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Mutation Champion: Bladder Cancer – Implications For Diagnostics and Therapy With Single Cell Sequencing


[↓] Bladder Cancer Ecosystem

[↓] Metastasis Inhibition through TIGIT targeting

[↓] Hexokinase 2 Assay For Cancer Diagnostics From Urine

[↓] Anti-tumor Cytotoxicity of Cancer Resident CD4+ T Cells

[↓] New Invasive Epithelial Cell Type

[↓] Data Mining For Independent Prognostic Factors

Bladder cancer is the twelfth most common malignancy around the world, bringing a tremendous social burden. The era of targeted therapy has mostly bypassed Muscle Invasive Bladder Cancer (MIBC), lagging behind other cancers. Significant improvements have been made only recently in the advent od Immune Checkpoint Inhibition (ICI) therapy development. This personalized medicine approach improves therapy outcomes and reduces unnecessary treatment of likely nonresponders. MIBC has one of the highest mutation rates among cancers. [1] This increases the difficulty of discerning which alterations are driver events, and this high mutation rate correlates with a higher degree of intra- and inter-tumoral heterogeneity (ITH), rendering the effectiveness of precision medicine a role of the dice between different patients.

Systemic cisplatin-based chemotherapy is a critical component of the treatment of localized and metastatic MIBC. The clonal evolution of MIBC raises the concern that chemotherapy will select for pre-existing tumor subclones resistant to chemotherapy. [2] This phenomenon likely explains the common pattern of tumor response followed by recurrence and progression. As with other types of cancer, a diverse range of T cell subtypes were identified, including pro-inflammatory cytotoxic CD8+ T cells and anti-inflammatory regulatory T cells (Treg), revealing notable T cell heterogeneity in bladder tumors. [3,4] Also, circulating tumor markers, including circulating tumor cells and ctDNA, provide an opportunity to analyze the sum-total of all tumor sites.

Single cell sequencing directly addresses the above problems and significantly increases the resolution of ITH, connecting cell type composition of the tumor to individual cancer and immune cell profiles. [5] In the last several years, this technology ushered a new age in studying MIBC and developing effective targeted therapies, elucidating the molecular underpinnings of ITH. [6,7] This molecular characterization facilitates our ability to decipher the essential pathways for MIBC initiation and progression, [8] to discover clinically useful biomarkers to guide treatment, [9] and to identify novel targets for ICI therapy, [10] as well as to predict clinical outcomes [11] . Here we show several examples of how single cell sequencing and data mining provides novel diagnostic platforms and treatment therapies for MIBC.

Bladder Cancer Ecosystem [↑]

Cystitis glandularis (CG) usually occurs in chronic inflammation leading to epithelial hyperplasia and metaplasia, which although otherwise benign, could lead to bladder cancer (BLCA) in patients with intestinal metaplasia and urinary tract accumulation. These studies have important implications for developing a cell therapy strategy for PD.

Luo et al. aimed to provide new biological knowledge about the cell composition of CG and the heterogeneity of different grades of bladder cancer. [8] Authors performed single cell RNA sequencing using Singleron cDNA library preparation kits and comprehensively described the expression characteristics of malignant epitheliums and immune cells, as well as the dynamic changes of cell percentages, and the heterogeneity of cell subtypes. They found that a macrophage cluster that highly expressed proinflammatory cytokine TNF, while T cells did not. Another macrophage cluster was shown to highly express immunosuppressive and angiogenic phenotype-related markers that promote tumor progression associated with CD163.

Figure 1. Changes of cell composition in microenvironment of different bladder samples. (Left) UMAP plot of immune cells and stromal cells, showing different cell types. (Right) T-cell pseudotime development trajectory.

Metastasis Inhibition through TIGIT targeting [↑]

Antitumor immunotherapy is one of the most effective tumor treatments after chemotherapy and radiotherapy, but its effective rate is almost impossible to exceed 50%. T-cell Immunoreceptor with Immunoglobulin and ITIM domain (TIGIT) plays a critical role in the regulation of immune responses as an immunoreceptor inhibitor checkpoint. TIGIT promotes the expression of interleukin 32 (IL-32), a novel cytokine implicated in regulating cancer development and inflammation, which was also shown to be highly expressed in bladder cancer.

