Early and continuing dispersal of tumor cells from the primary mass renders GBM refractory to complete surgical excision or targeted chemotherapy and directly leads to recurrence and dismal prognosis. Strategies aimed at containing the primary or recurrent tumor could significantly improve targeted delivery of chemotherapeutic agents and increase the likelihood of total surgical resection. To disperse, cells must first detach from the primary mass, a process that likely involves mechanisms that decrease cohesion between tumor cells [
]. Cells must also attach to substrates at strengths that optimize their motility and secrete factors to facilitate their interaction with parenchyma [
]. In addition, tumor cells must also become relatively compliant so as to deform and “squeeze” through pores in a meshwork of ECM components [
], and in the case of GBM, astrocytes within the normal brain parenchyma. Accordingly, strategies aimed at preventing tumor cell detachment, limiting motility, and inhibiting changes in compliance offer an effective approach to reduce dispersal. Ideally, such strategies should employ pharmacological agents that can cross the blood–brain barrier and that specifically target molecular pathways involved in mediating cohesion, adhesion, and compliance.
Cadherins, integrins and the extracellular matrix (ECM) are potential therapeutic targets, and various studies have identified drugs that can modulate their expression or function. For example, gamma-linolenic acid (GLA) up-regulates E-cadherin expression and inhibits invasion of lung, colon, breast, melanoma, and liver cancer [
]. Invasion suppression here was likely due to an increase in the strength of intercellular cohesion mediated by up-regulation of E-cadherin. 5-aza-deoxycitidine (5 AC) has also been shown to effectively inhibit invasion by up-regulating E-cadherin expression [
]. Because the down-regulation of E-cadherin is often associated with up-regulation of N-cadherin during epithelial-mesenchymal transition, drugs that can block N-cadherin expression have also been shown to be effective in blocking invasion. Biflorin, a novel o-naphtoquinone, has been shown to inhibit expression of N-cadherin and to block invasion of breast cancer cells [
]. Such drugs could be of potential benefit for glioblastoma given the correlation between increased N-cadherin expression in high-grade gliomas and tissue invasion [
]. Various integrins, including αvβ3 and αvβ5 have also been targets of anticancer therapy. Cilengitide, a cyclic pentapeptide, is a specific inhibitor of these integrins and has been shown to have anti-invasive activity in various glioma models [
]. Given the complexity and heterogeneity of the ECM, and the likelihood that glioma cells tune their integrin receptor fingerprint to match the local ECM microenvironment, drugs that modulate the ECM may prove effective in reducing dispersal. Many of these drugs, including various corticosteroids, target the ECM as a by-product of the drugs’ principal actions. Consequently, this activity may in part be beneficial to the drugs’ disease-modifying properties [
]. An example of such a drug is Dexamethasone (Dex). Dex is currently used to treat brain tumor-related edema associated with mass effect from Glioblastoma [
]. A by-product of the effects of Dex in glioblastoma is its ability to restore fibronectin matrix assembly (FNMA) and decrease detachment of tumor cells from cultured 3D spheroids [
]. However, due to the relatively high doses required, Dex has many side-effects, often limiting its long-term use. Identification of other drugs that can have similar effects but more specifically target pathways involved in modulating integrins and the ECM could be of therapeutic value.
The MAPK/ERK pathway has been identified as a commonly dysregulated pathway in several cancers, most notably in melanoma. Combined targeting of this pathway can have a synergistic effect in controlling tumor growth [
]. Clinical trials using various MEK inhibitors, such as trametinib [
], cobimetinib [
] and CI 1040 (PD184352) [
] have been shown to shrink some melanomas, specifically those with BRAF mutations. The MEK inhibitor PD0325901 has also demonstrated efficacy in melanoma cell lines independent of BRAF status [
]. Experimental models have demonstrated in vitro and in vivo efficacy of PD0325901 in controlling tumor growth in animal models of GBM [
], although studies have identified possible issues with limited access through the blood–brain barrier [
]. To our knowledge, there is only one ongoing phase-2 trial testing the effects of PD0325901 on tumor growth in patients with neurofibromatosis type −1 (NF1) or plexiform neurofibromas [
] NCT02096471), and none testing efficacy in GBM. The majority of these studies have focused mainly on inhibition of growth and on activation of apoptosis. Inasmuch as MEK inhibitors target pathways that can also influence actin organization and remodeling of the ECM, we asked whether PD0325901 could also serve to impact mechanisms that regulate dispersal of primary human GBM cells.
