Patients and primary tumors

Primary MPNSTs were obtained from the Hospital Infantil La Paz (NF1-18B) and the Hospital Universitari Germans Trias i Pujol (SP-10). Patient NF1-18B received chemotherapy and radiotherapy prior to tumor resection, whereas patient SP-10 did not receive any neoadjuvant treatment. The tumors were analyzed by the pathology department according to the standard protocols and a piece of each tumor was placed in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), 10% fetal bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (P/S, Gibco) at room temperature (RT). In the laboratory, samples were divided into portions and stored as follows: one portion was processed directly for cell line establishment, a second portion was used for mouse engraftment, a third portion was frozen for DNA, RNA, and/or protein extraction, and a fourth portion was cryopreserved using FBS and 10% dimethyl sulfoxide (DMSO). This study was approved by the IDIBELL Ethics Committee (#PR213/13) and all subjects gave written informed consent.

Cell lines establishment and culture

Tumor samples were minced into small fragments and digested with 100 U/mL collagenase (Sigma-Aldrich) and 1 U/mL dispase (Worthington Corporations) in DMEM medium (10% FBS and 1% P/S). After 18–24 h, the enzymes were removed and the digested tissue was filtered through a 40 µM filter to seed single cells into 6-well Corning® plates, which were initially incubated at 37ºC in 10% CO2. After a few passages, the MPNST cell lines were then expanded and maintained at 37ºC and 5% CO2. All cell lines were tested for mycoplasma prior to experimental procedures by a PCR amplification of the 16 S RNA of eight different mycoplasma species (results not shown).

The SP-01 and NF1-09 cell lines used as controls in this work were previously established and described [27], and the human foreskin fibroblast (HFF) cell line was obtained from the American Type Culture Collection (ATCC, BCRJ Cat#0275).

Animal care conditions

Five-week-old male Athymic Nude-Foxn1nu (Envigo) mice were housed in sterile cages with autoclaved bedding, food, and water and maintained on a 12-h light/12-h dark cycle daily.

PDOX establishment

Fresh tumor samples of 2–3 mm3 were implanted into 5-week-old athymic nude mice. Briefly, mice were anesthetized with isoflurane (continuous flow of 1–3% isoflurane/oxygen mixture, 2 L/min), and a subcutaneous pocket was made in the upper thigh with surgical scissors. A small incision was then made in the muscle to expose the sciatic nerve, where the piece of tumor was engrafted using Prolene 7.0 suture to grow around the epineurium. After implantation, tumor growth was monitored weekly by palpation and volume was measured with a caliper. When the tumor reached 1,000 to 1,500 mm3, the mice were sacrificed and the tumors were passed to other animals. After each passage, tumor samples were cryopreserved to provide a source of viable tissue. Mouse experiments were approved by the campus animal ethics committee and followed the procedures of the Association for Assessment and Accreditation of Laboratory Animal Care International.

DNA isolation and quantification

Puregene Core Kit A (Qiagen) was used to isolate DNA from frozen tumors and cell lines, according to the manufacturer’s recommendations. For tumor DNA extraction, TissueLyser (Qiagen) was first used to homogenize the tissue. DNA quantity of each sample was assessed using Qubit (Qubit™ dsDNA BR Assay Kit, Invitrogen) and quality was assessed using NanoDrop 1000 spectrophotometer (ThermoFisher Scientific). Visual inspection of the DNA integrity was also performed on 1% agarose gels.

Short tandem repeat (STR) authentication

DNA fingerprints were obtained using the AmpFISTR Identifiler Plus PCR Amplification Kit (Applied Biosystems) according to the manufacturer’s protocol. The kit amplifies 15 tetranucleotide STR loci and the sex-determining marker amelogenin in a single PCR amplification using 33 primers. Allele calls were made from peak plots by comparing peaks to known fragment sizes using GeneMapper 4.0 (Applied Biosystems). This analysis was performed on NF1-18B cell line passage 3 and SP-10 cell line passage 9.

Cell cycle analysis

A total of 5 × 105 cells from an 80% confluence plate were fixed with frozen 70% ethanol and stained with a mixture of phosphate-buffered saline 1X (PBS 1X, Gibco) + 1% FBS, propidium iodide (0.0625 mg/mL; Sigma-Aldrich), and RNAse A (10 µg/mL; Sigma-Aldrich) for 30–45 min at 37ºC. Each cell line was tested in triplicate. The HFF cell line was used as a diploid control. 20,000 events per sample were analyzed using a FACS Canto II cytometer (Becton Dickinson) and ModFit LT V.3.3.11 software. Goodness of fit to the model is expressed by the reduced chi-square (RCS).

Whole genome sequencing (WGS)

WGS was performed at BGI (Shenzhen, China). Briefly, libraries were prepared according to standard DNBseq protocols, sequenced on a BGISEQ-500 to a median of 881 million 150-bp paired-end reads per sample, and mapped to the GRCh38 genome using BWA-MEM [28]. WGS data were processed as described in [15].

