To better understand the events occurring during immunotherapy of liver metastases with effector cells, we have developed a clinically relevant animal model in which both effector-tumor cell interactions and survival can be evaluated. A cell line of human gastric carcinoma (HR) metastatic to the liver has been established from a patient's liver biopsy. HR cells (10 × 106) injected intrasplenically metastasize into the liver of immunosuppressed nude mice, with micrometastases detectable histologically by day 4 and macrometastases by day 7. The animals subsequently develop ascites and die between days 30 and 40 after tumor injection. To investigate early metastatic events in the liver, HR cells were transduced with a plasmid containing both the lacZ gene under the control of the CMV promoter and NeoR gene. Transfectants selected for neomycin resistance were lacZ gene positive and stained blue in the presence of a β-galactosidase substrate, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). These transfectants (HRLZ) remained lacZ gene positive for at least 25 passages in vitro. Injected intrasplenically, an HRLZ clone grew invasively in nude mice and formed liver metastases comparably to parental tumor cells. The number and localization of blue X-Gal-positive tumor cells were followed in liver tissues of animals sacrificed at various times, from 1 h to 28 days postinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels and sinusoids within 1 h after injection, and the progressive growth of micrometastases and macrometastases could be followed with precision by X-Gal staining. On day 3 after injection of HRLZ cells, numerous micrometastases were established containing 12–16 tumor cells. When these 3-day established HRLZ micrometastases were treated by the intrasplenic infusion of interleukin 2 (IL2)-activated human natural killer (NK) cells selected by IL2-induced adherence to plastic (A-NK) and systemic IL2, nearly all liver micrometastases were eliminated within 24 h after a single transfer of A-NK cells (P < 0.001). This xenogeneic model was also used for adoptive immunotherapy of 7-day established liver macrometastases with human A-NK cells injected intrasplenically and exogenous IL2 given i.p.. A significant decrease in the number of hepatic metastases and the weight of livers (P < 0.003) in comparison with those of control mice was observed. Survival of mice treated with a single dose of A-NK cells plus exogenous IL2 (60,000 international units/mouse twice daily for 5 days) was significantly increased (P = 0.001) as compared to control mice treated with Hanks' balanced salt solution or IL2 alone. In this xenogeneic model of liver metastasis, human A-NK cells were remarkably effective in eliminating both micro- and macrometastases and significantly prolonging survival of mice treated locoregionally with adoptive immunotherapy and IL2.

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Supported in part by Grant 5PO-1-CA47445-03 from the National Cancer Institute and by the Pathology Education and Research Foundation.

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