Furthermore, cell velocity upon DIS, CBX and combined treatment was analysed. adherent differentiated cells can be blocked to a similar extent by Carbenoxolone, as both cell populations form gap junctions, but the adherent differentiated cells are much more sensitive to Disulfiram treatment, which C via modulation of NF-B signalling C interferes with cell-substrate adhesion. Interestingly, inducing adhesion in tumour-initiating cells without differentiating them does not sensitize for Disulfiram. Importantly, combining Disulfiram, Carbenoxolone and the standard chemotherapeutic drug Temozolomide reduces tumour size in an orthotopic mouse model. Isolating GB cells from their direct environment within the brain represents an important addition to current therapeutic approaches. The blockage of cellular interactions via the clinically relevant substances Disulfiram and Carbenoxolone, has distinct effects on different cell populations within a tumour, potentially reducing motility and/or resistance to apoptosis. Introduction Glioblastoma (GB), formerly Glioblastoma multiforme, is the most common cancer of the central nervous system with poor prognosis exemplified by patient survival of about one year after diagnosis1. Despite intensive treatment involving mTOR inhibitor (mTOR-IN-1) tumour resection, radiation and chemotherapeutic treatment with Temozolomide (TMZ), GB inevitably recurs2. GB is a highly aggressive malignancy with rather unique features: while it only rarely metastasizes outside the neuraxis3, it is almost invariably found to be highly invasive upon presentation4. It is still debated whether GB should be viewed as a tumour within the brain or a systemic whole brain disease. The latter view had been particularly popular among early radiation oncologists5 and is currently gaining favour once more6. In extreme cases, GB can be lethal in the complete absence of tumour bulk4. The unfocused nature of this disease makes localized treatment, e.g. maximal safe surgery, particularly ineffective7. After excision of the tumour bulk, recurrence manifests within 2C3?cm of the resection cavity in more than 95% of cases4. The invading GB cells often associate with distinct anatomic structures, e.g. myelinated axons, basement membranes of blood vessels, other basement membrane-like structures, and the so-called secondary structures of Scherer8. These structures are known to confer increased resistance to apoptosis9,10 by inducing various pro-survival signalling cascades C a phenomenon we have previously referred to as AMAR, or adhesion-mediated apoptosis resistance11. Previous targeted therapies blocking individual adhesion receptors such as cilengitide (inhibitor of v3 and v5 integrins) have had limited efficacy in GB clinical trials12. The poor efficacy of targeted adhesion blocking therapies may be limited in part by redundancy in multiple adhesion receptor mediated signalling events, which confer AMAR across the disseminated GB microenvironment of Cd63 the brain. Therefore, a multi-targeted approach of blocking adhesion signalling in GB should minimize the conversation of tumour cells with their surroundings, reduce invasion and re-sensitize cancer cells for apoptosis. To test this hypothesis, we selected two forms of cellular conversation which have been shown to mTOR inhibitor (mTOR-IN-1) contribute to GB biology C cell-matrix interactions and gap junctions. Cell-matrix interactions are usually formed via integrin engagement that tethers the cell to its surroundings and activates complex intracellular signalling cascades11. We recently showed that invasive GB cells are associated with fibronectin that is secreted and processed by the tumour cells via plasminogen and matrix metallopeptidases13. Importantly, the creation of this new extracellular matrix (ECM)-based microenvironment was initiated upon a stress response resulting from mTOR inhibitor (mTOR-IN-1) the reduction of cell-cell interactions, which brought on NF-B activation13. Blocking NF-B activation via the nonspecific, but well-tolerated, inhibitor Disulfiram (DIS) reduces both tumour bulk and cellular invasion in an orthotopic mouse model13. This is also in line with previous data that suggest that Disulfiram-mediated inhibition of NF-B sensitizes colorectal cancer cells for cell death14. In contrast, gap junctions are formed between adjacent cells. They have been described to form transiently during invasion between GB cells and astrocytes as well as part of long-distance multicellular network structures15,16. Our own data, using the glycyrrhetinic acid-derivative Carbenoxolone (CBX) for the inhibition of gap junctions, also suggest that stable gap junctions contribute to the close cell-cell conversation associated with the tumour bulk, and that these structures clearly contribute to apoptosis resistance17. Therefore, we postulated that inhibition of cell-fibronectin conversation should mainly affect invasive/stressed cells, while blocking gap junctions should influence both invasive cells and tumour bulk – the latter more strongly however, as gap junctions are more stable in that context. To mimic the intratumoral heterogeneity, i.e. the presence of competing and supporting subpopulations of tumour cells that adds to the complexity of the disease18 in an experimental system, we used two genetically identical cell populations with different.