Extended Data Fig. 6: ER- mouse mammary tumour cells show equivalent metastasis in young and aged mice.
a, Additional data associated with Fig. 3g. TSAE1-mChLuc2 cells were isolated from the primary tumours or metastatic lungs, and tumour cell Pdgfa and Pdgfb expression assessed (RT-qPCR) and compared to expression in in vitro cultured cells (n = 3 biological repeats). b, PDGFC expression in ZR-75-1-mChLuc2 cells in vitro (n = 3 biological repeats) or isolated from primary tumours following orthotopic inoculation into NSG mice. RT-qPCR using a human-specific probe (n = 5 mice). c, 575,000 GFP+ CAFs were seeded with 200,000 TSAE1 or HRM1 tumour cells or 575,000 ZR-75-1 cells in 6-cm dishes in DMEM (10% FBS) and cultured for 48 hours. For monocultures, 400,000 TSAE1 or HRM1 cells or 750,000 ZR-75-1 cells were seeded. Single cell suspensions were stained with DAPI to exclude dead cells. 100,000-200,000 GFP-negative tumour cells were sorted on a Sony SH800 cell sorter and collected for RNA extraction. RT-qPCR analysis of Pdgfc/PDGFC expression (as detailed in Methods). Representative data of two (ZR-75-1) or three (TSAE1 and HRM1) independent repeats, with two independent CAF populations. d, 4T1 cells were inoculated orthotopically into young (n = 5 mice) or aged (>12-month, n = 5 mice) BALB/c mice. Left, tumour volume. Right, quantification of spontaneous metastasis to the lung, with representative images shown (scale bar, 5 mm). e, Pdgfc expression (RT-qPCR) in ER- AT-3 cells culture in vitro compared to ER+ TSAE1 cells. f, AT-3 cells were inoculated intravenously into young (n = 4 mice) or aged (18-month, n = 5 mice) C57BL/6 mice. Quantification of lung metastasis. Data are presented as mean values; a,b,d,f, ±SEM; a, one-way ANOVA with multiple comparisons; b,f (left), two-tailed Mann-Whitney U-test; d (left), two-way ANOVA with multiple comparisons; d, (right),f (right), two-tailed t-test.