Wu et al. implemented single-cell sequencing technology to demonstrate that Treg cells in bladder cancer tissues highly expressed TIGIT, demonstrating it as highly suitable therapeutic target. Moreover, targeting TIGIT had a dual effect, not only upregulating the immune response against bladder cancer but also inhibiting its metastasis. [10] This study revealed the existence of TIGIT+ IL-32+ Treg cells in bladder cancer tissues that not only suppressed antitumor immunoresponses but also promoted tumor metastasis and showed how this technology can be used to develop and verify new immunotherapeutic targets.

Figure 2. TIGIT+ Treg cells in bladder cancer tissues. (Left) Each dot represents a single nucleus colored by major cell types (epithelial cells, T cells, B cells, endothelial cells, fibroblasts, stromal cells, umbrella cells, and smooth muscle cells). (Right) Sample origin plot.

Hexokinase 2 Assay For Cancer Diagnostics From Urine [↑]

Compared with high-grade BC with a relatively poor prognosis, low-grade BC is at a high recurrence rate (50−70%) and a small number of patients (∼10%) will have their tumors progress to high-grade tumors. Urine assays (e.g., ImmunoCyt, BTA stat, NMP22 BladderChek, and UroVysion) provide noninvasive methods adjunct to cystoscopy but exhibit low sensitivity (16−50%) in diagnosing lowgrade tumors. Hexokinase 2 is the first step enzyme in the aerobic glycolytic metabolism, which urothelial cancer cells heavily depend on for energy and constitutes a promising biomarker for identifying high glycolytic exfoliated tumor cells (hgETCs) in urine by exploiting their metabolic abnormalities.

Here, Wang et al. describe a urine-based, noninvasive method for diagnosing BC with high accuracy, especially in low-grade tumors and early staged tumors without clinical evidence, by using HK2 as a marker. [12] This assay detects putative hgETCs (HK2high/pan-CK+/DAPI+ cells) in urine by on-slide fluorescence immunostaining of HK2, pan-CK (exclusion of leukocytes), and DAPI (a nucleus stain). The assay was developed by single cell sequencing, extensively validating the malignancy of HK2-derived hgETCs via genome-wide copy number variation (CNV) profiles or oncogenic driver mutations characteristic of BCs, leading to BC diagnosis.

Figure 3. Schematic depiction of HK2 assay development using single cell sequencing.

Anti-tumor Cytotoxicity of Cancer Resident CD4+ T Cells [↑]

T cells infiltrating solid tumors can either promote (i.e. Treg) or inhibit tumor growth (i.e. cytotoxic T cells). CD8+ T cells recognizing tumor associated antigen (TAA) can directly target and kill tumor cells by secreting cytotoxic molecules such as granzymes. Another example are IFNγ secreting Th1 cells that can stimulate multiple anti-tumor activities, such as enhancing tumor antigen presentation, promoting CD8+ T cell function, and polarizing macrophages to a pro-inflammatory phenotype. However, these anti-tumor T cells might be readily susceptible to suppression by the tumor microenvironment. The major caveat in studying such interactions is the tumor cell heterogeneity and lack of individual immune cell state/repertoire profiling data.

Oh et al. used single cell RNA and paired T cell receptor sequencing of human bladder tumors and non-malignant tissues from 7 patients to characterize tumor specific states of CD4+ and CD8+ cells. [4] Several states of cytotoxic CD4+ T cells expressing cytolytic effector proteins were identified, some of which are enriched in tumors. Cytotoxic CD4+ T cells were clonally expanded in tumors and could kill autologous tumors ex vivo. A gene signature of cytotoxic CD4+ T cells was predictive of a response to PD-1 blockade in an orthogonal RNA-seq dataset of metastatic bladder cancer patients treated with anti-PD-L1. these findings highlight the importance of CD4+ T cell heterogeneity and the relative balance between activation of cytotoxic CD4+ effectors and inhibitory regulatory cells for killing autologous tumors.

Figure 4. Trajectory analysis of Treg and Cytotoxic CD4+ cells differentiation between proliferative and non-proliferative states.

New Invasive Epithelial Cell Type [↑]

Phenotypic diversity and crosstalk within tumor cells are the key to understanding bladder cancer and therapeutic development. Approximately 70% of patients are initially diagnosed as non-muscle invasive bladder cancer (NMIBC stages T1, Tis, and Ta). However, high grade T1 and Tis patients exhibit a progressive course to develop into MIBC (T2-T4). Identification of patients who will progress to more aggressive disease may facilitate optimal treatment. Moreover, epithelial to mesenchymal transition (EMT) has also been widely reported to promote the transition from NMIBC to MIBC. The tumor microenvironment of bladder cancer consists of urothelial-derived malignant cells and nonmalignant stromal or immune cell types. It is increasingly evident that different nonmalignant cell types could interact or communicate with tumor cells to affect cancer progression and response to therapeutics.

