Mucinous Epithelial Cell Secretion Drives Mucinous Ascites Formation in Pseudomyxoma Peritonei Patients
SPRINGER. 2022: 520-521
View details for Web of Science ID 000789811800437
Patient-derived ex vivo TME-models and single-cell sequencing reveal transcriptional responses to immunotherapy.
AMER ASSOC CANCER RESEARCH. 2021
View details for Web of Science ID 000680263504448
Integrative single-cell analysis of allele-specific copy number alterations and chromatin accessibility in cancer.
Cancer progression is driven by both somatic copy number aberrations (CNAs) and chromatin remodeling, yet little is known about the interplay between these two classes of events in shaping the clonal diversity of cancers. We present Alleloscope, a method for allele-specific copy number estimation that can be applied to single-cell DNA- and/or transposase-accessible chromatin-sequencing (scDNA-seq, ATAC-seq) data, enabling combined analysis of allele-specific copy number and chromatin accessibility. On scDNA-seq data from gastric, colorectal and breast cancer samples, with validation using matched linked-read sequencing, Alleloscope finds pervasive occurrence of highly complex, multiallelic CNAs, in which cells that carry varying allelic configurations adding to the same total copy number coevolve within a tumor. On scATAC-seq from two basal cell carcinoma samples and a gastric cancer cell line, Alleloscope detected multiallelic copy number events and copy-neutral loss-of-heterozygosity, enabling dissection of the contributions of chromosomal instability and chromatin remodeling to tumor evolution.
View details for DOI 10.1038/s41587-021-00911-w
View details for PubMedID 34017141
An expanded universe of cancer targets.
2021; 184 (5): 1142–55
The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.
View details for DOI 10.1016/j.cell.2021.02.020
View details for PubMedID 33667368
Single Cell Analysis Can Define Distinct Evolution of Tumor Sites in Follicular Lymphoma.
Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled two nodal, synchronously-acquired tumor samples from ten follicular lymphoma patients using single cell RNA, B cell receptor (BCR) and T cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the two tumor sites in some patients, but in many patients the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene expression and cell surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in follicular lymphoma.
View details for DOI 10.1182/blood.2020009855
View details for PubMedID 33728464
- IDENTIFY IMMUNE CELL TYPES AND BIOMARKERS ASSOCIATED WITH IMMUNE-RELATED ADVERSE EVENTS USING SINGLE CELL RNA SEQUENCING BMJ PUBLISHING GROUP. 2020: A39
Identify biomarkers associated with immunotoxicities using single-cell RNAseq.
AMER ASSOC CANCER RESEARCH. 2020: 32
View details for Web of Science ID 000518188200037
Joint single cell DNA-seq and RNA-seq of gastric cancer cell lines reveals rules of in vitro evolution.
NAR genomics and bioinformatics
2020; 2 (2): lqaa016
Cancer cell lines are not homogeneous nor are they static in their genetic state and biological properties. Genetic, transcriptional and phenotypic diversity within cell lines contributes to the lack of experimental reproducibility frequently observed in tissue-culture-based studies. While cancer cell line heterogeneity has been generally recognized, there are no studies which quantify the number of clones that coexist within cell lines and their distinguishing characteristics. We used a single-cell DNA sequencing approach to characterize the cellular diversity within nine gastric cancer cell lines and integrated this information with single-cell RNA sequencing. Overall, we sequenced the genomes of 8824 cells, identifying between 2 and 12 clones per cell line. Using the transcriptomes of more than 28 000 single cells from the same cell lines, we independently corroborated 88% of the clonal structure determined from single cell DNA analysis. For one of these cell lines, we identified cell surface markers that distinguished two subpopulations and used flow cytometry to sort these two clones. We identified substantial proportions of replicating cells in each cell line, assigned these cells to subclones detected among the G0/G1 population and used the proportion of replicating cells per subclone as a surrogate of each subclone's growth rate.
View details for DOI 10.1093/nargab/lqaa016
View details for PubMedID 32215369
View details for PubMedCentralID PMC7079336
Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment.
