Cancer Models Research Articles

Thiazole, isatin and phthalimide derivatives tested in vivo against cancer models: a literature review of the last six years.

Cancer is a disease characterized by the abnormal multiplication of cells and is the second leading cause of death in the world. The search for new effective and safe anticancer compounds is ongoing due to factors such as low selectivity, high toxicity, and multidrug resistance. Thus, heterocyclic compounds derived from isatin, thiazole and phthalimide that have achieved promising in vitro anticancer activity have been tested in vivo and in clinical trials. This review focused on the compilation of promising data from thiazole, isatin, and phthalimide derivatives, reported in the literature between 2015 and 2022, with in vivo anticancer activity and clinical trials. A bibliographic search was carried out in the PUBMED, MEDLINE, ELSEVIER, and CAPES PERIODIC databases, selecting relevant works for each pharmacophoric group with in vivo antitumor activity in the last 6 years. In our study, 68 articles that fit the scope were selected and critically analyzed. These articles were organized considering the type of antitumor activity and their year of publication. Some compounds reported here demonstrated potent antitumor activity against several tumor types. This review allowed us to highlight works that reported promising structures for the treatment of various cancer types and also demonstrated that the privileged structures thiazole, isatin and phthalimide are important in the design of new syntheses and molecular optimization of compounds with antitumor activity.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

Isolation and Characterization of a Novel Mammary Adenocarcinoma, MCa-P1362, with Hormone Receptor Expression, Human Epidermal Growth Factor Receptor 2 Positivity, and Enrichment in Cancer and Mesenchymal Stem Cells

Preclinical models that display spontaneous metastasis are necessary to improve the therapeutic options for hormone receptor-positive breast cancers. Within this study, detailed cellular and molecular characterization was conducted on MCa-P1362, a newly established mouse model of metastatic breast cancer that is syngeneic in BALB/c mice. MCa-P1362 cancer cells express estrogen receptor, progesterone receptor, and the human epidermal growth factor receptor 2. MCa-P1362 cancer cells proliferate invitro and invivo in response to estrogen, yet do not depend on steroid hormones for growth and tumor progression. Analysis of MCa-P1362 tumor explants revealed the tumors contained a mixture of cancer cells and mesenchymal stromal cells. Through transcriptomic and functional analyses of both cancer and stromal cells, stem cells were detected within both populations. Functional studies demonstrated that MCa-P1362 cancer stem cells drove tumor initiation, whereas stromal cells from these tumors contributed to drug resistance. MCa-P1362 may serve as a useful preclinical model to investigate the cellular and molecular basis of breast tumor progression and therapeutic resistance.

Naringenin-Loaded Bovine Serum Albumin Nanoparticles Inhibit Reactive Oxygen Species (ROS) to Prevent Ulcer and Gastric Cancer

Gastric cancer is a malignance of digestive system and effective treatment measures are key to treatment of gastric cancer. In this experiment, we assessed the effect of bovine serum albumin nanoparticle (BASNP)-coated naringenin (NGN) on reactive oxygen species (ROS) in gastric cancer. After preparation of NGN-BASNP and animal model of gastric cancer, rats were administered with NGN-BASNP, ROS agonists and ROS inhibitors, when the model group was set. After one week of intervention, gastric ulcers in rats were measured and Hematoxylin-eosin (HE) staining was performed. Transwell chamber was used to detect cell invasion ability, while 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was detected proliferation ability of gastric cancer cells. Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot determined TNF-α mRNA expression and ROS. With about 100 nm diameter of NGN-BASNP nanoparticles, the nanoparticles presented in good shape. Compared with lesions in the model group, NGN-BASNP treatment greatly improved the condition, relieving the ulcer and decreasing the ulcer area (P &lt; 0.05). After adding ROS agonist, inhibitory effect of NGN-BASNP intervention was amplified (P &lt; 0.01) when the ROS level in the NGN-BASNP+ROS agonist group was decreased and TNF-α expression decreased. Moreover, NGN-BASNP effectively suppressed gastric cancer cell proliferation and migration but induced apoptosis. NGN-BASNP inhibited the expression of TNF-α mRNA and ROS level in gastric cancer cells, thereby alleviating gastric ulcer and delaying gastric cancer cell growth.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

