*2.9. Mutational Analysis of TP53, IDH1, IDH2, PI3KCA*

Genomic DNA were amplified by PCR (Taq PCR Master Mix (2x), EURx Ltd. (Gda ´nsk, Poland)). The fragments analyzed include: exon 4 of *IDH1* and *IDH2* genes, identified as mutation hot spots in chondrosarcoma; exon 20 of the *PI3KCA* gene; and exons 4 and 6 of the *TP53* gene. Reactions were carried out using the forward and reverse primers detailed in Supplemental Information and the different PCR products were detected by gel electrophoresis in 1.5% agarose, showing a single band. Samples were purified and sequenced by Macrogen Ltd. (Madrid, Spain) and were aligned with the reference sequences of the genes using SnapGene® 4.2.11 (GSL Biotech; available at snapgene.com).

#### *2.10. Library Construction and WES*

WES was performed by Macrogen (Seoul, Korea) using 1 μg of genomic DNA from each sample. DNAs were sheared with a Covaris S2 instrument and used for the construction of a paired-end sequencing library as described in the paired-end sequencing sample preparation protocol provided by Illumina. Enrichment of exonic sequences was then performed for each library using the Sure Select All Exon V6 kits following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA, USA). Exon-enriched DNA was pulled down by magnetic beads coated with streptavidin (Invitrogen, Carlsbad, CA, USA), followed by washing, elution, and additional cycles of amplification of the captured library. Enriched libraries were sequenced (2 × 101 bp) in an Illumina HiSeq4000 sequencer. WES results were processed using the bioinformatics software HD Genome One (DREAMgenics, Oviedo, Spain), certified with IVD/CE-marking (see Supplementary Materials for a comprehensive

description of the exome analysis, [33–47]). The datasets generated during the study are available in the European Nucleotide Archive repository [48].

#### **3. Results**

#### *3.1. Establishment of Patient-Derived Chondrosarcoma Cell Lines and Analysis of In Vivo Tumorigenic Potential*

Surgically resected tumor samples from 11 patients diagnosed of chondrosarcoma at the Hospital Universitario Central de Asturias (Spain) were processed to establish primary cultures. Cultures from two secondary chondrosarcoma, CDS06 (associated with a previous osteochondroma) and CDS11 (presenting Ollier disease), and one from a dedifferentiated chondrosarcoma (CDS-17), were able to growth long term in vitro (Table S1 show an overview of patient and tumor characteristics). These cell lines were able to form colonies in soft agar, an in vitro transformation assay to test the ability of the cells to grow in anchorage independent conditions (Table 1). In order to select the more tumorigenic populations within the cultures, the colonies able to grow in soft agar were recovered and placed back in adherent culture to continue with the corresponding cell line development. Recovered cell lines could be passaged at least 20 times (Table 1) and their identity with the original tumor was confirmed by STR genotyping (Table S2).


**Table 1.** Functional characterization of chondrosarcoma cell lines.

(§) Ability to growth forming colonies in embedded in soft agar. (\*) number of passages reached so far in adherent cultures. (\*\*) Tumor growth was follow for 1 and 2.5 months in subcutaneous and intra-bone experiments respectively. (‡) Ability of 3D spheroids to invade collagen matrices. (¶) There is a significant difference between the volumes of tumors generated by CDS17 and T-CDS17 cells (*p* = 0.043; two side Student's *t*-test. SD: Standard Deviation).

Two of the cell lines (CDS11, CDS17) were assayed for their ability to initiate tumor growth in vivo. Both of them were able to form small slow-growing tumors after subcutaneous (s.c.) inoculation in immunodeficient mice after 1 month (Figure 1A and Table 1). Following this, we generated a new cell line derived from a CDS17-xenograft tumor. Subsequent transplantation of this new cell line, T-CDS17, resulted in a more aggressive tumor growth (formation of significantly bigger tumors in similar latency periods), thus indicating that the tumor could be effectively propagated in vivo (Table 1). Histological analysis showed that the original CDS11 tumor was a malignant chondrosarcoma invading intra-trabecular bone matrix and presenting well differentiated and dedifferentiated areas. There was no inflammatory infiltrate and the dedifferentiated subcomponent displayed a mitotic index of 15 mitoses per 10 high power fields (HPF, 40X). The histology of tumors grown from the CDS11 line resembled that of the more undifferentiated/dedifferentiated areas of the original patient.

