Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients

Current treatments for clear cell renal cell cancer (ccRCC) are insufficient because two-thirds of patients with metastases progress within two years. Here we report the identification and characterization of a cancer stem cell (CSC) population in ccRCC. CSCs are quantitatively correlated with tumor aggressiveness and metastasis. Transcriptional profiling and single cell sequencing reveal that these CSCs exhibit an activation of WNT and NOTCH signaling. A significant obstacle to the development of rational treatments has been the discrepancy between model systems and the in vivo situation of patients. To address this, we use CSCs to establish non-adherent sphere cultures, 3D tumor organoids, and xenografts. Treatment with WNT and NOTCH inhibitors blocks the proliferation and self-renewal of CSCs in sphere cultures and organoids, and impairs tumor growth in patient-derived xenografts in mice. These findings suggest that our approach is a promising route towards the development of personalized treatments for individual patients.

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01/19/2020
Single Sequencing raw BCL files were converted to FASTQ using the BCL2FASTQ software (Illumina). FASTQ files were processed using a multistep python library available on GitHub (https://github.com/yanailab/celseq2). Using BowTie2, reads were mapped to the Hg19 reference genome, and raw reads were counted using a modified HTSeq-count script. Data were then analyzed using the Seurat package in R (http://satijalab.org/seurat/). HumanHT-12 v4 bead chip data were normalized and log-transformed in Partek Genomics Suite (Partek) and analyzed using the limma package87 in R. GO term enrichment and Kegg pathway analyses were performed with DAVID (see references in text). Expression of stem cell genes was analyzed using the GeneSpring GX software (Agilent), and supervised hierarchical clustering was performed in Genesis (see references in text).

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. The Gene Expression omnibus accession number is GSE89461 for Illumina bead chip experiments, GSE66270 and GSE66271 for gene expression from fresh frozen ccRCC tissues and GSE110680 for single-cell sequencing. Data with associated raw data are shown in Figures 4 and 5, as well as Supplementary Figures 4 and 5.
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For xenograft assays, we estimated that we would need at least three samples per treatment group to see a two-fold change in tumor volume, for a power of 80% and for the probability of type I error (!) = 0.05. Power calculations were performed in G*Power 3. No sample size calculations were performed for other experiments.
Data were not excluded from analysis.
Microarray data from FAC-sorted and sphere cultures were validated in an independent set of patient-derived primary cells to confirm deregulated gene expression. Kaplan-Meier Analysis from a limited number of ccRCC patients was confirmed in the TCGA data set. For all other experiments, we performed experiments in multiple patient-derived primary cells to ensure reproducibility.
For inhibitor assays in xenografts, subcutaneous tumors were allocated randomly to treatment and control groups.
Blinding was not performed.

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Antibodies were selected based on previous publications in ccRCC whenever possible. When antibodies were used for staining of organoids or spheres there specificity was validated on tissue section to account for correct cellular localization. Antibodies for FACS were titrated and IgG controls were used. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Outcomes Only primary patient-derived cells were used in this study Primary patient-derived cells were not authenticated.
Primary patient-derived cells were not tested for mycoplasma contamination.
Name any commonly misidentified cell lines used in the study and provide a rationale for their use.
Subcutaneous patient-derived xenografts were established from 4x4 mm ccRCC tissue cubes in adult nude (Nu/J) mice and all subsequent experiments were performed in adult NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, NOD scid gamma) mice. Both sexes were used.
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Animal experiments were carried out in accordance with the of the German Animal Protection Law and approved by the local responsible authorities. Epo complies to the EU guideline "European convention for the protection of vertebrate animals used for experimental and other scientific purposes. (EST 123)". Further we handle our animals according to the "Regulation on the protection of experimental scientific purposes or other Purposes used animals". Compliance with the above rules and regulations is monitored by the Landesamt fuer Gesundheit und Soziales (LAGeSo) which is the responsible regulatory authority monitoring the animal husbandry based on the German Animal Welfare Act. Approval was given after careful inspection of the site including bedding, feeding & water, ventilation, temperature & humidity, cleaning and hygiene concepts.
All patient characteristics are summarized in Supplementary Table 1 and 3. All patients undergoing nephrectomy at the Charite -University Hospital from 2011 to 2018 were included if tumor size and localization allowed for the collection of tissue. Participants were exluded if they had a known subtype other than ccRCC or if a subtype other than ccRCC was diagnosed post surgery.
The project was approved by the ethics committee of the Charite -University Hospital (EA1/134/12) and informed consent was obtained from all patients.
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