Patient’s history

The MFS cell line, named MF-R 3, was obtained from a 79-year-old man, previously diagnosed with high-grade MFS in another hospital, who underwent surgery at the Istituto Ortopedico Rizzoli for a grade 3 MFS second recurrence, localized at the left forearm (see MRI images, Fig. 1A). In the following months the patient presented vertebral metastases (see MRI Fig. 1A bottom right) and died a few months later. Histological diagnosis of MFS was performed by experienced sarcoma pathologists.

Fig. 1

Patient’s data and generation of MFS derived cell line. A Summary of clinical data of the patient (top), Sag T2 FAT Sat MRI of the forearm shows the recurred tumour mass (bottom left), while Sag T2 FAT Sat MRI of the spine (bottom right) shows multiple hyperintense metastatic lesions (blue arrows) with a pathological fracture in T6 (red arrow). B STR profiling data obtained by ATCC of the patient’s tumour sample (left) and MF-R 3 cells at passage 10 (hypothesized immortalization passage) (right)

The research protocol was approved by the Local Ethics Committees (AVEC and CEROM). The number of Ethical Committee approval is PG N. 0012825 of 12/11/2018.

Cell line isolation, culture, and authentication

The MF-R 3 cell line was obtained by mechanical fragmentation and enzymatic digestion of patient tumoural samples as described in De Vita et al. [19] with minor modifications. Specifically, the sample was treated with 2 mg/ml collagenase type I-A (Sigma-Aldrich, Merck Group) in D-PBS with Ca2+ Mg2+ (Life-Technologies) for 2 hours at 37°C in a humidified incubator. A total of 4 ml of this digestion buffer were used to digest 1 g of fresh tumour tissue. The obtained suspension was filtered with a 100 µm cell strainer, centrifuged for 10 min at 460 g, resuspended in fresh medium, and counted. Cells were seeded at 8*104 cells/cm2 density and then passaged once a week at 2*103 cells/cm2 density.

The 143B human osteosarcoma cell line was obtained by ATCC and was chosen as control in each experiment as an example of an extensively studied and characterised aggressive sarcoma cell line, unless differently specified.

All cell lines were cultured in DMEM high glucose supplemented with 10% lot-selected FBS, 1% L-glutamine, and 1% Penicillin/Streptomycin (all from Life Technologies). For the MFS cell line, amphotericin B (Lonza) was added to the medium in the first passages. All cells were cultured in a monolayer at 37 °C and 5% CO2 in a humidified incubator and were mycoplasma-free.

Cell line authentication was performed by ATCC through STR analyses (Cell Line Authentication kit, ATCC); MF-R 3 cells at passage 10 and cells obtained from another portion of the tumoural tissue present in the Department of Pathology and stored at the Musculoskeletal Tumor Biobank (BIOTUM) of the Istituto Ortopedico Rizzoli were sent to ATCC.

All the experiments have been conducted after passage 13, when the number of population doublings after the first passage offered a reliable first sign of MF-R 3 cells’ spontaneous immortalization.

Cellular proliferation assay

MF-R 3 and 143B cells were seeded at 2*103 cells/cm2 density in technical quintuplicate into 96-well plates and cultured for 7 days at standard conditions. At each time point (days 1, 2, 3, 4, and 7 after seeding), cells were fixed with 10% neutral buffered formalin (Diapath S.p.A) for 30 minutes at RT and stained for 30 minutes in the dark with 100 µl of 1% methylene blue solution in 0.01 M borate buffer pH 8.5. Cells were then washed three times with 0.01 M borate buffer and methylene blue was dissolved with 100 µl per well of ethanol 100%: HCl 0.1 M 1:1 (v/v) ratio for 3-5 minutes in the dark in agitation. When the staining solution appeared clearly dissolved, absorbance was read at λ=655 nm using a microplate reader with an empty plate as background signal (Synergy HT, Bio-Tek Instruments Inc.). Cell number was calculated from blank corrected absorbance interpolated against a reference standard curve.

