Chondrosarcomas are cartilage-forming, poorly vascularized ...

Chondrosarcomas are cartilage-forming, poorly vascularized tumors. They represent the second malignant primary bone tumor of adults after osteosarcoma but in contrast they are

resistant to chemotherapy and radiotherapy, surgical excision remaining the only therapeutic option. Few cell lines and animal models are available, and the mechanisms behind their

chemoresistance remain largely unknown. Our goal was to establish new cell lines and animal cancer models from human chondrosarcoma biopsies to study their chemoresistance.

A

1. From 10 chondrosarcoma biopsies, three tumoral cell lines and two animal models in nude mouse were established.

2. The new chondrosarcoma cell lines

are able to synthesize cartilaginous

matrix in chondrogenic 3D pellets.

Standard Chondro Osteo Adipo

N/A

Alcian blue Alizarin red S Nile red

N/A

N/A

CA

L78

C

H56

C

H03

M

SC

s

Human biopsy Human biopsy

X-rays HE

*

#

¶

X-rays HE X-rays HE Alcian blue

¶

# *

¶ ¶

# # * *

* Osteoid matrix ¶ Cartilaginous matrix # Tumoral area Blood vessels

Human biopsy Murine xenograft

X-rays HE

* ¶

#

Monolayer cells 3D Pellets

Three culture conditions were used:

Standard Chondrogenic Standard

21 days chondrogenic

medium:

complete medium +

dexamethasone

+ascorbate+TGF-β3

10-1

1

10

102

COL2A1

10-1

1

COMP

Expre

ssio

n level

** ** ##

** ##

ACAN

10-2

10-1

1

10

** ##

MS

Cs

Bio

psie

s

Pelle

ts

Cells

Bio

psie

s

Bio

psie

s

Cells

Cells

Chondrosarcoma Osteosarcoma Ewing

sarcoma

B

A: TaqMan® Low Density Array: screening of upregulated genes in

chondrosarcoma biopsies and chondrogenic 3D pellets

(red, apoptosis/cell cycle; purple, cartilage; *p<0.05, monolayer VS

biopsies and pellets

B: Quantitative RT-PCR: expression of three cartilage markers in

chondrogenic 3D pellets (**p<0.01, compared with the original biopsy,

;## p<0.01 compared with chondrogenic 3D pellets)

3. Chondrosarcoma cells express high levels of cartilage markers

in chondrogenic 3D pellets compared to standard monolayer culture.

CH

34

C

H56

C

H56

C

H03

C

H56

C

H34

C

H03

S

W

O27

PPARG DKK1 PHEX BMP4 BIRC5* E2F1 EGFR DNMT3B BCL2L1 TIMP2 PAX6 BAD* ITGB1 BAX PTCH1 BBC3 FGFR2 DLX5 SERPINA1* BGLAP WNT3A LPL ICAM1 CEBPD PDGFA SNAI1 MMP10 NFATC3* ATF4 TGFBR1 FOXD3 MMP13* XIAP* FGF4* NODAL* POU5F1* NANOG* SP7* FOS* SPP1* CXCR4* PTGS2 COL6A1* VEGFA* NFATC2* NEUROD1* BGN DCN ABCA5* MGP* SOX5* SOX6* COL2A1* COMP FN1* COL10A1* TIMP1 WNT1* IHH* SOX2 WIF1* HAPLN1* CHAD* ACAN* COL11A1* LECT1* COL9A1* IBSP FGFR3 BMP6 TNFSF11* PTH1R TGFB3 DMP1 PDGFB* MEPE IGF1 IGF2* MMP9

-0.57

-0.38

-0.19

0

+0.19

+0.38

+0.57

Bio

psie

s

Pelle

ts

Cells

A

Mafosfamide (µM)

0

20

40

60

80

100

10-1 1 10 102

0

20

40

60

80

100

10-1 1 10 102

Cisplatin (µM)

Via

ble

cells

(%

of contr

ol)

Doxorubicin (nM) 10 102 103 104

0

20

40

60

80

100

HOS

CH03

CH56

A673

Chondrosarcoma

Osteosarcoma

Ewing sarcoma

4.Chondrosarcoma cells cultured as monolayer are

sensitive to doxorubicin, mafosfamide and cisplatin.

Viability XTT assays: sensitivity

of the two new chondrosarcoma

cell lines cultured as monolayer

to three chemotherapeutic

agents compared to

osteosarcoma and

Ewing sarcoma cell lines

(3 day treatment).

CH03 monolayer cells CH03 chondrogenic 3D pellets CH03 standard 3D pellets

0

20

40

60

80

100

10-1 1 10 102

Mafosfamide (µM)

Via

ble

cells

(%

of contr

ol)

10 102 103 104

Doxorubicin (nM)

0

20

40

60

80

100

0

20

40

60

80

100

10-1 1 10 102

Cisplatin (µM)

5. In chondrogenic 3D pellets, chondrosarcoma cells are more resistant

to doxorubicin and mafosfamide, but not to cisplatin.

Viability trypan blue assays: sensitivity of CH03 chondrogenic 3D pellets to three chemotherapeutic

agents compared to standard 3D pellets and monolayer cells (3 day treatment).

CH03 standard 3D pellets CH03 chondrogenic 3D pellets

Contr

ol

DO

X 1

02nM

D

OX

10

3nM

DAPI Fluorescent DOX DAPI Fluorescent DOX

6. In chondrogenic 3D pellets, chondrosarcoma cells have an impaired

intra-nucleic incorporation of doxorubicin.

Accumulation of

doxorubicin (DOX):

fluorescence microscopy

on 3µm sections of

standard and

chondrogenic 3D CH03

pellets treated with DOX

for 3 days.

Insets:alcian blue

staining of untreated

3D pellets.

These results indicate that the cartilaginous matrix produced by chondrosarcoma cells may impair diffusion of chemotherapeutic drugs based on their hydrophobicity/water solubility and

thus contribute to chemoresistance. Although they lack angiogenesis and metastasis development, these chondrogenic 3D pellets represent valuable models for this complex pathology,

especially to study the link between chondrogenesis and chemoresistance.

Patient Gender Age Sub-type Cell line TP53 IDH1 IDH2 p16INK4A Tumorigenicity

BCSCH01 M 83 Conventional

central No - - - - Yes

BCSCH03 F 74 Dedifferentiated Yes c.318delC WT WT Deletion No


central No - - - - -

BCSCH30 M 46 Secondary No - - - - -


central Yes WT p.R132C WT Deletion No

BCSCH36 F 28 Secondary No - - - - -

BCSCH37 F 41 Secondary No - - - - -

BCSCH45 F 64 Conventional



central Yes WT WT p.R172S Deletion Yes



3D

Pellets

Chondrosarcomas are cartilage-forming, poorly vascularized ...

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