CCR2 signaling in breast carcinoma cells promotes tumor growth and invasion by promoting CCL2 and suppressing CD154 effects on the angiogenic and immune microenvironments.

Breast cancer is the second leading cause of cancer-related deaths for women, due mainly to metastatic disease. Invasive tumors exhibit aberrations in recruitment and activity of immune cells, including decreased cytotoxic T cells. Restoring the levels and activity of cytotoxic T cells is a promising anticancer strategy; but its success is tumor type dependent. The mechanisms that coordinate recruitment and activity of immune cells and other stromal cells in breast cancer remain poorly understood. Using the MMTV-PyVmT/FVB mammary tumor model, we demonstrate a novel role for CCL2/CCR2 chemokine signaling in tumor progression by altering the microenvironment. Selective targeting of CCR2 in the PyVmT mammary epithelium inhibited tumor growth and invasion, elevated CD8+ T cells, decreased M2 macrophages and decreased angiogenesis. Co-culture models demonstrated these stromal cell responses were mediated by tumor-derived CCL2 and CCR2-mediated suppression of the T-cell activating cytokine, CD154. Coculture analysis indicated that CCR2-induced stromal reactivity was important for tumor cell proliferation and invasion. In breast tumor tissues, CD154 expression inversely correlated with CCR2 expression and correlated with relapse free survival. Targeting the CCL2/CCR2 signaling pathway may reprogram the immune angiogenic and microenvironments and enhance effectiveness of targeted and immunotherapies.

Role of ALDH1A1 and HTRA2 expression in CCL2/CCR2-mediated breast cancer cell growth and invasion.

Chemokines mediate immune cell trafficking during tissue development, wound healing and infection. The chemokine CCL2 is best known to regulate macrophage recruitment during wound healing, infection and inflammatory diseases. While the importance of CCL2/CCR2 signaling in macrophages during cancer progression is well documented, we recently showed that CCL2-mediated breast cancer progression depends on CCR2 expression in carcinoma cells. Using 3D Matrigel: Collagen cultures of SUM225 and DCIS.com breast cancer cells, this study characterized the mechanisms of CCL2/CCR2 signaling in cell growth and invasion. SUM225 cells, which expressed lower levels of CCR2 than DCIS.com cells, formed symmetrical spheroids in Matrigel: Collagen, and were not responsive to CCL2 treatment. DCIS.com cells formed asymmetric cell clusters in Matrigel: Collagen. CCL2 treatment increased growth, decreased expression of E-cadherin and increased TWIST1 expression. CCR2 overexpression in SUM225 cells increased responsiveness to CCL2 treatment, enhancing growth and invasion. These phenotypes corresponded to increased expression of Aldehyde Dehydrogenase 1A1 (ALDH1A1) and decreased expression of the mitochondrial serine protease HTRA2. CCR2 deficiency in DCIS.com cells inhibited CCL2-mediated growth and invasion, corresponding to decreased ALDH1A1 expression and increased HTRA2 expression. ALDH1A1 and HTRA2 expression were modulated in CCR2-deficient and CCR2-overexpressing cell lines. We found that ALDH1A1 and HTRA2 regulates CCR2-mediated breast cancer cell growth and cellular invasion in a CCL2/CCR2 context-dependent manner. These data provide novel insight on the mechanisms of chemokine signaling in breast cancer cell growth and invasion, with important implications on targeted therapeutics for anti-cancer treatment.This article has an associated First Person interview with the first author of the paper.

Chemokine Signaling Facilitates Early-Stage Breast Cancer Survival and Invasion through Fibroblast-Dependent Mechanisms.

Ductal carcinoma in situ (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2+ macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2+ fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients.Implications: This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. Mol Cancer Res; 16(2); 296-308. ©2017 AACR.

Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment.

Triple negative breast cancers are an aggressive subtype of breast cancer, characterized by the lack of estrogen receptor, progesterone receptor and Her2 expression. Triple negative breast cancers are non-responsive to conventional anti-hormonal and Her2 targeted therapies, making it necessary to identify new molecular targets for therapy. The chemokine CCL2 is overexpressed in invasive breast cancers, and regulates breast cancer progression through multiple mechanisms. With few approaches to target CCL2 activity, its value as a therapeutic target is unclear. In these studies, we developed a novel gene silencing approach that involves complexing siRNAs to TAT cell penetrating peptides (Ca-TAT) through non-covalent calcium cross-linking. Ca-TAT/siRNA complexes penetrated 3D collagen cultures of breast cancer cells and inhibited CCL2 expression more effectively than conventional antibody neutralization. Ca-TAT/siRNA complexes targeting CCL2 were delivered to mice bearing MDA-MB-231 breast tumor xenografts. In vivo CCL2 gene silencing inhibited primary tumor growth and metastasis, associated with a reduction in cancer stem cell renewal and recruitment of M2 macrophages. These studies are the first to demonstrate that targeting CCL2 expression in vivo may be a viable therapeutic approach to treating triple negative breast cancer.

