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

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

Abstract 3987: Solid stress and elastic energy as measures of tumor mechanopathology

Abstract Introduction: Increased tissue stiffness is a widely accepted and actively studied biomechanical property of desmoplastic tumors, and has been linked to several hallmarks of cancer, such as growth, invasion and metastasis. The abnormal mechanics of tumors, however, are not limited to tissue stiffening. We recently demonstrated that solid stress represents a new mechanopathology that is consistently elevated in mouse and human tumors. The solid stress, transmitted by solid elements of the extracellular matrix, is distinct from interstitial fluid pressure. Therefore, tumors are not only more rigid than many normal tissues, but cancer cells also produce and are exposed to these physical forces. Composed of a combination of tension and compression, these forces are significant in tumors, but negligible in most normal tissues. Methods and Results: We developed the experimental and mathematical frameworks to provide (i) two-dimensional spatial map of solid stress in tumors (planar cut method), (ii) sensitive estimation of solid stress in small tumors with small magnitudes of solid stress, e.g., metastatic lesions (slicing method), and (iii) in situ quantification of solid stress in tumors, which retains the effects of the normal surrounding tissues (needle biopsy method). All three methods are based on the physical concept of releasing the solid stress in a controlled way with defined geometry, and then quantifying the stress-induced deformation by high-resolution ultrasonography or optical microscopy. Given the specific topography of the stress relaxation and the geometric and material properties of the tumour, the solid stress and discharged elastic energy is estimated using mathematical modeling. Applying these novel methods to multiple mouse cancer models in primary and metastatic settings has led to the following novel findings: (i) solid stress and elastic energy may be different between primary vs. metastatic settings, as they depend on both cancer cells and their microenvironment; (ii) tumor with higher elastic energy are not necessarily stiffer, and the stiffer tumors do not necessarily have higher elastic energy; (iii) solid stress increases with tumour size; and (iv) the normal tissue surrounding a tumour significantly contributes to the intratumoral solid stress. Conclusions: We developed three distinct methods to perform in situ and sensitive measurement of solid stress and obtain 2-D spatial map of solid stress in human and mouse tumors. Application of these methods in models of primary tumors and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; it increases with tumour size; and mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies. Citation Format: Hadi Nia, Hao Liu, Giorgio Seano, Meenal Datta, Dennis Jones, Nuh Rahbari, Joao Incio, Vikash Chauhan, Keehoon Jung, John Martin, Vasileios Askoxylakis, Tim Padera, Dai Fukumura, Yves Boucher, Francis Hornicek, Alan Grodzinsky, James Baish, Lance Munn, Rakesh Jain. Solid stress and elastic energy as measures of tumor mechanopathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2017-398

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

<p>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.</p

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

<p>(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.</p

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

<p>(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.</p

Metformin reprograms TAMs and reduces inflammation in tumors.

<p>(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) <i>in vitro</i>. Clinically relevant doses (0.05 mM) of metformin treatment reduces expression of M2 markers in macrophages <i>in vitro</i>, 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 <i>in vivo</i> (n = 3). Metformin treatment reduced expression of the M2 markers Arg-1 and IL-10 in TAMs <i>in vivo</i>. (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.</p

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

<p>(A) PSCs were incubated <i>in vitro</i> 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) <i>in vitro</i> (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.</p

Ly6C[superscript lo] monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy

Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6C lo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1+Ly6C lo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6C lo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6C lo monocytes recruit Ly6G + neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6C lo monocyte or Ly6G + neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6C lo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6C lo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies

Solid stress and elastic energy as measures of tumour mechanopathology.

Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is challenging. Here, we show that two-dimensional spatial mappings of solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and wide size ranges can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that: (i) solid stress depends on both cancer cells and their microenvironment; (ii) solid stress increases with tumour size; and (iii) mechanical confinement by the surrounding tissue significantly contributes to intratumoural solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to significant discoveries and new therapies