Cancer model• Liver metastasis I
H. Suemizu, M. Monnai, Y. Ohnishi, M. Ito, N. Tamaoki, and M. Nakamura. 2007. Identification of akey molecular regulator of liver metastasis in human pancreatic carcinoma using a novel quantitativemodel of metastasis in NOD/SCID/gammacnull (NOG) mice. Int J Oncol 31:741-51.
We developed a reliable new model system for assaying liver metastasis using NOD/SCID/γcnull (NOG) mice.
Seven human pancreatic cancer cell lines were examined for their ability to form diverse metastatic foci in thelivers of NOD/SCID and NOG mice. Capan-2 and PL45 showed no metastasis when seeded at up to 105 cells inboth strains, and no BxPC-3 metastasis was observed in NOD/SCID mice. The NOD/SCID mice model could
detect liver metastasis only in the AsPC-1 cell line when inoculated with more than 103 cells. In contrast, wheninoculated with only 102 MIA PaCa-2, AsPC-1 and PANC-1 cells, liver metastasis was evident in71.4% (5/7),
57.1% (4/7) and 37.5% (3/8) of the NOG mice, respectively. Capan-1 and BxPC-3 cells metastasized whenseeded at 103 cells in 50% (5/10) and in 12.5% (1/8) of the mice, respectively. Using the NOG mice model
system, we established a highly metastatic cell line, liver metastasized-BxPC-3 (LM-BxPC-3), from liver
metastatic foci formed by the relatively poorly metastatic parental BxPC-3 cell line. These results demonstrated
the feasibility of using the quantitative metastasis model to search for and develop new anti-cancer therapies, andnovel drugs against this and other key molecules.

Table 1 Liver metastasis after intrasplenic injection of human pancreatic cancer cells.
Cell line

MIA PaCa-2

AsPC-1

PANC-1

Capan-1

BxPC-3
Capan-2

Cell dose

Metastatic score in NOG mice(cells/head)

NOD/SCID

NOG%T/Lb(mean ± SD)

Liver surface areac(mm2, mean ± SD)

1 x 104

0/10 (0.0%)

10/10 (100.0%)

60.6 ± 13.9

1056.0 ± 338.0

1 x 103

0/7 (0.0%)

5/6 (83.3%)

ND

ND

1 x 102

0/6 (0/0%)

5/7 (71.4%)

ND

ND

1 x 104

8/9 (88.9%)

9/9 (100.0%)

48.2 ± 12.3

434.4 ± 77.6

1 x 103

2/8 (25.0%)

8/8 (100.0%)

ND

ND

1 x 102

0/6 (0.0%)

4/7 (57.1%)

ND

ND

1 x 104

0/10 (0.0%)

8/8 (100.0%)

26.6 ± 11.3

374.0 ± 68.5

1 x 103

0/6 (0.0%)

6/8 (75.0%)

ND

1 x 102

0/7 (0.0%)

3/8 (37.5%)

ND

ND

1 x 104

0/10 (0.0%)

9/10 (90.0%)

15.6 ± 5.3

425.6 ± 38.5

1 x 103

0/10 (0.0%)

5/10 (50.0%)

ND

ND

1 x 102

0/8 (0.0%)

0/8 (0.0%)

ND

ND

1 x 105

0/8 (0.0%)

8/8 (100.0%)

ND

ND

1 x 1040/8 (0.0%)

1/8 (12.5%)

0.0 ± 0.0

409.4 ± 37.3

1 x 105

0/8 (0.0%)

0/8 (0.0%)

ND

ND

1 x 10ND

0/10 (0.0%)

0.0 ± 0.0426.7 ± 39.1

1 x 105

0/8 (0.0%)

0/8 (0.0%)

ND

ND

1 x 104

ND

0/10 (0.0%)

0.0 ± 0.0

395.0 ± 36.1

4

PL45

Number of animal with liver metastasisa(metastasis/total)a Liver metastasis was evaluated 6 weeks after inoculation of 1 x 103, 104 and 10 5 cancer cells, and 8 weeks after inoculation of 102
cancer cells. b All liver images showing liver metastases in response to injection of 1 x 104 cancer cells were used to calculate thepercent tumor occupancy in the liver (T/L). c The surface area of the liver was calculated using all liver images obtained from micethat were injected with 1 x 104 cancer cells. ND, not done.