High-throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps.

The ability to accurately control fluid transport in microfluidic devices is key for developing high-throughput methods for single cell analysis. Making small, reproducible changes to flow rates, however, to optimize lysis and injection using pumps external to the microfluidic device are challenging and time-consuming. To improve the throughput and increase the number of cells analyzed, we have integrated previously reported micropumps into a microfluidic device that can increase the cell analysis rate to ∼1000 cells/h and operate for over an hour continuously. In order to increase the flow rates sufficiently to handle cells at a higher throughput, three sets of pumps were multiplexed. These pumps are simple, low-cost, durable, easy to fabricate, and biocompatible. They provide precise control of the flow rate up to 9.2 nL/s. These devices were used to automatically transport, lyse, and electrophoretically separate T-Lymphocyte cells loaded with Oregon green and 6-carboxyfluorescein. Peak overlap statistics predicted the number of fully resolved single-cell electropherograms seen. In addition, there was no change in the average fluorescent dye peak areas indicating that the cells remained intact and the dyes did not leak out of the cells over the 1 h analysis time. The cell lysate peak area distribution followed that expected of an asynchronous steady-state population of immortalized cells.

Fibrinogen, riboflavin, and UVA to immobilize a corneal flap--conditions for tissue adhesion.

PURPOSE: Laser-assisted in situ keratomileus (LASIK) creates a permanent flap that remains non-attached to the underlying laser-modified stroma. This lack of permanent adhesion is a liability. To immobilize a corneal flap, a protocol using fibrinogen (FIB), riboflavin (RF), and ultraviolet (UVA) light (FIB+RF+UVA) was devised to re-adhere the flap to the stroma. METHODS: A model flap was created using rabbit (Oryctolagus cuniculus) and shark (Squalus acanthias) corneas. Solutions containing FIB and RF were applied between corneal strips as glue. Experimental corneas were irradiated with long wavelength (365 nm) UVA. To quantify adhesive strength between corneal strips, the glue-tissue interface was subjected to a constant force while a digital force gauge recorded peak tension. RESULTS: In the presence of FIB, substantive non-covalent interactions occurred between rabbit corneal strips. Adhesiveness was augmented if RF and UVA also were applied, suggesting formation of covalent bonds. Additionally, exposing both sides of rabbit corneas to UVA generated more adhesion than exposure from one side, suggesting that RF in the FIB solution catalyzes formation of covalent bonds at only the interface between stromal molecules and FIB closest to the UVA. In contrast, in the presence of FIB, shark corneal strips interacted non-covalently more substantively than those of rabbits, and adhesion was not augmented by applying RF+UVA, from either or both sides. Residual RF could be rinsed away within 1 hour. CONCLUSIONS: Glue solution containing FIB and RF, together with UVA treatment, may aid immobilization of a corneal flap, potentially reducing risk of flap dislodgement.

The role of nonenzymatic glycation and carbonyls in collagen cross-linking for the treatment of keratoconus.

PURPOSE: Corneal cross-linking (CXL) is a treatment for keratoconus that eliminates the need for keratoplasty in most patients. However, its molecular mechanisms remain under study. Advanced glycation end products (AGEs) have been suggested by many studies as the causative strengthening agent during CXL, though no studies to date have directly tested this hypothesis. METHODS: Corneas of young rabbits and sharks were pretreated with pyridoxal hydrochloride and copper ions before CXL. Two known inhibitors of AGE formation, aminoguanidine and rifampicin, were applied during CXL in the treatment solution. Tensile strength tests were conducted after these experiments to detect diminished or accentuated corneal stiffening after CXL. SDS-PAGE was performed on type I collagen cross-linked in the absence and presence of AGE inhibitors. RESULTS: Pretreatment with pyridoxal hydrochloride resulted in significantly higher corneal stiffening after CXL. AGE inhibitors significantly diminished cross-linking as detected by both tensile strength measurements using whole corneas and gel electrophoresis of in vitro cross-linking of type I collagen in solution, in the presence and absence of the inhibitors. Rifampicin inhibited CXL more significantly than aminoguanidine in gel electrophoresis and tensile strength tests, confirming recent findings on its efficacy as an AGE inhibitor. CONCLUSIONS: Data presented here suggest that CXL is carbonyl dependent and involves the formation of AGE cross-links. Six possible cross-linking mechanisms are discussed.