Takahiro Yoshida

Preclinical and Clinical Evaluation of Intraductally Administered Agents in Early Breast Cancer

Intraductal administration of chemotherapeutic agents may reduce new breast cancer formation and offer a less toxic treatment regimen than intravenous therapy. Repairing the Ductwork Breast cancer is typically treated intravenously with chemotherapeutic drugs, poisons that permeate the entire body and cause toxic side effects, such as hair loss, pain, and nausea. Because most breast tumors originate in the cellular lining of the breast ducts, Stearns and colleagues designed a gentler, more local treatment regimen that gets right to the source: intraductal drug injection. The authors first tested intraductal treatment with five different chemotherapeutic agents, including paclitaxel and doxorubicin, on rats with mammary tumors, at doses comparable to what might be used in the clinic in actual patients. Compared to saline-treated or untreated control animals, the rats treated intraductally had fewer tumors in the mammary glands, with minimal side effects. Stearns et al. then enrolled 17 women in a phase 1 clinical trial to examine intraductal treatment using one chemical agent, pegylated liposomal doxorubicin. Their localized delivery to the breast ducts resulted in considerably lower systemic concentrations of the drug compared to intravenous administration, suggesting that the intraductal approach is a less toxic alternative to standard chemotherapy. This clinical trial also indicates that approved agents can be delivered to the breast ducts in an outpatient setting. Longer-term studies in more women will be necessary to determine the efficacy of intraductal chemotherapy. Intraductal treatment could be especially useful for women with premalignant lesions or those at high risk of developing breast cancer, thus drastically improving upon their other, less attractive options of breast-removal surgery or surveillance (termed “watch and wait”). It’s not yet routine practice, but direct treatment of the breast ductwork with cancer-fighting drugs promises to be a safer, less painful method for controlling cancer. Most breast cancers originate in the epithelial cells lining the breast ducts. Intraductal administration of cancer therapeutics would lead to high drug exposure to ductal cells and eliminate preinvasive neoplasms while limiting systemic exposure. We performed preclinical studies in N-methyl-N′-nitrosourea–treated rats to compare the effects of 5-fluorouracil, carboplatin, nanoparticle albumin-bound paclitaxel, and methotrexate to the previously reported efficacy of pegylated liposomal doxorubicin (PLD) on treatment of early and established mammary tumors. Protection from tumor growth was observed with all five agents, with extensive epithelial destruction present only in PLD-treated rats. Concurrently, we initiated a clinical trial to establish the feasibility, safety, and maximum tolerated dose of intraductal PLD. In each eligible woman awaiting mastectomy, we visualized one ductal system and administered dextrose or PLD using a dose-escalation schema (2 to 10 mg). Intraductal administration was successful in 15 of 17 women with no serious adverse events. Our preclinical studies suggest that several agents are candidates for intraductal therapy. Our clinical trial supports the feasibility of intraductal administration of agents in the outpatient setting. If successful, administration of agents directly into the ductal system may allow for “breast-sparing mastectomy” in select women.

Intraductal administration of a polymeric nanoparticle formulation of curcumin (NanoCurc) significantly attenuates incidence of mammary tumors in a rodent chemical carcinogenesis model: Implications for breast cancer chemoprevention in at-risk populations

Multiple lines of evidence support a role for curcumin in cancer chemoprevention. Nonetheless, despite its reported efficacy and safety profile, clinical translation of curcumin has been hampered by low oral bioavailability, requiring infeasible mega doses for achieving detectable tissue levels. We have engineered a polymeric nanoparticle encapsulated formulation of curcumin (NanoCurc) to harness its full therapeutic potential. In the current study, we assessed the chemoprevention efficacy of NanoCurc administered via direct intraductal (i.duc) injection in a chemical carcinogen-induced rodent mammary cancer model. Specifically, Sprague-Dawley rats exposed to systemic N-methyl-N-nitrosourea were randomized to receive either oral free curcumin at a previously reported mega dose (200mg/kg) or by direct i.duc injection of free curcumin or NanoCurc, respectively, each delivering 168 µg equivalent of curcumin per rodent teat (a ~20-fold lower dose per animal compared to oral administration). All three chemoprevention modalities resulted in significantly lower mammary tumor incidence compared with control rats; however, there was no significant difference in cancer incidence between the oral dosing and either i.duc arms. On the other hand, mean tumor size, was significantly smaller in the i.duc NanoCurc cohort compared with i.duc free curcumin (P < 0.0001), suggesting the possibility of better resectability for breakthrough cancers. Reduction in cancer incidence was associated with significant decrease in nuclear factor -κB activation in the NanoCurc treated mammary epithelium explants, compared to either control or oral curcumin-administered rats. Our studies confirm the potential for i.duc NanoCurc as an alternative to the oral route for breast cancer chemoprevention in high-risk cohorts.