Two-dimensional clonogenic assay

For each cell line, 100 cells/well in a 6-well plate were seeded and the colony number was evaluated after 7 days for 143B and after 10-14 days for the MF-R 3 cell line. Cells were fixed overnight in 70% Ethanol and stained for 15 minutes with 1% methylene blue solution in 0.01 M borate buffer pH 8.5. Colonies were then washed 3 times in diH2O and manually counted. Clonal efficiency was calculated as the percent ratio between the average number of counted colonies and the number of seeded cells.

Selective nucleolar silver-staining (AgNOR)

For the selective silver-staining of nucleoli, a confluent T150 flask of cells was trypsinized, pelleted, and then fixed overnight at 4°C in 10% neutral buffered formalin (Diapath S.p.A). The day after, cell pellets were washed three times with PBS and then resuspended in 100 µl of 1% low melting agarose (Lonza). The suspension was then poured into disposable cryomolds 7x7x5 mm and incubated at 4°C for at least 10 minutes to allow agar solidification. Once solid, samples were moved to histological cassettes for paraffin inclusion. For each sample, 5 µm-sections were deparaffinized and re-hydrated. The antigen retrieval step was performed in autoclave in citrate buffer at pH 6. Silver staining was carried out following the “one step” procedure described by Ploton et al. [27]; in brief, slices from the cell pellets were incubated for 13 min at 37°C in silver nitrate solution at 30% (w/v) in 2% gelatin, 1% aqueous formic acid. The slices were then washed several times in water, dehydrated, and mounted with coverslips. For each slide, 5 images were captured using a Diaplan Leitz microscope with a 40X objective, and nucleolar size was measured on an average of 20 cells per field using a custom-made macro on ImagePro Plus Analyser software. Quantification analyses were performed according to the guidelines of the “International Committee on AgNOR Quantitation” [28].

In Vitro wound healing assay

Cells were seeded at a density of 2.5*104/cm2 in a 6-well plate and grown until 100% confluence. The cell monolayer was wounded with a p200 pipette tip, washed with PBS, and kept in 2 ml of fresh medium. To monitor the wound closure, pictures were taken at 0, 16, 20, 24, 40, and 44 hours after the scratch at 40X magnification through an inverted Nikon Eclipse TE2000-U microscope equipped with a calibrated Nikon DS-Vi1- U3 CCD camera. The areas of wound closure were calculated with the Area auto-detection tool of NIS Elements D software (Nikon) on the captured images.

Invasion assay

Cell invasive potential was tested by seeding 2,5*104 cells in 1% FBS culture medium on the upper chamber of a PET transwell insert with 8 µm pores (Merck Group) in a 24-well plate. To simulate the presence of surrounding tissue, the upper side of the insert was previously coated with a thin layer of Type I collagen from rat tail tendon (Roche; 2 mg/ml in 0.1 % acetic acid, 8:1:1 v/v collagen:HEPES 10X:DMEM 10X to buffer pH to 7). Non-coated transwell control was performed for each experiment. Complete medium with 10% FBS was added in the lower compartment of each well to generate a chemotactic stimulus. The staining procedure has been performed as described in Galbiati et al. [29]. The number of invading cells was obtained by counting five random fields of each insert, at 100X magnification using an inverted Nikon Eclipse TE2000-U microscope.

Soft agar colony formation (SACF) assay

SACF assay has been performed as described by Kusakawa et al. [30], with minor adjustments. In brief, 24-well plates were used to dispense 500 µl per well of agar 0.5% [1:1 v/v Low Melting Agarose (NuSieve™ GTG™, Lonza) 1% in D-PBS:DMEM - Low glucose 2x (powder, Sigma Aldrich)] as the bottom agar portion. During the agar solidification step of 30 minutes at 4°C, cells were trypsinized, counted, and seeded as single cell suspension as follows: 2.5*103 cells/well were carefully suspended in 100 µl of DMEM - Low glucose 2x, then 50 µl of melted agar 1% in PBS was gently added to cells suspension. Immediately after, 150 µl of the mixture was dispensed in every single well. The agar solidification was allowed for 15 minutes at 4°C. Subsequently, 1 ml of fresh culture medium was added to each well and plates were placed in the incubator for 14 days. Six hundred microliters of fresh medium were replaced every 3/4 days, without disturbing the cells in the agar layer. After 2 weeks, colonies were counted under an inverted Nikon Eclipse TE2000-U microscope at 40X magnification using a home-made grid as track. Colony-forming efficiency has been calculated as in Kusakawa et al. [30].

