Tumor biology: use of tiled images in conjunction with measurements of cellular proliferation and death in response to drug treatments.

Tumor growth is dependent on the balance between cell proliferation and cell death, and these events occur heterogenously within an individual tumor. We present a methodology that provides integrative information about cell kinetics, cell death, and cell growth within individual tumors in animals treated with cytotoxic chemotherapeutic agents. Using HCT-116 and NCI-H460 cells, human colonic adenocarcinoma and non-small cell lung cells, respectively, traditional xenograft studies were performed. The tumor-bearing animals were treated with cyclophosphamide (Cytoxan), gemcitabine (Gemzar), or mitomycin C, and extensive analysis of the tumors was studied. Cell kinetics were evaluated by measuring the apoptotic and proliferation indices. The ability to image an entire tumor section using "tiling" by creating a large montage from many high-resolution images makes it possible to identify regional differences within areas of tumor and to demonstrate differences in these tumor regions after treatment with selected chemotherapeutic agents. Two specific areas within tumors have been identified: (a) areas of viable cells within the cell cycle, determined by bromodeoxyuridine and/or morphological characteristics determined by hematoxylin staining; and (b) areas of necrosis determined by the absence of bromodeoxyuridine and proliferating cell nuclear antigen-labeled cells coupled with morphological changes. By standardizing the tumor size to 100 mm2, different patterns of tumor responses to chemotherapeutic agents were determined. By creating such tiled images and by quantitating cell cycle kinetics, it is possible to gain a more complete understanding of tumor growth and response to treatment, leading to the development of more reliable methods for assessing the clinical behavior of anticancer drugs.

A fluorescent cellular adhesion assay using insect cell produced human VCAM1.

Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counterreceptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.

Novel small molecule alpha v integrin antagonists: comparative anti-cancer efficacy with known angiogenesis inhibitors.

Recent evidence supports the involvement of integrins in angiogenesis: blockade of alpha v beta 3 and alpha v beta 5 integrins disrupts angiogenesis leading to decreased blood vessel formation and hence decreased tumor growth. We hypothesized that av antagonists could inhibit tumor growth in tumor cells devoid of alpha v beta 3 integrins. We evaluated SM256 and SD983, novel small molecules that are specific av antagonists in mouse models of angiogenesis and tumorigenesis, and compared them with standards: TNP470, a fumagillin analog in the clinic, and flavopiridol, a cell cycle kinase inhibitor. In vitro SM256 was a selective alpha v beta 3 inhibitor with an IC50 = 4nM, and the affinity of SD983 against the mouse endothelial alpha v beta 3 integrin yielded an IC50 = 2nM and an IC50 = 54nM against alpha v beta 5. In the mouse Matrigel model of angiogenesis SM256 decreased blood vessel formation (hemoglobin content) with an ED50 = 0.055 ug/kg/day, tenfold more potent than TNP470. SG545, an ester of SD983, decreased blood vessel formation with an ED50 = 6 ug/kg/day, while flavopiridol ED50 = 18 ug/kg/day. In the mouse xenograft model, using human colon carcinoma RKO cells that do not express alpha v beta 3 but express alpha v beta 5, tumor growth was inhibited by SG545 (10 mg/kg/day) and flavopiridol (5 mg/kg/every other day) 40% and 70%, respectively (p < 0.05). Although the proliferative index (measured by BrdU incorporation) was not significantly changed with SM256, SG545 or flavopiridol (29-32%), the apoptotic index increased significantly (p < 0.05) in the SM256 and SG545-treated groups (2.3-2.7%) compared with controls (1.1%), suggesting increased cell death contributed to decreased tumor volumes. Neovascularization decreased with SM256 and SG545 treatment. The data demonstrate that potent selective av antagonist can target endothelial cells, tumor cells, inhibit angiogenesis and inhibit tumor growth.