Oncological Outcomes of Sequential Intravesical Gemcitabine and Docetaxel in Patients with Non-Muscle Invasive Bladder Cancer

Background: Bacillus Calmette-Guérin (BCG) unresponsive/relapsing patients with non-muscle invasive bladder cancer (NMIBC) who prefer bladder preservation over radical cystectomy (RC) or those who do not qualify for surgery may be offered intravesical therapies. Gemcitabine (GEM) combined with Docetaxel (DOCE) has been offered at Johns Hopkins Hospital (JHH). Objective: To evaluate experience with GEM/DOCE, to confirm safety of the regimen, to identify populations that may benefit most, and to consider the appropriate endpoints for judging efficacy of second line therapies. Methods: Thirty-three patients who received full induction GEM/DOCE since 2011, per the protocol adapted from U. Iowa, were identified and characterized. Multivariable logistic regression was used to determine factors associated with recurrence. Cox proportional hazard models evaluated risk factors for disease-free survival (DFS) and high-grade recurrence-free survival (HG-RFS). Results: There were no serious adverse effects of therapy. Across all patients, median follow-up time was 18.6 months with a median DFS of 6.5 months, 42% 1-year, and 24% 2-year DFS. Median HG-RFS was 17.1 months with 56% 1-year and 42% 2-year HG-RFS. Among patients initially presenting with HG-NMIBC, 46% (13/28) had HG recurrence. BCG unresponsive/relapsing patients (Nu200a=u200a25) displayed 49% 1-year HG-RFS and 34% 2-year HG-RFS. In total, there were 5 LG and 16 HG recurrences, with 5 progressions and 8 cystectomies among these. Conclusions: GEM/DOCE is a well-tolerated therapy that deserves further study as an alternative to immediate RC for highly selected patients with HG-NMIBC. BCG naïve patients responded more effectively than BCG unresponsive/relapsing patients. As anticipated, GEM/DOCE efficacy was improved for HG only patients.

Three-dimensional organoid culture reveals involvement of Wnt/β-catenin pathway in proliferation of bladder cancer cells

There has been increasing awareness of the importance of three-dimensional culture of cancer cells. Tumor cells growing as multicellular spheroids in three-dimensional culture, alternatively called organoids, are widely believed to more closely mimic solid tumors in situ. Previous studies concluded that the Wnt/β-catenin pathway is required for regeneration of the normal urothelium after injury and that β-catenin is upregulated in human bladder cancers, but no clear evidence has been advanced to support the idea that the Wnt/β-catenin pathway is directly involved in deregulated proliferation and the other malignant characteristics of bladder cancer cells. Here we report that the Wnt/β-catenin pathway activator, CHIR99021, promoted proliferation of established human bladder cancer cell lines when they were grown in organoid culture but not when they were grown in conventional adherent cultures. CHIR99021 activated Wnt/β-catenin pathway in bladder cancer cell lines in organoid culture. CHIR99021 also stimulated proliferation and the Wnt/b-catenin pathway in primary human bladder cancer organoids. RNAi-mediated knockdown of β-catenin blocked growth of organoids. The effects of CHIR99021 were associated with decreased expression of the urothelial terminal differentiation marker, cytokeratin 20. Our data suggest that the Wnt/β-catenin pathway is required for the proliferation of bladder cancer cells in three-dimensional organoid culture and provide a concrete example of why organoid culture is important for cancer research.

Preclinical Evaluation of Intravesical Cisplatin Nanoparticles for Non-Muscle-Invasive Bladder Cancer

Purpose: Prior clinical trials evaluating cisplatin for non–muscle-invasive bladder cancer (NMIBC) were stopped due to local and systemic toxicity. Currently, there is still a need for improved intravesical therapies, and nanoparticle-based CDDP may be efficacious without the toxicity of free cisplatin observed in the past. Experimental Design: Cisplatin nanoparticles (CDDP NPs) were developed using biocompatible poly(l-aspartic acid sodium salt; PAA), both with and without low and high grafting density of methoxy-polyethylene glycol (PEG). In vitro cytotoxicity studies confirmed activity of CDDP NPs and CDDP solution against a papillary bladder cancer cell line. Local toxicity was assessed by three weekly intravesical administrations of CDDP formulations. CDDP NPs and CDDP solution were evaluated for bladder absorption in murine models 1 and 4 hours after intravesical administration. In vivo efficacy was evaluated in an immunocompetent carcinogen model of NMIBC. Results: CDDP NPs showed decreased local toxicity, as assessed by bladder weight, compared with CDDP solution. Furthermore, >2 μg/mL of platinum was observed in mouse serum after intravesical administration of CDDP solution, whereas serum platinum was below the limit of quantification after intravesical administration of CDDP NPs. CDDP NPs provided significantly increased (P < 0.05) drug levels in murine bladders compared with CDDP solution for at least 4 hours after intravesical administration. In vivo, CDDP NPs reduced cancer cell proliferation compared with untreated controls, and was the only treatment group without evidence of invasive carcinoma. Conclusions: Cisplatin-loaded PAA NPs have the potential to improve intravesical treatment of NMIBC while reducing local and systemic side effects. Clin Cancer Res; 23(21); 6592–601. ©2017 AACR.

