Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a devastating metastatic disease for which better therapies are urgently needed. Macrophages enhance metastasis in many cancer types; however, the role of macrophages in PDAC liver metastasis remains poorly understood. Here we found that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting inflammatory monocytes to the liver. Mechanistically, we demonstrate that granulin secretion by metastasis-associated macrophages (MAMs) activates resident hepatic stellate cells (hStCs) into myofibroblasts that secrete periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment or genetic depletion of granulin reduced hStC activation and liver metastasis. Interestingly, we found that circulating monocytes and hepatic MAMs in PDAC patients express high levels of granulin. These findings suggest that recruitment of granulin-expressing inflammatory monocytes plays a key role in PDAC metastasis and may serve as a potential therapeutic target for PDAC liver metastasis.

Figure 1. Metastatic PDAC cells induce macrophage recruitment and activation of myofibroblasts in the liver

(a) Identification of pan-cytokeratin (CK)⁺ metastatic pancreatic cancer cells, hematopoietic cells (CD45⁺), macrophages (CD68⁺) and myofibroblasts (αSMA⁺) as predominant cell types at the hepatic metastatic microenvironment of pancreatic cancer by immunohistochemical analysis of human biopsies. Representative micrographs and quantification of the data are shown (n = 5 PDAC patients, n= 5 healthy subjects; five fields assessed per sample; mean ± s.e.m; two-tailed unpaired t-test). HL= healthy liver, LM= liver metastasis. (b) Established metastatic nodules in the liver were processed 12 days post intrasplenic implantation of 1x10⁶ KPC and analysed by flow cytometry. Composition of intrametastatic leukocytes is shown as a percentage of CD45+ cells using the following definitions: B cells (CD45⁺CD3^negB220⁺); T cells (CD45⁺B220^negCD3⁺), NK cells (CD45⁺B220^negCD3^negNK1.1⁺), Neutrophils (CD11b⁺Ly6G⁺F4/80^neg), MAMs (CD11b⁺F4/80⁺) (n= 4 healthy livers; n = 8 liver metastasis; data combine two independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (c) Representative immunofluorescence staining of myofibroblasts (αSMA⁺) clustering around metastatic KPC^luc/zsGreen (zsGreen) cells in the liver at 5 and 12 days after implantation. Histogram: quantification of myeloid cells (CD11b⁺) and myofibroblasts (αSMA⁺) cell frequency in livers during the course of metastasis formation. Nuclei were counterstained with DAPI (n = 6 mice per time point; four fields assessed per sample; data combine two independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (d) Representative Masson’s trichrome staining of tumour bearing livers at 5 and 12 days after implantation. Histogram: quantification of area occupied by fibrotic stroma (n = 6 mice per time point; four fields assessed per sample; data combine two independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (e) Schematic of the generation of chimeric mice resulting in tdTomatoRed positive BM derived macrophages and non-labelled resident Kupffer cells (KC). (f) Chimeric mice from (e) were intrasplenically implanted with 1x10⁶ Panc02 cells and livers were harvested after day 12. Immunofluorescence analysis of bone marrow derived tdTomatoRed⁺ cells in combination with F4/80 staining and αSMA staining in tumour bearing livers. Nuclei were counterstained with DAPI (data are from 6 mice per condition; one experiment). Scale bars = 100µm; ns, not significant.

