Bladder Cancer Xenograft Model Research Articles

Establishment of matched bladder cancer PDX and PDX-derived organoid model and evaluation of anti-tumor efficacy of abemaciclib.

Bladder cancer is one of the most common malignancies of the urinary system and there's a significant unmet need for new effective therapeutics for bladder cancer. The limited number of available models to study malignant bladder tumors is one of the obstructions in developing bladder cancer therapeutics. Patient-derived xenograft (PDX) and organoid (PDO) models are more representatives of human cancer than cell lines and cell line-derived xenograft (CDX) and are likely to be more promising and efficient in predicting drug response and finding new therapeutics. Three pairs of patient-derived xenograft (PDX) models of bladder cancer and their corresponding PDX-derived organoids (PDXOs) were successfully established. These models were utilized to assess the efficacy of abemaciclib. The sensitivity of the drug was determined through the Cell Counting Kit-8 (CCK8) assay in PDXO cultures, corroborated by the EdU incorporation assay. Additionally, the in vivo tumor growth was monitored in the matched PDX models. In vitro PDXO cultures and in vivo PDX tumor models consistently demonstrated that abemaciclib had varying degrees of inhibitory effects across different bladder cancer (BC) patients. Notably, our study further revealed that treatment with abemaciclib significantly modified the expression patterns of CyclinD1/CDK4. This was achieved by not only diminishing their expression levels but also by shifting their expression from a membrane-associated localization to the nucleus. Our research provided compelling evidence attesting to the reliability and potential of PDX and PDXO models in the realm of precision medicine. These models are instrumental in identifying patients who are likely to respond favorably to a specific drug treatment.

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.

Clofarabine Has a Superior Therapeutic Window as compared to Gemcitabine in Preclinical Bladder Cancer Models

Current standard-of-care systemic therapy options for locally advanced and metastatic bladder cancer (BC), which are predominantly based on cisplatin-gemcitabine combinations, are limited by significant treatment failure rates and frailty-based patient ineligibility. We previously addressed the urgent clinical need for better-tolerated BC therapeutic strategies using a drug screening approach, which identified outstanding antineoplastic activity of clofarabine in preclinical models of BC. To further assess clofarabine as a potential BC therapy component, we conducted head-to-head comparisons of responses to clofarabine versus gemcitabine in preclinical in vitro and in vivo models of BC, complemented by in silico analyses. In vitro data suggest a distinct correlation between the two antimetabolites, with higher cytotoxicity of gemcitabine, especially against several nonmalignant cell types, including keratinocytes and endothelial cells. Accordingly, tolerance of clofarabine (oral or intraperitoneal application) was distinctly better than for gemcitabine (intraperitoneal) in patient-derived xenograft models of BC. Clofarabine also exhibited distinctly superior anticancer efficacy, even at dosing regimens optimized for gemcitabine. Neither complete remission nor cure, both of which were observed with clofarabine, were achieved with any tolerable gemcitabine regimen. Taken together, our findings demonstrate that clofarabine has a better therapeutic window than gemcitabine, further emphasizing its potential as a candidate for drug repurposing in BC. Patient summaryWe compared the anticancer activity of clofarabine, a drug used for treatment of leukemia but not bladder cancer, and gemcitabine, a drug currently used for chemotherapy against bladder cancer. Using cell cultures and mouse models, we found that clofarabine was better tolerated and more efficacious than gemcitabine, and even cured implanted tumors in mouse models. Our results suggest that clofarabine, alone or in combination schemes, might be superior to gemcitabine for the treatment of bladder cancer.

