Institute of Molecular Cancer Research

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Bonde,Schwendener, Mete, Kumar

MODULATION OF THE TUMOR MICROENVIRONMENT BY BISPHOSPHONATES: BASIC BIOLOGICAL AND IMMUNOLOGICAL PROPERTIES AND EVALUATION AS A NOVEL APPROACH TO CANCER THERAPY

Background

Modulation of solid tumors with bisphosphonates and bisphosphonate-liposomes leads to growth inhibition and re-polarization to a growth-suppressing tumor microenvironment. We study the potential of this method by two ways:1) as a tool to study basic biological and immunological effects in the tumor microenvironment and 2) as cancer therapy approach in mouse tumor models. Our aim is to contribute to a better understanding of the complex processes of tumor development, growth and metastatic dissemination.

Introduction

Solid tumors are not only composed of malignant cells; they are complex organ-like structures comprising many cell types, including a wide variety of migratory hematopoietic (macrophages, neutrophils, myeloid-derived suppressor cells, immune cells) and resident stromal cells. Migration of these cell types into tumors has been interpreted as evidence for an immunological response of the host against a growing tumor. It is acknowledged that tumors are largely recognized as "self" and lack strong antigens. Instead, they have the property to manipulate the host immune system to prevent rejection and to facilitate their own growth and spread. This led to the proposal that hematopoietic cell infiltrates have a causal role in carcinogenesis. Clinical data collected from a wide range of solid tumors underscore these results, given that those high densities of leukocytic infiltrations, most notably tumor associated macrophages (TAM) and neutrophils (TAN), correlate with poor prognosis of the diseases.

TAMs are derived from circulating monocytes and are activated macrophages of the polarized type II (M2 macrophages), mainly induced by IL-4, IL-10, IL-13 and corticosteorids. Differential cytokine and chemokine production and coordinated temporal and spatial activities of these cells in the tumor stroma are key features of polarized macrophages that promote tumor angiogenesis and growth. Due to their tumorigenic role, M2-TAMs have been proposed as potential therapeutic targets. Similarly, TANs have been shown assume comparable polarized phenotypes that suppress anti-tumorigenic immune cells in the tumor microenvironment. To study the role of TAMs and TANs we use bisphosphonates and bisphosphonate-liposomes in various subcutaneous mouse tumor models. Tumor growth is monitored by immunohistochemistry, flow cytometry, microscopy and by genomic and proteomic approaches. Additionally, we use in vitro conditioned-medium co-culture models to identify emerging proteins translated in cancer and endothelial cells in response to macrophages.

Most conventional tumor therapies are flawed due to the genetic instability of cancer cells, which leads to drug resistance. Since macrophages and neutrophils are shown to be involved in assisting tumor properties e.g., polarization, invasion and angiogenesis, it is important to reveal the molecular mechanisms responsible for these events. Moreover, it is essential to identify new drug targets in signaling pathways in these cells and apply their inhibitors in liposomal formulations to specifically target myeloid cell types. To achieve this, we have established co-culture models of tumor cells and macrophages, which are used to screen inhibitors of inflammatory signaling pathways. We also exploit our co-culture models to identify nascent proteins translated in cancer and endothelial cells in response to macrophages. This project will shed light on the understanding of the role of the tumor microenvironment and for the development of new therapies.

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Figure 1. Recruitment of bone marrow-derived cells such as monocytes/ macrophages and neutrophils to the tumor microenvironment. Tumor and stromal cells mobilize tumor promoting myeloid cells to the peripheral blood through secretion of cytokines and chemokines followed by the mobilization and infiltration of these cells to the tumor microenvironment, where they assume tumorigenic properties. Bisphosphonate-liposomes inhibit tumor growth, either by depletion of M2-polarized TAMs or by causing neutrophil infiltration and skewing of N2-neutrophils to anti-tumorigenic N1-neutrophils, thereby creating an anti-tumorigenic microenvironment (Modified from Schmid & Varner, J. Oncol. 2010).