A DNA tetrahedron-based nanosuit for efficient delivery of amifostine and multi-organ radioprotection

Unnecessary exposure to ionizing radiation (IR) often causes acute and chronic oxidative damages to normal cells and organs, leading to serious physiological and even life-threatening consequences. Amifostine (AMF) is a validated radioprotectant extensively applied in radiation and chemotherapy medicine, but the short half-life limits its bioavailability and clinical applications, remaining as a great challenge to be addressed. DNA-assembled nanostructures especially the tetrahedral framework nucleic acids (tFNAs) are promising nanocarriers with preeminent biosafety, low biotoxicity, and high transport efficiency. The tFNAs also have a relative long-term maintenance for structural stability and excellent endocytosis capacity. We therefore synthesized a tFNA-based delivery system of AMF for multi-organ radioprotection (tFNAs@AMF, also termed nanosuit). By establishing the mice models of accidental total body irradiation (TBI) and radiotherapy model of Lewis lung cancer, we demonstrated that the nanosuit could shield normal cells from IR-induced DNA damage by regulating the molecular biomarkers of anti-apoptosis and anti-oxidative stress. In the accidental total body irradiation (TBI) mice model, the nanosuit pretreated mice exhibited satisfactory alteration of superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents, and functional recovery of hematopoietic system, reducing IR-induced pathological damages of multi-organ and safeguarding mice from lethal radiation. More importantly, the nanosuit showed a selective radioprotection of the normal organs without interferences of tumor control in the radiotherapy model of Lewis lung cancer. Based on a conveniently available DNA tetrahedron-based nanocarrier, this work presents a high-efficiency delivery system of AMF with the prolonged half-life and enhanced radioprotection for multi-organs. Such nanosuit pioneers a promising strategy with great clinical translation potential for radioactivity protection.

Chelator boosted tumor-retention and pharmacokinetic properties: development of 64Cu labeled radiopharmaceuticals targeting neurotensin receptor.

Accumulating evidence suggests that neurotensin (NTS) and neurotensin receptors (NTSRs) play key roles in lung cancer progression by triggering multiple oncogenic signaling pathways. This study aims to develop Cu-labeled neurotensin receptor 1 (NTSR1)-targeting agents with the potential for both imaging and therapeutic applications. A series of neurotensin receptor antagonists (NRAs) with variable propylamine (PA) linker length and different chelators were synthesized, including [64Cu]Cu-CB-TE2A-iPA-NRA ([64Cu]Cu-4a-c, i = 1, 2, 3), [64Cu]Cu-NOTA-2PA-NRA ([64Cu]Cu-4d), [64Cu]Cu-DOTA-2PA-NRA ([64Cu]Cu-4e, also known as [64Cu]Cu-3BP-227), and [64Cu]Cu-DOTA-VS-2PA-NRA ([64Cu]Cu-4f). The series of small animal PET/CT were conducted in H1299 lung cancer model. The expression profile of NTSR1 was also confirmed by IHC using patient tissue samples. For most of the compounds studied, PET/CT showed prominent tumor uptake and high tumor-to-background contrast, but the tumor retention was strongly influenced by the chelators used. For previously reported 4e, [64Cu]Cu-labeled derivative showed initial high tumor uptake accompanied by rapid tumor washout at 24h. The newly developed [64Cu]Cu-4d and [64Cu]Cu-4f demonstrated good tumor uptake and tumor-to-background contrast at early time points, but were less promising in tumor retention. In contrast, our lead compound [64Cu]Cu-4b demonstrated 9.57 ± 1.35, 9.44 ± 2.38 and 9.72 ± 4.89%ID/g tumor uptake at 4, 24, and 48h p.i., respectively. Moderate liver uptake (11.97 ± 3.85, 9.80 ± 3.63, and 7.72 ± 4.68%ID/g at 4, 24, and 48h p.i.) was observed with low uptake in most other organs. The PA linker was found to have a significant effect on drug distribution. Compared to [64Cu]Cu-4b, [64Cu]Cu-4a had a lower background, including a greatly reduced liver uptake, while the tumor uptake was only moderately reduced. Meanwhile, [64Cu]Cu-4c showed increased uptake in both the tumor and the liver. The clinical relevance of NTSR1 was also demonstrated by the elevated tumor expression in patient tissue samples. Through the side-by-side comparison, [64Cu]Cu-4b was identified as the lead agent for further evaluation based on its high and sustained tumor uptake and moderate liver uptake. It can not only be used to efficiently detect NTSR1 expression in lung cancer (for diagnosis, patient screening, and treatment monitoring), but also has the great potential to treat NTSR-positive lesions once chelating to the beta emitter 67Cu.