Clinical cancer research : an official journal of the American Association for Cancer Research
The tumor microenvironment (TME) consists of a heterogenous cellular milieu that can influence cancer cell behavior. Its characteristics havean impact on treatments such as immunotherapy. These features can be revealed with single-cell RNA sequencing (scRNA-seq). We hypothesized that scRNA-seq analysis ofgastric cancer (GC) together with paired normal tissue and peripheral blood mononuclear cells (PBMCs) would identify critical elements of cellular deregulation not apparent with other approaches.scRNA-seq was conducted on seven patients with GC and one patient with intestinal metaplasia. We sequenced 56,167 cells comprising GC (32,407 cells), paired normal tissue (18,657 cells) and PBMCs (5,103 cells). Protein expression was validated by multiplex immunofluorescence.Tumor epithelium had copy number alterations, a distinct gene expression program from normal, with intra-tumor heterogeneity. GC TME was significantly enriched for stromal cells, macrophages, dendritic cells (DCs) and Tregs. TME-exclusive stromal cells expressed distinct extracellular matrix components than normal. Macrophages were transcriptionally heterogenous and did not conform to a binary M1/M2 paradigm. Tumor-DCs had a unique gene expression program compared to PBMC DCs. TME-specific cytotoxic T cells were exhausted with two heterogenous subsets. Helper, cytotoxic T, Treg and NK cells expressed multiple immune checkpoint or costimulatory molecules. Receptor-ligand analysis revealed TME-exclusive inter-cellular communication.Single-cell gene expression studies revealed widespread reprogramming across multiple cellular elements in the GC TME. Cellular remodeling was delineated by changes in cell numbers, transcriptional states and inter-cellular interactions. This characterization facilitates understanding of tumor biology and enables identification of novel targets including for immunotherapy.
View details for DOI 10.1158/1078-0432.CCR-19-3231
View details for PubMedID 32060101
- Site to Site Comparison of Follicular Lymphoma Biopsies By Single Cell RNA Sequencing AMER SOC HEMATOLOGY. 2019
- Dynamic Immune Modulation Seen By Single Cell RNA-Sequencing of Serial Lymphoma Biopsies in Patients Undergoing in Situ Vaccination AMER SOC HEMATOLOGY. 2019
Functional genomics identifies predictive markers and clinically actionable resistance mechanisms to CDK4/6 inhibition in bladder cancer
JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH
2019; 38: 322
CDK4/6 inhibitors are a promising treatment strategy in tumor therapy but are hampered by resistance mechanisms. This study was performed to reveal predictive markers, mechanisms of resistance and to develop rational combination therapies for a personalized therapy approach in bladder cancer.A genome-scale CRISPR-dCas9 activation screen for resistance to the CDK4/6 inhibitor Palbociclib was performed in the bladder cancer derived cell line T24. sgRNA counts were analyzed using next generation sequencing and MAGeCK-VISPR. Significantly enriched sgRNAs were cloned and validated on a molecular and functional level for mediating resistance to Palbociclib treatment. Analysis was done in vitro and in vivo in the chorioallantois membrane model of the chicken embryo. Comparison of screen hits to signaling pathways and clinically relevant molecular alterations was performed using DAVID, Reactome, DGIdb and cBioPortal.In the screen, 1024 sgRNAs encoding for 995 genes were significantly enriched indicative of mediators of resistance. 8 random sgRNAs were validated, revealing partial rescue to Palbociclib treatment. Within this gene panel, members of Receptor-Tyrosine Kinases, PI3K-Akt, Ras/MAPK, JAK/STAT or Wnt signaling pathways were identified. Combination of Palbociclib with inhibitors against these signaling pathways revealed beneficial effects in vitro and in in vivo xenografts.Identification of potential predictive markers, resistance mechanisms and rational combination therapies could be achieved by applying a CRISPR-dCas9 screening approach in bladder cancer.
View details for DOI 10.1186/s13046-019-1322-9
View details for Web of Science ID 000476806800002
View details for PubMedID 31331377
View details for PubMedCentralID PMC6647307
- Single cell RNA sequencing of serial tumor and blood biopsies from lymphoma patients undergoing in situ vaccination AMER ASSOC CANCER RESEARCH. 2019
- Comprehensive characterization of gastric cancer at single-cell resolution AMER ASSOC CANCER RESEARCH. 2019
- Single cell RNA sequencing reveals multiple adaptive resistance mechanisms to regorafenib in colon cancer AMER ASSOC CANCER RESEARCH. 2019
scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data.
2019; 20 (1): 264
Single-cell RNA sequencing has enabled the characterization of highly specific cell types in many tissues, as well as both primary and stem cell-derived cell lines. An important facet of these studies is the ability to identify the transcriptional signatures that define a cell type or state. In theory, this information can be used to classify an individual cell based on its transcriptional profile. Here, we present scPred, a new generalizable method that is able to provide highly accurate classification of single cells, using a combination of unbiased feature selection from a reduced-dimension space, and machine-learning probability-based prediction method. We apply scPred to scRNA-seq data from pancreatic tissue, mononuclear cells, colorectal tumor biopsies, and circulating dendritic cells and show that scPred is able to classify individual cells with high accuracy. The generalized method is available at https://github.com/powellgenomicslab/scPred/.
