Abstract 5311: Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1)
Authors: Arima Yoshimi , Nobusue Hiroyuki , Sakai Shigeki , Kishi Kazuo , Takenouchi Toshiki , Kosaki Kenjiro , Saya Hideyuki 
Published: August 2019

Background: The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that negatively regulates the Ras signaling pathway by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. NF1 gene germline mutations cause various Neurofibromatosis type 1 (NF1) symptoms, including neurofibroma development. We are developing in vitro models that recapitulate the pathological and clinical properties of neurofibromas with the aim of developing therapeutic strategies to treat patients with NF1 gene-deficient tumors.

Methods: Neurofibroma cells and dedifferentiated fat (DFAT) cells were established from NF1 patient tumors. Tissue samples were obtained during tumor resection at our hospital from patients who met NIH clinical diagnostic criteria for NF1. Whole-blood specimens were also obtained for gene analysis. All patients provided written informed consent. The institutional review board at our university approved this aspect of our study. Tumor tissues were dissociated in DMEM containing collagenase. The neurofibroma cells at the bottom of the tube and the floating stromal adipocytes were collected separately after centrifugation. To establish DFAT cells, the stromal adipocytes were placed in a culture flask filled with 20% FBS-DMEM, and then the flask was inverted and incubated at 37 °C in a humidified atmosphere of 5% CO2. The stromal adipocytes floated up through the medium and adhered to the ceiling of the flask. After 1 week, the cells were firmly attached to the ceiling and had dedifferentiated. The DFAT cells as well as the neurofibroma cells can be passaged. The DFAT cells exhibited multipotent differentiation abilities into a variety of cell types.

Results: We established neurofibroma cells and DFAT cells from NF1-associated neurofibromas. We performed flow cytometry analysis and found that the cells derived from NF1 patients expressed SOX10, S100, and CD90, all of which are expressed in Schwann cells. We identified the NF1 mutations in patients by next-generation sequencing. Peripheral blood specimens from patients 1 and 2 were positive for c.1466A&gt;G, p.Tyr489Cys and c.3213_3214delAA, p.Ser1072Hisfs*16 mutations of NF1, respectively. We also identified NF1 mutations in the cells that we had established from tumors. In the tumor specimen of patient 1, we identified an additional somatic mutation, c.6772C&gt;T, p.Arg2258X of NF1 gene.

Conclusions: We established NF1 gene-deficient neurofibroma cells and NF1 gene-deficient DFAT cells from the tumor tissues from NF1 patients with NF1 gene mutations. These cells may well be useful in studying the pathophysiology of NF1 gene-deficient tumors as well as cell-based drug screening to facilitate the development of new treatments.

Note: This abstract was not presented at the meeting.

Citation Format: Yoshimi Arima, Hiroyuki Nobusue, Shigeki Sakai, Kazuo Kishi, Toshiki Takenouchi, Kenjiro Kosaki, Hideyuki Saya. Establishment of neurofibroma cells and dedifferentiated fat (DFAT) cells from tumor tissues from patients diagnosed with NF1 (Neurofibromatosis type 1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5311.