Figure 2. Macrophages promote myofibroblast activation and metastatic growth

(a - d) Liver metastasis was induced by intrasplenic implantation of 1x10⁶ KPC cells. Entire livers were harvested and analysed 12 days later. (a) Hematopoietic cells (CD45⁺), MAMs (CD45⁺CD11b⁺F4/80⁺), and IM (CD45⁺CD11b⁺Ly6C⁺F4/80^negLy6G^neg) from tumour bearing livers of wild type (WT) versus PI3Kγ^-/- (-/-) mice were evaluated by flow cytometry (n = 6 mice WT; n = 8 mice PI3Kγ^-/-; data combine two independent experiments; individual data points, horizontal lines represent mean ± s.e.m; two-tailed unpaired t-test). (b, c) Quantification of metastatic frequency (b) and average metastatic lesion size (c) in WT and PI3Kγ^-/- (-/-) mice by HE stained liver sections (n = 7 mice WT; n = 9 mice PI3Kγ^-/-; data combine two independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (d) Representative immunofluorescence staining and quantification of MAMs (F4/80⁺) and myofibroblasts (αSMA⁺) cell frequency in livers in WT and PI3Kγ^-/- (-/-). Nuclei were counterstained with DAPI (n = 6 mice WT; n = 8 mice PI3Kγ^-/-; four fields assessed per sample; data combine two independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (e - h) Liver metastasis was induced by intrasplenic implantation of 1x10⁶ KPC cells. Macrophages were depleted by clodronate liposome treatment after initial colonization of the liver had occurred. (e) Schematic illustration of the experiment. (f) Representative immunofluorescent staining and quantification of MAMs (F4/80⁺) and myofibroblasts (αSMA⁺) cell frequency in tumour bearing livers treated with liposomes containing PBS (PL) or clodronate (CL). Nuclei were counterstained with DAPI (n = 4 mice per condition; five fields assessed per sample; one experiment; mean ± s.e.m; two tailed unpaired t-test). (g, h) Evaluation of metastatic frequency (g) and area covered by metastatic cells (h) in tumour bearing livers of mice treated with PL or CL (n = 4 mice per condition; all metastatic nodules assessed from one section per sample; one experiment; individual data and mean ± s.e.m; two-tailed unpaired t-test). Scale bars = 100µm; ns, not significant.

Figure 3. Granulin secreted by macrophages activates hepatic stellate cells

(a) Quantification of αSMA (Acta2), and collagen 1a (Col1a) mRNA levels in primary hStCs stimulated with isogenic macrophage conditioned media (MCM) generated from wild type (WT) or granulin deficient (Grn^-/-) macrophages, and CM generated from Grn^-/- macrophages in the presence of recombinant granulin (rGranulin) as determined by qPCR (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (b) Quantification hStCs invasion towards MCM generated from WT or Grn^-/- deficient macrophages and MCM generated from Grn^-/- in the presence of rGranulin (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (c, d) same as (a, b), but MCM was generated using in vivo derived MAMs. Therefore liver metastasis was induced by intrasplenic implantation of 1x10⁶ KPC cells into isogenic WT + WT BM and WT + Grn^-/- BM chimeric mice (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (e) Representative immunofluorescence images of (d) showing Vybrant Dil (Em565) labelled murine hStCs showing invasion towards MCM generated from WT and Grn-/- MAMs and MCM generated from Grn-/- MAMs in the presence of recombinant granulin (rGranulin) (data are from three independent experiments). (f, g) Quantification of granulin mRNA levels by qPCR (f) and granulin secretion by ELISA (g) in primary unstimulated (M0) macrophages and macrophages stimulated with isogenic CM media generated from murine KPC and Panc02 tumour cells (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). Scale bar = 100 µm; ns, not significant.

Figure 4. Granulin is highly expressed in hepatic metastatic lesions and metastasis associated macrophages are the main source of granulin secretion

(a, b) Quantification of granulin mRNA levels (a) and granulin protein levels (b) in intrametastatic pancreatic cancer cells, immune cell depleted stromal cells (zsGreen^negCD45^neg), and MAMs (CD45⁺F4/80⁺) isolated by fluorescence activated cell sorting from established tumour bearing livers 12 days after intrasplenic implantation of 1x10⁶ KPC^luc/zsGreen cancer cells (a, data are from three pooled mice; one experiment; b, n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (c) Quantification of granulin and Tgfb mRNA levels in tissue resident (KC) and MAMs sorted from established metastatic lesions from chimeric WT mice harbouring tdTomatoRed+ BM as described in Fig. 1g (data are from three pooled mice; one experiment). (d) Spontaneous metastatic hepatic tumours derived from KPC mice were isolated and analysed. Representative images of immunohistochemistry staining for MAMs (CD68⁺) and granulin expression on serial tissue sections from metastatic lesions and healthy liver and quantification of the data (n = 5 control mice, n = 5 KPC mice, five fields assessed per sample; mean ± s.e.m.; two-tailed unpaired t-test). (e) Representative images of immunohistochemistry staining for MAMs (CD68⁺) and granulin expression on serial tissue sections from human metastatic PDAC lesions and healthy liver and quantification of the data (n = 5 healthy subjects, n = 5 PDAC samples; five fields assessed per sample; mean ± s.e.m.; two-tailed unpaired t-test). Scale bars = 100µm; asterisks indicate matched tissue areas; ns, not significant.

