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. 2021 Oct 27;12:6202. doi: 10.1038/s41467-021-26460-z

Fig. 2. Complement component 3 is upregulated in PM-LMSCs and M-LMSCs, and mediates the metastasis-promoting effect.

Fig. 2

A Heatmap of the top 20 mRNAs enriched in PM-LMSCs and M-LMSCs. n = 3 mice each. B C3 levels in the supernatants of cultured WT-LMSCs, A-LMSCs, PM-LMSCs, and M-LMSCs. Fifty thousand WT, A-, PM- or M-LMSCs were grown in 12-well plates for 24 h before supernatants were collected. n = 4 supernatants of independently cultured LMSCs. C Immunofluorescence of C3 and Nestin. PM-LMSCs were co-stained for C3 (red) and Nestin (green), and lung tissue of 10- to 11-week-old MMTV-PyMT mouse was co-stained with C3 (red) and Nestin (green). Scale bar (in vitro), 8 μm; scale bar (in vivo), 5 μm. The images were representative of three independent experiments. D Representative images of lung of MMTV-PyMT mice stained by immunofluorescence for C3 (red) and Nestin (green). Scale bar represents 25 μm. The images were representative of those generated from three mice each group. E C3 was depleted by RNAi. The RNAi efficiency was shown on the left. The WB were representative of two independent experiments. F Depletion of C3 blocked the metastasis-promoting activity of LMSCs. SCR or shC3 PM-LMSCs were co-administered with 4T1 cells into BALB/c mice and LMNs were counted on day 14. n = 5 mice each (Colonization model). 4T1 cells (4 × 105) were implanted into mammary gland fat pads of BALB/c mice. After 10 days, control PM-LMSCs or shC3 PM-LMSCs (1 × 105) were administered into these tumor-bearing mice by i.v. The lung metastatic nodules were counted on day 30. n = 5 mice each (Metastasis model). G C3 deletion in LMSCs reduced lung colonization. WT or C3−/− LMSCs were co-injected with 4T1 cells into BALB/c mice and LMNs were counted on day 14. n = 5 mice each (Colonization model). 4T1 cells (4 × 105) were implanted into mammary gland fat pads. After 10 days, WT or C3−/− LMSCs (1 × 105) were administered into these tumor-bearing mice by i.v. The LMNs were counted on day 30. n = 4 mice each (Metastasis model). H Recombinant C3a (mC3a) enhanced 4T1 metastasis. mC3a was administered i.v. into 4T1 tumor-bearing mice on days 1 and 7, and LMNs were counted on day 14. n = 5 mice each (left), n = 7, 6 mice (right). All the data are presented as mean values ± SD. ns, not significant; p < 0.05, significant, using a one-way ANOVA with Sidak’s post test for Fig. 2B, F, G; using an unpaired, two-tailed, Student’s t-test for 2H. Source data are provided as a Source Data file for Fig. 2B, E–H.