Supplementary Figure 3: Liver PDAC metastasis induces the recruitment of monocyte-derived macrophages, followed by the activation of resident hStCs.

(a,b) Experimental metastasis model by intrasplenic implantation of 1 × 10⁶ KPC cells. (a) Intrametastatic CD11b⁺ cells showing a marked expansion of the macrophage population (F4/80⁺, blue) in established macro-metastatic lesions (day 12, D12), while inflammatory monocytes (Ly6C⁺Ly6G^neg, red) predominantly increased during early micro-metastatic spreading (day 5, D5). Ctrl = 5 mice; Day 5 = 2 mice, Day 12 = 5 mice; data combine two independent experiments. (b) Representative immunofluorescence staining showing increased CD11b⁺ cells (upper) in micro-metastatic livers, followed by excessive accumulation of αSMA⁺ myofibroblasts in livers with macro-metastatic lesions. Macrophages (lower) are equally distributed in healthy livers (Kupffer cells) and show increase clustering in tumour bearing livers. Data are from 6 mice per time point; two independent experiments. (c) Representative Masson’s trichrome staining (MTS) of healthy control liver indicating absence of fibrotic stroma (lack of blue colour). Data are from 6 mice; two independent experiments. (d) Statistical comparison showing a positive correlation (Pearson) between increased numbers of αSMA⁺ myofibroblasts and area occupied by metastatic cancer cells in tumour bearing murine livers. solid line = best fit, dashed lines, 95% confidence intervals. Total n = 20 mice; four independent experiments. (e–g) Primary tumours and spontaneous metastatic hepatic tumours derived KPC mice. (e) Representative HE images showing the presence of an excessive stromal compartment at both sites (data are from 5 mice per condition, four fields assessed per sample). (f) Representative images of liver tumour sections stained for MAMs (F4/80⁺), pancreatic cancer cells (CK19⁺), or myofibroblasts (αSMA⁺) showing the presence of an excessive metastatic microenvironment mainly consisting of MAMs and myofibroblasts surrounding the tumour cells (data are from 5 mice per condition, four fields assessed per sample). (g) Flow cytometry analysis showing a marked expansion of the macrophage population (F4/80⁺) in metastatic livers (ML) compared to healthy livers (HL) (n = 5 mice per condition, data combine five independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (h–j) Chimeric WT + tdTomatoRed BM (WT + tdTR BM) mice. (h) Successful BM reconstitution was confirmed by flow cytometry analysing total circulating CD11b⁺Gr1⁺ cells. (i, j) Representative images showing co-localization of tdTomatoRed signal with F4/80 + MAMs (i), but not with αSMA + myofibroblasts in the metastatic lesion (below white line). Data are from 6 mice per condition; one experiment. Scale bars, b,c, f, 100 μm; e, 50 μm; i, j, 25 μm.