(CANCER RESEARCH 50, 159-163. January I. 1990] Enhancement of Monoclonal Antibody Uptake in Human Colon Tumor Xenografts following Irradiation1 H. Kalofonos, G. Rowlinson, and A. A. Epenetos2 Imperial Cancer Research Fund Oncology Group, Royal Postgraduate Medical School, Hammersmith Hospital, London W120HS, England ABSTRACT Indium-Ill-labeled Al Al tumor-associated monoclonal antibody raised against an antigen of colon adenocarcinoma was used to evaluate the effect of ionizing radiation on antibody uptake by the LoVo adeno carcinoma cell line grown as a xenograft in nude mice. Tumors were exposed to single doses of external X-irradiation of between 400 and 1600 cGy followed, 24 h later, by administration of specific or nonspecific antibody. Animals were sacrificed 3 days after antibody administration. At doses higher than 400 cGy, tumor uptake with both specific and nonspecific antibody was significantly increased. No difference in changes in tumor volume was observed between the groups receiving irradiation and the controls. Specific antibody uptake by tumors was always signif icantly higher than nonspecific having an approximate 4-fold binding advantage. Vascular permeability and the vascular volume of irradiated and control tumors was measured 24 and 72 h after irradiation, using iodine-125-labeled nonspecific antibody and labelling of the red blood cells in vivo with "TcO.t. At doses higher than 400 cGy, vascular permeability in the tumor 24 h after irradiation was significantly in creased (P < 0.05), while the vascular volume decreased (P < 0.001) compared to control values. However at 72 h after irradiation there was no difference between treated and control groups. The results obtained in this study suggest a potential value of external irradiation to increase monoclonal antibody uptake by tumors governed mainly by the increased vascular permeability of the tumor vasculature soon after the irradiation exposure. INTRODUCTION The usefulness of radiolabeled monoclonal antibodies for diagnosis (1) and therapy (2) of human tumors has been rec ognized, but a significant limitation for their application in vivo is the low absolute amount reaching the target (3). The local ization of monoclonal antibodies on the solid tumors is assumed to be governed by passive diffusion and binding on tumor cells. This depends on many factors such as antibody affinity, binding of the injected antibody by circulating antigen, density of the antigen expressed on tumor cells, vascular content of the tumor and accessibility of the antibody to solid tumors (4-6). It has been previously shown that a single dose of external irradiation can increase monoclonal antibody uptake by tu mors such as colorectal carcinoma (7), melanoma (8), and hepatoma (9), grown in mice and in an anecdotal hepatoma case reported by Order et al. (10). Ionizing radiation has been shown to change the vascular volume and the vascular perme ability to macromolecules in tumors. Paradoxically increased and decreased vascular volume have been observed depending upon the study and the time of observation after the irradiation (11, 12). The exposure of several different tissues to ionizing irradiation leads to a disruption of the vascular endothelium (13-15) and a quantitative increase in vascular permeability (15-18). The purpose of this study was to conduct a comprehensive evaluation of the effect of a single dose of external irradiation Received 3/20/89; revised 7/31/89; accepted 10/2/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by the Imperial Cancer Research Fund. 2To whom requests for reprints should be addressed, at ICRF Oncology Group, Hammersmith Hospital, DuCane Road, London W120HS. England. on the uptake of specific and nonspecific monoclonal antibody by a human colon adenocarcinoma xenograft in nude mice. A further aim was to assess and correlate the vascular volume and vascular permeability in this tumor model following irradiation. MATERIALS AND METHODS Animals and Tumors Twelve-week-old male athymic nu/nu (nude) mice, from a colony of mixed genetic background, were bred in the ICRF Animal Breeding Unit (South Mimms, Hertfordshire, UK). The mice were housed in sterile cages and maintained on irradiated diet and acidified water (pH 2.8). Xenografts were established s.c. by the inoculation under the skin of the flank of a single cell suspension of 5.106 cells in 0.1-ml medium of a human tumor cell line of a colon carcinoma designated LoVo (19). Tumor cells were grown in large tissue culture flasks in RPMI 1640 medium supplemented with 10% FCS.3 Cells were harvested by incu bation with 0.02% EDTA for 5 min and washed with serum free medium. Dimensions of tumors were measured before the irradiation as well as at the day of dissection to determine whether irradiation had a detectable effect on tumor volume. Tumor volume was determined from orthogonal measurements of three diameters using the formula: y = (4ir/3) x (dl x d2 x d3)/8 where V is the tumor volume, dl, d2, and d3 are the three different diameters. Mice were randomly allocated to treatment groups of five to six. The mean tumor volume in each group of mice was not signifi cantly different from the others. Monoclonal Antibodies AUA1. This is a murine IgG, raised in the conventional fashion by immunising BALB/c mice with the colon adenocarcinoma cell line LoVo (20). It recognizes a M, 35,000 antigen located on basolateral aspect of the cell membrane of a wide variety of neoplastic cells and a limited set of normal epithelial tissues (21). AUA1 is an epithelial specific antibody and reacts with the majority of human gastrointes tinal, ovarian and breast carcinomas, as well as proliferating epithelial cells in tissues such as normal colon. HMFG1. This is a murine IgG, and defines a tumor-associated antigen on a high molecular weight glycoprotein which is strongly expressed in lactating breast as well as in a range of neoplasms such as breast cancer, ovarian, and nonsmall cell lung cancer (22). This antibody does not react with LoVo adenocarcinoma and was used as a negative control. Radiolabeling of Antibodies Monoclonal antibodies were radiolabeled with '"In (0.04 M, carrier free, INS. 1; Amersham International, Amersham, UK) using DTPA, the cyclic anhydride (Sigma), as described by Hnatowich et al. (23). In the vascular studies the HMFG1 was labeled with '"I (IBS.3, Amer sham International, Amersham, UK), in order to determine the perme ability of tumor vasculature to IgG. lodination was carried out in iodogen coated tubes using an established technique (24). lodinated antibody was purified before each experiment by passage through a column with Sephadex G50 (Pharmacia, Sweden) to remove the free ' The abbreviations used are: PCS, fetal calf serum; FACS. fluorescence- activated cell sorting; FPLC, fast protein liquid chromatography: Ht. haematocrit; PBS, phosphate buffered saline: SDS-PAGE, sodium dodecyl sulfate-polyacryl- amide gel electrophoresis; VV, vascular volume; VP, vascular permeability. 159 on July 1, 2015. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from