Additional file 3 of Paracrine secretion of IL8 by breast cancer stem cells promotes therapeutic resistance and metastasis of the bulk tumor cells
Creators: name: Wu, Mingming
name: Zhang, Xiao
name: Zhang, Weijie
name: Yan, Linlin
name: Liu, Xiangtian
name: Zhang, Min
name: Pan, Yueyin
name: Lobie, Peter E.
name: Han, Xinghua
name: Zhu, Tao
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Cancer
subject: Plasma Physics
subject: Hematology

description: Additional file 2: Fig. S1. BCSC secretome confers tamoxifen resistance. (A) The schematic of BCSC conditioned medium and RNA-sequencing. (B) T47D cells were cultured in CM derived from ALDEFLUOR assay sorted BCSCs or differentiated cancer cells and treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. (C-D) MCF-7 (C) or T47D (D) cells were cultured in CM derived from the respective mammosphere-enriched BCSCs or parental cells and treated with a graded concentration of doxorubicin, docetaxel or 5-FU for 5 days. Cell viability was determined by MTT assay. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Two-way ANOVA test). Fig. S2. BCSC secretome confers metastasis. (A) In vitro wound-healing assay MCF-7 and SUM159 cells cultured with CM from parental cells or BCSCs. Scale bar: 300 μm. (B) Transwell migration and invasion assay of MDA-MB-231 cells cultured with CM from parental cells or BCSCs. Scale bar: 300 μm. (C) H&amp;E staining of lung metastasis derived from MDA-MB-231 cells cultured with CM from parental cells or BCSCs. Scale bar: 300 μm. (C) GO analysis of genes enriched in MDA-MB-231 cells cultured with BCSC Cm compared to that with control CM. (D) H&amp;E staining of lung sections derived from tail vein injected MDA-MB-231 cells cultured with control CM or BCSC CM. Scale bar: 300 μm. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Unpaired two-tailed Student’s t-test). Fig. S3. BCSC-derived IL8 drives tamoxifen resistance. (A) Densitometric analysis of the protein array of factors present in CM derived from mammosphere-enriched BCSCs or parental MCF-7 cells. (B) qRT-PCR quantification of IL8 mRNA levels in matched mammosphere-enriched BCSCs and parental MCF-7 or T47D cells. (C) ELISA quantification of secreted IL8 protein levels in CM from paired ALDEFLUOR assay sorted BCSCs or differentiated cancer cells derived from T47D cells. (D) Immunoblot showing CXCR1 and CXCR2 protein levels in matched mammosphere-enriched BCSCs and parental MCF-7 or T47D cells. (E) ALDEFLUOR assay of MCF-7 and T47D cells cultured with CM from BCSCs or parental cells. (F) MCF-7 or T47D cells were cultured with the CM derived from respective parental cells or mammosphere-enriched BCSCs in the presence or absence of 1 μg/ml IL8 neutralizing antibody and treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. (G) Immunoblot showing the IL8 protein levels in MCF-7 cells transfected with IL8 shRNA or vector. (H) MCF-7 cells were cultured with CM derived from parental cells or mammosphere-enriched shCONT-BCSCs or shIL8-BCSCs. (I) MCF-7 or T47D cells were cultured with the CM derived from respective parental cells or mammosphere-enriched BCSCs in the presence or absence of 100 nM AKT inhibitor IV (AIIV). (J) Kaplan–Meier plots of relapse-free survival (RFS) in ER-positive or TNBC breast cancer patients stratified according to their IL8 levels. The data was analyzed by Kaplan-Meier plotter software. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Unpaired two-tailed Student’s t-test in Fig. S3A-S3E, others Two-way ANOVA test). Fig. S4. Targeting IL8 overcomes tamoxifen resistance. (A) Immunoblot showing the ICAM1, MIF and CXCL12 protein levels in MCF-7 TamR cells transfected with the respective shRNA or vector. (B) The MCF-7 TamR cells transfected with vector or shRNAs to ICAM1, MIF or CXCL12 were treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. (C) The statistical result of Fig. 4E. (D) The matched MCF-7 or MCF-7 TamR cells in the presence or absence of 100 nM AIIV were treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. (E) IHC staining of pAKT in MCF-7 TamR derived tumors treated with vector or repertaxin. Scale bar: 100 μm. (F-G) The statistical result of Fig. 4K (F) and Fig. 4M (G). (H-I) IHC staining of Ki-67 or TUNEL in tumor treated with Fulv+Palb with or without Repertaxin. Scale bar: 100 μm. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Two-way ANOVA test in Fig. S4B and S4D, others one-way ANOVA followed by Tukey’s multiple comparison test). Fig. S5. BCSC-derived IL8 drives metastasis. (A) IL8 levels in breast cancer patients with different subtypes were analyzed in TCGA dataset. (B) The correlation between IL8 and EMT markers was analyzed in TCGA breast cancer dataset. (C) Transwell migration and invasion assay of MCF-7 and SUM159 cells transfected with IL8 shRNA or vector. Scale bar: 300 μm. (D) Transwell migration and invasion assay of MCF-7 and SUM159 cells cultured with CM derived from respective parental cells or mammosphere-enriched BCSCs in the presence or absence of 100 nM AIIV. Scale bar: 300 μm. (E) Transwell migration and invasion assay of MDA-MB-231 cells treated with 0.1 mM repertaxin. Scale bar: 300 μm. (F) Immunoblot analysis of CXCR1/2 levels in parental cells or BCSCs. (G) ALDEFLUOR assay of MDA-MB-231 cells cultured with recombinant IL8 or vehicle. (H) H&amp;E staining of lung sections derived from tail vein injected MDA-MB-231 cells treated with vehicle or IL8. Scale bar: 300 μm. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Unpaired two-tailed Student’s t-test). Fig. S6. β-CATENIN regulates the transcription of IL8. (A) MCF-7 cells were cultured with the CM derived from parental cells or mammosphere-enriched shCONT-BCSCs or shCTNNB1-BCSCs and treated with a graded concentration of tamoxifen for 5 days. Cell viability was determined by MTT assay. (B) Transwell migration and invasion assay of MCF-7 cells cultured with the CM derived from parental cells or mammosphere-enriched shCONT-BCSCs or shCTNNB1-BCSCs. Scale bar: 300 μm. (C) Schematic image of the regulation of BCSC-secreted IL8 in tamoxifen resistance and metastasis. All experiments were repeated at least three times. Results are shown as mean ± S.D. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001; ns, not significant (Two-way ANOVA test in Fig. S6A, one-way ANOVA followed by Tukey’s multiple comparison in Fig. SB).