Imaging and Downstaging Bladder Cancer with the 177Lu-Labeled Bioorthogonal Nanoprobe.

Efforts on bladder cancer treatment have been shifting from extensive surgery to organ preservation in the past decade. To this end, we herein develop a multifunctional nanoagent for bladder cancer downstaging and bladder-preserving therapy by integrating mucosa penetration, reduced off-target effects, and internal irradiation therapy into a nanodrug. Specifically, an iron oxide nanoparticle was used as a carrier that was coated with hyaluronic acid (HA) for facilitating mucosa penetration. Dibenzocyclooctyne (DBCO) was introduced into the HA coating layer to react through bioorthogonal reaction with azide as an artificial receptor of bladder cancer cells, to improve the cellular internalization of the nanoprobe labeled with 177Lu. Through magnetic resonance imaging, the targeted imaging of both nonmuscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) was realized after intravesical instillation of the multifunctional probe, both NMIBC and MIBC were found downstaged, and the metastasis was inhibited, which demonstrates the potential of the multifunctional nanoprobe for bladder preservation in bladder cancer treatment.

Elevating Second Near-Infrared Photothermal Conversion Efficiency of Hollow Gold Nanorod for a Precise Theranostic of Orthotopic Bladder Cancer.

The second near-infrared (NIR-II) window laser-activated agents have attracted broad interest in an orthotopic cancer theranostic. However, developing NIR-II photothermal agents (PTAs) with advanced photothermal conversion efficiency (PTCE) and tumor-specific response elevation remains a crucial challenge. Herein, a hollow gold nanorod (AuHNR) with a strong localized surface plasmon resonance (LSPR) peak in the NIR-II window was coated with MnO2 and chitosan to obtain AuHNR@MnO2@CS (termed AuMC) by a one-step method. Upon exposure to the tumor microenvironment (TME), the overexpressed GSH triggered degradation of the MnO2 layer to release Mn2+ and resulted in the PTCE elevation owing to exposure of the AuHNR. Consequently, photoacoustic and magnetic resonance imaging for accurate diagnosis, Mn2+-mediated chemodynamic therapy, and AuHNR elevating PT therapy for precise treatment could be achieved. Both in vitro and in vivo experiments confirmed the good performance of the AuMC on an orthotopic bladder cancer precise theranostic. This study provided NIR-II activated, TME-response PT conversion efficiency enhanced PTAs and offered a tumor-selective theranostic agent for orthotopic bladder cancer in clinical application.

Optimization and verification of orthotopic non-muscle invasive bladder cancer model in nude mice / 中国临床药理学与治疗学

AIM: To optimize an orthopedic non-muscle invasive bladder cancer (NMIBC) model in nude mouse by comparing four different ways of cellular transplantation, and to evaluate the efficacy of drug by bladder instillation, so as to provide a stable and efficient animal model for the treatment of bladder cancer. METHODS: After disruption of bladder mucosa by dilute acid-alkali or silver nitrate, T24 cells were instilled into the nude mouse bladder. T24 cells were injected directly into the bladder with mechanical injury of bladder mucosa. T24 cells were injected into the bladder wall. On the 14th day after making models, the nude mice were sacrificed. And the bladder mass and histopathological changes of tumor (including bladder) was observe to confirm the formation of orthopedic bladder cancer. The dynamic changes of orthopedic bladder cancer were observed after injecting T24 cells into the bladder wall. Gemcitabine was used to verify the applicability of the model of injecting T24 cells into the bladder wall in vivo. RESULTS: No tumor was found in the bladder after intravesical instillation of T24 cells with dilute acid-alkali or silver nitrate treatment. With mechanical injury of bladder mucosa, all nude mice had tumors after injection T24 cells. But the number of tumors varied and often occurred at multiple sites. The tumor was found in the bladder of all nude mice by injecting T24 cells into bladder wall, and there was only one tumor. The tumor showed slow linear growth within 15 days and rapid linear growth from day 18 to 31. In vivo efficacy evaluation, gemcitabine 150 mg/kg intravesical perfusion could significantly inhibit the growth of NMIBC in nude mice replicated by direct injection of T24 cells into the bladder wall, and the tumor inhibition rate was 97.1%. CONCLUSION: The orthotopic NMIBC model can not be established with the bladder mucosa injuried by dilute acid-alkali or silver nitrate treatment. The number and size of orthotopic bladder cancer are different by mechanical injury of bladder mucosa. Injection of T24 cells into the bladder wall of nude mouse can successfully establish the orthotopic NMIBC model, which can be used for the evaluation of NMIBC therapeutic drugs.

Liposome-Encapsulated Bacillus Calmette-Guérin Cell Wall Skeleton Enhances Antitumor Efficiency for Bladder Cancer In Vitro and In Vivo via Induction of AMP-Activated Protein Kinase.