Tumor, with tumor cells distributed diffusely in a mesenchymal matrix. No well-differentiated component was found in these tumors. Inflammation was also absent and tumors showed 9 mitoses per 10 HPF (Figure 1A). The CDS17 patient sample was a high-grade chondrosarcoma displaying the characteristic chondroid differentiation, with tumor cells presenting pericellular matrix and surrounded by a chondroid extracellular matrix. There was no inflammation present in this tumor and its mitotic index was 18 mitoses per 10 HPF. Tumors derived from CDS17 and T-CDS17 cells lines maintained chondroid differentiation, with tumor cells presenting pericellular halos and embedded in a chondroid basophilic extracellular matrix. There were no inflammatory infiltrates in these tumors and its mitotic index was 17 and 25 mitoses per 10 HPF for CDS17 and T-CDS17 respectively (Figure 1A).

**Figure 1.** In vivo tumorigenicty of chondrosarcoma cell lines. (**A**) Histological analysis (H&E staining) of original patient tumors and tumors developed 1 month after subcutaneous (s.c.) inoculation of CDS11, CDS17, and T-CDS17 cell lines in immunodeficient mice. Two different areas of the CDS11 patient tumor sample are shown. scale bars = 150 μm. (**B**) Radiologic examination (μCT scan) of tumors developed after intra-bone (i.b.) inoculation of CDS17 and T-CDS17 cells in immunodeficient mice. Coronal, sagittal and axial images are shown. Compared to a control leg, intra-medullar formation of tumor bone/osteoid formation (white arrows) is shown in mice inoculated with both cell lines. In addition, a mouse inoculated with T-CDS17 cells presented an extra-medullar lesion compatible with the radiographic features of osteochondrosarcoma (orange arrows). (**C**) H&E staining of an original patient sample (i), a control leg (ii), and tumors formed after i.b. inoculation of CDS17 (iii and iv) and T-CDS17 (v and vi) cells lines. Chondroid.like cells (yellow arrows) and areas of fibrillar (black arrow) and amorphous (orange arrow) osteo-chondroid matrix are indicated. (B: bone; RB: reactive bone).

In an attempt to create more faithful animal models CDS17 and T-CDS17 cells were inoculated intra-tibia in immunodeficient mice. Both CDS17 (1 out of 2 mice) and T-CDS17 (2 out of 2 mice) cells were able to generate tumor growth in this orthotopic location. Computerized tomography (CT) analysis at day 50 after inoculation (Figure S1) and microCT analysis at the end point of the experiment (day 80) (Figure 1B) revealed the formation of tumors resembling the radiological features of human chondrosarcomas. These tumors showed the presence of a dotted pattern chondroid-like matrix inside the bone marrow cavity. In addition, one of the T-CDS17 generated tumors also displayed extra-medullar tumor growth, forming an osteochondroid exostosis-like lesion (Figure 1B and Figure S1). Histological sections of these orthotopically grown tumors also resembled the main features of the patient sample (Figure 1C(i)), with chondrogenic tumor cells presenting pericellular halos and embedded in cartilaginous matrix (Figure 1C(iii,iv)). In addition, legs inoculated with tumor cells showed bone marrow cavities filled by dedifferentiated mesenchymal cells producing fibrillar and amorphous osteo-chondroid matrix and presenting extensive areas of reactive bone (Figure 1C(v,vi)). None of tumors presented inflammatory component and they showed mitotic indexes between 7 (CDS17) and 10 (T-CDS17) mitoses per 10 HP.