View details for DOI 10.1186/s13059-019-1862-5
View details for PubMedID 31829268
- Single Cell RNA Sequencing of Serial Tumor and Blood Biopsies from Lymphoma Patients on an in Situ Vaccination Clinical Trial AMER SOC HEMATOLOGY. 2018
Targeting the PI3K/AKT/mTOR Pathway in Bladder Cancer.
Methods in molecular biology (Clifton, N.J.)
2018; 1655: 335-350
The PI3K/AKT/mTOR signaling pathway shows frequent molecular alterations and increased activity in cancer. Given its role in the regulation of cell growth, survival and metastasis, molecules within this pathway are promising targets for pharmacologic intervention. Metastatic bladder cancer (BLCA) continues to have few treatment options. Although various molecular alterations in PI3K/AKT/mTOR signaling have been described in BLCA, clinical trials with small molecule inhibitors have not met their endpoints. In this article, we summarize results from preclinical studies and clinical trials that examined PI3K pathway inhibitors in BLCA focusing on technical challenges that might result in contradictory findings in preclinical studies. Based on published data from our group, we also address challenges that need to be overcome to optimize PI3K inhibition in BLCA and enable its successful translation into the clinic.
View details for DOI 10.1007/978-1-4939-7234-0_23
View details for PubMedID 28889395
- Characterization of colorectal liver metastasis at single-cell resolution reveals dynamic interplay in the tumor microenvironment AMER ASSOC CANCER RESEARCH. 2018
- Integrated single-cell DNA and RNA analysis of intratumoral heterogeneity and immune lineages in colorectal and gastric tumor biopsies AMER ASSOC CANCER RESEARCH. 2018
Parallel PI3K, AKT and mTOR inhibition is required to control feedback loops that limit tumor therapy.
2018; 13 (1): e0190854
Targeting the PI3K pathway has achieved limited success in cancer therapy. One reason for the disappointing activity of drugs that interfere with molecules that are important player in this pathway is the induction of multiple feedback loops that have been only partially understood. To understand these limitations and develop improved treatment strategies, we comprehensively characterized molecular mechanisms of PI3K pathway signaling in bladder cancer cell lines upon using small molecule inhibitors and RNAi technologies against all key molecules and protein complexes within the pathway and analyzed functional and molecular consequences. When targeting either mTORC1, mTOR, AKT or PI3K, only S6K1 phosphorylation was affected in most cell lines examined. Dephosphorylation of 4E-BP1 required combined inhibition of PI3K and mTORC1, independent from AKT, and resulted in a robust reduction in cell viability. Long-term inhibition of PI3K however resulted in a PDK1-dependent, PIP3 and mTORC2 independent rephosphorylation of AKT. AKT rephosphorylation could also be induced by mTOR or PDK1 inhibition. Combining PI3K/mTOR inhibitors with AKT or PDK1 inhibitors suppressed this rephosphorylation, induced apoptosis, decreased colony formation, cell viability and growth of tumor xenografts. Our findings reveal novel molecular mechanisms that explain the requirement for simultaneous targeting of PI3K, AKT and mTORC1 to achieve effective tumor growth inhibition.
View details for DOI 10.1371/journal.pone.0190854
View details for PubMedID 29357370
Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma.