Figure 1. Representative gross findings of liver
metastases of human pancreatic cancer cell lines.
Seven human pancreatic cancer cell lines: MIA PaCa2, AsPC-1, PANC-1, Capan-1, BxPC-3, Capan-2 andPL45 were intrasplenically implanted into NOG mice.
The mice were sacrificed 6 weeks later, and liver
metastases were enumerated immediately, without
prior fixation.

Figure 2. Establishment of a highly liver metastaticcell line. Primary tumors were generated by intrasplenic
injection of 1 x 105 BxPC-3 cells into NOG mice (a-c). Cells
from liver metastatic foci (open circles in Fig. 2 a) wereisolated and designated as LM-BxPC-3. Metastatic ability of
the LM-BxPC-3 cell line was evaluated by intrasplenic
injection with 1 x 105 cells (d-f).

Cancer model• Liver metastasis II
Hamada, K., M. Monnai, K. Kawai, C. Nishime, C. Kito, N. Miyazaki, Y. Ohnishi, M. Nakamura, and H.
Suemizu. 2008. Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shiscid IL2Rgammanull (NOG) mice. Int J Oncol 32:153-159.
To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed
a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgnull (NOG) mice. Elevenhuman colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers ofNOG mice as phenotypic and biological characters. When inoculated with 104 COLO320DM, HCT 116, HT-29,WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5%(7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 andSW1417 showed no metastasis when seeded at 104 cells even in NOG mice. The mRNA expression levels andgenetic mutations of N, H and K-RAS genes, which directly affects the levels of cellular RAS protein that would
be molecular target for FTI, was also examined in these six metastasizable human colon cancer cell lines asmolecular biological and genotypic characters. Only three cell lines had a point mutation in the RAS oncogene.
LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 to asp; G12D), and HCT 116 andLoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 to asp; G13D). Relative gene
expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6 to 5.0 folds lower than that ofLS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation ofFTI CH4512600 efficacy in in vivo. Using the NOG mouse liver metastasis model, we demonstrated theeffectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly
from 36.9 ± 2.9 to 50.3 ± 9.4 days.

Figure1. Representative gross findings of liver metastases of human colon cancer cell lines. Eleven human colon cancer
cell lines: CaCo2, COLO201, COLO320DM, HCT 116, HT-29, LoVo, LS123, LS174T, SW48, SW1417 and WiDr, wereintrasplenically implanted into NOG mice. The mice were sacrificed 6 weeks later, and liver metastases were enumerated immediately,
without prior fixation.

ab28days35daysFigure 2. Effects of FTI CH4512600 on the liver metastasis model. a) Chemical structure of FTI CH4512600. b)
HCT 116 cells (104 cells) transplanted into mouse liver was dissected at day 28 and day 35. FTI CH4512600 (50 mg/kg and 250mg/kg, daily) was administered for 3 weeks. Representative photos are shown here (n=3).

Survival ratio (%)

Body weight (g)3030252520201515--440714 21
14212828

3535

Days after administrationControl
CPT-11(75mg/kg)

120
100
80
60
40
200010203040 50

60

70

Survival days (day)CH4512600(50mg/kg)
CH4512600(250mg/kg)

Figure 3. Body weight and survival rate of HCT 116-transplanted mice. Survival rate of HCT 116-transplanted micefor vehicle control (n=8), CH4512600 (n=8) and CPT-11-treated mice (n=6). Administration was started 3 days after theinoculation (day 0). Black line: vehicle control, blue line: CPT-11 (75 mg/kg), red line: CH4512600 (50 mg/kg), light blue line:
CH4512600 (250 mg/kg). Statistical significance between control and 250 mg/kg of CH4512600 (P < 0.0001) and control and 75
mg/kg of CPT-11 (P = 0.0003) was calculated using SAS preclinical package ver.5.0.