The Mycobacterium Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS), the main immune active center of BCG, is a potent candidate non-infectious immunotherapeutic drug and an alternative to live BCG for use against urothelial carcinoma. However, its application in anticancer therapy is limited, as BCG-CWS tends to aggregate in both aqueous and non-aqueous solvents. To improve the internalization of BCG-CWS into bladder cancer cells without aggregation, BCG-CWS was nanoparticulated at a 180 nm size in methylene chloride and subsequently encapsulated with conventional liposomes (CWS-Nano-CL) using an emulsified lipid (LEEL) method. In vitro cell proliferation assays showed that CWS-Nano-CL was more effective at suppressing bladder cancer cell growth compared to nonenveloped BCG-CWS. In an orthotopic implantation model of luciferase-tagged MBT2 bladder cancer cells, encapsulated BCG-CWS nanoparticles could enhance the delivery of BCG-CWS into the bladder and suppress tumor growth. Treatment with CWS-Nano-CL induced the inhibition of the mammalian target of rapamycin (mTOR) pathway and the activation of AMP-activated protein kinase (AMPK) phosphorylation, leading to apoptosis, both in vitro and in vivo. Furthermore, the antitumor activity of CWS-Nano-CL was mediated predominantly by reactive oxygen species (ROS) generation and AMPK activation, which induced endoplasmic reticulum (ER) stress, followed by c-Jun N-terminal kinase (JNK) signaling-mediated apoptosis. Therefore, our data suggest that the intravesical instillation of liposome-encapsulated BCG-CWS nanoparticles can facilitate BCG-CW cellular endocytosis and provide a promising drug-delivery system as a therapeutic strategy for BCG-mediated bladder cancer treatment.

O2-generating MnO2 nanoparticles for enhanced photodynamic therapy of bladder cancer by ameliorating hypoxia.

Photodynamic therapy (PDT) is an emerging effective treatment for cancer. However, the great promise of PDT for bladder cancer therapy has not yet been realized because of tumor hypoxia. To address this challenge, we fabricated O2-generating HSA-MnO2-Ce6 NPs (HSA for human serum albumin, Ce6 for chlorin e6, and NPs for nanoparticles) to overcome tumor hypoxia and thus enhance the photodynamic effect for bladder cancer therapy. Methods: The HSA-MnO2-Ce6 NPs were prepared. We investigated the O2 generation of NPs in vitro and in vivo. The orthotopic bladder cancer model in C57BL/6 mice was established for in vivo study, and dual-modal imaging of NPs were demonstrated. Therapeutic efficacy of NPs for bladder cancer was evaluated. Results: HSA-MnO2-Ce6 NPs had an excellent performance in generating O2in vitro upon reaction with H2O2 at endogenous levels. Moreover, 1O2 generation was increased two-fold by using HSA-MnO2-Ce6 NPs instead of HSA-Ce6 NPs in the presence of H2O2 under 660 nm laser irradiation. In vitro cell viability assays showed that HSA-MnO2-Ce6 NPs themselves were non-toxic but greatly enhanced PDT effects on bladder cancer cells under laser irradiation. In vivo near-infrared (NIR) fluorescence and magnetic resonance (MR) imaging suggested the excellent bladder tumor-targeting property of HSA-MnO2-Ce6 NPs. O2 content in orthotopic bladder cancer was increased 3.5-fold after injection of HSA-MnO2-Ce6 NPs as compared with pre-injection. Given the excellent tumor-targeting ability and negligible toxicity, HSA-MnO2-Ce6 NPs were then used to treat orthotopic bladder cancer by PDT. The PDT with HSA-MnO2-Ce6 NPs showed remarkably improved therapeutic efficacy and significantly prolonged lifetime of mice as compared with controls. Conclusion: This study not only demonstrated the great potential of HSA-MnO2-Ce6 NPs for bladder cancer photodynamic ablation but also provided a new therapeutic strategy to overcoming tumor hypoxia.

The establishment of a growth-controllable orthotopic bladder cancer model through the down-regulation of c-myc expression.

To properly evaluate the biological effects of immunotherapy, it is critical to utilize a model of cancer in immune-competent mice. Currently, MBT-2 is the most common murine bladder cancer cell line used in orthotopic bladder cancer models, even though this cell type often has an inappropriate genetic mutation landscape. In these models, after tumors are detected with in vivo imaging, the mouse usually dies within two to three weeks due to post-renal azotemia caused by the rapidly growing mass. This event prohibits the evaluation of tumor behavior upon intravesical drug treatment. We explored whether an shRNA-induced decrease in the expression of the c-myc oncogene in MBT-2 cells could slow down their in vitro proliferation and in vivo tumor growth. We transduced MBT-2 cells with shRNA lentiviruses that bound c-myc, established MBT2.cMYCshRNA and confirmed the retardation of the growth of tumors implanted in C3H/He mice. Accordingly, this study suggests that this novel orthotopic bladder cancer model in immune-competent mice may be more appropriate for the analysis of the effects of the intravesical instillation of immunotherapeutic agents.