Tumor-associated macrophages regulate tumor cell malignancy by induction of epithelial to mesenchymal transition

Macrophages are important components of the tumor microenvironment and their cancer-promoting properties are widely acknowledged. Besides regulating the “angiogenic switch” and remodeling the extracellular matrix, a number of studies have suggested that macrophages orchestrate the migration and invasion of epithelial tumor cells. Epithelial-mesenchymal transition (EMT) is a well-characterized cellular process, through which cells down-regulate epithelial adherence molecules and acquire motile and invasive properties. In this project we are addressing the potential involvement of TAMs in the regulation of an EMT-associated phenotypic shift in tumor cells. We have used liposome-encapsulated clodronate to deplete macrophages in a murine F9-teratocarcinoma model. Gene expression analysis indicated a reduction in mesenchymal gene expression in macrophage depleted tumors. Our data suggest that macrophages can contribute to the regulation of an EMT-associated phenotypic shift in tumor cells. Using conditioned medium culturing we identified macrophage-derived TGF-β as the main regulator of the mesenchymal phenotype in F9-cells and mammary gland NMuMG-cells. Moreover, macrophage conditioned medium, as well as recombinant TGF-β, stimulated the invasive properties of the cells. The clinical relevance of our findings was addressed in a cohort of 491 non-small cell lung cancer patients by an immunohistochemical analysis. This study confirmed a significant correlation between CD68+ macrophage density, a pronounced mesenchymal tumor cell profile and tumor grade. In conclusion, this project has identified a regulatory role for TAMs in EMT-associated phenotypic shift of tumor cells.

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Figure 2. Immunohistochemical analysis identifies correlations between tumor cell expression of EMT markers and intra-tumoral CD68+ macrophage density. Whereas E-cadherin (red, left panel) and β-catenin (red, middle panel) localize to the plasma membrane of tumor cells in areas with low CD68+ macrophage (green) densities (upper row), expression of both proteins is compromised and partially lost in areas with high CD68+ densities (lower row). Conversely, fibronectin (red, right panel) expression is increased in areas with high CD68+ densities. Thus, intra-tumoral TAM density correlates with a mesenchymal tumor cell phenotype in F9-tumors. The nuclei are stained with DAPI (blue). Scale bar=0.02 mm.

Zoledronate-mediated modulation of the tumor microenvironment leads to impaired tumor growth

Zoledronate, an inhibitor of osteoclastic bone resorption, is commonly used to prevent and treat osteoporosis. There is emerging interest in the use of zoledronate as an anticancer agent based on preclinical evidence of its anti-tumor properties. Due to its high affinity for bone matrix, most models addressed the ability of zoledronate to reduce skeletal tumor burden and prevent bone metastases. However, whether zoledronate prevents tumor progression in soft tissue tumors and the mechanism of its antitumor effects is still under investigation.

To address these issues, we treated mice bearing syngeneic subcutaneous tumors with zoledronate and zoledronate-liposomes. A significant reduction in growth of Lewis lung (LLC) and colon carcinoma (MC38), but not B16 melanoma, tumors in mice was observed. We examined the effect of the drug on the tumor microenvironment focusing on tumor infiltrating myeloid cells. We saw an increase of CD11b+ myeloid cells in the tumor microenvironment as well as in spleen, blood and peritoneum of treated animals. It is known that solid tumors actively recruit myeloid cells and divert their functions toward an immune-suppressive and pro-tumorigenic M2-like phenotype. The inverse correlation between myeloid cell density and tumor growth in zoledronate-treated animals points to a  reprogramming of these cells: myeloid cells from treated tumors were found to acquire an M1 anti-tumorigenic phenotype, as shown by increased expression of pro-inflammatory and immunostimulatory and reduced expression of the immunosuppressive factors. Furthermore, these cells displayed an enhanced ability to stimulate proliferation of naive CD8+ T cells. Further characterization of these cells identified the neutrophils as increasingly accumulating myeloid cell types in tumors of zoledronate-treated animals. Accordingly, zoledronate was found to increase the production of neutrophil-attracting chemokines by cancer cells as well as tumor infiltrating myeloid cells. Further analysis of CD11b+Ly6G+ neutrophils and CD11b+Ly6G- monocytes/macrophages revealed that zoledronate exerts inflammatory and immunogenic transcriptional changes specifically in neutrophils, but not in macrophages. Another key finding was that recombinant TGF-β administration reduced therapeutic efficacy of zoledronate by reducing neutrophil infiltration. To improve the antitumor efficacy of zoledronate, we encapsulated the drug into liposomes, which significantly improved the antitumor efficacy of zoledronate by altering its pharmacokinetics and biodistribution profiles. Collectively, our findings reveal novel anti-tumorigenic properties of zoledronate that may assist in the design of more effective immunotherapeutic approaches for cancer.