We first determined whether primary human GBM cells used in this study are sensitive to PD0325901. We then assessed the effects of MEK inhibition on integrin activation
vis à vis restoration of FNMA and actin organization in both 2D and 3D cultures. We also quantified the effects of PD0325901 on spheroid mechanical properties including cohesion, stiffness and viscosity. We evaluated effects of PD0325901 in regulating the strength of cell-substrate adhesion, cell motility, dispersal of tumor cells from spheroids, and in an ex vivo dispersal assay. Finally, we determined whether PD0325901 could also influence the growth rate of both 2D and 3D cultures of GBM.
Therapies aimed at containing tumor cell dispersal could provide a powerful path towards extending the time of disease-free and overall survival of glioblastoma patients. Identifying drugs that can target molecular pathways involved in dispersal would provide valuable insight towards this goal. Our previous studies showed that Dexamethasone, an FDA approved drug to treat tumor-related edema in GBM, can also decrease in vitro and ex vivo dispersal of primary human GBM cells. It does so by activating α5β1 integrin and subsequent restoration of FNMA and re-organization of cortical actin into stress fibers. In turn, these changes engender an increase in the strength of intercellular cohesion, increased attachment of tumor cells to substrate, and reduced cell motility. The net effect is an overall reduction in dispersal [
]. The effects of Dex, however, are pleiotropic and the drug likely targets many pathways, which in part may explain the many side-effects associated with Dex treatment. Identifying drugs that are more specific in their targeting of dispersal-related pathways is therefore important.
In this study, we explore whether inhibition of the MAPK/ERK pathway, a critical regulator of processes underlying invasion and metastasis [
], could have similar effects on GBM dispersal. We tested the effects of the MEK inhibitor, PD0325901, on 4 primary GBM cell lines that were previously used to assess the effects of Dex on dispersal [
]. Studies have shown that certain GBM lines do not respond to MEK inhibitors [
]. We therefore assessed whether our lines are responsive to PD0325901 by determining whether treatment results in a decrease in the levels of phospho-ERK. All 4 lines responded to the drug. We previously established that the cell lines were all deficient in their capacity for FNMA [
]. In contrast to Dex, treatment with PD0325901 did not result in a significant increase in FNMA. However, treatment with the MEK inhibitor resulted in a remarkable change in cell shape and in the reorganization of actin from cortical into stress fibers. This was particularly evident when actin was visualized in 3D spheroids. Given that the actin cytoskeleton is a fundamental mediator of cell and tissue stiffness [
], we posited that a shift in actin organization would correspond to a change in tissue stiffness.
Stiffening of the ECM is considered to be a hallmark of fibrotic lesions and has been demonstrated to modulate cell invasion and migration [
]. The current study focused on whether aggregate stiffness and viscosity could modulate dispersal. We quantified stiffness and viscosity using methods based on ellipsoid relaxation, specifically after the deforming external force is removed [
]. The aggregate was modeled as a Kelvin-Voigt viscoelastic body [
]. Unexpectedly, PD0325901 treatment only resulted in a modest increase in aggregate stiffness but not of viscosity. However, when aggregates were generated in higher concentrations of fibronectin, both stiffness and viscosity increased significantly. This is important for several reasons. First, the fibronectin gene has been shown to be up-regulated in GBM [
]. Accordingly, tumors able to respond to PD0325901 and in the presence of high concentrations of fibronectin, could, in principle, become stiffer and more viscous. Stiffer tumors have previously been shown to be less invasive and to grow more slowly [
]. Few studies have addressed the issue of tumor viscosity and those that have focus on applications of magnetic resonance elastography in liver tumors where fibrosis is a key parameter. In those studies, tumor viscosity appeared to be higher in malignant tumors [
]. In GBM, however, fibrosis is not typically observed. In GBM spheroids, the increase in viscosity in response to PD0325901 treatment was likely due to higher binding energy between the activated α5β1 integrin and fibronectin. This would effectively increase the friction between cells and the ECM. This increase in friction could significantly reduce the capacity for dispersal of tumor cells from the primary mass.