Abstract C121: Development of CRB-701 (SYS6002): A novel site-specific, Nectin-4 targeting ADC

Abstract CRB-701 (SYS6002) is a clinical stage, next generation ADC targeting Nectin-4 wherein two monomethyl auristatin E (MMAE) moieties are site-specifically conjugated to a novel Nectin-4 targeting IgG1 antibody via a cleavable linker. Nectin proteins are cell adhesion molecules belonging to the immunoglobulin (Ig) superfamily that participate in cell-to-cell interactions and form adhesion junctions between cells. Nectin-4 is widely expressed in epithelial cancers including bladder, breast, lung, colorectal, pancreatic, and ovarian. By contrast the expression level of Nectin-4 is very low in healthy adult human tissues. Due to this differential expression, Nectin-4 has emerged as a robust tumor associated antigen (TAA) that has been clinically validated in urothelial carcinoma by Enfortumab Vedotin (EV). While approved, EV carries associated toxicity concerns including skin rash, peripheral neuropathy and ocular toxicities that can lead to EV discontinuations and lasting adverse effects. CRB-701 (SYS6002) has demonstrated high affinity Nectin-4 binding (~2ng/ml) across species and has selectivity for Nectin-4 among the Nectin family of proteins (>5000 ng/ml). The antibody retains potent in vitro activity against FcgRI and C1q (<10nM) conferring antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) activity in vitro. Compared to EV, CRB-701 (SYS6002) reduces free MMAE exposure by 4.8-9.7-fold when adjusted to ADC exposure (AUC) and there is a 2.2-7.4-fold reduction in MMAE in circulation based on Cmax in rats and non-human primates. Collectively these data reflect lower free MMAE in plasma, reducing the risk of MMAE-induced toxicities observed with EV in the clinic. This improved PK and safety profile coincides with a longer half-life of the ADC in plasma potentially reducing the frequency of administration to achieve efficacy. The internalization rate of CRB-701 (SYS6002) was 2-fold faster than EV when assessed in nectin-4 expressing cells. These data may explain the statistically significant reduction in tumor growth observed in a BL0597 patient derived xenograft model (PDX) of bladder cancer with low Nectin-4 protein expression by IHC (H score =50) when compared to EV at the same dose and frequency. Collectively these data suggest that CRB-701 (SYS6002) could demonstrate an improved therapeutic index and PK profile relative to EV and as such could achieve higher tolerated concentrations at lower dose frequency. The stability of the site-specific linker and the reduction in the drug-antibody ratio (DAR) could lead to a differentiated profile clinically that enables higher doses, longer treatment duration and a greater potential in drug combinations in nectin-4 positive tumors. CRB-701 (SYS6002) is currently being explored clinically in a phase 1 dose escalation with an anticipated completion date mid-2024. Citation Format: Rachael Brake, Zhaopeng Sun, Mo Dan, Lu Lv, Congcong Niu, Yang Zhang, Mingyue Shen, XiXin Hu, Xiwu Hui, Andrew Kolodziej. Development of CRB-701 (SYS6002): A novel site-specific, Nectin-4 targeting ADC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C121.

Fraxetin inhibits proliferation and induces apoptosis of bladder cancer through the Akt pathway in vitro and in vivo.

Fraxetin, a natural compound extracted from the Chinese herb Cortex Fraxini, is reported to boast extensive antitumor properties in various cancers. However, whether fraxetin exhibited an anticancer effect on bladder cancer remains unknown. In this study, cell counting kit-8 was utilized to detect cell viability. Flow cytometry analysis was performed for cell apoptosis analysis. Western blot analysis and real-time PCR were used to ascertain gene expression analysis. A mouse bladder cancer xenograft model was established and subjected to fraxetin treatment. Fraxetin reduced the viability of bladder cancer cells, induced apoptosis in vitro, and inhibited the growth of bladder cancer in vivo. Fraxetin inhibited the Akt pathway in J82 cells. In conclusion, the growth inhibitory properties of fraxetin against bladder cancer may be mediated via an Akt inhibitory effect and cell apoptosis promotion.

LncRNA BCLET variant confers bladder cancer susceptibility through alternative splicing of MSANTD2 exon 1.

