Supplementary Figure 4: The recruitment of monocyte derived macrophages is necessary for efficient pancreatic cancer metastasis.

1 × 10⁶ KPC-derived cells (a) or 1 × 10⁶ Panc02 cells (b- e) were intrasplenically injected into age and sex matched WT and PI3Kγ^−/− (−/−) mice. After 12 days, total livers were harvested and analysed by flow cytometry, immunohistochemistry, and immune fluorescence based methods. (a) Representative HE staining of liver tissue sections showing a marked decreased size of metastatic tumours in livers of PI3Kγ deficient (−/−) mice (data are from 7 WT and 9 PI3Kγ^−/− mice; data combine two independent experiments). (b) Percentage of intrametastatic macrophages among CD45⁺ cells quantified by flow cytometry. PI3Kγ deficiency (−/−) results in a marked reduction of MAMs compared to WT (n = 6 mice per condition; one experiment; individual data points, horizontal lines represent mean ± s.e.m.; two-tailed unpaired t-test). (c,d) Quantification of metastatic frequency (c) and average metastatic lesion size (d) in WT and PI3Kγ knockout mice (−/−) by hematoxylin and eosin (HE) stained liver sections (n = 6 mice per condition; all metastatic nodules assessed from one section per sample; one experiment; c, individual data points, horizontal lines represent mean ± s.e.m.; d, mean ± s.e.m.; two-tailed unpaired t-test). (e) Representative immunofluorescence staining and quantification of myofibroblasts (αSMA⁺) cell frequency in livers in WT and PI3Kγ knockout mice (−/−). Nuclei were counterstained with DAPI (n = 6 mice per condition; two fields assessed per sample; one experiment; mean ± s.e.m.; two-tailed unpaired t-test).