Preclinical models of bladder cancer: BBN and beyond.

Preclinical modelling is a crucial component of advancing the understanding of cancer biology and therapeutic development. Several models exist for understanding the pathobiology of bladder cancer and evaluating therapeutics. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced bladder cancer is a commonly used model that recapitulates many of the features of human disease. Particularly in mice, BBN is a preferred laboratory model owing to a high level of reproducibility, high genetic fidelity to the human condition, and its relative ease of use. However, important aspects of the model are often overlooked in laboratory studies. Moreover, the advent of new models has yielded a variety of methodologies that complement the use of BBN. Toxicokinetics, histopathology, molecular genetics and sex can differ between available models and are important factors to consider in bladder cancer modelling.

Personalized Nanovaccines Enhance Lymph Node Accumulation and Reprogram the Tumor Microenvironment for Improved Photodynamic Immunotherapy.

Tumor immunotherapy has emerged as an efficacious therapeutic approach that mobilizes the patient's immune system to achieve durable tumor suppression. Here, we design a photodynamic therapy-motivated nanovaccine (Dex-HDL/ALA-Fe3O4) co-delivering 5-aminolevulinic acid and Fe3O4 nanozyme that demonstrate a long-term durable immunotherapy strategy. After vaccination, the nanovaccine exhibits obvious tumor site accumulation, lymph node homing, and specific and memory antitumor immunity evocation. Upon laser irradiation, Dex-HDL/ALA-Fe3O4 effectively generates reactive oxygen species at the tumor site not only to induce the immunogenic cell death-cascade but also to trigger the on-demand release of full types of tumor antigens. Intriguingly, Fe3O4 nanozyme-catalyzed hydrogen peroxide generated oxygen for alleviating tumor hypoxia and modifying the inhibitory tumor microenvironment, thereby exhibiting remarkable potential as a sensitizer. The intravenous administration of nanovaccines in diverse preclinical cancer models has demonstrated remarkable tumor regression and inhibition of postoperative tumor recurrence and metastasis, thereby enabling personalized treatment strategies against highly heterogeneous tumors.

Sequence of androgen receptor-targeted vaccination with androgen deprivation therapy affects anti-prostate tumor efficacy

RationaleAndrogen deprivation therapy (ADT) is the primary treatment for recurrent and metastatic prostate cancer. In addition to direct antitumor effects, ADT has immunomodulatory effects such as promoting T-cell infiltration and...

Abstract A010: Cancer associated fibroblast dependency in a novel immunocompetent mouse model of bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy has proven to be crucial for the successful use of most kinase inhibitors. In bladder cancer, the PI3K/AKT/mTOR pathway is widely activated by mutation but untargeted. Our study focuses on developing an immunocompetent, CRISPR-edited bladder cancer mouse model that mirrors common PI3K/AKT/mTOR pathway mutations. We established effective genotype-kinase inhibitor pairings using CRISPR-edited organoids and cancer associated fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 Lox-Stop-Lox-Cas9/GFP transgenic mice and grown as organoids in matrigel. These organoids were co-edited for Trp53 and Rb1 KO, and further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib. Despite successful initial engraftment into mouse bladder, these edited cells regressed before tumor formation, and subcutaneous injection did not yield subcutaneous tumors. An alternate model featuring a 9p21 deletion with Trp53 KO also displayed similar regression post-engraftment into bladder or subcutaneous injections. However, co-injection with a previously established CAF line subcutaneously yielded palpable subcutaneous tumors that enlarged for 12 weeks. We then established a BBN bladder-tumor-derived CAF line, leading to the formation of tumors with squamous differentiation upon intramural co-injection with edited organoids. These models displayed immune-excluded architecture and a predominance of M2 over M1 macrophages. We also aimed to enhance the model's adaptability by introducing common PI3K/AKT/mTOR mutations, specifically TSC1/TSC2 KO, and Pik3ca hotspots, into our 9p21/Trp53 KO model. While TSC1/TSC2 KO lines exhibited elevated P-S6K signaling, they displayed resistance to Torin 1, Voxtalisib, and Everolimus—kinase inhibitors targeting mTORC1/2. In parallel, we employed CRISPR-mediated homologous recombination to successfully introduce a Pik3ca H1047R hotspot into the 9p21/Trp53 KO genotype, as evidenced by increased Phosphorylation of AKT compared to wild-type 9p21/Trp53 KO cell lines. This genotype resulted in approximately 80-fold selective increased sensitivity to pictilisib, a PI3K inhibitor compared to Pik3ca WT control. Conclusions: In this model, CAFs were required for successful engraftment of tumors into the bladder wall or subcutaneously. BUTU is a customizable model of bladder cancer and is suitable for direct targeting by kinase inhibitors while providing a platform for understanding the genotype-phenotype relationship in bladder cancer. We hope to use it as a tool to develop preclinical evidence on which to base precision medicine clinical trials. Citation Format: Philip H. Abbosh, Henkel Valentine. Cancer associated fibroblast dependency in a novel immunocompetent mouse model of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A010.