Low-density anchorage-independent growth of three-dimensional aggregates (myxospheres)

For 3D aggregate culture, cells were seeded at 600 cell/ml density in ultra-low attachment 6-well plates (Corning inc) in 2 ml of complete medium. Three-dimensional aggregates started to be visible to the naked eye after 1 week and passaged every 2 weeks. For passaging, aggregates were centrifuged and resuspended in 500 µl of fresh medium, then a single-cell suspension was obtained by pipetting the pellet with a 1 ml syringe with a 26G needle (BD Franklin Lakes). Erythrosine B stain was used to discriminate and count viable cells through Countess™ II FL Cell Counter (Life Technologies).

Transmission Electron Microscopy (TEM) analysis

For ultrastructural evaluation, MF-R 3 cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 for 1 h at room temperature. Afterwards, samples were postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4°C, dehydrated in an ethanol series, infiltrated with propylene oxide, and embedded in Epon resin. Ultrathin sections (80 nm thick) were stained with uranyl acetate and lead citrate (15 min each) and observed with a Jeol Jem 1011 transmission electron microscope operated at 100 kV. Images were captured using an Olympus digital camera and iTEM software.

Seeding of MF-R 3 on the chorioallantoic membrane (CAM)

Fertilized eggs were purchased from the Chick Farm company (Faenza, Italy) on day 1 post-fertilization and kept at 37°C with 56% humidity with scheduled rotation. On day 4 post-fertilization all unfertilized eggs were discarded. On day 5 post-fertilization, 2-3 ml of albumen were removed, and a window was opened on the eggs’ shell. To avoid contamination and damage to the embryo, the window was kept closed with a piece of parafilm and opened only in case of direct intervention or imaging. On day 8 post-fertilization, 4*105 MF-R 3 cells were mixed with LDEV Reduced Growth Factor Geltrex (1:1 v/v, 50 µl total final volume; Gibco™ Life Technologies) and seeded on the CAM. On day 15 post-fertilization, the chick embryos were euthanized, and the tumour masses were removed for further processing. A well distinguishable neoformation was visible in all the CAMs. Tissue samples were placed in the histological cassette and fixed in formalin (Merck Millipore) for at least 2 hours at +4°C and paraffin embedded.

Histology

4 µm thick histological sections were obtained by formalin-fixed, paraffin-embedded tumoural samples and stained with hematoxylin & eosin in an automatic slides stainer.

2D and 3D drug sensitivity assay

For 2D drug sensitivity tests, for both cell lines, 5*103 cells/well were seeded in black 96-well clear-bottom plates. After 24 hours, cells were treated with a series of 10-fold dilution of Epirubicin (Sigma-Aldrich, Merck Group) or Doxorubicin (Sigma Aldrich, Merck Group) in complete medium with final concentration ranging from 100 µM to 0 µM. Both drugs were resuspended in DMSO following the manufacturer’s instructions. In each experiment the maximum concentration of DMSO was tested for potential toxicity (0.1% for Epirubicin and 0.2% for Doxorubicin). Cell viability was assessed 72 hours after treatment using PrestoBlue™ (Invitrogen) diluted at 10% in complete medium and incubated for 2 hours at 37°C and 5% CO2 in a humidified incubator. Fluorescence measurement (ex 530 nm/em 590 nm) was performed using a microplate reader (Synergy HT, Bio-Tek Instruments Inc.).

For 3D drug sensitivity tests, both cell lines had 500 cells/well seeded in black Ultra-low Attachment 96-well plates with clear round bottom. Before treatment, spheroids’ formation was allowed for 5 days. Drug treatments were performed as described for 2D tests. Spheroids viability was assessed using CellTiter-Glo® 3D (Promega) following manufacturer’s protocol. Luminescence was measured using a microplate reader (Synergy HT, Bio-Tek Instruments Inc.).

Statistical analyses

The data have been analysed using GraphPad Prism 8.4.3. For each experiment, at least three biological and technical replicates were performed. Values of p lower than 0.05 were regarded as statistically significant.