Alternative splicing (AS)-related single nucleotide polymorphisms (SNPs) are associated with risk of cancers, but the potential mechanism has not been fully elucidated. Two-stage case-control studies comprising 1630 cases and 2504 controls were conducted to investigate the association between the AS-SNPs and bladder cancer susceptibility. A series of assays were used to evaluate the functional effect of AS-SNPs on bladder cancer risk. We observed that SNP rs558814 A>G located in lncRNA BCLET (Bladder Cancer Low-Expressed Transcript, ENSG00000245498) can decrease the risk of bladder cancer (odds ratio [OR] = 0.84, 95% confidence interval [CI] = 0.76-0.92, p = 3.26 × 10-4 ). Additionally, the G allele of rs558814 had transcriptional regulatory effects and facilitated the expression of BCLET transcripts, including BCLET-long and BCLET-short. We also found decreased BCLET expression in bladder cancer tissues and cells, and BCLET transcript upregulation substantially inhibited tumor growth of both bladder cancer cells and xenograft models. Mechanistically, BCLET recognized and regulated AS of MSANTD2 to participate in bladder carcinogenesis, preferentially promoting the production of MSANTD2-004. SNP rs558814 was associated with the expression of BCLET, which mainly increased the expression of MSANTD2-004 through AS of MSANTD2.

Hsa_circ_0008035 Knockdown Inhibits Bladder Cancer Progression through miR-1184/RAP2B Axis

Introduction: Circular RNAs (circRNAs) are related to the pathogenesis and progression of bladder cancer (BC). This research aimed to investigate the role and mechanism of hsa_circ_0008035 (circ_0008035) in BC progression. Methods: Circ_0008035, microRNA (miR)-1,184, and Ras-related protein 2B (RAP2B) levels were examined in BC via quantitative real-time polymerase chain reaction and Western blotting. Cell Counting Kit-8, colony formation, 5-ethynyl-2′-deoxyuridine staining, flow cytometry, caspase-3 assay kit, transwell, and tube formation assays were conducted to estimate the effects of circ_0008035 on the malignant phenotypes of BC tumors. The interaction between RNAs and genes was evaluated via a dual-luciferase reporter and RNA immunoprecipitation assays. A xenograft model of BC in nude mice was established to estimate the effect of circ_0008035 in BC in vivo. Results: Circ_0008035 and RAP2B levels were upregulated, while miR-1184 abundance was downregulated in BC tissues and cells. Circ_0008035 knockdown constrained cell proliferation, migration, invasion and angiogenesis but promoted apoptosis in vitro. And circ_0008035 silencing curbed xenograft tumor growth in vivo. Circ_0008035 acted as a miRNA sponge for miR-1184. Circ_0008035 increased RAP2B expression by sponging miR-1184. MiR-1184 downregulation relieved the effects of circ_0008035 knockdown on BC progression. And RAP2B knockdown partly reversed the effects of miR-1184 overexpression on BC progression. Conclusion: Circ_0008035-mediated BC progression via regulating the miR-1184/RAP2B axis, providing a potential target for BC treatment.

Bladder Cancer Progression Is Suppressed Through the Heart and Neural Crest Derivatives Expressed 2-Antisense RNA 1/microRNA-93-5p/Defective in Cullin Neddylation 1 Domain Containing 3 Axis.

MicroRNAs (miRNAs) are critical in progression of bladder cancer (BCa). miRNA-93-5p is increased in cancers and is positively correlated with an unfavorable prognosis. But its effects on BCa remain rarely understood. This investigation aimed to dig out miRNA-93-5p affecting biological behaviors of BCa. In this research, mRNA and protein expression in cancer cells were assessed via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell Counting Kit-8 (CCK-8), colony formation, scratch healing, and transwell assays were utilized to analyze cancer cell viability, colony-forming, migration, and invasion, respectively. Bioinformatics analysis predicted upstream regulatory genes and downstream target genes of miRNA-93-5p, with the targeting relationship being verified through a dual-luciferase assay. The BCa xenograft model in nude mice further investigated the effect of miRNA-93-5p and AND2-AS1 on tumor size and quality, and validated the relationship between HAND2-AS1/miRNA-93-5p/DCUN1D3. Our results displayed that miRNA-93-5p was increased in BCa cell lines. Knockdown miRNA-93-5p constrained BCa cell malignant phenotypes. HAND2-AS1 targeted miRNA-93-5p, thus restraining malignant progression of BCa cells. DCUN1D3 was found downstream of miRNA-93-5p. miRNA-93-5p modulated proliferation, migration, and invasion of BCa cells by targeting DCUN1D3. In vivo experiments disclosed that forced expression of lncRNA HAND2-AS1, and inhibited miRNA-93-5p regressed tumor growth. Meanwhile, the same as the results of cell experiments, the expression of miRNA-93-5p was downregulated, and DCUN1D3 expression was advanced in tumor tissues. To conclude, lncRNA HAND2-AS1 exerted anti-tumor effects and regulated BCa cell proliferation, invasion, and migration by targeting miRNA-93-5p/DCUN1D3.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

