Fig. 1.

Experimental set-up and gating strategy for flow cytometry analysis. a Schematics of experimental setup. 4T1 or 4T07 breast cancer cells were injected into the mammary fat pad of αSMA-RFP mice. 4T1 and 4T07 are sister cell lines derived from the same tumour, but have different metastatic profiles as denoted next to them in the schematic. CTCs = circulating tumour cells, met = metastases. b Flow cytometry gating strategy to obtain live cells devoid of lineage markers (Lin-), and subsequent gating on each CAF marker used for Boolean gating to define the CAF- and CAF+ populations. The gates for all markers were set using the fluorescent minus one (FMO) approach. The density plots show one representative 4T1 D7 tumour (D7: collected at day 7). FSC = forward scatter, SSC = side scatter