Immunofluorescence

Cells seeded in 12-well Corning® plates with coverslips (12 mm Ø) were fixed in 4% paraformaldehyde for 15 min. Cells were then permeabilized with PBS 1X + 0.1% Triton X-100 (Sigma-Aldrich) for 15 min and blocked with 10% goat serum (Gibco) for 30 min at RT. Antibodies anti-SOX9 (1:100; ab76997, Abcam), smooth muscle actin (SMA, 1:100, RB-9010-R7, ThermoFisher Scientific), S100B (1:1000, Z031129, Dako), H3K27me3 (1:1500, 9733, Cell Signaling), and SOX10 (1:50, 383R-14, Cell Marque) were diluted in PBS 1X-1% goat serum and incubated overnight (ON) at 4oC; then the cells were washed 3 times for 5 min with PBS 1X. Secondary antibodies Alexa Fluor 488 goat anti-mouse (1:1000, A11029; Invitrogen) and Alexa Fluor 568 goat anti-rabbit (1:1000, A11036; Invitrogen) were diluted in PBS 1X-10% goat serum and incubated for 1 h at RT. After three 5-minutes washes with PBS 1X, the nuclei were stained with DAPI (1:1000, ThermoFisher Scientific) for 10 min at RT. Finally, coverslips were mounted with Immu-Mount (ThermoFisher Scientific). Images were captured using a Nikon Eclipse 80i microscope and NIS-Elements Microscope Imaging software.

Population doubling time

Population doubling times (PDTs) were calculated using the MTT (3-(4,5-dimetrhylthiaziol-2-yl)-2,5-diphenyl-tetrazolium bromide, Sigma-Aldrich, #M2128-1) colorimetric cell viability assay [29].

Different numbers of cells for each cell line were seeded in six replicates in Corning® 96-well plates to reach 100% confluence after 8–9 days of culture: 1,100 cells/well for NF1-18B and 1,300 cells/well for SP-10. Every 24 h 0.5 mg/mL of MTT was added to each well. After 3 h of incubation, the formazan precipitate was diluted by adding 100 µL of a 1:3 solution of glycine buffer (0.1 M NaCl and 0.1 M glycine) and DMSO to each well. Absorbance was measured at 540 nm with VictorTM X5 2030 Multilabel Reader (PerkinElmer). PDT was calculated using an exponential growth equation with GraphPad Prism 6.

2-dimension (2-D) colony formation assay

Cells were seeded in triplicate at a density of 300 cells/well in 12-well Corning® plates. After 10 days, cells were fixed with ice-cold methanol for 10 min and stained with 0.1% crystal violet (80% H2Od + 20% methanol) for 15 min.

Hanging drop assay

200 cells in a 20 µL drop of DMEM were seeded on the top of the inverted lid of a 60 mm plate. 20 drops were placed in each plate and three plates were used for each cell line. 5 mL of PBS 1X was added to the bottom of the plate. The lid was inverted onto the PBS 1X filled bottom chamber and incubated at 37ºC in 5% CO2. Droplets were monitored at 24 and 48 h and images were captured with a Leica DM IL LED light microscope using the contrast phase mode from Leica Microsystems’s.

Wound healing assay

7 × 105 cells/mL were seeded (70 µL/well) into culture inserts (2 wells; Ibidi #80209) to reach confluence after 24 h, then the culture inserts were removed and the cells were cultured under standard conditions. Images were captured at 0, 4, 8, 12, and 24 h after removal of the inserts using a Leica DM IL LED light microscope at 10X magnification using the contrast phase mode from Leica Microsystems’s. Each cell line was seeded in triplicate.

In vivo tumorigenicity

3 × 106 tumor cells in 100 µL of PBS 1X were injected intramuscularly near both sciatic nerves of five-week-old female athymic nude mice (N = 2 for the SP-10; N = 3 for each of the two NF1-18B subpopulations). Animals were monitored weekly.

When tumors reached 1 cm in diameter, they were excised, cut into small fragments, and engrafted into mice (N = 4) both subcutaneously and orthotopically to assess tumor growth rate for each engraftment procedure. Tumors were measured twice weekly with a caliper, and tumor volume was calculated using the formula v = (pi/6 x L x W x W) [L: length; W: width]. All experiments with mice were approved by the IDIBELL Animal Care and Use Committee (#9111).

Immunohistochemistry analysis

Clinically relevant MPNST markers were analyzed in patient tumors, PDOX tumors, and cell lines. For the latter, cells were trypsinized and washed with PBS 1X, then equal volumes of human plasma and thrombospondin (Grifols) were added until a uniform pellet was obtained, which was embedded in paraffin.

Paraffin-embedded tumor and cell line Sections. (3 μm) were deparaffinized in xylene and gradually rehydrated through graded ethanol solutions. Antigen retrieval was performed by heating the tissue sections at 110ºC for 15 min in citrate buffer (pH = 6). Endogenous peroxidases were then blocked by incubation with hydrogen peroxide (3% H2O2 + 30% methanol + H2Od) for 15 min at 4ºC. Blocking was performed by incubation with 5% goat serum for 1 h. Primary antibodies anti-vimentin (1:200, 180052, Life Technologies), Ki67 (1:10 M7240, DAKO), SOX10 (1:50, EP268 Cell Marque), H3K27me3 (1:200, 9733, Cell Signaling), and S100B (1:300, Z031129, Dako) were incubated ON at 4ºC followed by 30 min at RT. Secondary antibodies EnVision + System HRP-conjugated polymer anti-rabbit or anti-mouse (DAKO) were incubated for 1 h at RT. Finally, detection was performed by incubation with diaminobenzidine (DAB) (DAKO) for 10 min and nuclei were counterstained with hematoxylin. Images were captured using a Nikon Eclipse 80i vertical microscope.