Metformin Reduces Desmoplasia in Pancreatic Cancer by Reprogramming Stellate Cells and Tumor-Associated Macrophages

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs.

Methods/results: In overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1β as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis.

Conclusion: Metformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.

Fig 1. Metformin treatment associates with reduced hyaluronan levels in human pancreatic cancers in overweight/obese patients.

(i) Representative histology images showing the effect of metformin on tumor hyaluronan levels in normal weight [Body mass index (BMI)<25)] or overweight/obese patients (BMI>25) (n = 22 controls, 7 metformin). (ii) Immunohistochemical analysis of total tumor hyaluronan levels. Metformin decreases the hyaluronan-positive area fraction (%) in overweight/obese patients. Data are presented as the mean ± standard error. * p < 0.05 vs. control in patients with BMI >25.

Fig 2. Metformin reduces ECM and AT1 expression in AK4.4 pancreatic cancer model in overweight/obese mice.

After 10 weeks of high-fat diet, AK4.4 tumors were orthotopically implanted in obese FVB mice. Animals were randomly assigned to metformin in drinking water (300 mg/kg) or no treatment at day 7 until day 21 when tumors were collected. (A-i) Representative immunohistochemistry images showing the effect of metformin on tumor hyaluronan and collagen-I levels in AK4.4 tumors (n = 3–4). (A-ii) Quantification of hyaluronan expression in AK4.4 tumors. Metformin decreases the hyaluronan-positive area fraction (%). (A-iii) Quantification of collagen-I expression in AK4.4 tumors. Metformin decreases the collagen-I-positive area fraction (%). (B-i) Representative immunohistochemistry images showing the effect of metformin on expression (co-localization) of hyaluronan and collagen-I levels in activated (αSMA-positive) pancreatic stellate cells (PSCs) in AK4.4 tumors (n = 3–4). (B-ii) Quantification of hyaluronan expression in PSCs. Metformin decreases the percentage of activated PSCs expressing hyaluronan. (B-iii) Quantification of collagen-I expression in PSCs. Metformin decreases the percentage of activated PSCs expressing collagen-I. (C-i) Representative Western blots for angiotensin-II receptor-1 (AT1) expression in AK4.4 tumors. ß-actin is used as a control for protein loading. (C-ii) Densitometric analysis of AT1 expression normalized to ß-actin. Metformin decreases the expression of AT1. Data are presented as the mean ± standard error. * p < 0.05 vs. control.

Fig 3. Metformin reduces collagen-I/hyaluronan production by pancreatic stellate cells.

(A) PSCs were incubated in vitro with metformin (1 mM) for 48h. (A-i) Representative immunocytochemistry images showing the effect of metformin on tumor hyaluronan and collagen-I levels in human pancreatic stellate cells (PSCs) in vitro (n = 2). (A-ii) Quantification of hyaluronan expression in PSCs. Metformin decreases the expression of hyaluronan in PSCs. (A-iii) Quantification of the expression of collagen-I in PSCs. Metformin decreases the expression of collagen-I in PSCs. αSMA denotes activated PSCs. (B) Representative Western blots for the expression of fibrosis-related markers and signaling proteins in PSCs treated with metformin at 0, 0.1, 1 and 10mM. Metformin decreases the expression of fibrosis-related markers and signaling proteins in PSCs. Densitometric analysis of protein expression normalized to ß-actin or total protein (in the case of phosphorylated proteins) is depicted as numbers below the representative bands. Data in A are presented as the mean ± standard error. *p < 0.05 vs. control.

Fig 4. Metformin reprograms TAMs and reduces inflammation in tumors.

(A-i) Representative immunocytochemistry images showing the effect of metformin metformin on the expression of F4/80 (immunofluorescence) in AK4.4 tumors (percentage of viable tumor area) (n = 4). (A-ii) Metformin-treated tumors (300 mg/kg in drinking water) had significantly reduced levels of F4/80-positive tumor-associated macrophages (TAMs). (B) Effect of metformin (0–0.2mM) on the gene expression (qPCR) of M1 (i) and M2 (ii) markers in RAW 264.7 cells (mouse leukaemic monocyte-macrophages) in vitro. Clinically relevant doses (0.05 mM) of metformin treatment reduces expression of M2 markers in macrophages in vitro, including Arg-1 and IL-10. (C) Effect of metformin on the gene expression (qPCR) of M1 (i) and M2 (ii) markers in TAMs isolated from PAN02 tumors in vivo (n = 3). Metformin treatment reduced expression of the M2 markers Arg-1 and IL-10 in TAMs in vivo. (D) Representative Western blots for the expression of signaling proteins in RAW 264.7 cells treated with metformin at 0, 0.05, 0.1, 0.2 and 0.4 mM. Metformin decreases the activation of signaling pathways and increased activation of AMPKα on RAW cells. Densitometric analysis of protein expression normalized to ß-actin or total protein (in the case of phosphorylated protein) is depicted as numbers below the representative bands. (E) Effect of metformin on the protein expression of major cytokines in AK4.4 tumors (n = 4–5) using multiplex protein array. Metformin treatment associated with reduced IL-1ß and CXCL1 expression in tumors. Data are presented as mean ± standard error in A, C and E. * p < 0.05, ** p < 0.01 vs. control.

Fig 5. Metformin reduces ECM remodeling, EMT, and metastasis in a PDAC mouse model.

(A) Expression of genes associated with extra-cellular matrix (ECM) remodeling, epithelial-to-mesenchymal transition (EMT) and inflammation in AK4.4 tumors from control and metformin-treated mice. Data normalized to control group. 3–4 samples per group pooled in one single PCR array plate. Metformin reduces the expression of pro-tumor genes and increases the expression of anti-tumor genes. (B-i) Representative Western blots showing the effect of metformin (300 mg/Kg) on MMPs and EMT markers in AK4.4 tumors. (B-ii) Densitometric analysis of protein expression normalized to ß-actin. Metformin decreases the expression of MMP-9 and vimentin and increases the expression of e-cadherin in AK4.4 tumors. (C) MMP activity in AK4.4 tumor protein extracts from control and metformin-treated mice (n = 3–4). Metformin decreases the activity of MMPs. (D) Effect of metformin on the percentage of mice affected (incidence) with mesenteric (peritoneal) and abdominal wall (retroperitoneal) metastasis in AK4.4 and PAN02 models (n = 3–8). Metformin reduced the percentage of mice that develop wall metastasis in the more metastatic model (PAN02 model) and induced a tendency for reduced wall as well as mesenteric metastasis in the less metastatic AK4.4 model. (E) Effect of metformin on the number (average) of mesenteric (peritoneal) and abdominal wall (retroperitoneal) metastasis per mouse in the AK4.4 and PAN02 models (n = 3–8). Metformin reduced the number of wall metastasis in the PAN02 model. There were also trends for fewer mesenteric metastasis in AK4.4 and PAN02 tumors. Data in B, C and E are presented as the mean ± standard error. *p < 0.05 vs. control.

Fig 6. Metformin reprograms pancreatic stellate cells (PSCS) and tumor-associated macrophages (TAMs), alleviates the fibro-inflammatory tumor microenvironment and reduces metastasis.

Metformin treatment reduces collagen-I and HA production by PSCs, leading to decreased fibrosis in PDACs. Metformin treatment also reduces cytokine production, infiltration and M2 polarization of TAMs, leading to decreased inflammation. This associated with improved desmoplasia and reduced extracellular matrix (ECM) remodeling, epithelial-to-mesenchymal transition (EMT), and metastasis.