Rapid development of novel treatment options demands valid preclinical screening models for urothelial carcinoma (UC). The translational value of high-throughput drug testing using 2-dimensional (2D) cultures is limited while for xenograft models handling efforts and costs often become prohibitive for larger-scale drug testing. Therefore, we investigated to which extent the chicken chorioallantoic membrane (CAM) assay might provide an alternative model to study antineoplastic treatment approaches for UC.The ability of 8 human UC cell lines (UCCs) to form tumors after implantation on CAMs was investigated. Epithelial-like RT-112 and mesenchymal-like T-24 UCCs in cell culture or as CAM tumors were treated with cisplatin alone or combined with histone deacetylase inhibitors (HDACi) romidepsin and suberanilohydroxamic acid. Tumor weight, size, and bioluminescence activity were monitored; tumor specimens were analyzed by histology and immunohistochemistry. Western blotting and quantitative real time polymerase chain reaction were used to measure protein and mRNA expression.UCCs were reliably implantable on the CAM, but tumor development varied among cell lines. Expression of differentiation markers (E-cadherin, vimentin, CK5, CK18, and CK20) was similar in CAM tumors and 2D cultures. Cellular phenotypes also remained stable after recultivation of CAM tumors in 2D cultures. Bioluminescence images correlated with tumor weight. Cisplatin and HDACi decreased weight and growth of CAM tumors in a dose-dependent manner, but HDACi treatment acted less efficiently as in 2D cultures, especially on its typically associated molecular markers. Synergistic effects of HDACi and subsequent cisplatin treatment on UCCs were neither detected in 2D cultures nor detected in CAM tumors.Our results demonstrate that the CAM assay is a useful tool for studying tumor growth and response to conventional anticancer drugs under 3D conditions, especially cytotoxic drugs as cisplatin. With some limitations, it might serve as a cost- and time-effective preclinical screening assay for novel therapeutic approaches before further assessment in expensive and cumbersome animal models.
View details for DOI 10.1016/j.urolonc.2017.05.003
View details for PubMedID 28551413
Wntless promotes bladder cancer growth and acts synergistically as a molecular target in combination with cisplatin.
To analyze the contribution of Wnt signaling pathway to bladder cancer growth in order to identify suitable target molecules for therapy.Expression of Wnt 2/4/7, LRP5/6, TCF1/2/4, LEF-1, and β-actin was detected by reverse transcription polymerase chain reaction in a panel of 9 and for Wntless (WLS) in 17 bladder cancer cell lines. Protein expression of WLS was detected in 6 cell lines. Wnt/β-catenin activity was analyzed using the TOPflash/FOPflash luciferase reporter assay. Expression level of β-catenin, WIF1, Dickkopf proteins (DKK), HSulf-2, sFRP4, and WLS was modulated by transfecting or infecting cells transiently or stably with respective shRNAs, siRNAs, or cDNAs. For protein detection, whole cell lysates were applied to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblots. Effects on cell growth were determined by cell viability assays and BrdU/APC incorporation/staining. For 3-dimensional tumor growth, the chicken chorioallantoic membrane model was used. Tumor growth was characterized by weight.Expression of molecular components and activation of the Wnt signaling pathway could be detected in all cell lines. Expression level of β-catenin, WIF1, DKK, WLS, and HSulf-2 influenced Wnt activity. Expression of WLS was confirmed in 17 cell lines by reverse transcription polymerase chain reaction and in 6 cell lines by immunoblotting. WLS positively regulates Wnt signaling, cell proliferation, and tumor growth in vitro and in vivo. These effects could be reversed by the expression of the Wnt antagonist WIF1 and DKK. Synergistic activity of cisplatin and WLS inactivation by genetic silencing could be observed on cell viability.The Wnt signaling pathway is ubiquitously activated in bladder cancer and regulates tumor growth. WLS might be a target protein for novel therapies in combination with established chemotherapy regimens.
View details for DOI 10.1016/j.urolonc.2017.04.015
View details for PubMedID 28501564
CDK4/6 Inhibitors in Cancer Therapy: A Novel Treatement Strategy for Bladder Cancer.
Bladder cancer (Amsterdam, Netherlands)
2017; 3 (2): 79-88
Patients with metastatic bladder cancer (mBC) treated with cisplatin-based chemotherapy have a limited median survival of only around 14 months . Despite over 30 years of basic and clinical research, until recently no therapeutic options beyond cisplatin-based therapy had entered clinical routine and, at least in the US, none of the tested agents had been approved for second-line treatment. This has changed with the advent of immune checkpoint blockade, including especially PD-1/PD-L1 inhibitors. The high response rates of 24% over a 14.4 month follow up led to the first US Food and Drug Administration (FDA) approval for a second line therapy for these patients, and it is likely that this marks the beginning of a new era in the systemic treatment of muscle-invasive bladder cancer [2-4]. The strong clinical need to improve the medical management of this disease for those patients, not responding to current therapy has led to an increased molecular understanding of bladder cancer and has forstered the development of many potential molecular manipulations and targeted strategies beyond the new immune-oncologic approaches. Among the molecular alterations indentified in bladder cancer, cell cycle deregulation appears to be a key driver of disease progression. Target-directed therapy against CDK4/6 is an emerging strategy to regain control of cell cycle deregulation. Here, we provide an overview of the current status of CDK4/6 inhibitors in cancer therapy, their potential use in mBC and the challenges for their clinical use.
