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Cited by 428Telomeres, Telomerase, and SenescenceCancer Cells and MetastasisTGF-β signaling in diseases

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Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

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Ortiz-Montero, Paola, Londoño-Vallejo, Arturo, Vernot, Jean-Paul·2017·Cell Communication and Signaling
DOI 10.1186/s12964-017-0172-3PMC PMC5418812
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Abstract

BACKGROUND: There is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines. METHODS: The MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231. RESULTS: Our results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features. CONCLUSIONS: This study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.

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Fig. 1 The SCM induces an EMT-like program in MCF-7 cells. a MCF-7 cells were cultured in media with 0,5% FBS (control) or SCM. The morphological evaluations were done at 48 and 96 h after SCM treatment. Representative images are shown. Arrowhead indicates cells with fibroblastoid morphology. Scale bar, 10 μm. b Representative plot of surface marker expression determined by flow cytometry (FACS) with CD44-FITC (mesenchymal marker) and CD24-PE (epithelial marker) monoclonal antibodies in MCF-7 cells stimulated with SCM during 5 days (n = 2). c CD44 expression determination by qRT-PCR; the values were normalized to GADPH and relative to control cells. Error bars represent SEM (n = 2). d Transwell migration (upper) and matrigel invasion (bottom) assays performed in MCF-7 cells control and treated with SCM by using 20% FBS as a quimioattractant in the lower compartment. The histograms show the number of cells present in the lower compartment as a measure of the ability of this cells to migrated (upper) and the number of cells present on the bottom surface of filters from at least 15 images as a measure of the ability to invade (bottom). Error bars indicate SEM. (**p < 0.01) (n = 2). e Growth kinetics of MCF-7 cells upon treatments with SCM. Equal numbers of cells were seeded by triplicate and cells were counted at the indicated time points. Error bars represent SEM. (***p < 0.001) (n = 2). f Representative FACS histograms showing the expression of Ki-67 in control and SCM-treated cells (black and grey histogram, respectively). Filled histogram corresponds to blanc and dotted histogram to isotype control
Fig. 1 The SCM induces an EMT-like program in MCF-7 cells. a MCF-7 cells were cultured in media with 0,5% FBS (control) or SCM. The morphological evaluations were done at 48 and 96 h after SCM treatment. Representative images are shown. Arrowhead indicates cells with fibroblastoid morphology. Scale bar, 10 μm. b Representative plot of surface marker expression determined by flow cytometry (FACS) with CD44-FITC (mesenchymal marker) and CD24-PE (epithelial marker) monoclonal antibodies in MCF-7 cells stimulated with SCM during 5 days (n = 2). c CD44 expression determination by qRT-PCR; the values were normalized to GADPH and relative to control cells. Error bars represent SEM (n = 2). d Transwell migration (upper) and matrigel invasion (bottom) assays performed in MCF-7 cells control and treated with SCM by using 20% FBS as a quimioattractant in the lower compartment. The histograms show the number of cells present in the lower compartment as a measure of the ability of this cells to migrated (upper) and the number of cells present on the bottom surface of filters from at least 15 images as a measure of the ability to invade (bottom). Error bars indicate SEM. (**p < 0.01) (n = 2). e Growth kinetics of MCF-7 cells upon treatments with SCM. Equal numbers of cells were seeded by triplicate and cells were counted at the indicated time points. Error bars represent SEM. (***p < 0.001) (n = 2). f Representative FACS histograms showing the expression of Ki-67 in control and SCM-treated cells (black and grey histogram, respectively). Filled histogram corresponds to blanc and dotted histogram to isotype controlCC BY · PubMed Central
Fig. 2 The treatment with IL6 and IL8 induced morphological and phenotypic changes associated with an EMT process. a MCF-7 cells stimulated with 50 ng/ml of IL6, IL8 or IL6 + IL8 cytokines. 5 ng/ml of TGF-β was used as a positive control and MCF-7 cultured in MEM-α medium supplemented with 0,5% FBS was used as a negative control. The evaluation was done after 96 h of cytokine treatment. Representative images are shown. Arrowhead indicates cells with fibroblastoid morphology. Scale bar, 10 μm. b Representative plot of fluorescence intensity of the EMT surface markers was determined by FACS using a CD44-FITC and a CD24-PE monoclonal antibody in MCF-7 cells stimulated for 5 days with cytokines (n = 2)
