Universita’ di Palermo
Centro di Oncobiologia
Sperimentale
Dipartimento di Oncologia Sperimentale e
Applicazioni Cliniche
FIBROBLAST’S: CO-ACTORS IN CANCER
Patrizia Cancemi, Nadia Ninfa Albanese, Luigi Minafra, Gianluca Di Cara, Maria Rita Marabeti, Ida Pucci-Minafra.
Dipartimento di Oncologia Sperimentale e Applicazioni Cliniche (DOSAC), Università di Palermo, Italy.
Centro di Oncobiologia Sperimentale (COBS), Casa di Cura di Alta Specialità La Maddalena, Palermo, Italy.
Fibroblasts are the major mesenchymal cell type committed to the matrix formation and renewal. They produce several soluble factors,
potentially active towards other mesenchymal and epithelial cells. Therefore, fibroblasts may influence tumorigenesis, through two
pathways: the remodelling of the ECM that occurs in carcinomas, and by paracrine factors that influence the growth of carcinoma cells.
Some authors have reported that factors derived from tumour fibroblasts stimulate tumour progression of non-tumorigenic epithelial cells.
Studies of fibroblasts associated to carcinomas have documented phenotypic modifications, including abnormal migratory behaviour in
vitro and altered expression of growth factors. In addition fibroblasts often recruit inflammatory cells involved in the stimulation of
angiogenesis, probably by the proteolytic release of sequestered angiogenic activators. Reciprocally, cancer cells may regulate the
biosynthetic activities of fibroblasts thus altering the ECM of the tumor, which in turn exerts some influence on neoplastic cell behaviour.
The aim of the present work was to extend our previous observations on the effects of microenvironment factors on neoplastic cell
behaviour (4), utilizing the well characterized breast cancer-derived cells, 8701-BC. The neoplastic cells were exposed to the fibroblasts
influences either in a co-culture system or through incubation with the fibroblast conditioned medium. In this report we show that the
fibroblasts affect neoplastic cell behaviour by: a) increasing cell proliferation rate; b) rising the migration and invasion properties of cells in
boyden chamber assays; c) inducing a transition of cytoskeletal filament expression. Besides, the responses of neoplastic cells were
studied by the proteomic approach, which showed a consistent modulation of sets of proteins, some of which were studied also at
transcriptional level. Conclusively data so far collected suggest that fibroblasts, rather than being host defenders may be co-actors in the
process of cancer progression.
Cancer cells
TGFb1,b2,b3
TGF -α
IGF1
Erb b
MIF
PtHRP
TGFb1
FGFs
IGF1
MMPs
VEGF
PDGF
TGFb1
ECM
KGF
VEGF
Fibroblasts
Il-1
Il-6
TNF
VEGF
CSF-1
MCP1-2-3
TGF
IGF1
TGF
CSF-1
bFGF
VEGF
Experimental procedures
Cocolture experiments
… for proteomics
Normal fibroblasts C29
Effects of fibroblasts
on 8701-BC on …
Analysis Software
… for invasion and
immunolocalization
5 days +FCS 10%
Endothelial cells
Macrophages
Chemotaxis
and chemio-invasion
through the Boyden Chamber
Breast cancer cells 8701/BC
Breast cancer cells 8701/BC
5 days +FCS 10%
Upper chamber
Soluble
factors
Soluble
factors
1.
Chemo-attractant
Cells and conditioned media were collected separately
Filter
+/- Matrigel
2.
Control cells were grown alone
+ conditioned medium
produced by breast cancer cells
Plastic
Transwell with 0.4 mm pore filters
.
Normal fibroblasts C29
Breast cancer cells8701/BC
8701-BC Control
8701-BC + MC Fibroblasts
0.7
Cocoltured
Control
0.8
O.D 492 nm
48h - FCS
…Growth
Lower chamber
+ conditioned medium
produced by fibroblasts
The software utilize several algorithm to estimate some spot
parameters such as optical density (pixel intensity), volume
(integration between OD and spot Area), percentage of volume
(Volume of a spot/sum of the spots).
58%
0.6
0.5
0.4
0.3
0.2
0.1
C-MYC
0
24hrs
48hrs
Effects of fibroblasts on 8701-BC proteome
Membraneassociated
proteins with
multiple activities
10%
Molecular
chaperones/heat
shock proteins
19%
Protein
degradation
3%
Cytoskeleton and
assocated proteins
22%
5d
Calcium binding
proteins
4%
8701-BC Control
100
8701-BC + MC fibroblasts
80
60
MMP-9
40
20
MMP-2
0
Chemotaxes
Western blot validation
of modulated proteins
ANX2 a
LEG1
ANXA2 sf
MIF
PSB4
SODM a
PDIA3 a
TPIS b
PGK 1 a
MDHM a
Up-regulated
PROF1 a
ACTB or ATCG fr
ACTB/G a
ACTB/G g
TPM2 a
MYL6
TPM3
10
Glycolitic enzymes…
LEG1
G3P2
1
Control
ANXA2LC
NPM a
ANXA1 b
S10A6
COX
EF1B
AK1BA a
ALDR a
AK1C3
ALDR b
THIO
G3P2 b
PPIA a
GRP 75
HSP27 a
HSP71
HSP74
ENOA
TBB5 a
TBA1 c
TBA1 b
TPM4 a
ACTB/G f
TYB4
ACTG
Down-regulated
Chemoinvasion
Detoxification and
redox proteins
10%
Metabolic enzymes
25%
Cococlture
7d
…Invasion
Average of cell
number/HPF 250X
Control
96hrs
Cocolture
0.1
ANXA2
ANXA1
Genomic approach: Superarray analysis
Unaffected
Cytoskeleton characterization
10%
8701-BC
Fibroblasts
Immunolocalization assay
Downregolated
Upregolated
CK8
12%
Western blot
8701-BC+
Fibroblasts
Up-regulated genes in cocolture
1200
8701-BC Control
8701/BC cells
+8701/BC MC
8701-BC Cocolture
800
IGFBP3
MELK
600
RPS4X
PSMD2
CELL-GROWTH
400
PFKP
GLYCOLITIC
METABOLISM
SYNTHESIS
200
CK8
fibroblasts
8701-BC
ESM1
MYBL2
PFKP
OXCT1
ERP70
STMN1
TGFB2
SERPINE1
TFRC
ITGB3
PGK1
PRC1
NUSAP1
PSMD2
BCL2
RPS4X
KIF3B
RAD21
MELK
CDK2
TRIP13
B2M
IGFBP3
TP53
0
Cytokeratin 8
1000
5.13 45000
5.06 42210
5.23 51750
500
400
300
Vimentin
600
5.06 42210
VIME
8701-BC+fibroblsts
Down-regulated genes in cocolture
8701/BC cells
+Fibroblasts MC
Actin
Actin
VIME
700
5.13 45000
5.23 51750
C-MYC
8701/BC cells
+8701/BC MC
Actin
Actin
ENOA
200
4.88 39550
100
GPSM2
BNIP3
FLT1
CTSC
EVL
FOS
VEGF
FBXO5
CD68
EXT1
BAX
RAB27B
CENPF
HMGB3
ASPM
HRB
TGFB1
ERBB2
CDC25B
KIAA1683
ARMC1
TMEFF1
LYRIC
PTDSS1
AKAP1
DEGS1
PSMD7
MAD2L1
MCCC1
CTNNB1
CDK4
0
fibroblas ts
8701-BC
8701-BC+fibrobls ts
4.88 39550
8701/BC cells
+Fibroblasts MC