NBI better visualize the tumors and optimize detection of early non-invasive stages of bladder cancer. On the contrary, CLE and OCT further characterize the detected lesions to improve accuracy in determining the grade and stage of the lesions. Fluorescent cystoscopy requires the administration of a contrast agent, which selectively binds to the cancer cells to improve visualization and differentiation of the cancer from normal tissue during resection procedures [15]. Photodynamic diagnosis/blue-light cystoscopy (BLC) is an FDA-approved procedure, which requires the intravesical administration of 5-aminolevulinic acid (5-ALA) or hexaminolevulinate (HAL) directly into the bladder [16‐19]. The dye is absorbed by the bladder tissue and after excitation by a light, it emits a red color allowing the better visualization of the tumor during the cystoscopy procedure. Previous studies indicates that BLC can detect bladder tumors more effectively than WLC, at both early and late stages [18‐22] and is now recommended as standard of care when available.

Cyclooxygenase-2 (Cox-2) is aberrantly expressed in bladder cancer and is one of the key proteins responsible for angiogenesis [23, 24] and tumorigenesis [25, 26]. The increased Cox-2 expression has also been reported to be correlated with tumor grade and poor clinical outcome for patients diagnosed with bladder cancer [27‐30]. The overexpression of Cox-2 in bladder cancer tissue can be used as a biomarker for the detection of bladder cancer and as a prognostic marker for outcome. Fluorescently labeled Cox-2 inhibitors used for targeted optical imaging could assist for the early detection of non-invasive disease before it metastasized. Fluorocoxib A is a rhodamine-conjugated analog of indomethacin that selectively targets Cox-2 in solid tumors [31]. Fluorocoxib A has been validated previously for the detection of LPS-induced inflammation in a rat model [31] and in Cox-2-expressing cancers in vitro [32] and in vivo [33, 34].

There are several models currently available for the study of bladder carcinogenesis, including genetically or carcinogens induced tumors in the rodents [35, 36]. In our study, we used a well-established BBN-induced mice bladder cancer model. BBN belongs to nitrosamines that is a highly carcinogenic group of compounds [37] known to induce hepatic, gastric, and bladder cancers [38, 39]. BBN is administered orally either in drinking water or by oral gavage at doses that range from 0.01–0.05% [40].

Current treatment options for patients diagnosed with bladder cancer is dependent on the stage and grade of cancer. After initial presentation and in office cystoscopy that demonstrates a concerning bladder lesion, patients are taken to the operating room at which time diagnostic and therapeutic TURBT is performed. Pathology results determines the presence of NMIBC versus MIBC. Therefore, an accurate understanding of the patient’s pathology and staging are critical in the provision of patients prognostic implications [4]. Unfortunately, WLC can fail to demonstrate occult bladder lesions, including CIS that are at high risk for the recurrence and/or progression. Thus, the new diagnostic tools are needed to improve detection during cystoscopy in order to distinguish non-invasive from muscle-invasive disease. This study demonstrates the improved visualization of Cox-2-expressing, carcinogen-induced early and late stage bladder cancer in an immunocompetent mouse model in vivo using the novel optical imaging agent, fluorocoxib A.

Interestingly, in our study we observed that mice exposed to 0.05% BBN in drinking water had increased water consumption as compared to mice from control groups as shown in Fig. 1c. Nitrosamines after ingestion are metabolized by the liver into several metabolites and excreted from the body through urine. In the bladder, the metabolites of nitrosamines come in contact with the urothelium and initiate the carcinogenic process resulting in DNA damage and development of both the early (NIMBC) and late stages (MIBC) of bladder cancer in mice [41, 42]. Bladder cancer in mice develops relatively early after BBN exposure with early-stage tumors developing after 12 weeks of exposure and late-stage tumors presenting after 18 weeks of exposure.

