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

An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis

Subcutaneous xenografts have been used for decades to study hepatocellular carcinoma (HCC). These models do not reproduce the specific pathophysiological features of HCCs, which occur in cirrhotic livers showing pronounced necroinflammation, abnormal angiogenesis, and extensive fibrosis. As these features are critical for studying the role of the pathologic host microenvironment in tumor initiation, progression, and treatment response, alternative HCC models are desirable. Here, we describe a syngeneic orthotopic HCC model in immunocompetent mice with liver cirrhosis induced by carbon tetrachloride (CCl4) that recapitulates key features of human HCC. Induction of significant hepatic fibrosis requires 12 weeks of CCl4 administration. Intrahepatic implantation of murine HCC cell lines requires 30 minutes per mouse. Tumor growth varies by tumor cell line and mouse strain used. Alternatively, tumors can be induced in a genetically engineered mouse model. In this setting, CCl4 is administered for 12 weeks after tail-vein injection of Cre-expressing adenovirus in Mst1–/–Mst2F/– mice and results in development of HCC tumors (hepatocarcinogenesis) concomitantly to liver cirrhosis

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma

Background and Aims: Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. Approach and Results: We recapitulated these clinical outcomes using orthotopic—grafted or induced—murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8–positive (CD8+) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2–positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. Conclusions: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy–resistant and anti-PD-1 therapy–responsive HCC models

Placental growth factor promotes tumour desmoplasia and treatment resistance in intrahepatic cholangiocarcinoma.

OBJECTIVE Intrahepatic cholangiocarcinoma (ICC)-a rare liver malignancy with limited therapeutic options-is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. DESIGN We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. RESULTS PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC.ConclusionPlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC

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