Introduction
It has been shown that lysosomes, acting presumably via their acid hydrolases, are involved in a variety of cytoplasmic degradative changes during physiological processes [1, 2] and regression of mammary tumors [3, 4]. Acid phosphatase (AP) has also been used to monitor cell death and cell lysis [5,6,7,8]. Activities of lysosomal hydrolases were demonstrated to be more marked in cancer cells than in homologous normal tissue [9, 10]. Ductal carcinoma
In this report, AP, the marker enzyme for lysosomes [17], was used to visualize these organelles in normal and neoplastic breast tissues. We detected higher AP activity and histochemistry in DCIS associated with IDC compared to IDC alone.
Materials & methods
Tissue Collection & Processing
Normal and malignant breast tissues used in this study were obtained from the Cooperative Human Tissue Network (Philadelphia, PA). Two cases of IDC associated with DCIS and one case of IDC were studied. The patients were 58, 67, and 73 years old at the time of the biopsies. Normal breast tissue from each patient was also examined and considered as controls. Tissues were fixed overnight in 4 % paraformaldehyde at 4°C, placed in Tissue Tek OCT (Miles, Elkhart, IN), frozen in liquid N2, cut as 5 μm sections onto poly-L-lysine coated slides (Sigma, St. Louis, MO), and stored at -20°C until they were stained.
Lysosomal Localization
Lysosomes were localized from slides of frozen sections using a histochemical assay for AP (Sigma) as previously described [18]. Briefly, prior to fixing the slides, 0.6 mL of sodium nitrite solution were added to 0.6 mL of fast garnet GBC solution, mixed by inversion; allowed to stand for 2-4 min; and added to 23 mL of dH2O. Three mL of acetate solution and 3 mL of naphthol AS-BI phosphoric acid solution (provides naphthol AS-BI phosphate, the substrate) were added to the previous solution. Slides were fixed in citrate-acetone-formaldehyde solution at room temperature, in Coplin jars, for 30 sec and rinsed with dH2O for 1 min. Slides were then incubated for 1 h at 37°C in naphthol AS-BI phosphate and fast garnet stain in acetate buffer. Slides were rinsed with tap H2O for 2 min, dried for 15 min, counterstained with methylene blue for 1 min, rinsed with dH2O, and mounted in CrystalMount (Biomeda Corp., Foster City, CA). The presence of acid phosphatase was indicated by distinct red-violet focal precipitates, which were resolved by light microscopy.
Results and discussion
Normal myoepithelial cells showed a moderate amount of lysosomes (Figs.
Infiltrating Ductal Carcinoma (IDC).
Infiltrating Ductal Carcinoma (IDC). a, b) Faint histochemical activity (dark granules) is seen in the epithelial cells from normal breast tissue and is negative in the connective tissue.. c) The florid complex and irregularly shaped epithelial bridges resulted in superimposed micropapillary features. d) Enhanced enzyme activity is evident in the epithelial elements. X 200, a and c; X 400, b and d.
IDC Associated with Ductal Carcinoma in situ (DCIS).
IDC Associated with Ductal Carcinoma in situ (DCIS). a, b) Very few AP positive granules (dark granules) are seen in the normal breast. Stromal fibroblasts contain no enzyme activity. c, d) Cords of tumor cells are strongly positive while the stromal tissue is unstained (clear areas). X 200, a and c; X 400, b and d.
IDC Associated with DCIS.
IDC Associated with DCIS. a, b) Normal breast myoepithelia has is sparsely stained for AP. c) Strong enzyme activity is evident throughout the cytoplasm of the tumor cells. The AP positive granules (dark areas) are much larger than those in the control tissue (a, b). The majority of the tumor cells are AP positive, suggesting that they are undergoing cell death. d) Intense AP activity in the cords of tumor cells. Note that AP staining is absent in the stroma. X 200, a and c; X 400, b and d.
Histochemical reactions for acid phosphatase were demonstrated in all cases of breast cancer that we examined. The reaction was most constant in the epithelial cells and negligible in connective tissues. This observation is in agreement with previous reports [19, 20]. It seems that increased acid phosphatase activity is a constant feature in neoplastic transformation [21], and this was noted in all the cases of breast carcinomas we studied. Acid phosphatase activity has been used to indicate that lysosomes participate in the execution of cell death in a variety of tissues [22, 23] and in the regression of mammary carcinomas [24, 25]. Apoptosis could represent a barrier to the progression of invasive cancer [26, 27]. Our observations support this hypothesis in that we detected more AP activity in DCIS relative to IDC. Moreover, a recent study found that apoptosis comprises only 11 cells per mm2 of tissue in 288 cases of invasive breast cancer [28]. Acid phosphatase has been used as a marker of metastatic bone disease and response to treatment in breast cancer patients [29]. In this light, our findings suggest that the level of AP activity in breast tumors may be correlated to the amount of apoptosis that is going on in the cells.