Abstract PR006: EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic

Abstract Background: EG-70 (detalimogene voraplasmid) is a novel, investigational, non-integrating, non-viral gene therapy specifically engineered to elicit local stimulation of anti-tumor immune response in the bladder and to drive durable efficacy in patients with BCG-unresponsive high-risk non-muscle-invasive bladder cancer (NMIBC), while mitigating the risk of systemic toxicities from immune stimulation. Previous preclinical data indicate that intravesical instillation (IVI) of EG-70 results in local expression of transgene products in murine and non-human primate models. Here we describe the latest preclinical data to support the mechanism of action of EG-70 and Phase 1 clinical study results for EG-70 in the treatment of BCG-unresponsive NMIBC. Methods: Preclinical: A murine surrogate formulation of EG-70 (mEG-70) was administered locally to the bladder by IVI in C57BL/6 mice. Efficacy was assessed in a luciferase-expressing MB49 model of orthotopic bladder cancer. Following confirmation of cancer cell implantation by in vivo imaging, mice received two weekly IVIs of mEG-70. Immune profiling was evaluated by assessing protein expression levels and cell compositions (via MSD, flow cytometry, and immunohistochemistry). Immune cell depletion studies were conducted using specific antibodies directed against CD4 or CD8 antigen to assess T-cell contribution to efficacy. Clinical: In a Phase 1/2 study in high-risk BCG-unresponsive NMIBC with carcinoma in situ (CIS) (LEGEND; NCT04752722), EG-70 was assessed in four separate dose groups. Results: Preclinical: In a mouse model of orthotopic bladder cancer, IVI of mEG-70 caused striking remodeling of the tumor microenvironment from an immunosuppressive phenotype to a pro-inflammatory milieu supportive of tumor clearance. This was demonstrated by a significant decrease in myeloid cells and IL-4 cytokine levels, an increase in NK-cells and T-cells, and an increase in pro-inflammatory cytokines in tumor tissue. Accordingly, administration of mEG-70 was associated with marked reduction in tumor burden and significant improvement of survival. The anti-tumor response was largely immune-mediated as demonstrated by the critical role of T-cells, durable clearance of tumors, and protection against subsequent tumorigenic re-challenge, both locally in the bladder as well as distally, after flank implantation. Clinical: Results from the Phase 1 portion of the LEGEND study show that IVI of EG-70 was well-tolerated in patients with high-risk BCG-unresponsive NMIBC with CIS across all dose levels, with an overall complete response rate of 73%. Conclusions: The latest preclinical findings demonstrate that EG-70 has the potential to deliver immunostimulatory payloads locally to the bladder. The mechanism of action described preclinically has been translated into the clinic, in the Phase 1 portion of the LEGEND study in patients with BCG-unresponsive NMIBC with CIS. Citation Format: Marie-Line Goulet, Shauna M Dauphinee, Daniel Veilleux, Kristine S Louis, David Lazure, Sarah Stevenson, Darius Bilimoria, Fazmina Zamzameer, Ximin Chen, Sébastien Sublemontier, Sahar Amirkhani, Carlos Fleet, Richard Bryce, Matthew Bui, Shreyas Joshi, Susan J. Kalota, Anthony Cheung, James Sullivan. EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR006.

