Tumor microenvironment tenascin-C promotes glioblastoma invasion and negatively regulates tumor proliferation

Abstract

Background: Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recent research on cancer stroma indicates that the brain microenvironment plays a substantial role in tumor malignancy and treatment responses to current antitumor therapy. In this work, we have investigated the effect of alterations in brain tumor extracellular matrix tenascin-C (TNC) on brain tumor growth patterns including proliferation and invasion.

Methods: Since intracranial xenografts from patient-derived GBM neurospheres form highly invasive tumors that recapitulate the invasive features demonstrated in human patients diagnosed with GBM, we studied TNC gain-of-function and loss-of function in these GBM neurospheres in vitro and in vivo.

Results: TNC loss-of-function promoted GBM neurosphere cell adhesion and actin cytoskeleton organization. Yet, TNC loss-of-function or exogenous TNC had no effect on GBM neurosphere cell growth in vitro. In animal models, decreased TNC in the tumor microenvironment was accompanied by decreased tumor invasion and increased tumor proliferation, suggesting that TNC regulates the "go-or-grow" phenotypic switch of glioma in vivo. We demonstrated that decreased TNC in the tumor microenvironment modulated behaviors of stromal cells including endothelial cells and microglia, resulting in enlarged tumor blood vessels and activated microglia in tumors. We further demonstrated that tumor cells with decreased TNC expression are sensitive to anti-proliferative treatment in vitro.

Conclusion: Our findings suggest that detailed understanding of how TNC in the tumor microenvironment influences tumor behavior and the interactions between tumor cells and surrounding nontumor cells will benefit novel combinatory antitumor strategies to treat malignant brain tumors.

Keywords: TNC; glioblastoma; patient-derived GBM neurospheres; tumor microenvironment.

Fig. 1.

Expression of tenascin-C (TNC) in glioblastoma (GBM) neurosphere cells. (A). TNC protein was highly expressed in GBM1A and GBM 1B cells. The multiple immunoreaction bands indicate multisplicing forms of TNC. (B). TNC was detected in the conditioned medium of GBM neurosphere cells, suggesting that TNC may elicit biological function via autocrine or paracrine loop. (C). GBM neurosphere cells were transfected with lentivirals containing nonsilencing shRNA sequence (NS) or TNC shRNA together with a green fluorescent protein coding frame. After transfection, the neurosphere cells were plated as single cells and observed under fluorescence microscopy. Approximately 80%–90% of the transfected cells were GFP+. Bar = 100 µm. (D). Western blot analysis confirmed significant downregulation of TNC in cells receiving 2 distinct TNC shRNAs (TNCKD1 and TNCKD2) compared with control transfected cells (NS). (E). TNC expression in the conditioned medium of TNC knockdown GBM neurosphere cells was also decreased. (F). Immunocytostaining of TNC in GBM neurosphere cells. TNC was highly expressed in control cells, whereas the staining was much weaker in TNC knockdown cells. Bar = 20 µm.

Fig. 2.

Tenascin-C (TNC) loss-of-function facilitates GBM neurosphere cell adhesion. (A). Phase contrast microphotographs of GBM neurosphere cells grown on laminin-coated (10 µg/mL) surfaces for 4 hours. Control cells loosely attached to the laminin-coated surfaces, TNC knockdown cells spread widely on laminin-coated surfaces. When the TNC knockdown cells were plated on TNC-coated surfaces, there was no difference in cell adhesion between control and TNC knockdown cells. Bar = 50 µm (B). Quantitative assays of GBM neurosphere cell adhesion on laminin- or TNC-coated surfaces. Cells were plated for 4 hours; nonadherent cells were washed away; and the remaining adherent cells were measured by MTT assays. Compared with control NS cells, more than 3-5 fold TNC knockdown cells remained on laminin-coated surfaces; whereas TNC-coated surfaces blocked cell adhesion. (C). Control and TNC knockdown cells were plated on Matrigel-coated transwells in growth factor-free GBM medium; the bottom wells were filled with 10% FCS containing DMEM medium. After 24 hours, the invading cells were quantified by counting cells in 5 selected fields per transwell. TNC knockdown cells were less invasive than control cells. (D). TNC knockdown in GBM neurosphere cells did not affect cell growth as indicated by cell number counting in cultures after 3 and 6 days. (*P < .05; ***: P < .001).

Fig. 3.

