Supplementary Figure 2: FACS-based quantification of immune cell infiltrating the metastatic site in PDAC.

Experimental metastasis model by intrasplenic implantation of 1 × 10⁶ KPC-derived pancreatic cancer cells. After 12 days, liver tissues were isolated, enzymatically digested and resulting single cell suspensions were stained for flow cytometry analysis. Naïve livers were used as controls (healthy). (a) Representative flow cytometry dot plots showing gating strategy used to quantify intrametastatic B cells (CD45⁺B220⁺), T cell (CD45⁺CD3⁺), NK cells (CD45⁺NK1.1⁺CD3^negB220^neg), Neutrophils (CD45⁺CD11b⁺F4/80^negLy6G⁺Ly6C⁺, inflammatory monocytes (CD45⁺CD11b⁺F4/80^negLy6G^negLy6C⁺) and macrophages (CD45⁺CD11b⁺F4/80⁺). Only viable cells (SYTOX^neg) were used (data are from 5 healthy livers or 8 liver metastasis; repeated two times with similar results). (b) Quantification of CD45⁺ hematopoietic cells detected in healthy control livers (Ctrl) and metastatic tumour bearing livers (LM) (n = 5 Ctrl mice; n = 8 LM mice; data combine two independent experiments; individual data points, horizontal lines represent mean ± s.e.m.; two-tailed unpaired t-test). (c) Percentage of intrametastatic immune cells among total viable cells gated according to (a) in healthy control livers (Ctrl) and metastatic tumour bearing livers (LM) (individual data points, horizontal lines represent mean ± s.e.m.; two-tailed unpaired t-test). Data shown combine two independent experiments; total mice: n = 5 Ctrl mice, n = 6 LM mice (B220, NK1.1, CD3); n = 5 Ctrl mice; n = 9 LM mice (Ly6G, Ly6C, F4/80). (d) Representative flow cytometry dot plots showing gating strategy to analyse CCR2 (CD192) expression levels. Intrametastatic inflammatory monocytes (CD45⁺CD11b⁺F4/80^negLy6G^negLy6C⁺) and macrophages (CD45⁺CD11b⁺F4/80⁺) express CCR2 (data are from 5 Ctrl mice or 8 LM mice; repeated two times with similar results). ns, not significant.