1. Cancer-stroma interactions: role of cancer-associated fibroblasts and mast cells in breast carcinogenesis A Malfettone 1, G Simone 2, R Rossi 3, C Salvatore 2, R Daprile 2, A Paradiso 1, L Resta 3, A Mangia 1 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 FIRENZE 7- 9 Settembre 2009

2. 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.

3. AIM OF THE STUDY In order to assess if there are significant differences related to different histological compartment of carcinoma,  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.

4. MATERIALS AND METHODS 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. adjacent non-involved 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. Tissue Specimens

5. Immunohistochemistry I formalin fixed and paraffin embedded tissues Immunohistochemistry was performed on formalin fixed and paraffin embedded tissues utilizing standard procedure for sampling, fixation and paraffin embedded. De-paraffinized serial sections in a humid chamber overnight at 4°C with two murine monoclonal antibodies against CD34 (1:50, QBEnd⁄10, Novocastra) and α- SMA (1:150, 1A4, Santa Cruz) to detect normal stromal fibroblasts and CAF, respectively. 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 α- SMA (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.

6. Tissue MCs count After deparaffinization, sections contiguous to those immunohistochemically assessed for fibroblats (T, PT, PM) stained with toluidine blue for 10 min, then rinsed in distilled water, and mounted. 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. 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.

7. 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. 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. 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.

8. Distribution of stromal  SMA+ fibroblasts Figure 1.  SMA+ fibroblasts from invasive breast cancer. A: PM; B: PT; C: 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). A B CRESULTS

9. Distribution of stromal CD34+ fibroblasts Figure 2. CD34+ fibroblasts from invasive breast cancer. A: PM; B: PT; C: T 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). A B C

10. Quantitative analysis of MCs Metachromatic Figure 3. Metachromatic MCs from invasive breast cancer. A: PM; B: PT; C: T Number of intact MCs T tissues () 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). A B C

11. Interaction between fibroblasts and MCs  etachromatic  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. Figure 4. Adjacent sections of PM breast tissue Metachromatic A: Metachromatic MCs B: CD34+ fibroblasts A B A B Figure 5. Adjacent sections of T tissue Metachromatic A: Metachromatic MCs B:  SMA+ fibroblasts

12. 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.