Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: ( A ) Locus of the identified DMC in intron 1 of IL34 (GRCm38) overlapping an enhancer (ENSEMBL) ( B ) Locus of the CpGs cg01782798 and cg26831220 in the intron 1 of IL34 (GRCh37) overlapping an enhancer (GeneHancer);( C and D) Scatterplot comparing the IL34 transcription and methylation level of the CpGs;( E and F ) Kaplan-Meier plots for overall survival stratified in 2 groups (TCGA KIRC cohort). low: patient group with low methylation level;high: patient group with high methylation level. ( G ) Summary of results. HR (95%CI): Hazard Ratio with 95%Confidence Interval;p value: log-rank test p-value;empirical p value: prognostic performance of the 2 CGs were test against 1-CG random signatures.

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Methylation

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: Expression analysis conducted in orthotopic implanted tumors generated from IL34-overexpressing RENCA cells. Expression of markers in IL34-overexpressing tumors for ( A ) total macrophages (F4/80 and CSF1R), ( B ) M1-type macrophages (IL12a and IL1β) and ( C ) M2-type macrophages (MMR and ARG1) compared to control tumors. Graph show mean ± SEM. T-test. *p<0.05; ***p<0.001. ( D ) Correlation relationship between IL34 and MMR (type 2 macrophage marker). Statistical test: Spearman correlation test. ( E ) Semi-quantitative analysis of MMR protein expression in mouse tumors generated from IL34-overexpressing and control RENCA cells from western blots (Quantification of band intensities compared to vinculin and using a reference sample). On the right, a representative WB of MMR in IL34-overexpressing and control tumors. T-test. ****p<0.0001. ( F ) Quantification of MMR-positive cells in the kidney tumors. On the right, representative images of primary tumors stained by IF using an anti-MMR fluorescent antibody (red) and DAPI (blue) for DNA counterstaining. Scale bar, 100 µm. T-test. ****p<0.0001. ( G ) Quantification of MMR-positive cells in lung metastasis after tail vein injection. On the right, representative images of lung metastases stained by IF using an anti-MMR fluorescent antibody (red) and DAPI (blue) for DNA counterstaining. Scale bar, 100 µm. T-test. ****p<0.0001.

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Expressing, Generated, Control, Marker, Western Blot, Staining, Injection

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: ( A ) Expression analyses of IL34 gene expression in amplified RENCA cell lines (Kidney, Tail and Lung) where IL34 was strongly upregulated compared to Parental cells (Series 0). Of note, the increase of expression is cell-passage dependent and, thus, related to progression toward a more aggressive phenotype. Graph shows mean ± SEM. ( B ) IL34 expression in primary tumor cells and in tumor cells from lung metastasis compared to the parental cell line. ( C ) ELISA assay of IL34 in supernatants in late passaged (S5) cell lines compared to control (Parental S0) of 1 million cells. Graph shows mean ± SEM. ( D ) Correlation of IL34 expression to overall survival (OS) in patients from TCGA KIRC cohort ((HR(log-rank)=1.857 (1.38-2.498)). ( E ) IL 34 expression correlated to Disease Free Survival (DFS) in patients from TCGA KIRC cohort. ((HR(log-rank)=2.056 (1.315-3.214)) ( F ) Correlation of Fuhrman grade with IL34 gene expression in the TCGA KIRC cohort. ( G ) qPCR analyses of IL34 expression in healthy kidney and in tumors from patients (UroCCR). Results as mean and SEM. Analysis by two-tailed paired t-test. P=0.0006. ( H ) Evaluations of IL34 expression in TMA from RCC patients (UroCCR cohort) (Scoring by a pathologist, D. Ambrosetti). ANOVA followed with tukey’s multiple comparisons. **p<0.01; ****p<0.0001. ( I ) Histological staining of IL34 in tumors from patients (UroCCR). Scale bar = 50µm. ( J ) Disease Free Survival (DFS) from the TMA IL34 scoring (UroCCR Cohort)HR(log-rank)=1.878 (1.056-3.342).