Lai et al. characterized tumor cell heterogeneity in bladder cancer on the single cell level. [13] Basal-like tumor cells were found to possess epithelial-to-mesenchymal transition (EMT) features, including enrichment of EMT-associated genes. Basal-like cells further exhibited a potential to generate luminal daughter cells, making them possible precursors for local invasion. In addition, aggressive cells with upregulation of stemness-related genes were observed in a muscle-invasive T3-stage tumor. The findings suggest that invasive cell subsets within bladder cancers may serve a significant role in disease progression.

Figure 5. Epithelial and other cell types in bladder cancer.

Data Mining For Independent Prognostic Factors [↑]

Data mining of single cell sequencing supports lab data by validation or potential biomarker discovery. You et al. performed RNA sequencing on human tumor tissues to identify candidate biomarkers in NMIBC, and then selected genes with prognostic significance by analyzing public datasets from multiple cohorts of bladder cancer patients. [14] They found that SKA3 was associated with NMIBC pathophysiology and poor survival.

Zhao et al. explored The Cancer Genome Atlas (TCGA) and the published single cell sequencing datasets of over 50 000 bladder cancer cells of several cohorts with the aim to investigate Monocarboxylate transporter 4 (MCT4) as a bladder cancer biomarker. [15] This study showed that high MCT4 protein expression is an independent prognostic factor for bladder cancer patients who had undergone radical cystectomy.

Figure 6. Cell type composition from (Left) [15] (Right) [14]

References [↑]

[1] Li et al. Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer. Gigascience, 2012. [PubMed]

[2] Faltas et al. Clonal evolution of chemotherapy-resistant urothelial carcinoma. Nat. Genet., 2016. [PubMed]

[3] Sacher et al. Cytotoxic CD4+ T Cells in Bladder Cancer-A New License to Kill. Cancer Cell, 2020. [PubMed]

[4] Oh et al. Intratumoral CD4+ T Cells Mediate Anti-tumor Cytotoxicity in Human Bladder Cancer. Cell, 2020. [PubMed]

[5] Yu et al. Single-Cell Transcriptomic Map of the Human and Mouse Bladders. J. Am. Soc. Nephrol., 2019. [PubMed]

[6] Duan et al. G-Protein Subunit Gamma 4 as a Potential Biomarker for Predicting the Response of Chemotherapy and Immunotherapy in Bladder Cancer. Genes (Basel), 2022. [PubMed]

[7] Xu et al. Immunosuppressive tumor-associated macrophages expressing interlukin-10 conferred poor prognosis and therapeutic vulnerability in patients with muscle-invasive bladder cancer. J. Immunother. Cancer., 2022. [PubMed]

[8] Luo et al. Single-Cell Transcriptome Comparison of Bladder Cancer Reveals Its Ecosystem. Front. Oncol., 2022. [PubMed]

[9] Lee et al. Single-cell RNA sequencing reveals the tumor microenvironment and facilitates strategic choices to circumvent treatment failure in a chemorefractory bladder cancer patient. Genome. Med., 2020. [PubMed]

[10] Wu et al. Targeting TIGIT Inhibits Bladder Cancer Metastasis Through Suppressing IL-32. Front. Pharmacol., 2022. [PubMed]

[11] Zhou et al. Development and validation of an intra-tumor heterogeneity-related signature to predict prognosis of bladder cancer: a study based on single-cell RNA-seq. Aging (Albany NY), 2021. [PubMed]

[12] Wang et al. Single-Cell Sequencing-Enabled Hexokinase 2 Assay for Noninvasive Bladder Cancer Diagnosis and Screening by Detecting Rare Malignant Cells in Urine. Anal. Chem., 2020. [PubMed]

[13] Lai et al. Single-cell RNA sequencing reveals the epithelial cell heterogeneity and invasive subpopulation in human bladder cancer. Int. J. Cancer., 2021. [PubMed]

[14] You et al. Integrative Transcriptome Profiling Reveals SKA3 as a Novel Prognostic Marker in Non-Muscle Invasive Bladder Cancer. Front. Oncol., 2021. [PubMed]

[15] Zhao et al. Integrative Analysis Identified MCT4 as an Independent Prognostic Factor for Bladder Cancer. Cancers (Basel), 2021. [PubMed]