Transcript Poster SIAPEC

FIRENZE
7- 9 Settembre 2009
Cancer-stroma interactions:
role of cancer-associated fibroblasts and mast cells
in breast carcinogenesis
A Malfettone1, G Simone2, R Rossi3, C Salvatore2,
R Daprile2, A Paradiso1, L Resta3, A Mangia1
1
Clinical Experimental Oncology Laboratory, National Cancer Institute-Bari, Italy
2 Pathology Department, National Cancer Institute-Bari, Italy
3 Pathological Anatomy Department, University of Bari, Italy
BACKGROUND
 Carcinogenesis is influenced and controlled by cellular
interactions derived from a complex relationship between
epithelial, stromal, and extracellular matrix components.
 Studies in human breast, lung, colon and prostate cancer
have identified “reactive stroma” that is characterised by
increased microvessel density, inflammatory cells and
fibroblasts with an “activated” phenotype.
 Recent evidence shows that fibroblasts and inflammatory
elements may not be passive bystanders but might have an
important role in modifying tumor growth and cancer
progression.
AIM OF THE STUDY
 In order to assess if there are significant differences related
to different histological compartment of carcinoma, we
investigated the distribution of a subpopulation of activated
fibroblasts called carcinoma-associated fibroblasts (CAFs),
tumor infiltrating mast cells (MCs), and their simultaneous
interaction in invasive human breast cancers.
MATERIALS AND METHODS
Tissue Specimens
 Expression of α-smooth muscle actin (αSMA), CD34 stromal
fibroblasts by immunohistochemistry, and accumulation of
intact methacromatic MCs with toluidine blue staining, was
examined in 30 breast cancers.
 Tumor (T), peritumor (PT) and adjacent non-involved (PM)
tissues from the same patient have been investigated in order to
identify and to quantify CAFs and intact MCs. Ten of the 30
total cases were also studied with electron microscopy.
Immunohistochemistry
 Immunohistochemistry was performed on formalin fixed and
paraffin embedded tissues utilizing standard procedure for
sampling, fixation and paraffin embedded.
 De-paraffinized serial sections of 4m were incubated in a
humid chamber overnight at 4°C with two murine monoclonal
antibodies against CD34 (1:50, QBEnd⁄10, Novocastra) and αSMA (1:150, 1A4, Santa Cruz) to detect normal stromal
fibroblasts and CAF, respectively.
 The bound antibody was visualized using a biotinylated
secondary antibody, peroxidase-labelled streptavidin, and
DAB substrate-chromogen (LSAB2 System-HRP; Dako). The
slides were counterstained with H&E. For negative controls,
the primary antibody was omitted and replaced by PBS.
Tissue MCs count
 After deparaffinization, sections contiguous to those
immunohistochemically assessed for fibroblats (T, PT, PM)
were stained with toluidine blue for 10 min, then rinsed in
distilled water, and mounted.
 The sulfated proteoglycans in secretory granules of MCs
have a metachromatic property, so toluidine blue stained
MCs could be detected. Visual counting of intact MCs was
performed in 5 to 8, non-overlapping, 200X microscopic
fields, covering both peripheral and central tissue regions.
Statistical analysis
 Percentage of fibroblasts was assessed by counting stromal
cells stained positively for α-SMA or CD34 with respect to total
cells, in three fields at 400X in each specimen. The mean and
standard error of the mean (SEM) of immunoreactive
fibroblats in T, PT and PM tissue were then calculated and
compared, using a two-tailed, unpaired Student’s t-test for
statistical significance.
 The mean and SEM of MCs in each tissue section were
calculated and compared, by one-way ANOVA. All pairwise
multiple comparisons were done by Bonferroni's multiple
comparison test. Differences were considered statistically
significant when p<0.05.
RESULTS
Distribution of stromal SMA+ fibroblasts
A
B
C
Num ber of αS MA+ fibroblas ts
(400X m ag nific ation)
Figure 1. SMA+ fibroblasts from invasive breast cancer. A: PM; B: PT; C: T
100
***
90
80
***
70
60
50
40
30
*
20
10
0
PM
PT
T
SMA+
fibroblasts
were
prevalently present in the stroma of
all T tissues (66,8% ± 2,1) with
respect to PT (11,9% ± 4,2) and PM
regions (1,8% ± 1,8) (PM vs PT,
p<0.05; PM vs T and PT vs T,
p<0.001, for both compared
groups).
Distribution of stromal CD34+ fibroblasts
C
B
A
Num ber of C D34+ fibroblas ts
(400X m ag nific ation)
Figure 2. CD34+ fibroblasts from invasive breast cancer. A: PM; B: PT; C: T
100
90
80
70
60
50
40
30
20
10
0
***
CD34+
fibroblasts
were
statistically
present
in
the
connective tissue of all PM (80,2%
± 1,9) and PT (68,6% ± 4,6) with
respect to T tissues (5,0% ± 4,8)
(PM vs T and PT vs T, p<0.001 for
both compared groups).
***
PM
PT
T
Quantitative analysis of MCs
B
A
C
Figure 3. Metachromatic MCs from invasive breast cancer. A: PM; B: PT; C: T
Num ber of m as t c ells
(200X m ag nific ation)
60
***
50
***
40
**
30
20
10
0
PM
PT
T
Number of intact MCs gradually
increased
during
cancer
progression, from PM regions
(14.7% ± 0.9), PT (25.2% ± 1.5) to
T tissues (38.3% ± 3.1) (PM vs PT,
p<0.01; PM vs T and PT vs T,
p<0.001 for both compared
groups).
Interaction between fibroblasts and MCs
Figure 4. Adjacent sections
of PM breast tissue
A: Metachromatic MCs
B: CD34+ fibroblasts
A
B
Figure 5. Adjacent sections
of T tissue
A: Metachromatic MCs
B: SMA+ fibroblasts
A
B

Direct physical interactions between metachromatic MCs and fibroblasts occur
mostly in first phases of tumorigenesis and decrease in tumors, perhaps
because intimate interactions, with respect to paracrine effects, do not appear
to be an indispensable part of the signaling process.

When MCs activity is not properly regulated, the result may be the influence of
stromal proliferation, differentiation and activation from normal fibroblasts to
CAFs.
CONCLUSIONS
 CAFs can apparently originate under the action of several
activating factors and MCs, also in human breast cancer, are a
potential source of some of those factors.
 CAFs are significantly increased in T, in parallel with a
higher density of MCs in T and PT with respect to PM tissues of
each patient.
 Our preliminary findings suggest that MCs, since the early
steps of tumorigenesis, may contribute to breast cancer stromal
remodelling, characterized by a loss of CD34+ fibroblasts and
subsequent progressive fibroblasts activation in CAFs.