Fig. 2 The treatment with IL6 and IL8 induced morphological and phenotypic changes associated with an EMT process. a MCF-7 cells stimulated with 50 ng/ml of IL6, IL8 or IL6 + IL8 cytokines. 5 ng/ml of TGF-β was used as a positive control and MCF-7 cultured in MEM-α medium supplemented with 0,5% FBS was used as a negative control. The evaluation was done after 96 h of cytokine treatment. Representative images are shown. Arrowhead indicates cells with fibroblastoid morphology. Scale bar, 10 μm. b Representative plot of fluorescence intensity of the EMT surface markers was determined by FACS using a CD44-FITC and a CD24-PE monoclonal antibody in MCF-7 cells stimulated for 5 days with cytokines (n = 2)CC BY · PubMed Central
Fig. 3 The treatment with IL6 and IL8 increases the migration capacity of MCF-7 cells. a Wound healing assays performed in MCF-7 cells treated with cytokines. Representative micrographs (10×) were taken at 0, 6 and 12 h showing an increased migration capacity of treated MCF-7 cells. Black dotted line indicated the area of the wound. b Quantification of the area of wound healing assay from at least 10 images by using the image J program. Error bars indicate SEM. (**p < 0.01). c Transwell migration assay towards 20% FBS performed in cytokine-treated and control MCF-7 cells. Representative micrographs (20×) were taken from the membrane filter (bottom surface of filters) stained with Crystal violet. d Quantification of transwell migration assay by counting the number of cells present in the lower compartment. Error bars indicate SEM. (*p < 0.05). e Transwell matrigel invasion assay towards 20% FBS performed in cytokine-treated and control MCF-7 cells. The histograms show the number of cells present on the bottom surface of filters from at least 15 images. Error bars indicate SEM. (***p < 0.001) (n = 2)
Fig. 3 The treatment with IL6 and IL8 increases the migration capacity of MCF-7 cells. a Wound healing assays performed in MCF-7 cells treated with cytokines. Representative micrographs (10×) were taken at 0, 6 and 12 h showing an increased migration capacity of treated MCF-7 cells. Black dotted line indicated the area of the wound. b Quantification of the area of wound healing assay from at least 10 images by using the image J program. Error bars indicate SEM. (**p < 0.01). c Transwell migration assay towards 20% FBS performed in cytokine-treated and control MCF-7 cells. Representative micrographs (20×) were taken from the membrane filter (bottom surface of filters) stained with Crystal violet. d Quantification of transwell migration assay by counting the number of cells present in the lower compartment. Error bars indicate SEM. (*p < 0.05). e Transwell matrigel invasion assay towards 20% FBS performed in cytokine-treated and control MCF-7 cells. The histograms show the number of cells present on the bottom surface of filters from at least 15 images. Error bars indicate SEM. (***p < 0.001) (n = 2)CC BY · PubMed Central
Fig. 4 The treatment with IL6 and IL8 induced gene expression and functional changes associated with an EMT program. Gene expression levels of EMT-associated transcription factors Zeb1, Twist 1 and Twist 2 (a) and Snail 1 and Snail2/Slug (b) in MCF-7 cells stimulated with cytokines as indicated and determined by qRT–PCR. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM. (*p < 0.05; **p < 0.01; ***p < 0.001) (n = 2). c Adhesion of MCF-7 cells to immobilized-fibronectin after cytokine treatment. Representative micrographs (20×) taken from the plates stained with Crystal violet. The number of adherent cells was counted. Error bars indicate SEM. (*p < 0.05; ** p < 0.01)
Fig. 4 The treatment with IL6 and IL8 induced gene expression and functional changes associated with an EMT program. Gene expression levels of EMT-associated transcription factors Zeb1, Twist 1 and Twist 2 (a) and Snail 1 and Snail2/Slug (b) in MCF-7 cells stimulated with cytokines as indicated and determined by qRT–PCR. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM. (*p < 0.05; **p < 0.01; ***p < 0.001) (n = 2). c Adhesion of MCF-7 cells to immobilized-fibronectin after cytokine treatment. Representative micrographs (20×) taken from the plates stained with Crystal violet. The number of adherent cells was counted. Error bars indicate SEM. (*p < 0.05; ** p < 0.01)CC BY · PubMed Central
