Integrin α3β1 Can Function to Promote Spontaneous Metastasis and Lung Colonization of Invasive Breast Carcinoma

Growth of primary tumors and total tumor burden. On day 0, 5,000 luciferase-expressing 4T1 wild-type, α3si, and α3Rx cells were implanted in the fourth mammary fat pad of female Balb/C mice. A, BLI of the cells on day 10 (when α3si tumor cell burden appeared reduced compared with controls) and day 31 (when α3si tumor cell burden appeared similar to wild-type). B, total apparent tumor burden, measured as log photon flux, for the entire timecourse of the experiment. The α3si tumor cell burden was significantly less than wild-type or α3Rx on days 6 and 10 (*, P < 0.001 vs. wild-type, P < 0.01 vs. α3Rx, ANOVA with Tukey posttest; n = 10 mice per group). The slight reduction in photon flux observed on day 35 was due to the loss of some of the mice with the highest tumor burdens between day 31 and day 35. C, tumor volumes measured by caliper. The mean volume of the α3si tumors was significantly less than the volumes of both the wild-type and α3Rx tumors from day 20 through day 31 (*, P < 0.01 on day 20, and P < 0.05 on days 24–31, ANOVA with Tukey posttest, except for day 27, when α3si was significantly different from α3Rx, but not wild-type).

Spontaneous metastasis is significantly impaired in α3 integrin-silenced 4T1 cells. A, lung colonization as quantified in ex vivo BLI images (see Supplementary Fig. S2B for representative images). The α3si 4T1 cell colonization was significantly reduced compared with wild-type or α3Rx cell colonization (*, P < 0.05 vs. wild-type, P < 0.001 vs. α3Rx, ANOVA with Tukey posttest; n = 10–14 mice per group; bars indicate mean ± SEM). B, wild-type, α3si, and α3Rx 4T1 cells were cell surface biotinylated, and α3β1 integrin was recovered by immunoprecipitation from Triton X-100 lysates, as in Fig. 1. For each set, the lane marked “parental” corresponds to the original cell line implanted into mammary fat pad at the beginning of the assay, and the lanes numbered 1 to 3 correspond to sublines recovered from lung explants at the end of the assay. Quantification of band intensities by LI-COR blot imager is indicated under each lane (in arbitrary intensity units). C, the relationship between primary tumor volume and lung colonization measured by BLI is graphed for each cell type. Values for R² and P from Pearson correlation tests are shown for each graph. In no case was there a significant correlation between primary tumor volume and lung colonization.

Experimental lung metastasis is significantly impaired in α3 integrin-silenced 4T1 cells. On day 0, 50,000 wild-type, α3si, or α3Rx luciferase-expressing 4T1 cells were injected by tail vein into female Balb/C mice. A, BLI imaging on day 0, immediately after tail vein injection and on day 14, about halfway through the assay. B, total tumor burden (log photon flux) as measured by BLI. Metastatic colonization by the α3si cells was significantly reduced compared with wild-type or α3Rx cells from day 12 onward (*, P < 0.001 vs. wild-type and α3Rx cells, on days 12–20; #, P < 0.05 vs. wild-type and P < 0.001 vs. α3Rx on day 22; and *, P < 0.001 vs. α3Rx on day 26, ANOVA with Tukey posttest, n = 15 mice per group). C, survival to endpoint for mice bearing wild-type, α3si, or α3Rx 4T1 cells. Survival of mice with α3si 4T1 cells was significantly increased compared with mice with wild-type or α3Rx 4T1 cells (P < 0.0001 vs. wild-type and P = 0.0127 vs. α3Rx, Mantel–Cox log-rank test).

Gross and histologic analysis of experimental lung metastasis. A, lungs recovered 18 to 28 days after injection from mice bearing wild-type, α3si, or a3Rx 4T1 cells were paraformaldehyde fixed and photographed using a dissecting microscope before paraffin embedding. Pulmonary metastases appear as pale tan nodules on the surfaces of lungs (arrows point to examples). Fewer nodules were evident on lungs from mice with α3si cells. B, quantification of tumor nodules per 4× field in H&E-stained sections from paraffin-embedded lungs. The α3si 4T1 cells formed fewer nodules than either wild-type or α3Rx 4T1 cells. The wild-type cells formed more nodules than the α3Rx cells, indicating a partial restoration of α3 function for the α3Rx cells in this assay. (*, P < 0.01, ANOVA with Tukey posttest, n = 3 mice per group, 3 slides per mouse, 3 fields per slide, for a total of 27 fields per cell type). Shown at right are representative photomicrographs of lungs from mice injected with (C) wild-type, (D) α3si, or (E) α3Rx 4T1 cells. Asterisks indicate examples of tumor nodules within the pulmonary parenchyma. H&E, bars = 200 μm.

Impaired proliferation of α3si 4T1 cells. Wild-type, α3si, and α3Rx 4T1 cells were plated in 2% FBS in wells coated with 1 μg/mL LM-332, 1 μg/mL LM-511, 10 μg/mL collagen I, or in uncoated wells. One set of wells was assayed by WST-1 on day 0 (the day of plating) to measure cells input, and the remaining wells were assayed on day 2. Bars represent mean ± SEM for 6 wells per cell type per condition, normalized to day 0 values. The growth of the α3si cells was significantly less than that of either the wild-type or α3Rx cells on all substrates tested (*, P < 0.001, ANOVA with Tukey posttest).