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. 2018 May;109(5):1480-1492.
doi: 10.1111/cas.13569. Epub 2018 Apr 15.

Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth

Masami Yamamoto  1 , Sachiyo Nomura  2 , Akihiro Hosoi  3 , Koji Nagaoka  3 , Tamaki Iino  3 , Tomohiko Yasuda  2   4 , Tomoko Saito  5 , Hirokazu Matsushita  3 , Eiji Uchida  4 , Yasuyuki Seto  2 , James R Goldenring  6 , Kazuhiko Kakimi  3 , Masae Tatematsu  7 , Tetsuya Tsukamoto  8
Affiliations

Established gastric cancer cell lines transplantable into C57BL/6 mice show fibroblast growth factor receptor 4 promotion of tumor growth

Masami Yamamoto et al. Cancer Sci. 2018 May.

Abstract

Previously no mouse gastric cancer cell lines have been available for transplantation into C57BL/6 mice. However, a gastric cancer model in immunocompetent mice would be useful for analyzing putative therapies. N-Methyl-N-nitrosourea (MNU) was given in drinking water to C57BL/6 mice and p53 heterozygous knockout mice. Only 1 tumor from a p53 knockout mouse could be cultured and the cells s.c. transplanted into a C57BL/6 mouse. We cultured this s.c. tumor, and subcloned it. mRNA expression in the most aggressive YTN16 subline was compared to the less aggressive YTN2 subline by microarray analysis, and fibroblast growth factor receptor 4 (FGFR4) in YTN16 cells was knocked out with a CRISPR/Cas9 system and inhibited by an FGFR4 selective inhibitor, BLU9931. These transplanted cell lines formed s.c. tumors in C57BL/6 mice. Four cell lines (YTN2, YTN3, YTN5, YTN16) were subcloned and established. Their in vitro growth rates were similar. However, s.c. tumor establishment rates, metastatic rates, and peritoneal dissemination rates of YTN2 and YTN3 were lower than for YTN5 and YTN16. YTN16 established 8/8 s.c. tumors, 7/8 with lung metastases, 3/8 with lymph node metastases and 5/5 with peritoneal dissemination. FGFR4 expression by YTN16 was 121-fold higher than YTN2. FGFR4-deleted YTN16 cells failed to form s.c. tumors and showed lower rates of peritoneal dissemination. BLU9931 significantly inhibited the growth of peritoneal dissemination of YTN16. These studies present the first transplantable mouse gastric cancer lines. Our results further indicate that FGFR4 is an important growth signal receptor in gastric cancer cells with high FGFR4 expression.

Keywords: C57BL/6 mouse; FGFR4; animal model; gastric cancer; immunocompetent.

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Figures

Figure 1
Figure 1
Establishment of mouse gastric cancer cell lines. Mice were given drinking water ad libitum containing 30 ppm N‐methyl‐N‐nitrosourea (MNU) on alternate weeks for a total exposure of 5 weeks and killed at 40 weeks. Fresh gastric tumor tissues were washed and primary cultured, removing fibroblasts. Cells were injected into C57BL/6 mouse s.c. The established tumor was excised and cultured. From the 2 dishes, 2 single cell cloned cell lines each were established. The 2 cell lines from 1 dish were named YTN2 and YTN3. The other 2 cell lines from the other dish were named YTN5 and YTN16
Figure 2
Figure 2
Primary tumor from which cell lines were established. (A) Hematoxylin and eosin staining (10×). (B) Higher magnification (40×). Tumor shows a well‐differentiated phenotype. (C) Pepsinogen 1 was negative in the tumor. (D) Class III mucin was positive in the tumor and thus the tumor was classified as a pyloric phenotype
Figure 3
Figure 3
Microscopic features of growing cell lines in vitro. (A) YTN2, (B) YTN3, (C) YTN5, (D) YTN16. (E) Growth curves of the cell lines. Growth rates were not different among the cell lines. (F) PCR for WT p53 and mutant allele of p53. The subcloned cell lines were lacking in WT p53, suggesting LOH
Figure 4
Figure 4
Growth curves of s.c. tumor with representative macroscopic mouse s.c. tumors. Growth curves of (A) YTN2, (B) YTN3, (C) YTN5, (D) YTN16. Macroscopic mouse cutaneous tumors of (E) YTN3, (F) YTN16
Figure 5
Figure 5
Microscopic appearance of s.c. tumor, lung metastasis, lymph node metastasis, and peritoneal dissemination of YTN2, YTN3, YTN5, and YTN16. (A‐D) S.c. tumor of YTN2‐16. (E‐H) Lung metastasis of YTN2‐16. (I‐K) Lymph node metastasis of YTN2, YTN5, YTN16. (L‐N) Peritoneal dissemination of YTN2, YTN5, YTN16. Lymph node metastasis was not detected in YTN3. Small peritoneal dissemination was macroscopically detected for YTN3, without confirmation microscopically. Microscopic morphology was not remarkably different among the cell lines
Figure 6
Figure 6
Fgfr4 disrupted YTN16. (A) F7 has an insertion of adenine in exon 3 of Fgfr4, and Fgfr4 is disrupted by a frameshift termination. (B) F87 has a deletion of adenine in exon 3 of Fgfr4, and Fgfr4 is disrupted by a frameshift termination. (C) Growth rates of YTN16, F7 and F87 were not different in vitro. (D‐F) Microscopic morphology of YTN16, F7, and F87 showed no difference in vitro. (G‐I) F7 and F87 do not form s.c. tumors. (J‐L) F7 and F87 formed very small foci of peritoneal dissemination in 2 out of 5 mice. YTN16 formed large nodules in 5 out of 5 mice in the same period. (M‐O) YTN16 formed large nodules of peritoneal dissemination. F7 and F87 formed only very small peritoneally disseminated tumors in macroscopically positive mice only. Arrows: small peritoneally disseminated tumor
Figure 7
Figure 7
BLU9931 treated YTN16. (A,B) Growth of s.c. transplanted YTN16 was blunted with BLU9931 30 mg/kg b.i.d. treatment. (C,D) Growth of peritoneal dissemination of YTN16 is remarkably inhibited with BLU9931. (E,G) Microscopic appearance of peritoneal dissemination of YTN16 (E) without BLU9931 and (G) with BLU9931 treatment for 3 weeks. (F,H) Microscopic features of s.c. tumor of YTN16 (F) without BLU9931 and (H) with BLU9931 treatment for 3 weeks. YTN16 tumor under treatment with BLU9931 does not form glandular structures. (I,K) pSTAT3, (J,L) pERK, (M,N) pAKT expression in s.c. tumor of YTN16 treated (I,J,M) without or (K,L,N) with BLU9931 treatment. Expression of all 3 markers was weaker in BLU9931‐treated tumor compared to the original YTN16 tumor. (J) pErk was positive in glandular cells in the invasion front in non‐treated YTN16 tumor. (M) pAkt was positive in glands in non‐treated YTN16 throughout, and was (N) weakly positive in the few glands present in BLU9931‐treated tumor. (E‐N) Insets show high magnification
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