Both macrophages and hypoxia play critical role in regulating invasion of gastric cancer in vitro

Figures & data

Table I. Source of gastric cancer cell lines.

Cell lines	Source	Histopathological subtype	Tumorigenic in mice	Catalog number	Origin
AGS	original tumor	Intestinal adenocarcinoma	Yes	CRL-1739	American Type Culture Collection (ATCC)
HGC-27	metastatic lymph node	Intestinal adenocarcinoma	Yes	94042256	European Collection of Cell Cultures (ECACC)
Hs-746T	metastatic tumor in left leg	Intestinal adenocarcinoma	Yes	HTB-135	American Type Culture Collection (ATCC)
NCI-N87	metastatic tumor in the liver	Intestinal adenocarcinoma	Yes	CRL-5822	American Type Culture Collection (ATCC)

Figure 1. Gastric cancer cells were grown with macrophages in Matrigel. (A) 3D picture screened by 3D dynamic migration imaging system. (B) AGS, HGC-27, Hs-746T and NCI-N87 gastric cancer cell cultured with macrophages. (a) image of gastric cancer cells with macrophages under normal light, ×100 magnification; (b) image of gastric cancer cells stained with fluorescence, ×100 magnification; (c) combined image, ×100 magnification.(N = 3).

Figure 2. Effect of macrophages on the mobility of gastric cancer cells under normal and hypoxic conditions. The cells movement rates were accelerated due to macrophages in AGS, HGC-27, Hs-746T and NCI-N87 cell lines under normal condition. Hypoxia induced increase in migration rate in AGS and HGC cell lines, decrease in Hs-746T and NCI-N87 cell lines without macrophages. When the cancer cells with macrophages cultured together under hypoxia condition, the cells movement speeds were increased in AGS, HGC-27 and NCI-N87 cell line compared with normal condition with macrophages, but decreased in Hs-746T cell line.*P < 0.05; **P < 0.001.(N = 3).

Table II. ADAM8, ADAM9, MMP9, TIMP3, VEGF-1 and IL-8 mRNA relative expression in gastric cancer cell lines.

	m RNA relative expression (Target gene/GAPDH, 2^-ΔCt)
	ADAM8 ( × 10^24)	p-value	ADAM9 ( × 102^1)	p-value	MMP9 ( × 10^25)	p-value	TIMP3 ( × 102^4)	p-value	VEGF-1 ( × 10^22)	p-value	IL-8 ( × 10^25)	p-value
AGS	1.3 ± 0.1		1.3 ± 0.0		0.2 ± 0.0		0.7 ± 0.0		1.7 ± 0.0		24.6 ± 4.5
HGC-27	0.3 ± 0.0	0.803	0.2 ± 0.0	0.066	0.1 ± 0.0	0.999	149.3 ± 16.5	0.001*	1.0 ± 0.0	0.145	0.1 ± 0.0	0.522
Hs-746T	32.6 ± 5.2	0.001*	7.5 ± 0.1	0.001*	641.5 ± 20.0	0.001*	0.9 ± 0.0	0.591	23.7 ± 0.5	0.001*	829.8 ± 43.1	0.001*
NCI-N87	6.3 ± 0.9	0.252	1.7 ± 0.0	0.417	136.5 ± 14.1	0.014*	1.0 ± 0.1	0.909	6.5 ± 0.1	0.001*	127.1 ± 23.5	0.042*

*Significant at the p < 0.05 level as compared to AGS cell line (n = 6).

Figure 3. Effect of macrophages on ADAM8, ADAM9, MMP9, TIMP3, VEGF-A and IL-8 expression in gastric cancer cell lines under normal or hypoxic conditions, log(mRNA relative expression). Different gastric cancer cell lines were cultured with or without macrophages either in normal or hypoxic conditions. Expressions of the above mentioned genes were measured with realtime RT-PCR method. Under normal conditions, macrophage co-culture upregulated all six genes in gastric cancer cells. Hypoxia had effects depending on cell line, but in general hypoxia tended to decrease MMP9 expression and increase TIMP3 expression in gastric cancers cells grown with or without macrophages. *P < 0.05; **P < 0.001, as compared to AGS cell line. # the relative mRNA results is significant at P < 0.05 level.(N = 4).

Figure 4. Correlations between ADAM8 and TIMP3 expressions in cancer cells and changes in invasion rate either by macrophages or by hypoxia. (A) ADAM8 expression in different cancer cell lines (dots) correlates with their response in invasion rate as the cancer cells are co-cultured with macrophages (p = 0.006, N = 4). (B) TIMP3 expression in different cancer cell lines (dots) correlates negatively with changes in invasion rate induced by hypoxia (p = 0.007, N = 4).

Figure 5. A western blot of ADAM8 protein expression in macrophages or AGS cells grown alone or in co-culture. ADAM8 bands are shown near 75 kD and the possible proform near 90 kD.