Combination of T-DM1 and platinum-based chemotherapy in patient-derived xenograft models of muscle-invasive bladder cancer.

Objective(s):To assess the efficacy and safety of T-DM1, as an anti-HER2 antibody-drug conjugate (ADC), alone and in combination with two platinum-based chemotherapy regimens in patient-derived xenografts (PDXs) of muscle-invasive bladder cancer (MIBC) established in immunodeficient mice. Materials and Methods:After treatment initiation, tumor size was measured twice a week. Percent of tumor growth inhibition (TGI) and tumor response rates were calculated as efficacy endpoints. To evaluate treatment toxicity, relative body weight (RBW) was calculated for each group. For comparison of TGIs between treatment groups, the Kruskal-Wallis test was used. Also, the significance of the overall response (OR) rate between placebo groups with treatment groups was analyzed using Fisher’s exact test. Immunohistochemistry and fluorescence in situ hybridization techniques were used to evaluate the level of HER2 expression.Results:Our data showed that T-DM1 alone induced a moderate antitumor activity. While chemotherapy regimens induced a slight TGI when administered alone, interestingly, they showed strong antitumor activity when administered combined with T-DM1. The OR rates were higher when T-DM1 was combined with chemotherapy regimens than T-DM1 alone. When compared with the placebo group, the OR rates of combination groups were statistically significant. Our data also showed that the administered dose of each drug was well tolerated in mice. Conclusion:The combination of T-DM1 and platinum-based chemotherapy may represent a new treatment option for bladder tumors with even low HER2 expression, and could also provide substantial novel insight into tackling the challenges of MIBC management.

NAT10-mediated mRNA N4-acetylcytidine modification promotes bladder cancer progression.

BackgroundDysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA translation efficiency through RNA acetylation. However, the role of NAT10‐mediated acetylation modification in bladder cancer remains elusive. MethodsThe clinical value of NAT10 was estimated according to NAT10 expression pattern based on TCGA data set and the tumor tissue array. Acetylated RNA immunoprecipitation sequencing was utilized to explore the role of NAT10 in mRNA ac4C modification. Translation efficiency and mRNA stability assay were applied to study the effect of NAT10‐deletion on target genes. The nude mouse model and genetically engineered mice were conducted to further verify the characteristics of NAT10 in promoting BLCA progression and regulating downstream targets.ResultsNAT10 was essential for the proliferation, migration, invasion, survival and the stem‐cell‐like properties of bladder cancer cell lines. NAT10 was responsible for mRNA ac4C modification in BLCA cells, including BCL9L, SOX4 and AKT1. Deficient NAT10 in both xenograft and transgenic mouse models of bladder cancer reduced the tumor burden. Furthermore, acetylated RNA immunoprecipitation sequencing data and RNA immunoprecipitation qPCR results revealed that NAT10 is responsible for a set of ac4C mRNA modifications in bladder cancer cells. Inhibition of NAT10 led to a loss of ac4C peaks in these transcripts and represses the mRNA's stability and protein expression. Mechanistically, the ac4C reduction modification in specific regions of mRNAs resulting from NAT10 downregulation impaired the translation efficiency of BCL9L, SOX4 and AKT1 as well as the stability of BCL9L, SOX4.ConclusionsIn summary, these findings provide new insights into the dynamic characteristics of mRNA's post‐transcriptional modification via NAT10‐dependent acetylation and predict a role for NAT10 as a therapeutic target in bladder cancer.Highlights NAT10 is highly expressed in BLCA patients and its abnormal level predicts bladder cancer progression and low overall survival rate. NAT10 is necessary and sufficient for BLCA tumourigenic properties.NAT10 is responsible for ac4C modification of target transcripts, including BCL9L, SOX4 and AKT1.NAT10 may serve as an effective and novel therapeutic target for BLCA.