Figure 5. Granulin depletion prevents myofibroblast activation and PDAC metastasis

Liver metastasis was induced by intrasplenic implantation of 5x10⁵ KPC cells into WT and granulin deficient (Grn^-/-;) mice (a – e) and chimeric WT + WT BM and WT + Grn^-/- BM mice (f – l). Entire livers were harvested and analysed 12 days later. (a) Representative images of HE staining of liver sections (data are from 6 WT and 7 Grn-/- mice; one experiment). (b) Metastatic frequency (n = 6 WT mice, n = 7 Grn^-/- mice; all metastatic nodules assessed from one section per sample; one experiment; individual data points, horizontal lines represent mean ± s.e.m; two-tailed unpaired t-test). (c) Metastatic area (n = 6 WT mice, n = 7 Grn^-/- mice; all metastatic nodules assessed from one section per sample; one experiment; mean ± s.e.m; two-tailed unpaired t-test). (d) Representative immunofluorescent staining and quantification of MAMs (F4/80⁺) and myofibroblasts (αSMA⁺) cell frequency in tumour bearing livers. Nuclei were counterstained with DAPI (n = 6 WT mice, n = 7 Grn^-/- mice; four fields assess per sample; one experiment; mean ± s.e.m; two-tailed unpaired t-test). (e) qPCR analysis of multiple hStCs activation markers in intrametastatic myofibroblasts isolated from established metastatic lesions (data are from three pooled mice per condition; one experiment). (f) Metastatic frequency (n = 5 WT + WT BM mice, n = 6 WT + Grn^-/- BM mice; all metastatic nodules assessed from one section per sample; one experiment; individual data points, horizontal lines represent mean ± s.e.m; two-tailed unpaired t-test). (g) Metastatic area (n = 5 WT + WT BM mice, n = 6 WT + Grn^-/- BM mice; all metastatic nodules assessed from one section per sample; one experiment; mean ± s.e.m; two-tailed unpaired t-test). (h) Representative immunofluorescence staining and quantification of MAMs (F4/80⁺) and myofibroblasts (αSMA⁺) cell frequency in tumour bearing livers. Nuclei were counterstained with DAPI (n = 5 WT + WT BM mice, n = 6 WT + Grn^-/- BM mice; four fields assessed per sample; one experiment; mean ± s.e.m; two-tailed unpaired t-test). (i) qPCR analysis of multiple hStCs activation markers in intrametastatic myofibroblasts isolated from established metastatic lesions (data are from five pooled mice per condition; one experiment). (j, k) Representative IHC staining and quantification of Ki67+ tumour cell frequency (j) and cleaved caspase 3+ cell numbers (k) in metastatic livers (n = 5 WT + WT BM mice, n = 6 WT + Grn^-/- BM mice; five fields assessed per sample; one experiment; mean ± s.e.m; two-tailed unpaired t-test). (l) qPCR analysis of multiple M2- and M1- macrophage associated genes in MAMs isolated from metastatic tumours developed in WT + WT BM and WT + Grn^-/- BM mice (data are from 6 pooled mice per condition; one experiment). Scale bars = 100μm; ns, not significant.

Figure 6. Myofibroblast secreted periostin enhances pancreatic cancer cell growth.