Fig. 5 MCF-7 cells treated with IL6 and IL8 exhibit self-renewal properties. a Sphere formation assay in the presence of defined medium (EGF and FGF) in MCF-7 cells that were previously treated or not (Control) with SCM (a and b) or with cytokines (c and d) during 5 days. Representative micrographs (10×) show sphere formation after 8 days of induction. Scale bar, 100 μm. The total number of spheres per well larger than 100 μm was counted at day 8. Error bars indicate SEM. (**p < 0.01) (n = 2)
Fig. 5 MCF-7 cells treated with IL6 and IL8 exhibit self-renewal properties. a Sphere formation assay in the presence of defined medium (EGF and FGF) in MCF-7 cells that were previously treated or not (Control) with SCM (a and b) or with cytokines (c and d) during 5 days. Representative micrographs (10×) show sphere formation after 8 days of induction. Scale bar, 100 μm. The total number of spheres per well larger than 100 μm was counted at day 8. Error bars indicate SEM. (**p < 0.01) (n = 2)CC BY · PubMed Central
Fig. 6 MCF-7 cells treated with IL6 and IL8 exhibit differentiation potential characteristics and express reprogramming factors. a Differentiation to mesenchymal cell lineages was induced by using specific induction media as described in Materials and Methods and evaluated after 10 days of induction. Osteoblasts (ALP activity), adipocytes (oil red-O) and chondrocytes (Safranin O) differentiation was determined with specific staining. Representative images are shown. Scale bar, 10 μm. b Gene expression levels of reprogramming-associated transcription factors in MCF-7 cells stimulated with cytokines as indicated and determined by qRT–PCR. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM (*p < 0.05) (n = 2)
Fig. 6 MCF-7 cells treated with IL6 and IL8 exhibit differentiation potential characteristics and express reprogramming factors. a Differentiation to mesenchymal cell lineages was induced by using specific induction media as described in Materials and Methods and evaluated after 10 days of induction. Osteoblasts (ALP activity), adipocytes (oil red-O) and chondrocytes (Safranin O) differentiation was determined with specific staining. Representative images are shown. Scale bar, 10 μm. b Gene expression levels of reprogramming-associated transcription factors in MCF-7 cells stimulated with cytokines as indicated and determined by qRT–PCR. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM (*p < 0.05) (n = 2)CC BY · PubMed Central
Fig. 7 The pro-inflammatory cytokines IL6 and IL8 are required for the maintenance of the migration capacity and stem cell properties in MCF-7 cells. MCF-7 cells were incubated with SCM with or without 1 μg/ml of neutralizing monoclonal antibodies against IL6 (Ref: MAB206, R&D systems) and IL8 (Ref: AF-208-NA, R&D systems). a Wound healing assays showing a decrease in the migration capacity of treated MCF-7 cells with the neutralizing antibodies. The histograms show the quantification of the area of wound healing from at least 10 images taken at 12, 24 and 36 h, using the image J program. Error bars indicate SEM. (***p < 0.001). b Sphere formation assay in the presence of defined medium (EGF and FGF) in MCF-7 cells that were previously treated or not (Control) with SCM alone or in the presence of neutralizing antibodies. The total number of primary, secondary and tertiary spheres per well larger than 100 μm was counted at day 5. Error bars indicate SEM. (***p < 0.001) (n = 2). c Representative micrographs (10×) showing primary, secondary and tertiary generations of mammospheres. Scale bar, 100 μm
Fig. 7 The pro-inflammatory cytokines IL6 and IL8 are required for the maintenance of the migration capacity and stem cell properties in MCF-7 cells. MCF-7 cells were incubated with SCM with or without 1 μg/ml of neutralizing monoclonal antibodies against IL6 (Ref: MAB206, R&D systems) and IL8 (Ref: AF-208-NA, R&D systems). a Wound healing assays showing a decrease in the migration capacity of treated MCF-7 cells with the neutralizing antibodies. The histograms show the quantification of the area of wound healing from at least 10 images taken at 12, 24 and 36 h, using the image J program. Error bars indicate SEM. (***p < 0.001). b Sphere formation assay in the presence of defined medium (EGF and FGF) in MCF-7 cells that were previously treated or not (Control) with SCM alone or in the presence of neutralizing antibodies. The total number of primary, secondary and tertiary spheres per well larger than 100 μm was counted at day 5. Error bars indicate SEM. (***p < 0.001) (n = 2). c Representative micrographs (10×) showing primary, secondary and tertiary generations of mammospheres. Scale bar, 100 μmCC BY · PubMed Central
