Various cancer cell lines have been previously demonstrated to generate spheroids when placed in hanging drop culture, including the lung cancer cell line Lewis Lung Carcinoma [1
], human fibrosarcoma HT-1080 [31
], cells of the glioblastoma U87-MG series [3
], and breast cancer MCF-7 cells [33
]. For these lines, aggregates either spontaneously formed spheroids or were induced to do so by embedding cells in extracellular matrix. Two of the three prostate cancer cell lines used in this study, AT-2 and JHU-3, spontaneously formed spheroids when placed in hanging drop culture. However, in order to generate sufficiently large spheroids for measurement of aggregate cohesion, it was necessary to admix normal rat fibroblasts with the MLL cells at a ratio of 1:4. The inclusion of fibroblasts provided sufficient motility within the aggregate to elicit the shape change required for aggregates to become spherical. Accordingly, we also admixed fibroblasts with AT-2 or JHU-3 cells so as to be able to compare aggregate cohesion between lines. We demonstrated an inverse relationship between aggregate cohesion and invasive index. We also showed that aggregate cohesion is independent of size and of the applied force [23
], confirming that the TST measurements reflect real differences in cohesion between the three cell lines.
We also established a correlation between aggregate cohesion and capacity for FNMA. Earlier studies have ascribed a role for FNMA as a mediator of strong tissue cohesion in various cell lines including Chinese Hamster Ovary (CHO) cells [15
], and cells derived from glioblastoma tumors [3
]. This is the first demonstration that prostate cancer cells can vary in their ability to assemble a fibronectin matrix and that this correlates with aggregate cohesion, a property previously demonstrated to significantly influence cell detachment [3
], aggregate spreading onto a substrate [34
], and invasive capacity [1
Alpha2Beta1 integrin, the receptor for collagen and other matrix molecules, is significantly down-regulated in poorly differentiated breast cancer [35
], and has been demonstrated to suppress metastasis in mouse and human models of breast and prostate cancer [36
]. We found a similar pattern of expression for α5β1; aggressive MLL cells expressing approximately 7-fold fewer receptors on their surface than JHU-3 cells. This could explain why MLL cells are deficient in their capacity to assemble a fibronectin matrix. Accordingly, we transfected MLL cells with α5 cDNA and bulk-selected a population of cells of increased α5β1 expression (MLL-X5C5). This resulted in increased FNMA, increased aggregate compaction, higher cohesion, and reduced invasive capacity. Compaction and cohesion could be blocked by incubation of MLL-X5C5 cells with the 70 kDa fragment of fibronectin, a fragment previously demonstrated to interfere with FNMA [37
]. Accordingly, it was not possible to perform 3D invasion assays in the presence of the fragment in order to determine whether blocking matrix assembly results in rescue of the invasive phenotype. However, transfection of MLL cells with a chimeric integrin construct in which the cytoplasmic domain of α5 integrin was switched to that of α2 integrin (MLL-X5C2) did not increase aggregate cohesion or decrease invasion. This chimeric construct does not promote FNMA, rather, the fibronectin becomes localized in punctata and fibers do not extend between cells [30
]. This further confirms that an intact matrix, assembled into fibers that extend between cells, is necessary to generate the force required to increase aggregate cohesion and discourage detachment of tumor cells and their subsequent invasion.
We propose that loss of the fibronectin matrix can promote invasion by facilitating the detachment of cancer cells from the tumor mass. Accordingly, loss of α5β1 expression or function represents a possible early mechanism whereby cells can proceed further down the metastatic pathway. Once cells have detached, they become free to move and undergo intravasation. Interestingly, loss of α2β1 integrin is associated with increased intravasation of breast cancer cells [36
]. Since α2β1 is a receptor for collagen and other matrix molecules, it is also possible that loss of this receptor can also give rise to a decrease in tumor cohesion through decreased integrin-collagen (or other ECM) interactions in a similar fashion as the cohesion mediated through the interaction of α5β1 integrin and fibronectin [15
]. Therefore, the combined loss of α5β1 and α2β1 could, in principle, markedly promote metastasis by controlling two key steps in the metastatic cascade: cell detachment and intravasation. The studies described above focus on integrin heterodimers that tend to be down-regulated in more aggressive cancers. Other integrin heterodimers have been shown to be over-expressed in aggressive tumors. AlphavBeta3 [38
] and αvβ5 [39
], for example, are currently being explored in clinical trials as potential targets of integrin antagonists [40
]. Our study suggests that it may also be possible to reduce invasion and metastasis by developing integrin agonists that could act to reactivate integrin expression or function. This has already been demonstrated for glioblastoma cells, where reactivating FNMA by dexamethasone, the MEK-inhibitor PD98059, or the benzoquinone ansamycin antibiotic Geldanamycin, led to a significant increase in aggregate cohesion and reduced aggregate dispersal velocity [3
]. More potent and FDA-approved MEK inhibitors, such as AZD6244, are currently being used in clinical trials for melanoma. Here we show that treatment of MLL cells with AZD6244 resulted in restoration of FNMA by MLL cells and that this manifested in a marked increase in tumor aggregate cohesion. Interestingly, drug treatment did not result in differences in the ability of single cells to migrate through an 8 μm filter, but rather, reduced the ability of tumor cells to detach from the 3D mass.
In conventional 2D cell culture, AZD6244 treatment of MLL cells resulted in a marked reorganization of the actin cytoskeleton and enhanced adhesion to the substrate, processes indicative of integrin activation. A recent report in melanoma cells also showed that AZD6244 induced actin reorganization and promoted integrin-mediated adhesion to substrate [41
]. Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix [42
]. Extended to 3D culture, increased affinity of integrin receptors for substrate could also contribute to the overall increase in aggregate cohesion observed here. On the one hand AZD6244 appears to promote aggregate cohesion, while on the other hand, it also appears to increase affinity of integrins for substrate and could, in principle also promote migration of cells away from the aggregate. These two opposing forces are physically interdependent. A previous study established an interplay between cell-cell and cell-substratum adhesion in mediating aggregate spreading [34
] and it is likely that a similar relationship exists for aggregates of MLL cells. A shift in the balance favoring FNMA-mediated cell-cell cohesion is likely the case here.