Phase 2 trial of crizotinib in Japanese patients with advanced NSCLC harboring a MET gene alteration: a Co-MET study.

MET exon 14 skipping mutations occur in 3-4% and MET high amplifications occur in < 1% of patients with non-small-cell lung cancer (NSCLC). Crizotinib, a selective ATP-competitive small-molecule inhibitor of c-Met, ALK, and ROS1 tyrosine kinases, has shown activity in cancer models with various types of MET activation. The Co-MET study is a single-arm phase 2 trial to assess the safety and efficacy of crizotinib in MET inhibitor-naïve patients with advanced NSCLC harboring MET exon 14 skipping mutation (cohort 1) or high MET gene copy number of ≥ 7 (cohort 2). The primary endpoint was the objective response rate (ORR) per RECIST v1.1 by independent radiology review in cohort 1. The key secondary endpoints were the duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. A total of 28 patients (23 in cohort 1 and 5 in cohort 2) were enrolled between March 2018 and February 2020. The primary endpoint was met as the ORR (90% confidence interval: CI) in cohort 1 was 38.1% (20.6-58.3). Median DoR, PFS, and OS (95% CI) were 7.6 (1.9-NE), 5.7 (2.1-11.3), 9.1 (4.0-19.9) months, respectively, in cohort 1. ORR in cohort 2 was 40.0% (18.9-92.4). The safety signals were generally consistent with the known safety profile of crizotinib. Crizotinib showed a clinical activity similar to that of tepotinib and capmatinib in patients with NSCLC harboring MET exon 14 skipping mutations. UMIN000031623.

Abstract A019: TRIM29 enhances the invasion of bladder cancer cells by modulating the intermediate filament network and focal adhesions

Abstract Background: Bladder cancer patient treatment and outcomes are determined by the tumor’s ability to invade and metastasize. Our previous work has shown that TRIM29, also known as ATDC, is regulated by TP63 in basal bladder cancers, where it promotes cancer invasion and metastasis. We also observe that TRIM29 predominately localizes to intermediate filaments in invasive bladder cancer cells. Hypothesis: TRIM29 promotes the invasive progression of bladder cancer by regulating K14-containing intermediate filaments and focal adhesions. Methods: To test this hypothesis, we utilized cell culture models of bladder cancer, live cell imaging and 2D and 3D invasion assays to examine the role of TRIM29 in the regulation of K14, focal adhesions and the migration and invasion and bladder cancer cells. Results: We observed a physical interaction between TRIM29 and K14 filamentous structures in bladder cancer cells. We also found that TRIM29 and K14 regulate focal adhesion stability during bladder cancer invasion. Finally, we found that both K14 and the focal adhesion protein, zyxin (ZYX), are essential for bladder cancer migration and invasion, acting downstream of TRIM29. Conclusions: Our findings highlight a novel role for TRIM29 in regulating the cytoskeleton and focal adhesion dynamics during tumor cell invasion and suggest a potential therapeutic pathway for treating bladder cancer. Citation Format: Yin Wang, Nicole A. Jerome, Allan Kelleher, Marian L. Henderson, Mark Day, Pierre A. Coulombe, Phillip L. Palmbos. TRIM29 enhances the invasion of bladder cancer cells by modulating the intermediate filament network and focal adhesions [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A019.

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.