Fig. 8 The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or (c) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or (e) cytokines, as indicated. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM. (*p < 0.05; **p < 0.01; ***p < 0.001) (n = 2). The incubation of SCM with neutralizing anti-IL6 and anti-IL8 antibodies suppresses SA-βGal activity (d) and reduce the expression of senescent markers P16, P21 and P53 (f) determined by qRT-PCR. Error bars represent SEM. (**p < 0.01; ***p < 0.001) (n = 2)
Fig. 8 The treatment with IL6 and IL8 induces senescence in MCF-7 cells. a Representative images of MCF-7 cells treated with SCM during 10 days or (c) cytokines (50 ng/ml) during 5 days and stained for SA-β-GAL. Scale bar, 10 μm. b Gene expression profile of p16, p21 and p53 in MCF-7 cells stimulated with SCM or (e) cytokines, as indicated. The values were normalized to GADPH and relative to control cells (dotted lines). Error bars represent SEM. (*p < 0.05; **p < 0.01; ***p < 0.001) (n = 2). The incubation of SCM with neutralizing anti-IL6 and anti-IL8 antibodies suppresses SA-βGal activity (d) and reduce the expression of senescent markers P16, P21 and P53 (f) determined by qRT-PCR. Error bars represent SEM. (**p < 0.01; ***p < 0.001) (n = 2)CC BY · PubMed Central
Fig. 9 MDA-MB-231 cells exhibited incomplete/aberrant stem-like cell characteristics. a Sphere formation assay in the presence of defined medium (EGF and FGF) for the MCF-7 and MDA-MB-231 cell lines. Representative micrographs (10, 20 and 40×) show sphere formation after 9 days of induction. Scale bar, 100 μm (b). Differentiation assay of MCF-7 and MDA-MB-231 cells. Differentiation was evaluated after 10 days of induction. Osteoblasts (ALP activity), adipocytes (oil red-O) and chondrocytes (Safranin O) differentiation was determined with specific staining. Representative images are shown. Scale bar, 10 μm. c RUNX2 and APL gene expression determined at day 3, 6 and 9 of differentiation by qRT–PCR. The values were normalized to GADPH and relative to control cells. Error bars represent SEM (*p < 0.01; (**p < 0.001) (n = 2)
Fig. 9 MDA-MB-231 cells exhibited incomplete/aberrant stem-like cell characteristics. a Sphere formation assay in the presence of defined medium (EGF and FGF) for the MCF-7 and MDA-MB-231 cell lines. Representative micrographs (10, 20 and 40×) show sphere formation after 9 days of induction. Scale bar, 100 μm (b). Differentiation assay of MCF-7 and MDA-MB-231 cells. Differentiation was evaluated after 10 days of induction. Osteoblasts (ALP activity), adipocytes (oil red-O) and chondrocytes (Safranin O) differentiation was determined with specific staining. Representative images are shown. Scale bar, 10 μm. c RUNX2 and APL gene expression determined at day 3, 6 and 9 of differentiation by qRT–PCR. The values were normalized to GADPH and relative to control cells. Error bars represent SEM (*p < 0.01; (**p < 0.001) (n = 2)CC BY · PubMed Central

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Authors
Ortiz-Montero, P., Londoño-Vallejo, A., & Vernot, J.-P.
Affiliations
Universidad Nacional de Colombia (CO)
Corresponding author
Vernot, Jean-Paul
ORCID
Vernot, Jean-Paul
Venue
Cell Communication and Signaling
Publisher
Springer Science and Business Media LLC
Published
2017-05-04
Volume
15
Issue
1
Pages
17-17
ISSN
1478-811X (ISSN-L)
DOI
10.1186/s12964-017-0172-3
PMC
PMC5418812
OpenAlex
W2611479744
Cited by
428
References
88
Citations by year
2026: 58 · 2025: 87 · 2024: 57 · 2023: 35 · 2022: 47 · 2021: 63 · 2020: 37 · 2019: 20 · 2018: 21 · 2017: 3
FWCI
16.40
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Cell Communication and Signaling (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Francisco José de Caldas, Colciencias (Contract No RC-692–2013)
  • DIEB and Faculty of Medicine, Universidad Nacional de Colombia. (30174)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Telomeres, Telomerase, and Senescence
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
Keywords
senescence-associated secretory phenotype, interleukin-6, interleukin-8, senescence, breast cancer, MCF-7 cells, MDA-MB-231 cells, epithelial-mesenchymal transition, cancer stemness, tumorigenicity, autocrine loop, inflammation, multilineage differentiation, CD44, CD24
MeSH terms
Breast Neoplasms, Humans, Inflammation, Phenotype, Interleukin-6, Interleukin-8, Cellular Senescence, Culture Media, Conditioned, MCF-7 Cells, Carcinogenesis

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