(a) Top gene ontology (GO) functions of secreted proteins enriched in human myofibroblasts (Mf) following in vitro education with macrophage conditioned media (CM). (b) Fold change rank of identified proteins associated with extracellular matrix organisation. Data were obtained from one experiment assessing two biologically independent samples (Green = periostin). (c) Colony formation assay of primary murine KPC cells, murine Panc02, and human Panc1 cells in the presence or absence of Mf CM and periostin neutralising antibody (anti-Periostin). Representative images reflecting colonies and single cells are displayed for KPC cells (n = 3 independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (d) Immunohistochemical stainings of periostin in human liver biopsies (n = 5 healthy subjects, n= 5 PDAC; five fields assessed per sample; mean ± s.e.m; two-tailed unpaired t-test) and in spontaneous metastatic liver tumours collected from KPC mice (n = 5 mice per condition; five fields assessed per sample, mean ± s.e.m; two-tailed unpaired t-test). Representative micrographs and quantification of the data. HL = healthy liver, LM = liver metastasis. Scale bars = 100μm.

Figure 7. Macrophage derived granulin induces periostin expression by hepatic stellate cells in vitro and in vivo.

(a) Evaluation of periostin (Postn) mRNA expression levels in primary hStCs following stimulation with CM collected from BM WT or Grn^-/- macrophages in the presence or absence of recombinant granulin (rGranulin) (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (b, c) Evaluation of periostin (Postn) mRNA levels by qPCR (b) and periostin protein levels by ELISA (c) in primary hStCs following stimulation with CM collected from in vivo derived WT or Grn^-/- MAMs, in the presence or absence of recombinant granulin (rGranulin) (n = 3 independent experiments; mean ± s.e.m.; two-tailed unpaired t-test). (d, e) Evaluation of periostin deposition and fibrotic stroma formation in metastatic livers of control WT, WT mice treated with clodronate liposomes (CL), PI3Kγ^-/-, Grn^-/-, and WT + Grn^-/- BM mice 12 days after intrasplenic implantation of KPC cells. (d) Representative immunofluorescence staining and quantification of periostin deposition. Nuclei were counterstained with DAPI. (e) Representative Masson’s trichrome staining (MTS) and quantification of area occupied by fibrotic stroma (n = 4 mice per condition; four fields assessed per sample; data combine five independent experiments; mean ± s.e.m; two-tailed unpaired t-test). (f) Quantification of periostin (Postn) mRNA expression levels by qPCR in intrametastatic myofibroblasts isolated from tumour bearing livers of WT and Grn^-/- mice, and chimeric WT + WT BM and WT + Grn^-/- BM mice (data are from six pooled mice per condition; one experiment). Scale bars = 100μm.

Figure 8. Metastatic PDAC patients have increased circulating inflammatory monocytes that express high levels of granulin

(a) Peripheral mononuclear cells were isolated from healthy subjects and metastatic PDAC patients. Representative dot plot of inflammatory monocytes (IM; CD14^hiCD16^neg) and resident monocytes (RM; CD14^dimCD16^hi) after gating for the CD45⁺CD3^negB220^negCD19^negSytox^neg cell population (data are from 6 different PDAC patients and 6 different healthy subjects). (b) Quantification of a. Percentage of monocyte populations in healthy subjects and metastatic PDAC patients (n = 6 different healthy; n = 6 different PDAC samples; individual data points, horizontal lines represent mean ± s.e.m.; two tailed unpaired t-test). NS, not significant. (c) Quantification of granulin mRNA levels in inflammatory monocytes (IM) isolated from metastatic PDAC patients and healthy subjects (HS) as described in (a, b) (n = 3 different healthy, n = 4 different PDAC samples; mean ± s.e.m; two-tailed unpaired t-test). (d) Quantification of granulin mRNA levels in circulating IM sorted from KPC mice with pathological confirmed liver metastasis or tumour free litter mates (data are from four pooled mice per condition; one experiment). (e) Schematic depicting the role of macrophage-derived granulin in activation of hStCs and in PDAC liver metastasis. Inflammatory monocytes are recruited to the liver by metastatic pancreatic tumour cells through a PI3Kγ-dependent mechanism. Once in the metastatic tissue, differentiated macrophages stimulate the activation and recruitment of resident hStCs through granulin secretion resulting in excessive accumulation of myofibroblasts. Granulin-induced myofibroblasts release high levels of the extracellular matrix protein periostin, thereby enhancing survival and growth of metastatic pancreatic cancer cells in a hostile environment. Interruption of this sequence, by either preventing macrophage accumulation or by abolishing granulin expression in recruited macrophages, limits metastatic growth of pancreatic cancer cells.