Abstract A021: The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer

Abstract Biological sex significantly affects bladder cancer incidence and outcome. Although men have much greater incidence of bladder cancer than women, females tend to be diagnosed with more advanced tumors and have worse outcomes. The mechanisms driving these differences are likely multifactorial, with biological factors playing an important role, however, much of the biology underlying these differences is unknown. The immune system is a critical regulator of cancer biology, and immune features have been associated with bladder cancer grade, stage, progression, and response to therapies. Sex dramatically affects the immune system including anti-tumor immunity. However, how sex and immunity intersect to affect clinical outcomes in bladder cancer patients remains unclear. CD8+ T cells are a key part of anti-tumor immune responses and the importance of inhibitory receptors such as programmed cell death-1 (PD-1), is now well established as a mechanism restricting T cell responses in MIBC. However, there are a variety of other regulatory pathways that restrict their anti-tumor function, which have yet to be investigated. One such pathway involves alterations in protein glycosylation, which can affect T cell function at many levels. The activity of the glycosyltransferase N-acetylglucosaminyltransferase V (MGAT5), responsible for complex N-glycan branching, has been shown to regulate T cell function in autoimmunity and chronic viral infections, however, its role in regulating anti-tumor CD8+ T cell responses has yet to be explored. We assessed the activity of MGAT5 in circulating and tumor-infiltrating CD8+ T cells in mice using a panel of cell lines in a syngeneic orthotopic model of muscle-invasive bladder cancer (MIBC). We found that in all tumor models, MGAT5 activity was upregulated in tumor infiltrating CD8+ T cells compared with circulating cells. Furthermore, using high parameter flow cytometry, we assessed the activity of MGAT5 in CD8+ T cells from mice with bladder cancer induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and in patients with MIBC. In both mice and humans, we found that sex affects MGAT5 activity in memory CD8+ T cells, with females exhibiting decreased MGAT5 activity in these cells compared to males. The functional significance of altering MGAT5 activity in T cells was also assessed in vivo. To do this, chimeric mice were generated by mixed bone-marrow transplant resulting in mice with loss of Mgat5 in T cells. Bladder cancer was then induced using BBN, and morbidity (defined as either 10% weight loss in one week, 10% weight loss post-BBN, or humane endpoint), as a proxy for mortality, was assessed over time. In this model, male mice were protected from BBN-induced morbidity compared to female mice, and this protection was abrogated by homozygous or heterozygous loss of Mgat5 in T cells. Overall, our data suggest that sex-dependent regulation of MGAT5 activity in CD8+ T cells may be an important underlying factor affecting outcomes in bladder cancer. Citation Format: Morgan E. Roberts, Igor Moskalev, Peter C. Black. The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A021.

Abstract PR008: Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy

Abstract Background: We present the development of NECTIN4-CAR T cells targeting bladder cancer and evaluate the ability of thiazolidinedione, a class of anti-diabetic drugs, to specifically upregulate NECTIN4 expression and expand the therapeutic window of NECTIN4-CAR T cells. In addition, we assess efficacy of NECTIN4-CAR T cell therapy against a model of bladder cancer with acquired resistance to enfortumab vedotin (EV). Methods: NECTIN4-targeting chimeric antigen receptor (CAR) constructs were designed and cloned into a second-generation EQ-28ζ CAR backbone construct. Human primary T cells were transduced with lentiviral NECTIN4-CAR constructs and co-cultured with a panel of bladder cancer cell lines with a range of NECTIN4 expression and against NECTIN4 knockout cells to assess specificity. Tumor cell count over time was monitored in vitro using an IncuCyte S3. Bladder cancer cell lines (RT112, UMUC-1, HT1197) were treated with PPARγ agonists (rosiglitazone) and antagonists (T0070907), and total and surface NECTIN4 protein levels were assessed. Anti-tumor effects of the NECTIN4-CAR T were evaluated in vitro and in vivo, either with or without rosiglitazone pre-treatment. Finally, RT112 bladder cancer cells were exposed to “cycles” of escalating doses of EV to generate EV-resistant cell line models, and anti-tumor efficacy against EV-resistant lines was evaluated. Results: NECTIN4-targeting CAR T cells displayed potent and NECTIN4-specific cytotoxic activity against bladder cancer cell lines. The efficacy of NECTIN4-CAR T cells was strongly correlated with NECTIN4 expression. Treatment with PPARγ agonists and antagonists was shown to upregulate and downregulate NECTIN4 expression, respectively. Treatment with the PPARγ agonist rosiglitazone induced NECTIN4 expression in a PPARγ-dependent and dose responsive manner. NECTIN4 expression was increased in bladder cancer xenografts established in immunodeficient NSG mice that were treated with systemic rosiglitazone. Priming with rosiglitazone enhanced sensitivity to NECTIN4-CAR T cells in vitro, and the combination of rosiglitazone pre-treatment and NECTIN4-CAR T cell therapy showed enhanced tumor control in bladder cancer xenograft models. We generated RT112 cells that exhibited resistance to EV in vitro and in vivo, which retained NECTIN4 expression. Interestingly, EV-resistant RT112 cells remained sensitive to NECTIN4-CAR T cell therapy both in vitro and in vivo. Conclusions: We engineered a NECTIN4-targeting CAR T cell therapy that is highly antigen-specific and demonstrates antitumor activity against multiple bladder cancer models, including EV resistant cells. Modulating the PPARγ pathway increases NECTIN4 expression, which we leveraged by repurposing an FDA-approved anti-diabetes drug rosiglitazone to increase targeting and anti-tumor efficacy of NECTIN4-CAR T cells. These preclinical results lay the groundwork for CAR T therapy development in bladder cancer and suggest rational drug combinations that can expand the therapeutic window of NECTIN4-targeting therapies. Citation Format: Kevin Chang, Henry M. Delavan, Jun Zhu, Corynn Kasap, Elizabeth Yip, Roshan Lodha, Sheng-You Liao, Sima Porten, Terence Friedlander, Vadim Koshkin, Felix Feng, Arun Wiita, John Lee, Carissa E. Chu, Jonathan Chou. Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR008.