Gypenoside-Induced Apoptosis via the PI3K/AKT/mTOR Signaling Pathway in Bladder Cancer.

Gynostemma pentaphyllum (Thunb.) Makino (G. pentaphyllum) is a natural herbal drug that has been widely used to treat many diseases. The antitumor effects of G. pentaphyllum were first described in the illustrated catalog of plants. Gypenosides are the major active components of G. pentaphyllum, and they have been widely reported to possess antitumor effects in prostate cancer, gastric cancer, hepatocellular carcinoma, colon cancer, lung cancer, and breast cancer. However, research on the use of gypenoside in the treatment of bladder cancer has not been conducted. In this study, we explored the potential molecular mechanisms of gypenosides in the treatment of bladder cancer using network pharmacology and experimental validation. First, we used a network pharmacology–based method to identify both the effective components of gypenosides and the molecular mechanism underlying their antibladder cancer effects. The results were further confirmed by molecular docking, CCK8 and colony formation assays, and cell cycle and cell apoptosis analyses. Additionally, a mouse xenograft model of bladder cancer was used to investigate the antitumor effect of gypenosides in vivo. We identified 10 bioactive ingredients and 163 gene targets of gypenosides. Network exploration suggested that VEGFA, STAT3, and PI3KCA may be candidate agents for the antibladder cancer effect of gypenosides. In addition, analysis of the Kyoto Encyclopedia of Genes and Genomes pathway revealed that the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway may play a crucial role in the mechanism of action of gypenosides against bladder cancer. Molecular docking revealed that gypenosides combine well with PI3K, AKT, and mTOR. As expected, gypenosides displayed apoptosis-inducing properties in bladder cancer cells by inactivating the PI3K/AKT/mTOR signaling pathway in vitro. Furthermore, gypenosides significantly (P < 0.05) inhibited the growth of bladder cancer cells in vivo. Mechanistically, gypenosides induced the apoptosis of bladder cancer cells via inactivation of the PI3K/AKT/mTOR signaling pathway.

A Sampling of Highlights from the Literature.