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Figure 3. Model of neutrophil re-polarization induced by zoledronate. Upon in vivo administration, zoledronate preferentially accumulates in bone where it inhibits tumor-induced osteoclast activity and bone resorption. Thereby, zoledronate prevents the release of bone matrix-embedded growth factors (e.g. TGF-β, IGF-1, BMPs) that, induce enhanced expression of neutrophil-attracting chemokines, and thus cause increased accumulation of neutrophils in tumors. Zoledronate skews neutrophil polarization away from the pro-tumorigenic and immunosuppressive N2-like phenotype that is known to be regulated by TGF-β. Accordingly, TGF-β administration reverses the anti-tumor effects. Free zoledronate primarily accumulates in bones while liposomal encapsulation of the drug improves its bioavailability in extraskeletal tumor sites that leads to stronger inhibition of tumor growth.

Liposomes as drug carriers

Our laboratory has a continuous interest in liposome technology and development of liposomal formulations with antitumor and antiviral drugs, peptide vaccines, macrophage depletion and lipophilic contrast agents. For a comprehensive overview of our activities including recent publications we recommend a visit to the website of the Laboratory of Liposome Research.

Publications

Richter, Kirsten; Perriard, Guillaume; Behrendt, Rayk; Schwendener, Reto A; Sexl, Veronika; Dunn, Robert; Kamanaka, Masahito; Flavell, Richard A; Roers, Axel; Oxenius, Annette (2013). Macrophage and T cell produced IL-10 promotes viral chronicity. PLoS Pathogens, 9(11):e1003735.

da Costa, Maria Helena Bueno; Sant'anna, Osvaldo A; Quintilio, Wagner; Schwendener, Reto Albert; de Araujo, Pedro Soares (2012). A Rational Design for the Nanoencapsulation of Poisonous Animal Venoms in Liposomes Prepared with Natural Phospholipids. Current Drug Delivery, 9(6):637-644.

Warchol, Mark E; Schwendener, Reto A; Hirose, Keiko (2012). Depletion of resident macrophages does not alter sensory regeneration in the avian cochlea. PLoS ONE, 7(12):e51574.

Linkermann, Andreas; Bräsen, Jan H; De Zen, Federica; Weinlich, Ricardo; Schwendener, Reto A; Green, Douglas R; Kunzendorf, Ulrich; Krautwald, Stefan (2012). Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor-α-induced shock. Molecular Medicine, 18(1):577-586.

Panigrahy, D; Edin, M L; Lee, C R; Huang, S; Bielenberg, D R; Butterfield, C E; Barnés, C M; Mammoto, A; Mammoto, T; Luria, A; Benny, O; Chaponis, D M; Dudley, A C; Greene, E R; Vergilio, J A; Pietramaggiori, G; Scherer-Pietramaggiori, S S; Short, S M; Seth, M; Lih, F B; Tomer, K B; Yang, J; Schwendener, R A; Hammock, BD; Falck, J R; Manthati, V L; Ingber, D E; Kaipainen, A; D'Amore, P A; Kieran, M W; Zeldin, D C (2012). Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice. Journal of Clinical Investigation, 122(1):178-191.