Abstract B012: BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors

Abstract Background: Bladder cancer (BC) remains a common and deadly disease, with 83,190 cases and 32,350 deaths projected in the U.S. in 2024. We previously showed bromodomain and extra-terminal protein inhibitors (BETi) are potent in multiple preclinical models of BC. Our bulk RNA-seq and RT-qPCR data showed BETi significantly reduces expression of homologous recombination (HR) genes, such as RAD51 and RBBP8, which could activate alternative DNA repair pathways including non-homologous end joining (NHEJ) and theta-mediated end joining (TMEJ). Specifically, DNA breaks are repaired through TMEJ by polymerase theta (Polθ) utilizing microhomology templates. Recently published data revealed Polθ inhibition is synthetically lethal when other DDR genes are concomitantly lost or inhibited. Thus, in this study, we sought to elucidate whether BETi-induced HR gene repression impacts TMEJ and POLQ expression, and to identify combinations with BETi that best leverage Polθ-mediated synthetic lethality. Methods: 5637 and J82 BC cells were pretreated with birabresib for 24 h and then transfected via electroporation with a Cas9 ribonucleoprotein, a gRNA targeting LBR2 and a 996 bp HR donor. Transfected cells were treated with birabresib for 24H, and DDR pathway function was assessed by digital droplet PCR. BC cells were synchronized at the G1/S transition with 2 mM thymidine incubated with birabresib alone. After 48 h, cells were harvested, and changes to target gene expression (i.e., POLQ) was assessed by RT-qPCR. Next, the same cells were treated with 8 ascending doses of the BETi birabresib (0.1–100 μM) alone or in combination with either the PARP inhibitor olaparib, CtIP inhibitor triapene, or RAD51 inhibitor RI-1 (10 nM–200 μM). After 72-96 h, CellTiter-Glo™ measured cell viability, and Compusyn v1 calculated combination index (CI) scores where &amp;lt;1.0 indicated synergism and &amp;gt;1.0 indicated antagonism. RT-qPCR was conducted to evaluate expression changes to POLQ after combination treatment. Results: After 48 h, birabresib significantly reduced TMEJ in both 5637 and J82 BC cells (49% reduction, P=0.008 and 41% reduction, P=0.004 both n=3), but did not significantly impact HR or NHEJ. POLQ mRNA expression was most reduced in synchronized 5637 and J82 BC cells after treatment with birabresib alone (46% reduction, P=0.006, n=3, and 38% reduction, P=0.001, n=3). Birabresib combined with olaparib was the most synergistic combination in both 5637 and J82 BC cells (CI = 0.83 and 0.29), but birabresib combined with triapene was also synergistic (CI = 0.91 and 0.90). In J82 cells, birabresib significantly reduced POLQ expression (27% reduction, P=0.008, n=3), but olaparib did not. In both 5637 and J82 cells combined birabresib and olaparib increased POLQ expression (41% increase and 5% increase, ns, n=3). Combined birabresib and triapene displayed a similar trend (46% increase and 17% increase, ns, n=3). Conclusions: These preliminary data support further exploration of POLQ expression increases caused by combined BETi+DDRi treatment in preclinical models of BC. Citation Format: Ryan M. Kemper, Bhavika C. Chirumamilla, Dennis A. Simpson, Manfred Meng, Gaorav P. Gupta, Daniel J. Crona. BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B012.

