IL-8/CXCR1/2 signalling promotes tumor cell proliferation, invasion and vascular mimicry in glioblastoma

Abstract

BackgroundGlioblastoma multiforme (GBM) is one of the lethal malignant tumors of the central nervous system. Despite advances made in understanding this complex disease, little has been achieved in improving clinical efficacy towards it. Factors such as chemokines play important role in shaping the tumor microenvironment which in turn plays a significant role in deciding course of tumor progression. In this study, we investigated the role of chemokine IL-8 in glioblastoma progression with particular emphasis on immunomodulation, cellular proliferation, invasion and vascular mimicry.MethodsRole of IL-8 in GBM immunology was determined by correlating the expression of IL-8 by immunohistochemistry with other immune cell markers such as CD3 and CD68. Effect of high IL-8 expression on overall survival, the difference in expression level between different GBM subgroups and anatomic structures were analyzed using other databases. Two GBM cell lines –U-87MG and LN-18 were used to study the impact of targeting IL-8-CXCR1/2 signalling using neutralizing antibodies and pharmacological antagonist. Reverse transcriptase–polymerase chain reaction and immunocytochemistry were used to determine the expression of these axes. Impact on cell viability and proliferation was assessed by MTT, proliferation marker-ki-67 and clonogenic survival assays. Multicellular tumor spheroids generated from GBM cell lines were used to study invasion in matrigel.ResultsWeak Positive correlation was observed between IL-8 and CD3 as well as between IL-8 and CD68. High IL-8 expression in GBM patients was found to be associated with dismal survival. No significant difference in IL-8 expression between different molecular subgroups of GBM was observed. In vitro targeting of IL-8-CXCR1/2 signalling displayed a significant reduction in cell viability and proliferation, and spheroid invasion. Furthermore, the presence of CD34-/CXCR1+ vessels in GBM tissues showed the involvement of IL-8/CXCR1 in vascular mimicry structure formation.ConclusionThese results suggest a direct involvement of IL-8-CXCR1/2 axes in GBM progression by promoting both cell proliferation and invasion and indirectly by promoting neovascularization in the form of vascular mimicry.Electronic supplementary materialThe online version of this article (10.1186/s12929-018-0464-y) contains supplementary material, which is available to authorized users.

Vessel co-option in glioblastoma: emerging insights and opportunities

“…The authors showed that endothelial IL-8 increased GSCs invasiveness and growth [46,47]. Moreover, another report demonstrated that the IL-8-CXCR1/2 axis induces GBM proliferation, invasion, and vascular mimicry [48].…”

Methods for in vitro modeling of glioma invasion: Choosing tools to meet the need

“…The sandwich invasion assay, a 3D spheroid invasion model, places a tumor neurosphere in between two layers of matrix then follows cells as they spread out from the central sphere (Figure 3b; Goertzen, Eymael, & Magalhaes, 2018;Markwell et al, 2019;Sharma, Singh, Siraj, & Saxena, 2018). Diverse ECMs can be incorporated into this assay, but most studies utilize either collagen I or Matrigel due to ease, rapid gelification, and commercial availability (Markwell et al, 2019;Naakka et al, 2019;Sharma et al, 2018). An acellular ECM layer is deposited into the bottom of a well then allowed to solidify (Goertzen et al, 2018;Markwell et al, 2019;Nath & Devi, 2016;Sharma et al, 2018).…”

“…Diverse ECMs can be incorporated into this assay, but most studies utilize either collagen I or Matrigel due to ease, rapid gelification, and commercial availability (Markwell et al, 2019;Naakka et al, 2019;Sharma et al, 2018). An acellular ECM layer is deposited into the bottom of a well then allowed to solidify (Goertzen et al, 2018;Markwell et al, 2019;Nath & Devi, 2016;Sharma et al, 2018). Next, neurospheres are suspended in the ECM mixture and placed atop the solidified ECM layer.…”

“…As the top layer solidifies, neurospheres sink down to the interface between the two ECM layers, hence the sandwich assay title as spheres are "sandwiched" between ECM layers. If the cells undergo collective invasion, this takes on a fried egg appearance where the invasive front spreads out like the egg white and the central sphere remains in the center similar to the egg yolk (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis, Colson, & Grinstaff, 2017). While theoretically spheres should spread evenly in all directions, it is not uncommon to find that cells spread out preferentially along the interface between ECM layers (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis et al, 2017).…”

“…If the cells undergo collective invasion, this takes on a fried egg appearance where the invasive front spreads out like the egg white and the central sphere remains in the center similar to the egg yolk (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis, Colson, & Grinstaff, 2017). While theoretically spheres should spread evenly in all directions, it is not uncommon to find that cells spread out preferentially along the interface between ECM layers (Goertzen et al, 2018;Markwell et al, 2019;Sharma et al, 2018;Tevis et al, 2017). Although this assay artifact may limit certain studies, there is evidence that in vivo invasion also occurs along 2D planes within the body (Holland et al, 2000;Louis, 2006;Onishi, Ichikawa, Kurozumi, & Date, 2011;Weigelin, Bakker, & Friedl, 2012 (Goertzen et al, 2018;Sand et al, 2013).…”