Zhang, Yi; Zhang, Ruihua; Zhang, Huafeng; Liu, Jing; Yang, Zhuoshun; Xu, Pingwei; Cai, Wenqian; Lu, Geming; Cui, Miao; Schwendener, Reto A; Shi, Huang-Zhong; Xiong, Huabao; Huang, Bo (2012). Microparticles released by Listeria monocytogenes-infected macrophages are required for dendritic cell-elicited protective immunity. Cellular & Molecular Immunology, 9(6):489-496.

Nasser, M W; Qamri, Z; Deol, Y S; Ravi, J; Powell, C A; Trikha, P; Schwendener, R A; Bai, X F; Shilo, K; Zou, X; Leone, G; Wolf, R; Yuspa, S H; Ganju, R K (2012). S100A7 Enhances Mammary Tumorigenesis through Upregulation of Inflammatory Pathways. Cancer Research, 72(3):604-615.

Vuarchey, C; Kumar, S; Schwendener, R A (2011). Albumin coated liposomes: a novel platform for macrophage specific drug delivery. Nanotechnology Development, 1(1):e2.

Zattoni, M; Mura, M L; Deprez, F; Schwendener, R A; Engelhardt, B; Frei, K; Fritschy, J M (2011). Brain infiltration of leukocytes contributes to the pathophysiology of temporal lobe epilepsy. Journal of Neuroscience, 31(11):4037-4050.

Bijnsdorp, I V; Schwendener, R A; Schott, H; Fichtner, I; Smid, K; Laan, A C; Schott, S; Losekoot, N; Honeywell, R J; Peters, G J (2011). Cellular pharmacology of multi- and duplex drugsconsisting of ethynylcytidine and 5-fluoro-2'-deoxyuridine. Investigational New Drugs, 29(2):248-257.

Schott, H; Goltz, D; Schott, T C; Jauch, C; Schwendener, R A (2011). N(4)-[Alkyl-(hydroxyphosphono)phosphonate]-cytidine-new drugs covalently linking antimetabolites (5-FdU, araU or AZT) with bone-targeting bisphosphonates (alendronate or pamidronate). Bioorganic & Medicinal Chemistry, 19(11):3520-3526.

Egilmez, N K; Harden, J L; Virtuoso, L P; Schwendener, R A; Kilinc, M O (2011). Nitric oxide short-circuits interleukin-12-mediated tumor regression. Cancer Immunology, Immunotherapy, 60(6):839-845.

Yang, H; Kim, C; Kim, M J; Schwendener, R A; Alitalo, K; Heston, W; Kim, I; Kim, W J; Koh, G Y (2011). Soluble vascular endothelial growth factor receptor-3 suppresses lymphangiogenesis and lymphatic metastasis in bladder cancer. Molecular Cancer, 10:36.

Treiger Borema, S E; Schwendener, R A; Osso, J A; de Andrade , H F; Nascimento, N (2011). Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages. International Journal of Antimicrobial Agents, 38(4):341-347.

Schwendener, R A; Schott, H (2010). Liposome formulations of hydrophobic drugs. In: Weissig, V. Liposomes, Methods and Protocols, Vol. 1: Pharmaceutical Nanocarriers. New York, NY, USA, 129-138. ISBN 978-1-60327-359-6.

Schwendener, R A; Ludewig, B; Cerny, A; Engler, O (2010). Liposome-based vaccines. In: Weissig, V. Liposomes, Methods and Protocols, Vol. 1: Pharmaceutical Nanocarriers. New York, NY, USA, 163-175. ISBN 978-1-60327-359-6.

Westwood, J A; Haynes, N M; Sharkey, J; McLaughlin, N; Pegram, H J; Schwendener, R A; Smyth, M J; Darcy, P K; Kershaw, M H (2009). Toll-like receptor triggering and T-cell costimulation induce potent antitumor immunity in mice. Clinical Cancer Research, 15(24):7624-7633.