Characterization of CD44⁺/CD24^-/low Breast Cancer Cell Adhesion to Bone Marrow Endothelium

K. Luers Henson¹ and M. Burdick¹

¹Ohio University

Breast cancer tumor initiating cells have a putative phenotypic characterization of CD44⁺/CD24^-/low and are indicated in cancer metastasis to bone marrow. Several microenvironmental factors are believed to play a role in this site-specific metastasis including E-selectin, CXCR4, and CXCR4 ligand SDF-1α. Seven commercially available breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, T47D, ZR-75-1, BT-20, Hs578t) were analyzed via flow cytometry and immunofluorescence microscopy for CD44 and CD24 expression. Two of these cell lines (MDA-MB-231 and Hs578t) were found to have subpopulations that exhibited the proposed molecular surface expression of breast cancer tumor initiating cells.

Additionally, cell lines were evaluated via flow cytometry for expression of CXCR4, a regulator of cell adhesion to bone marrow endothelium. CXCR4 expression and functional analyses were performed in varying concentrations of chemokine SDF-1α. Flow chamber adhesion assays were employed to examine the role of SDF-1α and E-selectin in tethering and rolling adhesive interactions of CD44⁺/CD24^-/lowbreast cancer cells to endothelial cells under physiological flow conditions. Our findings suggest that resident molecules within the bone marrow microenvironment influence the migration of breast cancer cell subpopulations.

Extracellular Matrix Components and Tumor Factors Induce a Proangiogenic Dendritic Cell Phenotype

F. Benencia¹ and A. Kalyani Venkatesh²

¹College of Osteopathic Medicine and Russ College of Engineering, Ohio University, ²Russ College of Engineering, Ohio University

Dendritic cells (DCs) are professional antigen-presenting cells which have the capacity to generate specific immune responses. Interestingly, it has been shown that DCs present in the microenvironment of different tumors are poorly immunogenic and collaborate with tumor growth by generating angiogenic factors. We hypothesize that tumor factors and extracellular matrix components collaborate in developing this particular DC phenotype. We decided to investigate this using the 4T1 model of syngeneic breast cancer in BALB/c mice. First, by means of flow cytometry analysis, we determined that DCs isolated from the microenvironment of breast cancer express low levels of costimulatory molecules CD80, CD86, CD40, and MHC-II. Further, we determined that these cells express several angiogenic molecules at the level of RNA.

Thus, the profile of these 4T1 tumor-associated DCs is that of proangiogenic leukocytes. Interestingly these cells express high levels of CD29 and CD49f, which generate a complex that specifically binds to laminin.

This extracellular matrix component is present in the microenvironment of many tumors. When bonafide DCs were cultured on laminin-coated surfaces they downregulated the expression of costimulatory molecules. As determined by ELISA analysis, these cells were also able to produce large amounts of VEGF, a paradigmatically angiogenic molecule. These properties seemed to be a result of the interplay between the Akt and MEK signaling pathways as defined by inhibition studies. Finally, we were able to determine that when these cells were cultured on laminin, in the presence of tumor cell conditioned media, they acquire a phenotype closely resembling that of tumor-associated DCs.

Characterization of Glycolipid-Mediated Adhesion of Cancer Cells to Vascular Endothelium

J. Marshall¹, S.M. Wood¹, C. Abram¹, and M. Burdick^1
1Ohio University

One of the least understood aspects of cancer is the process of metastasis, or spreading from the original tumor site to a new organ. Cancer cells break off from the primary tumor, travel through the bloodstream, attach to the endothelium, and eventually pass through the blood vessel wall to create a new colony in a distant tissue site. However, the molecular mediators of these steps are largely unknown. It is believed that the latter two steps occur through a model parallel to that of leukocyte migration to sites of inflammation, a phenomenon critically dependent on E-selectin located on blood vessel endothelial cells. We hypothesize that head and neck squamous carcinoma cells (HNSCCs) express ligands that can bind to E-selectin, thereby mediating metastasis. Flow cytometry shows that HNSCCs express sialofucosylated molecules that could act as E-selectin ligands, and flow chamber adhesion assays confirm that HNSCCs bind to cytokine-stimulated endothelial cells in a wholly E-selectin-dependent manner. Enzyme treatment further reveals that sialofucosylated glycolipids are major adhesive ligand(s) for E-selectin. Determining which of the numerous candidate glycolipids play the largest role in adhesion will give insight into the mechanisms of metastasis and allow for further advances in metastasis prevention, diagnosis, and treatment.