Lactic acid is a metabolic checkpoint for Treg cells (from Jynto via Wikimedia Commons)The intratumoral ratio of CD8+ T cells to regulatory T (Treg) cells impacts responsiveness to PD-1 blockade. Kumagai et al. find the ratio of CD8+ T cells to Treg cells is low in highly glycolytic tumors, including MYC-amplified tumors and liver metastases, and that expression of PD-1 is lower in CD8+ T cells than Treg cells. This effect is mediated by tumor-derived lactic acid and leads to resistance to PD-1 blockade in mouse models of Myc-overexpressing colon cancer and intrahepatic tumors. Blocking lactic-acid production or its uptake by Treg cells overcomes this resistance, suggesting potential new targets for cancer immunotherapy.Kumagai S, …, Nishikawa H.Cancer Cell2022 Feb 14;40:201–18.e9.Pancreatic cancer can be protected from CAR T cells by N-glycans (from Neelima Shah and Edna Cukierman via NCI Visuals Online)Chimeric antigen receptor (CAR) T-cell therapy has limited efficacy against solid tumors. Greco et al. identify expression of N-linked glycans as a targetable barrier to killing of solid tumors by CAR T cells. Knockout of mannoside acetyl-glucosaminyltransferase 5 and exposure to 2-deoxy-D-glucose (2DG), both of which disrupt N-glycan expression, render T3M-4 human pancreatic cancer cells sensitive to killing by CD44v6-targeted CAR T cells in vitro. Treatment with 2DG enhances tumor-cell killing by CAR T cells in xenograft models of pancreatic, ovarian, bladder, and lung cancer, suggesting a new approach to improve CAR T-cell therapy for solid tumors.Greco B, …, Casucci M.Sci Transl Med2022 Jan 19;14:eabg3072.Blocking “don’t eat me” signals increases macrophage phagocytosis (from QuidsUp)Modulation of macrophage phagocytosis is a tumor escape mechanism. Li et al. find a role for signaling lymphocytic activation molecule (SLAM) family receptors (SFR) in limiting macrophage phagocytosis by hematopoietic tumors. Specifically, SLAMF3 and SLAMF4 are identified as “don’t eat me” receptors on macrophages. Deficiency in SFRs, with and without CD47 deficiency, increases phagocytosis of hematopoietic cells (i.e., “self”) and malignant cells, highlighting a novel signaling pathway that can modulate the function of macrophages in the tumor microenvironment.Li D, …, Dong Z.Sci Immunol2022 Jan 21;7:eabj5501.HLA-A*03 is associated with poor response to immune checkpoint blockade (from Pdeitiker via Wikimedia Commons)Human leukocyte antigens (HLA) play a vital role in immunity, and thus, could impact immunotherapy efficacy. Using multiple large-scale datasets, Naranbhai et al. identify HLA-A*03 as a biomarker of response to immunotherapy but not response to other types of cancer treatment. Expression of this HLA associates with worse progression-free survival and lower responses to immune checkpoint inhibitors, which was validated in several independent cohorts of patients with various tumor types.Naranbhai V, …, Carrington M. Lancet Oncol 2022 Jan 1;23:172–84.Understanding of the NSCLC immune landscape continues to deepen (from Librepath via Wikimedia Commons)Deepening understanding of the immune landscape of non–small cell lung cancer (NSCLC) can help identify biomarkers of response to immunotherapy. Using single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), Leader et al. find a strong correlation between PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages; they refer to these cells collectively as the lung cancer activation module (LCAM). The LCAMhi phenotype correlates with tumor mutational burden, TP53 mutations, and response to anti–PD-L1 therapy but not chemotherapy, suggesting that it could be a new biomarker of NSCLC response to immunotherapy.Leader AM, …, Merad M. Cancer Cell 2021 Dec 13;39:1594–609.e12.TRIB3 regulates CD8+ T-cell exclusion in the tumor microenvironment (from Max Pixel)Colorectal cancer resistance to immune checkpoint therapy is partly attributable to a lack of tumor-infiltrating T cells. Shang et al. report that TRIB3 expression in tumor cells correlates with CD8+ T-cell exclusion in mouse models of colorectal cancer and human samples. TRIB3 expression inhibits expression of the T cell–recruiting chemokine CXCL10 by activating STAT3, which inhibits transcription of STAT1 and subsequently CXCL10. Inhibition of the P300 acetylase, which acetylates TRIB3 and prevents E3 ligase–mediated degradation, improves CD8+ T-cell infiltration and sensitizes colorectal tumors to checkpoint blockade in vivo, suggesting a therapeutic strategy to improve immunotherapy efficacy in immunologically “cold” colorectal tumors.Shang S, …, Hua F. Sci Transl Med 2022 Jan 5;14:eabf0992.

Long Non-Coding RNA PCAT19 Regulates the Malignant Progression of Bladder Cancer through the miR-335-5p/IER2 Axis.

