Extracellular acidification alters lysosomal trafficking in human breast cancer cells

Abstract

Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pH(e)) resulted in a distinct shift of lysosomes from the perinuclear region to the cell periphery irrespective of the HMECs' degree of malignancy. With decreasing pH, larger lysosomal vesicles were observed more frequently in highly metastatic breast cancer cells, whereas smaller lysosomes were observed in poorly metastatic breast cancer cells and HMECs. The number of lysosomes decreased with acidic pH values. The displacement of lysosomes to the cell periphery driven by extracellular acidosis may facilitate exocytosis of these lysosomes and increase secretion of degradative enzymes. Filopodia formations, which were observed more frequently in highly metastatic breast cancer cells maintained at acidic pH(e), may also contribute to invasion.

Figure 1

(a) Representative immunofluorescence staining of hLAMPs in tumor sections of breast carcinoma patients. The LAMP fluorescence is displayed in red; the fluorescence of the cell nuclei is displayed in green. The image on the left shows a (i) normal duct (FOV 80 x 80 m). The central image shows (ii) hyperplasia (160 x 160 m). The image on the right shows a (iii) ductal carcinoma (80 x 80 m). Histological grading of the tumor sections was verified by hematoxylin and eosin staining of neighboring sections of the same tumor. (b) Western blots probing for (i) hLAMP-1 or (ii) hLAMP-2 in cell lysates from HMECs and human breast cancer cell lines used in this study. Left to right lane: MCF-12A, MCF-7, MDA-MB-231, and MDA-MB-435 cells. hLAMP-1 and hLAMP-2 antibodies revealed immunoreactive bands between 100 and 140 kDa that are typical of mature, highly glycosylated LAMP proteins. Immunofluorescence staining of hLAMP-1/hLAMP-2 in tumor sections as well as Western blots for hLAMP-1/hLAMP-2 prove that this protein is ubiquitously expressed in breast tissues and human breast cancer cell lines with different degrees of malignancy.

Figure 2

Representative immunofluorescence staining of invasive/metastatic MDA-MB-231 breast cancer cells exposed to (a) pH 7.4 (control), (b) pH 6.8, or (c) pH 6.4. Examples from three images (80 x 80 m) are shown for each pH to demonstrate that in control cells (pH 7.4), lysosomes were mainly perinuclear, whereas in cells at acidic extracellular pH (pH 6.8 and pH 6.4), lysosomes were more scattered and shifted toward the cell periphery.

Figure 3

(a) Snapshots displaying the image analysis window of our in-house software to demonstrate the boundary detection of the nucleus (white line) and the lysosomes (yellow lines) performed by the software. The image on the left is a (i) raw fluorescence image of a single MDA-MB-231 cell (80 x 80 m) and the image on the right shows (ii) the same image after boundary detection by the software. (b) The frequency distribution of (i) lysosome-to-nucleus distance and (ii) lysosomal diameter in MDA-MB-231 breast cancer cells exposed to pH 7.4 (upper panel, control), pH 6.8 (central panel), or pH 6.4 (lower panel). Individual cells (typically from one to two cells per image) from four independent stainings per pH value were analyzed.

Figure 4

Box-and-whisker plots of changes in lysosomal displacement with extracellular pH in (a) MCF-12A, (b) MCF-7, (c) MDA-MB-231, and (d) MDA-MB-435 cells. The box represents the range between the third quartile and the first quartile of the respective distribution displaying the length of the interquartile range, which is 50% of the data. The median of the distribution is indicated by the horizontal line in the box. *Represents P < .05. **Represents P < .01, pH 6.8 vs 7.4 and pH 6.4 vs 7.4 (Mann-Whitney U-test; if D ≠ 0, P values for D > 0, D < 0).

Figure 5

Box-and-whisker plots of changes in lysosomal diameter with extracellular pH in (a) MCF-12A, (b) MCF-7, (c) MDA-MB-231, and (d) MDA-MB-435 cells. The box represents the range between the third quartile and the first quartile of the respective distribution displaying the length of the interquartile range, which is 50% of the data. The median of the distribution is indicated by the horizontal line in the box. *Represents P < .05. **Represents P < .01. ***Represents P < .001, pH 6.8 vs 7.4 and pH 6.4 vs 7.4 (Mann-Whitney U-test; if D ≠ 0, P values for D > 0, D < 0).

Figure 6

Box-and-whisker plots of changes in lysosomal number with extracellular pH in (a) MCF-12A, (b) MCF-7, (c) MDA-MB-231, and (d) MDA-MB-435 cells. The box represents the range between the third quartile and the first quartile of the respective distribution displaying the length of the interquartile range, which is 50% of the data. The median of the distribution is indicated by the horizontal line in the box. *Represents P < .05. **Represents P < .01. ***Represents P < .001, pH 6.8 vs 7.4 and pH 6.4 vs 7.4 (Mann-Whitney U-test; if D ≠ 0, P values for D > 0, D < 0).

Figure 7

Representative (a) differential interference contrast images (80 x 80 m) and (b) dansyl group fluorescence images (80 x 80 m) of living MDA-MB-435 human breast cancer cells perfused in the microscopy-compatible cell perfusion system using cell culture medium at (i) pH 7.4 (control), (ii) pH 6.4 for 4 hours, and (iii) pH 6.4 for 6 hours. Highly glycosylated lysosomal proteins of these cells were biosynthetically prestained prior to the experiment with the dansyl group carrying glucosamine 6-O-dansyl-GlcNH₂, as previously described [40]. Some cells displayed filopodia that contained lysosomes as well (b(ii), arrows).

Figure 8

Quantitation of the number of filopodia per cell (upper panel) and the length of filopodia (lower panel) in invasive/metastatic human breast cancer cells (a) MDA-MB-231 and (b) MDA-MB-435 at (i) pH 7.4 (control), (ii) pH 6.8, and (iii) pH 6.4. 100 cells from four independent experiments were measured per cell line and pH employing DIC images. In both metastatic breast cancer cell lines, the number of cells with a high number of filopodia as well as the number of long filopodia increased gradually with decreasing pH. No filopodia were observed in MCF-12A HMECs and poorly invasive human breast cancer cells MCF-7.