Breast cancer is the most common cancer and second leading cause of death in women. Neoadjuvant chemotherapy is increasingly used to "shrink" tumors prior to surgery and enable breast conservative approaches;however, long-term survival remains poor, in part due to factors that limit delivery of cytotoxic drugs to tumor tissue. Abnormal tumor blood vessels and altered transendothelial permeability and increased interstitial fluid pressures (IFP), conspire to limit delivery of macromolecular cytotoxic drugs. Thus, approaches that aim to alter tumor vessel hemodynamics and vascular permeability would effectively increase tissue accumulation of chemotherapeutic agents by overcoming high IFP and promoting convection driven uptake of large macromolecular agents into tissues. To this end, we have discovered a novel, endogenous pathway regulating vascular permeability that remains functional in tumor vessels. Whereas transforming growth factor beta 1 (TGF?1) restricts normal vascular permeability, inhibition of the type I TGF??receptor Alk5 expressed in vascular cells, enhances vascular permeability in normal as well as tumor vasculature (Sounni et al. manuscript submitted). Thus, we propose to administer an Alk5 inhibitor, in combination with macromolecular chemotherapeutic agents to improve breast tumor perfusion and accumulation of conventional chemotherapeutic agents. Paradoxically, while increases in tumor perfusion and improved penetration of cytotoxic agents have also been achieved by 'normalization'of tumor vasculature by blocking vascular endothelial growth factor (VEGF) to reduce vascular permeability, these effects are transient and largely limited to immature vessels more frequently associated with early stage tumors. As tumors progress, tumor vessels mature and become refractory to VEGF blockade. Importantly, we have also observed that TGFb1-mediated vascular stabilization remains functional in more mature tumor vessels, and further predict that Alk5 blockade will improve cytotoxic drug penetration in both early as well as late stage tumors. We will compare accumulation of Doxil in mammary tumor-bearing mice treated with Alk5 inhibitor as opposed to those treated with anti-VEGF antibody (DC101), and assess vascular permeability and accumulation of Doxil in both early and late stage tumors in MMTV-PymT mice. Moreover, while previous studies have used MR imaging data to asses breast cancer responses after cytotoxic drug administration, we will demonstrate that MR imaging of macromolecular contrast media (MMCM) in the presence or absence of Alk5 blockade correlates with Doxil accumulation and thus can be used to predict macromolecular drug distribution and to identify tumors most likely to benefit from cytotoxics combined with agents that improve vascular permeability. In addition, we will assess the anti-tumor impact of improved Doxil accumulation following Alk5 blockade by monitoring histopathologic characteristics of mammary adenocarcinomas, as well as tumor burden, tumor latency (to endpoint), and frequency of pulmonary metastasis, and further demonstrate that MR imaging based predictions of drug accumulation also correlates with therapeutic responses. We will also examine responses of orthotopically implanted human breast tumor cells in mice treated with Alk5 inhibitor plus Doxil versus Doxil as monotherapy, to demonstrate enhanced efficacy of Doxil in human tumor cell killing. Together these studies will establish the effectiveness of 1) improved cytotoxic drug accumulations in the presence and absence of ALK5 inhibitors at different tumor stages, 2) MRI predictions of tumor microvascular permeability and cytotoxic drug accumulation, and 3) enhanced tumor responses and reduced cytotoxicity from improved chemotherapeutic drug accumulation in tumors. Thus, by utilizing new predictive MRI correlations and exploiting a novel endogenous pathway regulating vascular permeability that remains functional in breast cancer, the delivery of chemotherapeutic agents and subsequent response of both early and late stage breast tumors will be radically improved.

Public Health Relevance

The major goal of our project is to examine whether specific short-term inhibition of ALK5 in vivo alters hemodynamics and tissue perfusion of mammary adenocarcinomas such that delivery of Molecular Resonance (MR) imaging compounds is improved and/or delivery of chemotherapeutic agents is enhanced, thus providing a survival advantage. Realization of this goal will improve diagnostic imaging and drug delivery into tissues containing vasculature that limits efficient tissue perfusion. The ability to transiently alter vessel stability and open vascular beds to facilitate intravenous or potentially intra-organ delivery of diagnostic or therapeutic agents would represent a significant advance in disease therapy and/or diagnostic imaging.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZCA1-SRLB-G (M1))
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Alley, Michael C
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Oregon Health and Science University
Anatomy/Cell Biology
Schools of Medicine
United States
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Ruffell, Brian; Chang-Strachan, Debbie; Chan, Vivien et al. (2014) Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells. Cancer Cell 26:623-37
Affara, Nesrine I; Ruffell, Brian; Medler, Terry R et al. (2014) B cells regulate macrophage phenotype and response to chemotherapy in squamous carcinomas. Cancer Cell 25:809-21
Medler, Terry R; Coussens, Lisa M (2014) Duality of the immune response in cancer: lessons learned from skin. J Invest Dermatol 134:E23-8
Ansari, Celina; Tikhomirov, Grigory A; Hong, Su Hyun et al. (2014) Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy. Small 10:566-75, 417
Anand, Sudarshan; Coussens, Lisa M (2014) Manipulating microRNAs to regulate macrophage polarization in gliomas. J Natl Cancer Inst 106:
Ruffell, Brian; Affara, Nesrine I; Cottone, Lucia et al. (2013) Cathepsin C is a tissue-specific regulator of squamous carcinogenesis. Genes Dev 27:2086-98
Ganesan, Anusha-Preethi; Johansson, Magnus; Ruffell, Brian et al. (2013) Tumor-infiltrating regulatory T cells inhibit endogenous cytotoxic T cell responses to lung adenocarcinoma. J Immunol 191:2009-17
Ruffell, Brian; Affara, Nesrine I; Coussens, Lisa M (2012) Differential macrophage programming in the tumor microenvironment. Trends Immunol 33:119-26
Ruffell, Brian; Au, Alfred; Rugo, Hope S et al. (2012) Leukocyte composition of human breast cancer. Proc Natl Acad Sci U S A 109:2796-801
Erez, Neta; Coussens, Lisa M (2011) Leukocytes as paracrine regulators of metastasis and determinants of organ-specific colonization. Int J Cancer 128:2536-44

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