Ex vivo culture of tumor cells from N-methyl-N-nitrosourea-induced bladder cancer in rats: Development of organoids and an immortalized cell line

OBJECTIVEnWe ex vivo cultured primary tumor cells from N-methyl-N-nitrosourea (MNU)-induced bladder tumors in rats and established an immortalized cell line from them.nnnMATERIALS AND METHODSnBladder tumors in rats were induced by instillation of MNU into the murine bladder. Primary tumor cells were prepared by the cancer-tissue originated spheroid method. An immortalized cell line was established by co-culture with fibroblasts. The cultured tumor cells were molecularly and functionally characterized by quantitative real-time polymerase chain reaction, Western blot, growth assay, and transwell migration assay.nnnRESULTSnPrimary tumor cells were successfully prepared as multicellular spheroids from MNU-induced bladder tumors. The differentiation marker expression patterns observed in the original tumors were largely retained in the spheroids. We succeeded in establishing a cell line from the spheroids and named it T-MNU-1. Although basal markers (CK14 and CK5) were enriched in T-MNU-1 compared to the spheroids, T-MNU-1 expressed both luminal and basal markers. T-MNU-1 was able to migrate through a transwell.nnnCONCLUSIONSnTumor cells in MNU-induced bladder tumors were successfully cultured ex vivo as organoids, and an immortalized cell line was also established from them. The ex vivo models offer a platform that enables analysis of intrinsic characteristics of tumor cells excluding influence of microenvironment in MNU-induced bladder tumors.

M1 Macrophages Are Predominantly Recruited to the Major Pelvic Ganglion of the Rat Following Cavernous Nerve Injury

INTRODUCTIONnNeurogenic erectile dysfunction is a common sequela of radical prostatectomy. The etiology involves injury to the autonomic cavernous nerves, which arise from the major pelvic ganglion (MPG), and subsequent neuroinflammation, which leads to recruitment of macrophages to the injury site. Currently, two macrophage phenotypes are known: neurotoxic M1 macrophages and neuroprotective M2 macrophages.nnnAIMnTo examine whether bilateral cavernous nerve injury (BCNI) in a rat model of erectile dysfunction would increase recruitment of neurotoxic M1 macrophages to the MPG.nnnMETHODSnMale Sprague-Dawley rats underwent BCNI and the MPG was harvested at various time points after injury. The corpora cavernosa was used to evaluate tissue myographic responses to electrical field stimulation exxa0vivo. Quantitative real-time polymerase chain reaction was used to examine the gene expression of global macrophage markers, M1 macrophage markers, M2 macrophage markers, and cytokines and chemokines in the MPG. Mathematical calculation of the M1/M2 index was used to quantify macrophage changes temporally. Western blot of MPG tissues was used to evaluate the protein amount of M1 and M2 macrophage markers quantitatively. Immunohistochemistry staining of MPGs for CD68, CD86, and CD206 was used to characterize M1 and M2 macrophage infiltration.nnnMAIN OUTCOME MEASURESnCorpora cavernosa responsiveness exxa0vivo; gene (quantitative real-time polymerase chain reaction) and protein (western blot) expressions of M1 and M2 markers, cytokines, and chemokines; and immunohistochemical localization of M1 and M2 macrophages.nnnRESULTSnBCNI impaired the corporal parasympathetic-mediated relaxation response to electrical field stimulation and enhanced the contraction response to electrical field stimulation. Gene expression of proinflammatory (Il1b, Il16, Tnfa, Tgfb, Ccl2, Ccr2) and anti-inflammatory (Il10) cytokines was upregulated in the MPG 48 hours after injury. M1 markers (CD86, inducible nitric oxide synthase, interleukin-1β) and M2 markers (CD206, arginase-1, interleukin-10) were increased after BCNI in the MPG, with the M1/M2 index above 1.0 indicating that more M1 than M2 macrophages were recruited to the MPG. Protein expression of the M1 macrophage marker (inducible nitric oxide synthase) was increased in MPGs after BCNI. However, the protein amount of M2 macrophage markers (arginase-1) remained unchanged. Immunohistochemical characterization demonstrated predominant increases in M1 (CD68+CD86+) macrophages in the MPG after BCNI.nnnCONCLUSIONnThese results suggest that an increase in M1 macrophage infiltration of the MPG after BCNI is associated with impaired neurogenically mediated erectile tissue physiology exxa0vivo and thus has significant implications for cavernous nerve axonal repair. Future studies are needed to demonstrate that inhibition of M1 macrophage recruitment prevents erectile dysfunction after CNI.