Fluorescence cystoscopy allows the visualization of the accumulated fluorescent contrast agents in the cancerous cells. Previous studies indicate improved diagnostic outcomes for patients with NMIBC using a fluorescent cystoscopy than WLC [43, 44]. There are several limitations to this method including rapid photo-bleaching during the cystoscopy procedure and a high false-positive rate (up to 30%) [45, 46]. In addition, the currently used non-selective photosensitizing agents (e.g. 5-ALA) during fluorescent cystoscopy procedures detect inflammation [45, 47], which contributes to the increased false positive detection rates. These limitations drive the need for the synthesis and validation of novel, cancer-specific contrast imaging agents that will improve the detection of the neoplastic lesions in the bladder resulting in improved patient prognosis. Fluorocoxib A, which is a Cox-2-selective imaging agent, is a prime candidate for the detection of bladder cancer during cystoscopy procedures due to its high stability and non-toxic effects. Previous preclinical studies indicate that fluorocoxib A has demonstrated to be highly specific and selective for detection of Cox-2-expressing head and neck, bladder, non-melanoma skin, and colorectal cancers [31‐34].

In this study, the significant 3- and 7-fold TNR increases in the uptake of fluorocoxib A were observed in bladders dissected from mice in Group 2 – 12wks BBN and Group 3 - 18wks BBN, respectively, when compared to bladder of mice from Group 1 – 18wks H₂O (control) that also correlated with the progression of bladder cancer (Fig. 2). Moderate uptake of fluorocoxib A was observed in both the liver and muscle tissue as shown in Fig. 2c and d due to the metabolism and excretion route of fluorocoxib A [31]. These results demonstrate the specificity of fluorocoxib A uptake by the cancerous bladder tissue when compared to normal urothelium (Group 1 – 18wks H₂O) where no fluorocoxib A uptake was detected. Furthermore, the range of fluorocoxib A signal between the groups indicates the ability of fluorocoxib A to detect different stages of bladder tumors, including hyperplasia and CIS vs carcinoma lesions that was confirmed by histology analysis (Fig. 3). All bladder tissue from mice in Group 3 (18wks BBN) displayed signs of inflammation and/or CIS/Hyperplasia in developed bladder carcinomas as shown in a Table 2 and Fig. 5. These results confirm the progression of bladder carcinogenesis induced by BBN exposure and incidence rates are consistent with previously published BBN studies [37, 38, 42]. Ki67 is a cellular marker associated with cell proliferation and often correlates with patient prognosis and aids in determining clinical course of treatment. The progression of bladder carcinogenesis was confirmed by the presence of increased expression of Ki67 positive cells in bladder urothelium from mice treated with BBN (Group 2 – 12wks BBN and Group 3 – 18wks BBN) (Fig. 3). Uroplakin-1A (UP1a) is a member of a group of cell-surface proteins and is highly expressed in normal bladder urothelium. The downregulation of UP1a indicated a loss of normal urothelium and changes in morphology of the bladder tissues as the cancer progressed as was also confirmed in our study (Fig. 3).

Cox-2 plays a key role in modulating cellular proliferation, apoptosis, and tumor invasion. The increased Cox-2 expression has also been reported to be correlated with tumor grade and poor clinical outcome for patients diagnosed with bladder cancer [27‐30]. In our study we confirmed that developed bladder carcinomas in mice had significantly higher Cox-2 expression when compared to normal urothelium, bladder inflammation, and hyperplasia in bladders from BBN-treated mice (Fig. 4). There was a significant 3- and 9-fold increase in Cox-2 expression in bladder tissues in mice for Group 2 – 12wks BBN and Group 3 – 18wks BBN, respectively, when compared to Group 1 – 18wks H₂O (Fig. 4c). These results also correlate with the increased uptake of fluorocoxib A and Cox-2 expression in the bladders of mice that were positive for CIS/Hyperplasia with inflammation and carcinomas with CIS/Hyp and inflammation (Fig. 5). The detected COX-2 in bladder cancer tissue by fluorocoxib A could be used as a biomarker not only for the detection of bladder cancer, but also as a prognostic factor for poor clinical outcome of patients diagnosed with COX-2-expressing bladder cancers.

The specific and increased fluorocoxib A uptake in the BBN-induced bladder carcinomas in mice in vivo correlated with the progression of bladder carcinogenesis and with increased Cox-2 expression. Currently, conventional imaging technologies for the detection of bladder cancer have several limitations, primarily lacking the ability to detect early stage bladder cancer and poor visualization of tumor margins during resection procedures. The development of novel techniques and imaging agents, such as fluorocoxib A, could potentially greatly improve patient prognosis by aiding in both the diagnostic and treatment procedures.