Self-assembled tumor-targeting hyaluronic acid nanoparticles for photothermal ablation in orthotopic bladder cancer.

Bladder cancer is one of the most frequent malignancies in the urinary system. Radical cystectomy is inevitable when bladder cancer progresses to a muscle-invasive disease. However, cystectomy still causes a high risk of death and a low quality of life (such as ureter-abdomen ostomy, uroclepsia for ileal-colon neobladder). Therefore, more effective treatments as well as bladder preservation are needed. We developed self-assembled tumor-targeting hyaluronic acid-IR-780 nanoparticles for photothermal ablation in over-expressing CD44 (the receptor for HA) bladder cancer, which show high tumor selectivity, high treatment efficacy, good bioavailability, and excellent biocompatibility. The nanoparticles demonstrated a stable spherical nanostructure in aqueous conditions with good mono-dispersity, and their average size was 171.3±9.14nm. The nanoparticles can be degraded by hyaluronidase when it is over-expressed in bladder cells; therefore, they appear to have a hyaluronidase-responsive "OFF/ON" behavior of a fluorescence signal. HA-IR-780 NPs also showed high photothermal efficiency; 2.5, 5, 10 and 20µg/mL of NPs had a maximum temperature increase of 11.2±0.66°C, 18.6±0.75°C, 26.8±1.11°C and 32.3±1.42°C. The in vitro cell viability showed that MB-49 cells could be efficiently ablated by combining HA-IR-780 NPs with 808nm laser irradiation. Then, in vivo biodistribution showed the HA-IR-780 NPs are targeted for accumulation in bladder cancer cells but have negligible accumulation in normal bladder wall. The photothermal therapeutic efficacy of HA-IR-780 NPs in the orthotopic bladder cancer model showed tumors treated with NPs had a maximum temperature of 48.1±1.81°C after 6min of laser irradiation. The tumor volume was approximately 65-75mm3 prior to treatment. After 12days, the tumor sizes for the PBS, PBS plus laser irradiation and HA-IR-780 NPs-treated groups were 784.75mm3, 707.5mm3, and 711.37mm3, respectively. None of the tumors in the HA-IR-780 NPs plus laser irradiation-treated group were visible to the naked eye. A toxicity study showed HA-IR-780 NPs (2.5-20mg/kg, i.v.) were nontoxic and safe for in vivo applications. HA-IR-780 nanoparticles address current clinical challenges, treating locally aggressive lesions and preserving the bladder. They have enormous potential to improve the bladder cancer treatment strategies in clinic. STATEMENT OF SIGNIFICANCE: 1) Bladder cancer is one of the most frequent malignancies in the urinary system. Radical cystectomy is inevitable while bladder cancer progress to muscle-invasive disease. 2) We developed self-assembled tumor-targeting hyaluronic acid-IR-780 nanoparticles for photothermal ablation in over-expressing CD44 (the receptor for HA) bladder cancer. 3) Photothermal therapeutic efficacy of HA-IR-780 NPs in orthotopic bladder cancer model showed tumors were completely ablated. 4) HA-IR-780 nanoparticles address current clinical challenges, treating locally aggressive lesions as well as for bladder preservation.

Establishment and CT Diagnosis of Orthotopic Rat Model of Bladder Cancer / 中国实验动物学报

Objective To establish an orthotopic rat model of bladder cancer induced by N-methyl.nitrosourea (MNU)and to evaluate the diagnostic value of CT(computed tomography)scanning in this experimental model.Methods 50 SD rats were randomly divided into the control group(group A)with 15 rats and experimental group(group B)with 35 rats.Each rat of group B was treated with 2 mg MNU per dose every other week,totally 4 doses,by per urethra administration.Meanwhile,each rat of group A was treated with normal saline.Then,at the 14th week,all the rats were evaluated by CT scanning and pathological examination.Results Abnormal changes were detected in each of the 28 rats in group B by CT scanning,and manifested as local mass,thickening bladder wall accompanied with heterogeneous density.Bladder cancer was diagnosed by pathology.However,no bladder tumor was detected by CT scanning and pathologicalexamination in group A.Conclusion A rat model of orthotopic bladder cancer can be established by per urethra administration of MNU.CT scanning is a reliable diagnostic technique concerning this model.Furthermore,this technique can render US a more suitable rat model for further experimental studies on chemotherapy and radiotherapy of bladder Cancer.