Treatment also resulted in the localization of p-FAK at sites of cell-substrate attachment. This is consistent with the observed resistance to flow-induced substrate detachment of GBM cells, and to decreased motility. Since cells require intermediate levels of cell-ECM adhesion to be optimally motile [
], an increase in the strength of cell-ECM adhesion past this point might stabilize adhesion to substrate to a point that significantly reduces cell movement, and consequently, dispersal. Decreased motility also appears to be associated with a significant decrease in dispersal velocity of GBM aggregates. Since PD0325901 treatment did not restore FNMA, it is likely that decreased motility rather than increased cohesion is the physical mechanism that restrains the detachment of tumor cells from the mass. Indeed, cells at the leading edge of treated aggregates appear to attach tightly to substrate causing cells behind them to pile up, again pointing to reduced motility as the primary restraint for detachment. For three of the four primary GBM lines, PD0325901 also significantly reduced the ability of single GBM cells to disperse through an astrocyte-seeded scaffold. It is not possible to differentiate between the effects of PD0325901 on decreased motility and ability to disperse through the scaffold, however, it is possible that on a single cell level, the re-organization of actin into stress fibers may have effectively rendered cells less compliant and inhibited their capacity to sufficiently deform and squeeze through pores established by the physical environment established by the scaffold. It is important to note that for GBM-4, treatment did not reduce z-axis dispersal. It is possible that in this line, compliance was not effected by treatment, thus allowing cells to penetrate into the scaffold.
Lastly, MEK inhibitor treatment also appears to significantly reduce growth rate of these primary GBM lines in both conventional 2D and in 3D cultures. Other studies have demonstrated in vivo efficacy of PD0325901 in reducing tumor growth in preclinical orthotopic models of glioblastoma [
]. Our study provides compelling evidence that PD0325901 can also reduce dispersal. Growth and dispersal contribute significantly to recurrence. Accordingly, the drug has the potential to significantly delay the onset of recurrence in GBM.
Identifying agents that can contain the primary or recurrent tumor could significantly improve targeted delivery of chemotherapeutic agents and increase the likelihood of total surgical resection. We have previously identified Dexamethasone (Dex) as a potential candidate to reduce dispersal of GBM [
]. Interestingly, the doses required to elicit a dispersal inhibitory response are significantly lower than those typically used to reduce edema [
]. Clinically, MEK inhibitors are generally well tolerated. Commonly occurring toxicities include rash, diarrhea, fatigue, peripheral oedema and acneiform dermatitis. Life-threatening toxicities associated with MEKi are extremely rare. Long-term use is possible providing that adverse events are monitored and dose or treatment schedules are modified, as required [
]. The measureable outcome for MEK inhibitor studies focus on their ability to reduce tumor size. Here, we show an added benefit of one MEK inhibitor as a potential deterrent of tumor cell dispersal. Whereas Dexamethasone readily crosses the blood–brain barrier, some MEK inhibitors, including trametinib, have demonstrated limited brain distribution due to association with the P-glycoprotein efflux transporters found at the blood–brain barrier [
]. Perhaps a strategy in which MEK inhibitors are used as interstitial chemotherapy, followed by continued administration of low-dose Dex, could significantly improve prognosis of this devastating disease.
The authors gratefully acknowledge the efforts of Dr. Joseph Kramer, Director, Confocal Imaging Facility, Rutgers-Robert Wood Johnson Medical School, for assistance in image acquisition and analysis.
SS was supported by an NIH Institutional Research and Academic Career Development Award K-12 GM093854. MY received funding from the NSF-CMMI. LL was supported by grants from NSF-DMS and from the AFOSR. The study was also supported from research funds from the Department of Surgery-Robert Wood Johnson Medical School to RAF.
Availability of data and materials
SS performed and analyzed the dispersal velocity and aggregate spreading assays. DJ performed the immunoblot and immunofluorescence assays. IE generated and analyzed the cell attachment data. PB, CC and JC performed the tissue surface tensiometry assays for measurement of aggregate cohesion and analyzed the data. AM performed the experiments for measurement of aggregate stiffness and viscosity. MY designed the in-house image capture and analysis algorithm and also analyzed the shape relaxation images for extraction of the stiffness and viscosity data. CV conceived, performed and analyzed the cell motility assay. MW generated and analyzed the ex vivo dispersal data. DS, JDZ, LL and HL conceived, designed, and mathematically solved the solution for extraction of stiffness and viscosity. They also supervised the project at the Departments of Biomedical Engineering (DS, JDZ) and Mechanical and Aerospace Engineering (LL, HL), respectively. RAF conceived the study, supervised the overall project, statistically analyzed the data, generated the figures, interpreted the results, and wrote the manuscript. All authors reviewed and edited the manuscript for intellectual content and have approved the final version.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Tumor samples were obtained with approval of the Rutgers-Robert Wood Johnson Medical School Institutional Review Board under protocol #CINJ 001208. Samples were anonymized and the IRB waived the need for written consent. Cell lines GBM-1, GBM-2, GBM-3 and GBM-4 were developed directly from these tumor samples and are therefore covered under the same IRB protocol.