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Expressing, Gene Expression, Amplification, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: ( A to D ) OS and DFS of patients stratified according to IL34 expression (TCGA KIRC Cohort) and their metastatic status (OS: M0 HR(log-rank)=1.985 (1.359-2.9);M1[NS] HR(log-rank)=1.368 (0.834-2.242) –DFS: M0 HR(log-rank)=3.542 (1.874-6.694);M1[NS] HR(log-rank)=1.38 (0.731-2.606)). ( E ) IL34 plasmatic levels in patients before and after sunitinib treatment (SUVEGIL-TORAVA cohorts) ( F ) IL34 mRNA expression in mice implanted with 786-O cells and treated with sunitinib. Specific primers were used for human (left panel) and mouse (right panel) IL34. Results presented as mean±SEM. *p<0.05. ns:Non significant. Unpaired t test.

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Expressing

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: ( A and B ) Validation of IL34 expression by qPCR and western-blot in RENCA cells transduced with lentiviral vector (pLenti CMV/TO-mIL34v1-Puro) encoding for mouse IL34. ( C ) IL34 ELISA assay from supernatants of transduced cells. ( D ) Tumor weights of IL34 overexpressing and control tumors. ( E and F ) Validation of IL34 expression in IL34 overexpressing tumors by qPCR (T-test. ***p<0.001) and Western Blot. ( G ) Correlation between IL34 mRNA and CSF1R mRNA (global macrophage marker). And Spearman correlation test. ( H ) CSF1 mRNA expression in IL34 overexpressing and control tumors. ( I ) Quantification of the metastasis area in lung sections after tail vein injection of IL34 overexpressing or control cells. T-test (ns).

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Biomarker Discovery, Expressing, Western Blot, Transduction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Control, Marker, Injection

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: ( A ) validation of IL34 knock-down by ELISA in conditioned media of RENCA cells. ( B ) Proliferation assay with crispr IL34-ko (IL341a, IL341b) and control cells (LacZ) with no significant differences (Dunnett’s multiple comparison tests on all timepoints). ( C ) Mathematical modeling of primary tumor growth kinetics for 7 different animals.

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: Biomarker Discovery, Knockdown, Enzyme-linked Immunosorbent Assay, Proliferation Assay, CRISPR, Control, Comparison

Journal: bioRxiv

Article Title: A multi-layered systems approach for renal cell carcinoma

doi: 10.1101/2020.01.13.904235

Figure Lengend Snippet: Effect of IL34 silencing in vivo . ( A ) Weight from kidney bearing tumors after orthotopic implanted IL34 knock-out (Crispr-Cas9) or control (LacZ) cells represented as mean+-SEM. The pictures on the right shows images of implanted tumors. ANOVA followed by Dunnett’s multiple comparisons test. ***p>0.001; ****p<0.0001. ( B ) Mouse survival (HR(log-rank)=8.09 (1.7-38.497)) and ( C ) number of mice without lung metastasis (HR(log-rank)=3.839 (0.967-15.244)). ( D ) Mathematical modeling issued of tumors from sub-capsular implant of IL 34 Crispr or control (LacZ) cells. Volume of primary tumors and the number of lung metastasis were quantified by MRI and fitted to generate simulations for tumor growth and metastasis formation, using a population mixed-effects approach. ( E ) Metastasis density after tail-vein injections of IL34-silenced and control (LacZ) cells. The graph shows the means of the metastasis area in lung sections relative to the tissue area ± SEM. Examples of macroscopic images of lungs, and lung metastasis from GFP+ RENCA cells are shown. Statistical analysis: ANOVA followed by Dunnett’s multiple comparisons test. *p<0.05; **p<0.01. ( F ) Quantification of MMR-positive cells in lung metastasis after the tail vein injection. Representative images of IF staining for MMR (red) and DAPI(blue) are shown. Scale bar = 100µm. Statistics: ANOVA followed by Dunnett’s multiple comparisons test. ****p<0.0001

Article Snippet: ELISA were performed according to the manufacturer’s protocols: human IL34 (R&D systems, D3400), human SAA2 (DLDEVELOP, DL-SAA2-Hu-96T), Human CFB (abcam, ab137973).

Techniques: In Vivo, Knock-Out, CRISPR, Control, Injection, Staining