Abstract IA001: Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer

Abstract Two major concerns in clinical management of bladder cancer are: (i) the high frequency of recurrence; and (ii) the morbidity and mortality associated with progression to invasive disease (invasive urothelial carcinoma, IUC). To gain a better understanding of these issues we have used the murine BBN (N-butyl-N-(4-hydroxylbutyl) nitrosamine) experimental model of bladder cancer, as well as tissue samples from normal human bladders and from bladders of patients at various stages of bladder cancer formation and progression. We have documented in the BBN model a CIS-like stage, prior to invasion, in which clonal expansion from a single cell occupies a significant fraction of the urothelium. With this experimental murine model alongside observations from patient samples and data from The Cancer Genome Atlas (TCGA), we have ascertained that a key event in progression of pre-invasive to invasive carcinoma is the silencing of the Sonic hedgehog (Shh) member of the Hedgehog gene family. Silencing-associated invasion appears due to the loss of SHH-induced differentiation signals, which when present slow growth and block invasion. Despite the consistent loss of SHH expression in invasive murine and human bladder cancers, however, TCGA data show few if any mutations in the SHH gene of IUC cells. Instead, our studies of patient samples and cancer-derived cell lines reveal that the loss of SHH expression is due to tighter chromosomal packaging and reduced accessibility of a bladder-specific enhancer located ~200 kb upstream of the SHH transcription start site. We have found that inhibitors of certain chromatin-modifying enzymes, including an FDA-approved drug, are capable of re-activating SHH expression, and are testing the possibility that these drugs may block or reverse invasion of urothelial carcinoma. The high frequency of bladder cancer recurrence is thought to result from field cancerization, that is, an increased probability of new cancer formation in apparent normal urothelium, even after successful resection of the primary tumor. To understand the basis for this field effect, we compared apparent normal urothelium from cancer patients to truly normal urothelium from bladders never affected by malignancy. We found that apparent normal samples display a dramatically increased basal character in transcriptomic assays, using as a metric of basalization an index constructed from single cell RNA-seq analysis of normal urothelium. We and others have demonstrated previously by lineage tracing and cell ablation studies in the BBN model that IUC arises from basal stem cells of the urothelium. The basalization of urothelium distant from the primary tumor that we observe thus may account for the increased probability of cancer formation in the apparent normal urothelium of bladder cancer patients. We have found that several FDA-approved antagonists of EGF signaling can reverse this basalization in the mouse BBN model as well as in organoid culture, and that these drugs dramatically reduce the formation of tumors following BBN exposure. Citation Format: Philip A. Beachy, Kris B. Prado, Aaron M. Kershner, Karim Mrouj, Bernard M. Kiss, Hua Dong, Ozgu Aydogdu, Ye Tian, Joseph C. Liao. Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA001.

Breaking the Tumor Chronic Inflammation Balance with a Programmable Release and Multi-Stimulation Engineering Scaffold for Potent Immunotherapy.

Tumor-associated chronic inflammation severely restricts the efficacy of immunotherapy in cold tumors. Here, a programmable release hydrogel-based engineering scaffold with multi-stimulation and reactive oxygen species (ROS)-response (PHOENIX) is demonstrated to break the chronic inflammatory balance in cold tumors to induce potent immunity. PHOENIX can undergo programmable release of resiquimod and anti-OX40 under ROS. Resiquimod is first released, leading to antigen-presenting cell maturation and the transformation of myeloid-derived suppressor cells and M2 macrophages into an antitumor immune phenotype. Subsequently, anti-OX40 is transported into the tumor microenvironment, leading to effector T-cell activation and inhibition of Treg function. PHOENIX consequently breaks the chronic inflammation in the tumor microenvironment and leads to a potent immune response. In mice bearing subcutaneous triple-negative breast cancer and metastasis models, PHOENIX effectively inhibited 80% and 60% of tumor growth, respectively. Moreover, PHOENIX protected 100% of the mice against TNBC tumor rechallenge by electing a robust long-term antigen-specific immune response. An excellent inhibition and prolonged survival in PHOENIX-treated mice with colorectal cancer and melanoma is also observed. This work presents a potent therapeutic scaffold to improve immunotherapy efficiency, representing a generalizable and facile regimen for cold tumors.