Extended Data Figure 3: Characterization of recruited TMP195-activated myeloid cells.

Tumour-bearing MMTV-PyMT mice were randomly placed into treatment groups and received daily i.p. injections of either vehicle (DMSO) or 50 mg kg⁻¹ of TMP195 for 5 days. Whole tumours were processed into single cells and flow cytometry was performed. a, Gating strategy for MHCII⁺CD11b^lo versus MHCII⁺CD11b^hi. b, Representative mammary tissue macrophages (MTMs) versus tumor associated macrophages (TAMs) plots of 5 vehicle-treated and 5 TMP195-treated mice with quantitation. Graphs show the results from 5 independent experiments in which there were 3–5 animals per treatment group. c–e, To identify pre-existing versus new tumour macrophages, mice were pretreated with dextran labelled with Alexa555, which is ingested by macrophages. Then mice were treated for 5 days with vehicle or TMP195. Mice were injected with dextran labelled with Alexa594 before being euthanized. Whole tumours were processed into single cells and flow cytometry was performed. d, There is an increase in new, but not pre-existing macrophages, in tumours from TMP195-treated animals. Representative graph from two separate experiments with n = 3 per treatment group (unpaired t-test). e, Of note, the new macrophages are MHCII⁺CD11b^hi (mammary tissue macrophages; MTMs). t-test *P < 0.05. f, Mice received one i.p. injection of either vehicle (DMSO) or 50 mg kg⁻¹ of TMP195. The following day mice were i.v. injected with CD11b⁺ cells labelled with CFSE. Mice were then treated for an additional 5 days with vehicle or TMP195. g, Whole tumours were processed into single cells and flow cytometry was performed. 13 mice from 3 different experiments are shown. There is a significant increase in recruitment of i.v. injected CD11b⁺CFSE⁺ monocytes to tumours in TMP195-treated mice. Graphs show the results from 2 independent experiments (unpaired t-test). All graphs show mean and error bars represent s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.