Bladder cancer is a common urological tumor, and its development process is complicated. Many long non-coding RNAs (lncRNAs), including PCAT19, have a regulatory role in cell development and gene expression, and is widely involved in the occurrence and development of a variety of cancers. We studied the changes in cell function through MTT assay, flow cytometer, transwell assay, etc. Several molecular assays are employed to explore these molecular mechanistic aspects, such as luciferase reporter, reverse transcription quantitative polymerase chain reaction, and Western blot. In addition, we also constructed a mouse bladder cancer xenograft model to verify the function of the PCAT19/miR-335-5p/IER2 signal axis in vivo. PCAT19 is upregulated in bladder cancer tissues, while miR-335-5p is downregulated, and the expression of PCAT19 and miR-335-5p is negatively correlated. The survival rate of bladder cancer patients in the PCAT19 upregulated group or miR-335-5p downregulated group was lower. After overexpression of PCAT19, the amount of miR-335-5p in cell is decreased, and the IER2 expression is increased. It can significantly promote the vitality, proliferation, migration and invasion capabilities of bladder cancer cells, and significantly inhibit cell apoptosis. Studies on molecular mechanisms show that PCAT19 can regulate the miR-335-5p/IER2 signal axis both in vivo and in vitro. PCAT19, which is upregulated in bladder cancer tissue, interacts with miR-335-5p to regulate the expression of downstream target gene IER2, forming a complete PCAT19/miR-335-5p/IER2 signal regulation axis, and ultimately affect the malignant progression of bladder cancer both in vivo and in vitro.

Identification of Activated Protein Kinase Cα (PKCα) in the Urine of Orthotopic Bladder Cancer Xenograft Model as a Potential Biomarker for the Diagnosis of Bladder Cancer.

Bladder cancer has a high recurrence rate; therefore, frequent and effective monitoring is essential for disease management. Cystoscopy is considered the gold standard for the diagnosis and continuous monitoring of bladder cancer. However, cystoscopy is invasive and relatively expensive. Thus, there is a need for non-invasive, relatively inexpensive urinary biomarker-based diagnoses of bladder cancer. This study aimed to investigate the presence of activated protein kinase Cα (PKCα) in urine samples and the possibility of PKCα as a urinary biomarker for bladder cancer diagnosis. Activated PKCα was found to be present at higher levels in bladder cancer tissues than in normal bladder tissues. Furthermore, high levels of activated PKCα were observed in urine samples collected from orthotopic xenograft mice carrying human bladder cancer cells compared to urine samples from normal mice. These results suggest that activated PKCα can be used as a urinary biomarker to diagnose bladder cancer. To the best of our knowledge, this is the first report describing the presence of activated PKCα in the urine of orthotopic xenograft mice.

Depression Induced by CUMS Leads to Bladder Cancer Development and Local Tumor Immunosuppression in Mice.

Depression is a common mental disease in bladder cancer patients, leading to a loss of happiness, an increase in the suicide rate, and higher mortality. However, the influence of depression on bladder tumor tissue remains unknown. In this current study, a subcutaneous bladder cancer xenograft model was established on male C57 mice with mouse bladder cell line MB49. Chronic unpredictable mild stress (CUMS) was established to simulate depression in bladder cancer patients. The depression caused by CUMS was confirmed by testing sucrose preference and plasma cortisol and adrenocorticotropic hormone (ACTH) levels. Then, we measured and weighed tumors to demonstrate the promotion of tumor growth by CUMS. Immune-related cells and molecules were examined to reveal the mechanism. There is a significant decrease of CD8+/CD4+T cells ratio, NK cells, IL-2, and IFN-γ and a significant increase of T regs, IL-6, IL-1β, TNF-α, IL-10, and PGE2 in CUMS group, indicating the inhibition of immunity in the tumor microenvironment. Our results supported the perspective that depression exacerbated bladder cancer and revealed a possible mechanism. We suggest attaching importance to the psychological health of bladder cancer patients to prevent a worse prognosis induced by depression.

Development of a bispecific immune engager using a recombinant malaria protein

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

Centromere protein U enhances the progression of bladder cancer by promoting mitochondrial ribosomal protein s28 expression.

