Associate Professor Rensselaer Polytechnic Institute, United States
Introduction:: Neurofibromatosis type I (NF1) is a tumor-predisposition disease. NF1 affects 1 in 3000 people and has a wide range of symptoms including altered skin pigmentation to learning disabilities to the formation of various tumors.1 One tumor type in NF1 is the plexiform neurofibroma (PNF), a benign tumor that initiates from Schwann cells (SCs) and forms in the peripheral nervous system. PNFs, despite being benign, can cause disfigurement, pain, and organ impaction. Additionally, PNFs have a 10-15% chance to transform into malignant peripheral nerve sheath tumors (MPNSTs), the precise causes of which are not understood.1 Recent advances in drug treatments have significantly benefitted NF1 patients, but there is still no known cure for PNFs, and the treatment options for MPNSTs are limited. Altered cell and tissue mechanics are linked to tumor initiation, growth, and metastasis in some cancers2, but these biomechanics have not been studied for NF1. Here, in an effort to identify much-needed cancer biomarkers for NF1 patients, we initiated these biomechanical studies by characterizing healthy and tumor tissues from mouse models of PNF and MPNST.
Materials and Methods:: Plexiform neurofibromas were generated spontaneously in the DRG of NF1 haplo-insufficient (Nf1+/-) mice, where targeting of the second Nf1 allele in Schwann cells is by introduction of Cre recombinase on the Krox20 gene (Nf1Fl/-; Krox20Cre/+). NF1-associated and non-NF1-associated (sporadic) MPNSTs were generated with injections of a CRISPR-Cas9 system (including sgRNA targeting Nf1 and Trp53 packaged into adenoviral particles3) into the sciatic nerve of Nf1+/- and WT mice. Tumor progression was continuously monitored, and mice were humanely euthanized when tumors reached 1 cm. As healthy control tissues, sciatic nerves (SN) and dorsal root ganglion (DRG) of healthy WT adult mice were similarly biopsied. All mouse work was performed in accordance with IACUC approved protocols and under the supervision of a resident veterinarian. Biopsied tumors and control nerve tissues were transferred to a protease inhibitor cocktail ahead of mechanical testing using atomic force microscopy (AFM) and histological analysis performed on tissue sections following immunofluorescence or hematoxylin and eosin (H&E) standard protocols.
Results, Conclusions, and Discussions:: Results and Discussion: We characterized native environments for Schwann cells the dorsal root ganglion and sciatic nerves. Our atomic force microscopy measurements of the tissues revealed a bimodal distribution of stiffness with peaks from 50-200 Pa and 500-700 Pa. Initial mechanical data from an NF1 associated tumor and non-NF1 tumor was taken revealing the NF1 tumor had a significantly greater stiffness (3900 Pa) than healthy environments (490 and 390 Pa) and then the tumor from a wildtype mouse (606 Pa). Additionally, NF1 tumors displayed varying stiffness based on region within the tumor rather than a uniform stiffness as was found in the wildtype tumor. The NF1 tumor was softest at the tumor core (559 Pa), displaying a similar stiffness to the healthy tissues and tumor, followed by a middling stiffness in the intermediate region (1083 Pa), and the stiffest region at the tumor edge (8474 Pa). Combining these measurements with H&E images we will generate stiffness and structure profiles for all tumor types. These initial results point to the potential importance of the NF1 genotype in the formation and behaviors of theses tumors. Conclusions: Our initial NF1 tumor displayed different stiffnesses than both healthy tissues and tumors from wildtype mice suggesting an importance of the NF1 genotype in tumor development and progression. We also have identified the initial mechanics Schwann cells face in the peripheral nerve environments of either sciatic nerves or dorsal root ganglion. This work plays a crucial role in understanding the mechanism driving the mechanobiology of tumor formation in NF1 as we elucidate how the tumorigenic Schwann cells ultimately result in the PNF and MPNSTs. Understanding the mechanical properties of both healthy and tumor tissues may help better identify tumorigenic and malignant factors in NF1 and provide a biomarker for NF1 tumor progression.
Acknowledgements (Optional): : Acknowledgements: New Investigator Award (K. Mills) from the DoD CDMRP Neurofibromatosis Research Program (NF180070). NF1 fibroblasts and Schwann cells are from Dr. Peggy Wallace at the University of Florida.
References (Optional): : References: 1. Gutmann, D. H. et al. Nat. Rev. Dis Primers 3, (2017). 2. Plodinec, M. et al. Nat. Nanotech 7, (2012). 3. Huang, J. et al. Nat. Commun.8, 15999 (2017)