80

Cancer model• Multiple myeloma
Y. Miyakawa, Y. Ohnishi, M. Tomisawa, M. Monnai, K. Kohmura, Y. Ueyama, M. Ito, Y. Ikeda, M.
Kizaki, and M. Nakamura. 2004. Establishment of a new model of human multiple myeloma using
NOD/SCID/gammac(null) (NOG) mice. Biochem Biophys Res Commun 313:258-262.
We developed a new experimental animal model of human multiple myeloma using immunodeficient
NOD/SCID/cnullc (NOG)mice. A human myeloma cell line, U266, was intravenously inoculated into 20 NOG
mice, all of which developed hind leg paralysis and distress around 6 weeks after transplantation. Pathologicalstudies showed that only the bone marrow was infiltrated with U266 cells, and no cells were present in otherorgans. Osteolytic lesions in cortical bones and loss of trabecular bones were prominent in U266-transplanted
NOG mice. In contrast, U266 cells were not detected in CB17scid or NOD/SCID mice 6 weeks after intravenous
inoculation. Human IgE, produced by U266 cells, was detected in the serum of U266-transplanted NOG mice byELISA. The results indicated that this hu-myeloma NOG model might be useful for studying the pathogenesis ofmyeloma and related osteolytic lesions,and are suggestive of its applicability to the future development of newdrugs.

CB17scid

NOD/SCIDNOGFigure 1. Histological analyses of bone marrow in CB17scid, NOD/SCID, and NOG mice after U266 myeloma celltransplantation. Five CB17scid, 5 NOD/SCID, and 20 NOG mice were intravenously injected with U266 cells after 2.4Gy
irradiation. Six weeks after transplantation, all mice were sacrificed and their bone marrow from the sternum was analyzed histologically.
There is no infiltration of U266 cells into the bone marrow of CB17scid (A, B) and NOD/SCID (C, D), but massive infiltration of U266
cells is observed in NOG (E, F), accompanied by osteolytic lesions (arrows in E,F). HE staining, magnification 100 x (A, C, and E), 200x (B, D, and F).

AControl

B
Figure 2. Clinical manifestation and histologicalanalyses of U266-transplanted NOG mice. (A) U266transplanted NOG mice but not control mice revealed hind legparalyses around 6 weeks after transplantation. (B) Massive
infiltration of U266 cells is observed in the lumbar bone lesions.
U266 cells invade into the spinal cavity and muscles,
accompanied by the osteolytic lesions. However, U266 cells are
not histologically observed in other organs of NOG mice; (C)liver, (D) lungs, (E) spleen, and (F) eosinophilic osteoclasts areobserved near the cortical bones of lumbar spines. HE staining.

U266

CDE

FA

C

B
PB

BoneBM

U266
Anti-human CD45

Anti-human CD45

Figure 3. FACS and histological analyses of U266-transplanted NOG mice. (A) Peripheral blood of NOG mice wasanalyzed 6 weeks after U266 cell transplantation using a PE-conjugated anti-human CD45 antibody by FACS. Dotted lines, isotypematched negative control. (B) Bone marrow of NOG mice was analyzed 6 weeks after U266 cell transplantation and approximately 84%
was positive for human CD45. (C) Bone marrows of U266-transplanted NOG mice are stained with an anti-human IgE antibody.

Serum hIgE (ng/ml)

A
350300**250200Figure 4. Serum levels of human IgE and histologicalstudies in U266-transplanted NOG mice aftertreatment with doxorubicin. (A) Human IgE concentrations

150100500ControlDoxBControlDoxin serum of NOG mice 4 weeks after transplantation with U266
cells were analyzed by ELISA. Eight NOG mice were injectedwith 1 mg/kg doxorubicin (DOX) per day for 3 days, 1 week after
transplantation (n= 8). Control mice were injected with PBS in thesame schedule (n = 8). Values are given as means±SD of eightmice. Student’s t test was performed to evaluate the statisticalsignificances. **p < 0:01. (B) Histological studies of sternum ofU266-transplanted NOG mice after treatment with or without
doxorubicin in the same schedule as in (A).