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Plexiform neurofibromas (Pnfs) are benign peripheral nerve sheath tumors that are major features of the human genetic syndrome, neurofibromatosis type 1 (NF1). Pnfs are derived from Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1+/− milieu and thus are variably lacking in the key Ras-controlling protein, neurofibromin (Nfn). As these SCs are embedded in a dense desmoplastic milieu of stromal cells and abnormal extracellular matrix (ECM), cell–cell cooperativity (CCC) and the molecular microenvironment play essential roles in Pnf progression towards a malignant peripheral nerve sheath tumor (MPNST). The complexity of Pnf biology makes treatment challenging. The only approved drug, the MEK inhibitor Selumetinib, displays a variable and partial therapeutic response. Here, we explored ECM contributions to the growth of cells lacking Nfn. In a 3D in vitro culture, NF1 loss sensitizes cells to signals from a Pnf-mimicking ECM through focal adhesion kinase (FAK) hyperactivation. This hyperactivation correlated with phosphorylation of the downstream effectors, Src, ERK, and AKT, and with colony formation. Expression of the GAP-related domain of Nfn only partially decreased activation of this signaling pathway and only slowed down 3D colony growth of cells lacking Nfn. However, combinatorial treatment with both the FAK inhibitor Defactinib (VS-6063) and Selumetinib (AZD6244) fully suppressed colony growth. These observations pave the way for a new combined therapeutic strategy simultaneously interfering with both intracellular signals and the interplay between the various tumor cells and the ECM.

A methodology for the characterization of kinetochore-containing (CREST+) micronuclei (MN), based on the use of antikinetochore antibodies (derived from CREST patients) and indirect immunofluorescence, was applied to mouse bone marrow erythrocytes. The proposed protocol allows us to obtain fluorescent signals of good quality and highly reproducible data. The clastogenic agent mitomycin C (MMC; 1 mg/kg body wt) and the two aneugenic compounds chloral hydrate (CH; 200 mg/kg body wt) and colchicine (COL; 1 mg/kg body wt) were used to verify the sensitivity of this approach to chemicals with different mechanisms of action. These compounds were tested at a 20 h time interval from treatment and all of them were able to significantly increase (P less than 0.001) the frequency of MN in polychromatic erythrocytes. Of the MN observed in preparations from control animals, 45% were CREST+ and this percentage increased significantly (P less than 0.001) after treatment with CH or COL. On the contrary, only 22% CREST+ MN were found after treatment with MMC (statistical comparison with the control value: P less than 0.001). The CREST characterization of MN induced in vivo in mouse bone marrow allows us to infer the origin of MN formation, thus contributing to the identification of aneugenic agents.

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