Bladder cancer is one of the most common types of cancer. Most gene mutations related to bladder cancer are dominantly acquired gene mutations and are not inherited. Previous comparative transcriptome analysis of urinary bladder cancer and control samples has revealed a set of genes that may play a role in tumor progression. Here we set out to investigate further the expression of two candidate genes, centromere protein U (CENPU) and mitochondrial ribosomal protein s28 (MRPS28) to better understand their role in bladder cancer pathogenesis. Our results confirmed that CENPU is up-regulated in human bladder cancer tissues at mRNA and protein levels. Gain-of-function and loss-of-function studies in T24 human urinary bladder cancer cell line revealed a hierarchical relationship between CENPU and MRPS28 in the regulation of cell viability, migration and invasion activity. CENPU expression was also up-regulated in in vivo nude mice xenograft model of bladder cancer and mice overexpressing CENPU had significantly higher tumor volume. In summary, our findings identify CENPU and MRPS28 in the molecular pathogenesis of bladder cancer and suggest that CENPU enhances the progression of bladder cancer by promoting MRPS28 expression.

HMGN5 promotes IL-6-induced epithelial-mesenchymal transition of bladder cancer by interacting with Hsp27.

Bladder cancer (BC) is one of the most common cancers worldwide, with a high rate of recurrence and poor outcomes. High-mobility group nucleosome-binding domain 5 (HMGN5) is overexpressed in many cancers and could cause carcinogenesis in BC. By protein-protein-interaction (PPI) analysis, we found that heat shock protein 27 (Hsp27), also a crucial functional factor in BC carcinogenesis, is significantly related to HMGN5. Hsp27 is required for IL-6-mediated EMT via STAT3/Twist signaling in prostate cancer. Here, we hypothesize that HMGN5 may interact with Hsp27 to affect IL-6-induced EMT and invasion in BC via STAT3 signaling. In the present study, we found that HMGN5 and Hsp27 are highly expressed in BC tissues and positively correlated with each other. HMGN5 interacts with Hsp27 in vitro, to modulate the cell invasion and EMT in BC. Moreover, HMGN5 could modulate IL-6-Hsp27-induced EMT and invasion in BC cells by regulating STAT3 phosphorylation and STAT3 targeting of the Twist promoter. HMGN5 interacts with Hsp27 to promote tumor growth in a human BC xenograft model in nude mice. In summary, HMGN5 interacts with Hsp27 to promote IL-6-induced EMT, therefore promoting invasion in BC and contributing to the progression of BC.

Diagnosis of bladder cancer using 18F-labeled α-methyl-phenylalanine tracers in a mouse model.

Although 2-18F-fluoro-2-deoxy-glucose (18F-FDG) has established roles in the diagnosis of a variety of cancers, it has limited value in the detection of primary/recurrent lesions in the bladder, mainly because of interference by the pooled radioactivity in the urine. Our previous study revealed promising properties of L- and D-2-18F-α-methyl-phenylalanine (2-18F-FAMP) as radiotracers; i.e., their rapid blood clearance and low renal accumulation. In the present study we evaluated the utility of L- and D-2-18F-FAMP for imaging bladder cancer in a mouse model. We used the human bladder cancer cell line HT1376 to prepare a bladder cancer xenograft model in mice bearing both orthotopic and subcutaneous tumors. Biodistribution and PET imaging studies were performed at 1 and 3h after injection of L-2-18F-FAMP or D-2-18F-FAMP. 18F-FDG was used as a control. At 1h after injection, greater accumulations of both L-2-18F-FAMP and D-2-18F-FAMP were observed in the orthotopic tumors compared to 18F-FDG. The orthotopic tumor-to-muscle ratio of D-2-18F-FAMP was significantly higher than that of 18F-FDG (p < 0.01), because of the rapid blood clearance of D-2-18F-FAMP. L-2-18F-FAMP showed the highest subcutaneous tumor-to-muscle ratio (p < 0.01) due to its high subcutaneous tumor uptake. Compared to L-2-18F-FAMP, D-2-18F-FAMP exhibited faster clearance and lower kidney accumulation. In the PET imaging studies, L- and D-2-18F-FAMP both clearly visualized the orthotopic bladder tumors at 1h after injection. Our study showed that L-2-18F-FAMP and D-2-18F-FAMP have the potential to detect bladder cancer.