Function of Hyaluronic Acid and Hyaluronic Acid Synthases in Head and Neck Cancer Cell Adhesion

C. Abram¹ and M. Burdick¹

¹Ohio University

In order to more effectively treat and prevent metastasis, or the spreading of cancer from the original tumor site, an understanding of the molecular mechanisms of this process is necessary.  Metastasis occurs when cancer cells break free from the tumor and travel through the bloodstream.  As these cells are flowing through the bloodstream, they adhere to endothelial cells, thus initiating metastatic invasion.  Hyaluronic acid (HA), a molecule located on the surface of cells, is believed to play a significant role in cell adhesion.  It is produced in cells by hyaluronic acid synthases (HAS), a membrane protein of which there are three known types: HAS-1, HAS-2, and HAS-3. The presence and effect of HA and HAS in head and neck cancers (HNC) is being explored with this project. HA is a biological polymer of high molecular weight. Because it forms a meshwork located on the cell surface, it can physically block other cell adhesion molecules, including CD44 and glycolipids.  CD44 has different functions as a ligand of E-selectin and other ECM proteins. CD44 is a receptor for HA as well. Glycolipids, smaller molecules located on the cell surface, also mediate adhesion to endothelial cells. Because HA blocks CD44 directly by binding to it, and covers glycolipids through steric hindrance, it could possibly be inhibiting cell adhesion by preventing these molecules from engaging their respective receptors.  Therefore, by removing HA from the surface of the cancer cells, these cells should bind more readily to endothelial cells.  Furthermore, by removing HAS, the HA on the surface of the cells should be eliminated, thus causing more adhesion.  Western blotting results show the presence of HA, and RT-PCR results show that all three types of HAS mRNA are expressed in varying concentrations in HNC. If this model is correct, it would show a possible mechanism for the metastasis of head and neck cancer, which could be targeted and treated.

Y. Choi¹, P. Sundd¹, S. Rogers¹, D. Goetz¹, and D. F. Tees¹

¹Ohio University

Leukocyte sequestration in lung capillaries is a key step in the inflammatory response to lung infection. P-selectin and ICAM-1 have well defined roles in leukocyte adhesion in systemic venules but their role in pulmonary capillaries is still unclear. Here, a novel in vitro Micropipette Cell Adhesion Assay (MCAA) used P-selectin, ICAM-1 or BSA coated capillary-sized glass microvessels as an in vitro model for a pulmonary capillary. Leukocytes were aspirated into adhesion molecule-coated micropipettes of varying diameters. Cell velocities and activation times were determined under pressures representative of lung capillaries. Neutrophil velocities in MCAA were significantly lower on P-selectin than BSA and decreased with increasing P-selectin concentration. Pre-treating P-selectin-coated microvessels with an anti-P-selectin mAb resulted in a increase in velocity. ICAM-1 expressed alone or with P-selectin, does not mediate adhesion of unstimulated neutrophils in pulmonary capillary geometry. These results demonstrate that P-selectin at low density mediates leukocyte adhesion in the pulmonary capillary geometry. The effect of micropipette size on activation time in the presence of these adhesion molecules will also be described. This work was supported by grant BES-0547165 from NSF.

A Sandwich ELISA for Wnt5a Detection in Fluid Phase Systems

C. Kummitha¹, K. Mayle¹, M. Christman II¹, S. Deosarkar¹, A. Schwartz¹, R. Malgor¹, K. McCall¹, L. Kohn¹, and D. J. Goetz¹

