S. Yasumura et al., IMMUNOTHERAPY OF LIVER METASTASES OF HUMAN GASTRIC-CARCINOMA WITH INTERLEUKIN 2-ACTIVATED NATURAL-KILLER-CELLS, Cancer research, 54(14), 1994, pp. 3808-3816
To better understand the events occurring during immunotherapy of live
r metastases with effector cells, we have developed a clinically relev
ant animal model in which both effector-tumor cell interactions and su
rvival can be evaluated. A cell line of human gastric carcinoma (HR) m
etastatic to the liver has been established from a patient's liver bio
psy. HR cells (10 x 10(6)) injected intrasplenically metastasize into
the liver of immunosuppressed nude mice, with micrometastases detectab
le histologically by day 4 and macrometastases by day 7. The animals s
ubsequently develop ascites and die between days 30 and 40 after tumor
injection. To investigate early metastatic events in the liver, HR ce
lls 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 t
he presence of a beta-galactosidase substrate, -bromo-4-chloro-3-indol
yl-beta-D-galactopyranoside (X- Gal). These transfectants (HRLZ) remai
ned lacZ gene positive for at least 25 passages in vitro. Injected int
rasplenically, an HRLZ clone grew inva sively in nude mice and formed
liver metastases comparably to parental tumor cells. The number and lo
calization of blue X-Gal positive tumor cells were followed in liver t
issues of animals sacrificed at various times, from 1 h to 28 days pos
tinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels
and sinusoids within 1 h after injection, and the progressive growth o
f micrometastases and macrometastases could be followed with precision
by X-Gal staining. On day 3 after injection of HRLZ cells, numerous m
icrometastases were established containing 12-16 tumor cells. When the
se 3-day established HRLZ micrometastases were treated by the intraspl
enic infusion of interleukin 2 (IL2)-activated human natural killer (N
K) cells selected by IL2-induced adherence to plastic (A-NK) and syste
mic IL2, nearly all liver micrometastases were eliminated within 24 h
after a single transfer of A-NK cells (P < 0.001). This xenogeneic mod
el was also used for adoptive immunotherapy of 7-day established liver
macrometastases with human A-NK cells injected intrasplenically and e
xogenous IL2 given i.p.. A significant decrease in the number of hepat
ic metastases and the weight of livers (P < 0.003) in comparison with
those of control mice was observed. Survival of mice treated with a si
ngle 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 solutio
n or IL2 alone. In this xenogeneic model of liver metastasis, human A-
NK cells were remarkably effective in eliminating both micro- and macr
ometastases and significantly prolonging survival of mice treated loco
regionally with adoptive immunotherapy and IL2.