Decrease of tenascin-C (TNC) in the tumor microenvironment regulates GBM go-or-grow. (A). Immunofluorescent staining of TNC in tumor xenografts derived from control and TNCKD1 GBM neurosphere cells. Hatched areas are enlarged in the two panels on the right; “c” stands for center, “b” stands for border. TNC was highly expressed in control tumor extracellular spaces, both center and border (upper panel). In TNC knockdown xenografts, weak TNC expression was found in the extracellular space along tumor borders, and TNC expression in tumor cores was extremely low (lower panel). (B). Quantification of tumor size based on H&E staining. Xenografts from TNC knockdown cells were significantly bigger than those of control cells. (C and D). H&E staining and fluorescent images showed that TNC knockdown xenografts were less invasive than controls. In TNC knockdown xenografts, tumor cells were confined within distinct tumor borders; whereas in control tumors, cells migrated into adjacent normal brain tissues. (E). Tumor invasion was quantified by counting the number of migrating tumor cells (GFP+) beyond the tumor mass under fluorescent microscopy in 8 randomly selected fields per tumor. Control cells clearly displayed an invasive phenotype as there were 70% more migrating tumor cells beyond tumor borders (***P < .001). Bar = 200 µm.

Fig. 4.

Tenascin-C (TNC) downregulation in the tumor microenvironment promotes intracranial tumor growth. (A). Co-immunostaining of Ki67 and TNC in NS and TNC knockdown xenografts. Compared with control, TNC xenografts showed lower TNC expression with relatively more Ki67+ cells, indicating that TNC knockdown increased tumor cell proliferation in vivo. (B). Quantification of Ki67 staining revealed ∼32% higher proliferation index in TNC knockdown xenografts compared with that of control xenografts (***P < .001). (C). Cleaved caspase-3 immunofluorescent staining showed no significant cell death in control and TNC knockdown xenografts. Bar = 50 µm.

Fig. 5.

Alteration of tenascin-C (TNC) in tumor extracellular spaces influences tumor cell-stromal cell interactions. (A). Laminin staining of intracranial tumors showed that TNC knockdown tumors had fewer blood vessels, but enlarged tumor blood vessel lumen, compared with control. (B). Distribution and morphology of tumor microglia stained with microglial marker Iba1. In control xenografts, the morphology of microglia in tumor center and border was similar, both with long processes, resembling the inactivated, ramified microglia in the brain. In TNC knockdown tumors, microglia in tumor border were similar to those in control tumors with long processes. However, in the center of TNC knockdown tumors, the microglia morphology resembled those activated amoeboid-like microglia with fewer processes. (C). Immunostaining and quantification of active microglia in NS and TNC knockdown xenografts. The tumors were stained with an anti-murine MHCII antibody, which recognizes active microglia in xenografts. MHCII+ cells/field were counted under fluorescent microscopy. Compared with control, TNC knockdown xenografts harbored ∼2 times more MHCII+ cells (N = 5, ***P < .001). (D). The adhesion of rat brain endothelial (RBE) cells was significantly reduced on TNC-coated surfaces compared with that of fibronectin- and laminin- coated surfaces. Phase contrast microphotograph showing RBE cell morphology after being plated on fibronectin (FN) or TNC coated surfaces for 4 hours. (E). RBE cell adhesion was quantified by MTT assays. Compared with fibronectin and laminin, RBE cells adhered poorly on TNC-coated surfaces. (F). Transwells were coated with laminin, fibronectin, or TNC (10 µg/mL). Compared with laminin- and fibronectin- coated surfaces, RBE cells migrated slowly on TNC-coated transwells (***P < .001).

Fig. 6.

Simultaneous targeting of brain tumor cells by combining tenascin-C (TNC) knockdown with temozolomide. (A). Hundred thousand/well NS and TNCKD GBM neurosphere cells were plated in 6 well/plates and treated with temozolomide at 3 µM for 2 days. Phase-contrast microphotographs indicate TNC KD GBM neurosphere cells were more sensitive to temozolomide by showing small neurosphere size and dead cells. (B). NS and TNCKD GBM neurosphere cells were treated with temozolomide at indicated concentrations for 2 days; live cells were quantified by trypan blue exclusion staining and counting. Compared with NS cells, TNCKD GBM neurosphere cells were ∼10 times more sensitive to temozolomide treatment by showing significant cell death (55%) at 3 µm, whereas NS cells showed significant cell death (47%) at 30 µm temozolomide (***P < .001).