MP54-12 MOLECULAR CHARACTERIZATION OF N-METHYL-N-NITROSOUREA-INDUCED BLADDER UROTHELIAL TUMOR IN RATS

Silvia Selinski, Meinolf Blaszkewicz, Katja Ickstadt, Dortmund, Germany; Holger Gerullis, Oldenburg, Germany; Thomas Otto, Neuss, Germany; Emanuel Roth, Frank Volkert, Lutherstadt Wittenberg, Germany; Daniel Ovsiannikov, Koblenz, Germany; Thomas Reckwitz*, Oliver Moormann, Dortmund, Germany; Gergely Banfi, Peter Nyirady, Budapest, Hungary; Sita Vermeulen, Nijmegen, Netherlands; Montserrat Garcia-Closas, Bethesda, MD; Jonine Figueroa, Edinburgh, United Kingdom; Alison Johnson, Burlington, VT; Margaret Karagas, Hanover, NH; Manolis Kogevinas, Barcelona, Spain; Nuria Malats, Madrid, Spain; Molly Schwenn, Augusta, ME; Debra Silverman, Stella Koutros, Nathaniel Rothman, Bethesda, MD; Lambertus Kiemeney, Nijmegen, Netherlands; Jan Hengstler, Klaus Golka, Dortmund, Germany

MP54-06 THREE-DIMENSIONAL ORGANOID CULTURE REVEALS INVOLVEMENT OF WNT/β-CATENIN PATHWAY IN PROLIFERATION OF BLADDER CANCER CELLS

(IHC) on tissue microarrays were used to confirm tissue sampling and the gene expression analysis. RESULTS: Unsupervised consensus clustering yielded four distinct consensus clusters (CC) or subtypes: CC1-Basal, CC2Luminal, CC3-Immune and CC4-Scar-like. The CC1-Basal and CC2Luminal subtypes expressed genes consistent with a basal-like (KRT5/ 6, KRT14) and a luminal-like (GATA3, PPARG) phenotype, respectively. The CC3-Immune subtype had the highest immune activity, including T-cell infiltration and checkpoint molecules (CTLA4, CD80). Finally, the CC4-Scar-like profile was consistent with non-neoplastic scar samples, expressing genes associated with wound healing / scarring (MYH11, SHH, CNN1). Approximately the same numbers of pre-NAC basaland luminal-like samples remained static (CC1 or CC2) with respect to subtype or were highly immune-infiltrated (CC3) in the post-NAC setting. In the post-NAC setting, luminal-like pre-NAC samples were more likely adopt a scar-like character (CC4). CONCLUSIONS: This study expands our knowledge of cisplatin-resistant MIBC by suggesting molecular subtypes to understand the biology of these tumors. Although these molecular subtypes imply consequences for adjuvant treatments, this ultimately needs to be tested in clinical trials.

Organoid culture of bladder cancer cells

An organoid is a miniature three-dimensional (3D), multicellular, “stem-cell derived genetically-encoded self-assembly programmed structure” which exhibits characteristics, e.g., cell-cell interactions, tissue polarity, hypoxia, drug penetration, and nutrition gradients, that recapitulate the in vivo state of the original tissue better than cell lines that are maintained in two-dimensional (2D) adherent cultures. Because precursor stem cells needed to form organoids can be edited to express desired genetic variants or gene mutations or can be derived from complex genetic backgrounds, organoids become powerful tools to model diseases-from Zika virus infection and single gene disorders like cystic fibrosis to complex conditions like cancer. Patient-derived organoids (PDOs) have recently emerged as robust preclinical models for their abilities to recapitulate patient drug responses in the clinic, and they might therefore be integrated into precision medicine programs. PDO cultures have been established for many types of cancer, including colorectal, prostate, breast, pancreas, lung, and liver cancers. As for urothelial cancer, our group succeeded in preparing PDOs from surgically resected human bladder cancer tissue by applying the cancer-tissue originated spheroid (CTOS) method [1]. In this editorial, we introduce our recent findings and those of others using bladder cancer organoids and discuss future applications of them for discovery and precision medicine.