¹Ohio University

Wnt5a is a noncanonical member of the family of Wnt signaling molecules. It has been linked to a multiplicity of physiological and pathological processes including cell migration and growth, vascular remodeling, cancer, atherosclerosis and chronic inflammation. Wnt5a's role in pathology suggests its expression level could have diagnostic and/or prognostic value. In this study we developed a sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA) for detecting Wnt5a. We used a rabbit anti-human Wnt5a antibody as the capture antibody and attempted to detect recombinant Wnt5a (rmWnt5a) spiked into buffer. We used two systems to detect captured rmWnt5a: (1) goat anti-Wnt5a and horseradish peroxidase (HRP) conjugated F(ab')₂donkey anti-goat IgG as detection and enzyme-linked antibodies, respectively, and (2) biotinylated goat anti-Wnt5a and HRP-streptavidin as detection antibody and enzyme-linked avidin, respectively. Although sandwich ELISA using both of these systems failed to detect rmWnt5a in HBSS+, 1% BSA, the addition of polyethylene glycol (PEG) during the binding stage of rmWnt5a afforded the detection of rmWnt5a. The use of PEG during the binding stages of rmWnt5a and the detection antibody resulted in maximal detection of rmWnt5a. The dose response curve, using biotinylated goat anti-mouse Wnt5a and HRP streptavidin, is linear, has a slope of 0.039 and an intercept of 0.1603 with an R²of 0.9934. With PEG, this is a specific and sensitive sandwich ELISA to detect rmWnt5a in buffers.

Detection of VCAM-1 Positive Endothelial Cells in Whole Blood Using Real-Time PCR

S. P. Deosarkar¹, P. Bhatt¹, C. J. Lewis¹, and D. J. Goetz¹

¹Ohio University

Detection and characterization of circulating endothelial cells (CECs) could serve as a diagnostic tool in assessing endothelial damage and severity of vascular disease. In the present study, we explored the use of real-time PCR to detect endothelial cells and determine their activation state. For this purpose we used MCAM, an endothelial marker, and VCAM-1, an adhesion molecule known to be increased on endothelium at sites of vascular disease. As a first step in the development, we studied the effect of various inflammatory cytokines on VCAM-1 and MCAM mRNA expression by human umbilical vein endothelial cells (HUVEC). None of the cytokines studied had a significant effect on MCAM mRNA expression by HUVEC while several significantly increased VCAM-1 expression. We next spiked cytokine activated and resting HUVEC into separate samples of whole blood and analyzed the RNA via real time PCR. We observed that the level of MCAM mRNA detected correlated with the number of HUVEC spiked into the whole blood and that significantly higher levels of VCAM-1 mRNA was detected in blood spiked with cytokine activated HUVEC relative to the level detected in blood spiked with resting HUVEC. We defined an Endothelial Cell Activation Index (ECAI) which represents the extent of activated endothelial cells present in whole blood. For our studies, ECAI ranged between 1224±301 and 1940±605. Combined, these findings indicate that a MCAM and VCAM-1 real-time PCR assay may be used to detect activated endothelial cells in whole blood.

E-Selectin Ligands of Breast Cancer Cells
V. S. Shirure¹ and M. Burdick^1
1Ohio University
Hematogenous metastasis is the bloodborne spread of a malignant tumor to secondary sites different from the tumor origin. We hypothesized that E-selectin ligands (ESLs) expressed by breast cancer cells mediate adhesion to vascular endothelium, thus plays a crucial role in metastasis. In dynamic flow adhesion experiments, MDA-MB-468 and BT-20 breast cancer cells adhered to interleukin-1β activated human umbilical cord vein endothelial cells (HUVEC) but not to anti-E-selectin monoclonal antibody treated activated HUVEC. Characterization of breast cancer cell ESLs using flow cytometry showed that BT-20 cells but not MDA-MB-468 cells were positive for Slex and Slea. It was also found that both cell lines were positive for CD44, an ESL when appropriately glycosylated. CD44 dynamic antigen capture assay showed that tumor cell CD44 is an ESL, however perfusion of protease treated breast cancer cells over activated
HUVEC implied the possible role of glycolipid ligands. Perfusion of E-selectin transfected cells over
purified glycolipids from either breast cancer cell line indicated that glycolipids are ESLs in both. Sialidase treated breast cancer cells failed to adhere to activated HUVEC indicating a sialylation requirement for ESL function. Further characterization of breast cancer glycolipids and sialylated CD44 as ESLs can open new windows for diagnostic and prognostic developments.