Additional file 1 of Unveiling CXCR2 as a promising therapeutic target in renal cell carcinoma: exploring the immunotherapeutic paradigm shift through its inhibition by RCT001
Creators: name: Montemagno, Christopher
name: Jacquel, Arnaud
name: Pandiani, Charlotte
name: Rastoin, Olivia
name: Dawaliby, Rosie
name: Schmitt, Thomas
name: Bourgoin, Maxence
name: Palenzuela, Héliciane
name: Rossi, Anne-Laure
name: Ambrosetti, Damien
name: Durivault, Jerome
name: Luciano, Frederic
name: Borchiellini, Delphine
name: Le Du, Julie
name: Gonçalves, Leticia Christina Pires
name: Auberger, Patrick
name: Benhida, Rachid
name: Kinget, Lisa
name: Beuselinck, Benoit
name: Ronco, Cyril
name: Pagès, Gilles
name: Dufies, Maeva
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1. Supplementary Table S1. High ELR+CXCL expression is associated with poor PFS and OS on TKI in RCC patients. The levels of ELR+CXCLs (CXCL1, CXCL2, CXCL3, CXCL5, CXCL8) mRNA expression in metastatic RCC patients under TKI (sunitinib, axitinib) first-line treatment were measured by NGS. The value of the third quartile of ELR+CXCL expression was chosen as the cut-off value and correlated with PFS and OS. The Kaplan-Meier method was used to generate survival curves and analyses of censored data were performed using Cox models. The statistical significance (p-values) is given. Supplementary Table S2. Primer sequences used in the qPCR analyses. “m” for mouse primer. Supplementary Table S3. Antibody used for immune cells characterization in tumor. Supplementary Figure S1. High ELR+CXCL expression is associated with poor PFS and OS on TKI in RCC patients. The levels of CXCLs (CXCL1, CXCL2, CXCL3, CXCL5, CXCL8) mRNA expression in metastatic RCC patients under first-line TKI (sunitinib, axitinib) treatment were measured by NGS. The value of the third quartile value of CXCLs expression was chosen as the cut-off value. We created a score reflecting the overexpression of each ELR+CXCL (score 0= no ELR+CXCL overexpressed, score 6= all 6 ELR+CXCL overexpressed). The score was correlated with PFS (a) and OS (b). The Kaplan-Meier method was used to generate survival curves and analyses of censored data were performed using Cox models. Statistical significance (p-values) is indicated. Supplementary Figure S2. RCT001 synthesis and characterization. Protocol for the synthesis of RCT001. 1H NMR spectrum and HPLC chromatogram assessing the purity of the compound &gt; 99.9%. Supplementary Figure S3. SX-682 is a classical antagonist of CXCR2. a-b. CXCR2 kinetic activation (0 to 30 min) was measured by the λmax shift after stimulation by RCT001 (500 nM), CXCL1 (100 nM, a) or CXCL7 (100 nM, b) c. CXCR2 Kinetic activation (0 to 60 min) was measured by the λmax shift after stimulation by CXCL8 (100nM), and 30 min after treatment with the CXCR2 inhibitor SX-682 (500 nM, MedChemExpress, #HY-119339). Supplementary Figure S4. RCT001 inhibits angiogenesis and viability of RCC cells in vitro. a. Cell proliferation assay of serum-depleted endothelial cells treated with CXCL8 (75 ng/mL) in the presence of RCT001 (0.1 mM) or SX682 (CXCR2 inhibitor, 0.1 and 10 µM, MedChemExpress, #HY-119339) or ladarixin (CXCR2 inhibitor, 0.1 and 10 µM, MedChemExpress, #HY-19519) for 72 h. c. Cell proliferation assay of serum-depleted endothelial cells treated with VEGF (75 ng/mL) in the presence of RCT001 (0.1 to 0.5 mM) for 72h. d-e. RCC cell viability A498 (d) and 786-O (e) treated with RCT001 for 48 h. f. Immunoblot analysis of the expression of pAKT and pERK1/2 in RCC cells treated with RCT001 (2.5 mM) for 24 or 48 h. g. CXCL8 (75 ng/ml) dependent migration of RCC cells was analyzed using Boyden chamber assays in the presence/absence of RCT001 (0.1, 0.25 or 0.5 µM). Results are presented as the mean of three independent experiments ± SD. Statistics were performed using the ANOVA test: **p &lt; 0.01, *** p &lt; 0.001 vs control conditions, #p&lt; 0.01, ###p &lt; 0.001 vs CXCL8 conditions, &amp;p&lt; 0.05, &amp;&amp;&amp;p &lt; 0.001 vs CXCL 8+ 0.1µM RCT001 conditions. Supplementary Figure S5. RCT001 has no effect on macrophage-like viability. M0, M1 or M2-like macrophages were treated with RCT001 (2.5 µM) for 48 h. Cell death was determined by cytometry. Results are presented as the mean of three independent experiments ± SD. Statistical significance was determined using an unpaired t-test. Supplementary Figure S6. RCT001 inhibits polarization of M2-like macrophages. Purified monocytes from healthy human donors (n=4) were differentiated into M0-like macrophages (5 days with CSF1) and then polarized into M0-like macrophages (CSF1), or M2-like macrophages (IL4) or M1-like macrophages (LPS) for 48 h, in the presence/absence of RCT001 (2.5µM). a-c. M0-like macrophages were polarized into M0 or M2-like macrophages in the presence of RCT001. a-b. Specific M2 macrophage membrane-markers (CD206, CD209, CD200R, CD163) were evaluated by flow cytometry. c. Specific M2 macrophage mRNA markers (TIMP3, CCL17, CCL18, CCL22, CCL23, CCL24) were evaluated by qPCR. d. M0-like macrophages were polarized into M0 or M1-like macrophages in the presence of RCT001. Specific M1 macrophage mRNA markers (CXCL11, CCL20) were evaluated by qPCR. Results are presented as the mean of four independent experiments ± SD. Statistics were performed using the ANOVA test: *p &lt; 0.05, **p &lt; 0.01, *** p &lt; 0.001. Supplementary Figure S7. Reduction of CXCR2 expression inhibits M2-like macrophages polarization. Purified monocytes from healthy human donors were differentiated into M0-like macrophages (5 days with CSF1) and then transfected with siRNA against CXCR2 (siCXCR2) or control (siCT) for 48 h and polarized into M2-like macrophages (IL4) for 48 h. Specific M2 macrophage membrane markers (CD206, CD209, CD200R, CD163) were evaluated by cytometry. Results are presented as the mean of three independent experiments ± SD. Statistics were performed with an unpaired t-test: *p &lt; 0.05,*** p &lt; 0.001. Supplementary Figure S8. RCT001 efficiently inhibits RCC growth and synergizes with ICIs. RENCA cells were injected subcutaneously. Once tumor reached 30 mm3, mice were divided into different groups. Tumor volume was measured every day. The results are given as the mean values ± sd. a. Control (n=10), RCT001 alone (n=10, 20 mg/kg), anti-PD-1 (n=10, 100 mg) and the combination of RCT001 + PD-1 (n=10). b. Control (n=10), RCT001 suboptimal concentration (n=10, 5 mg/kg), anti-PD-1 (n=10, 100 mg) and the combination of RCT001+ PD-1 (n=10). Statistics were performed using the ANOVA test: *p &lt; 0.01, **p &lt; 0.01, *** p &lt; 0.001.