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Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness

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Abstract

Tumour microenvironment plays a critical role in cell invasion and metastasis. To investigate the role of cancer-associated fibroblasts (CAFs) in melanoma cell invasiveness, we used 3D spheroid invasion assay. The effect of conditioned media from normal fibroblasts and CAFs cultivated alone or co-cultivated with melanoma cells on BLM or A2058 melanoma spheroid invasion was analysed. We found that conditioned media from CAFs and CAFs co-cultured with melanoma cells, especially, promote invasion and migration, without significant effect on melanoma cell proliferation. We further analysed the expression of pro-invasive cytokines IL-8 and IL-6 in media and found that melanoma cells are dominant producers of IL-8 and fibroblasts are dominant producers of IL-6 in 2D monocultures, while co-cultivation of CAFs with melanoma cells induces production/secretion of IL-6 and IL-8 into the media. The analyses of IL-6 levels in 3D cultures and human melanoma samples, however, revealed that at least in some cases IL-6 is also produced directly by melanoma cells. Analysis of the role of IL-6 and IL-8 in CAF-induced melanoma invasion, using neutralising antibodies, revealed that simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. In summary, these experiments indicate the important role of CAFs and IL-8 and IL-6 cytokines in melanoma cell invasiveness.

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Acknowledgments

Authors are grateful to Marie Charvátová, Marie Jindráková and Radana Kavková for excellent technical assistance. This publication is a result of the project implementation: “The equipment for metabolomic and cell analyses”, Registration Number CZ.1.05/2.1.00/19.0400, supported by Research and Development for Innovations Operational Programme (RDIOP) co-financed by European regional development fund and the state budget of the Czech Republic. Salary of Dr Mateu was covered by project GLYCOPHARM. This study was also supported by the Grant Agency of the Czech Republic (Project Nos. 304/02/1333 and 16-05534S), the Charles University (project of Specific University Research and PRVOUK-27, UNCE 204013, Grant Agency of Charles University Grant 629312), the Ministry of Education, Youth and Sports of CR within the National Sustainability Program II (Project BIOCEV-FAR Reg. No. LQ1604), the project BIOCEV (CZ.1.05/1.1.00/02.0109) and the Kellner Family Foundation Principal Investigator grant.

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Authors and Affiliations

  1. Department of Cell Biology, Charles University in Prague, Viničná 7, Prague, Czech Republic

    Njainday Pulo Jobe, Daniel Rösel & Jan Brábek

  2. 1st Faculty of Medicine, Institute of Anatomy, Charles University, U Nemocnice 3, 12800, Prague 2, Czech Republic

    Barbora Dvořánková, Ondřej Kodet, Lukáš Lacina, Rosana Mateu & Karel Smetana

  3. Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (BIOCEV), Průmyslová 595, 25242, Vestec u Prahy, Czech Republic

    Njainday Pulo Jobe, Daniel Rösel, Barbora Dvořánková, Ondřej Kodet, Lukáš Lacina & Jan Brábek

  4. Department of Dermatovenerology, 1st Faculty of Medicine, Charles University and General University Hospital, U Nemocnice 2, 12808, Prague 2, Czech Republic

    Ondřej Kodet

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  1. Njainday Pulo Jobe
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Correspondence to Karel Smetana or Jan Brábek.

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Supplementary Fig. 1

Blocking IL-6 and IL-8 in conditioned media fully inhibits CAF-induced invasion. WM3629 (a) and WM3670 (b) melanoma spheroids were embedded in collagen, and incubated with conditioned media (top panel) and conditioned media supplemented with IL-6 and IL-8 blocking antibody (bottom panel) at a concentration of 200 ng/ml, each. The graphs (c, d) show relative invasion after 48 hours. CC HFP3 and CC Mel Fib is conditioned media from co-culture of melanoma cells with normal fibroblasts and CAFs, respectively. These results are representative of three independent experiments. Scale bar 400 µm. *p < 0.05 (TIFF 3623 kb)

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Jobe, N.P., Rösel, D., Dvořánková, B. et al. Simultaneous blocking of IL-6 and IL-8 is sufficient to fully inhibit CAF-induced human melanoma cell invasiveness. Histochem Cell Biol 146, 205–217 (2016). https://doi.org/10.1007/s00418-016-1433-8

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