View details for DOI 10.3233/BLC-170105
View details for PubMedID 28516152
CDK4/6 Inhibition Controls Proliferation of Bladder Cancer and Transcription of RB1
JOURNAL OF UROLOGY
2016; 195 (3): 771-779
The retinoblastoma signaling network is frequently altered in advanced bladder cancer. We investigated the potential of CDK4/6 as a therapeutic target and determined biomarkers for patient stratification.Genetic alterations were analyzed using public databases, including TCGA (The Cancer Genome Atlas), COSMIC (Catalogue of Somatic Mutations in Cancer) and CCLE (Cancer Cell Line Encyclopedia). Effects of the CDK4/6-inhibitor PD-0332991 or LY2835219 were examined in 10 bladder cancer cell lines by immunoblot, cell viability, apoptosis and cell cycle progression. Efficacy of the PD-0332991 and cisplatin combination was analyzed using the combination index. Gene expression level was determined by quantitative polymerase chain reaction. Cytomegalovirus promoter regulated recombinant retinoblastoma was used for reconstitution. Three-dimensional xenografts were grown on chicken chorioallantoic membrane and analyzed by measuring tumor weight and immunohistochemical expression of total retinoblastoma and Ki-67.PD-0332991 treatment decreased the proliferation of retinoblastoma positive bladder cancer cell lines and was synergistic in combination with cisplatin. PD-0332991 or LY2835219 treatment decreased the phosphorylation, total protein and transcript level of retinoblastoma. Treatment resulted in a decrease in E2F target gene expression (CCNA2 and CCNE2) and cell cycle progression from G0/G1 to the S-phase but did not affect apoptosis. In retinoblastoma negative cells reconstituted with recombinant retinoblastoma PD-0332991 affected only phosphorylation and not the total retinoblastoma level. These cells remained resistant to treatment. In 3-dimensional retinoblastoma xenografts, treatment resulted in reduced tumor weight and decreased expression of total retinoblastoma and Ki-67.We provide preclinical evidence that CDK4/6 inhibition is a potential therapeutic strategy for retinoblastoma positive bladder cancer that probably acts by negatively regulating retinoblastoma transcription.
View details for DOI 10.1016/j.juro.2015.08.082
View details for Web of Science ID 000370054500090
View details for PubMedID 26318986
Mutant PIK3CA controls DUSP1-dependent ERK 1/2 activity to confer response to AKT target therapy
BRITISH JOURNAL OF CANCER
2014; 111 (11): 2103-2113
Alterations in the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway are frequent in urothelial bladder cancer (BLCA) and thus provide a potential target for novel therapeutic strategies. We investigated the efficacy of the AKT inhibitor MK-2206 in BLCA and the molecular determinants that predict therapy response.Biochemical and functional effects of the AKT inhibitor MK-2206 were analysed on a panel of 11 BLCA cell lines possessing different genetic alterations. Cell viability (CellTiter-Blue, cell counts), apoptosis (caspase 3/7 activity) and cell cycle progression (EdU incorporation) were analysed to determine effects on cell growth and proliferation. cDNA or siRNA transfections were used to manipulate the expression of specific proteins such as wild-type or mutant PIK3CA, DUSP1 or CREB. For in vivo analysis, the chicken chorioallantoic membrane model was utilised and tumours were characterised by weight and biochemically for the expression of Ki-67 and AKT phosphorylation.Treatment with MK-2206 suppressed AKT and S6K1 but not 4E-BP1 phosphorylation in all cell lines. Functionally, only cell lines bearing mutations in the hotspot helical domain of PIK3CA were sensitive to the drug, independent of other genetic alterations in the PI3K or MAPK signalling pathway. Following MK-2206 treatment, the presence of mutant PIK3CA resulted in an increase in DUSP1 expression that induced a decrease in ERK 1/2 phosphorylation. Manipulating the expression of mutant or wild-type PIK3CA or DUSP1 confirmed that this mechanism is responsible for the induction of apoptosis and the inhibition of tumour proliferation in vitro and in vivo, to sensitise cells to AKT target therapy.Conclusion or interpretation:PIK3CA mutations confer sensitivity to AKT target therapy in BLCA by regulating DUSP1 expression and subsequent ERK1/2 dephosphorylation and can potentially serve as a stratifying biomarker for treatment.
View details for DOI 10.1038/bjc.2014.534
View details for Web of Science ID 000345597700010
View details for PubMedID 25349966
View details for PubMedCentralID PMC4260039