Establishment and characterization of a new hypoxia-resistant cancer cell line, OCUM-12/Hypo, derived from a scirrhous gastric carcinoma

Abstract

Background: Many kinds of solid tumour have heterogeneously a hypoxic environment. Tumour hypoxia reported to be associated with more aggressive tumour phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. However, the mechanisms by which hypoxia affects the aggressive phenotypes remain unclear.

Methods: We established a scirrhous gastric carcinoma cell line (OCUM-12) from ascites associated with scirrhous gastric carcinoma, and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) was cloned from OCUM-12 cells by continuous exposure to 1% oxygen.

Results: Histologic findings from orthotopic tumours derived from parent OCUM-12 cells and daughter OCUM-12/Hypo cells revealed poorly differentiated adenocarcinoma with extensive fibrosis that resembled human scirrhous gastric cancer. Necrotic lesions were frequently detected in the OCUM-12 tumours but were rarely found in the OCUM-12/Hypo tumours, although both types had multiple hypoxic loci. Apoptosis rate of OCUM-12 cells was increased to 24.7% at 1% O(2), whereas that of OCUM-12/Hypo was 5.6%. The OCUM-12/Hypo orthotopic models developed multiple metastases to the peritoneum and lymph nodes, but the OCUM-12 models did not. OCUM-12/Hypo cells showed epithelial-to-mesenchymal transition and high migratory and invasive activities in comparison with OCUM-12 cells. The mRNA expression levels of both E-cadherin and zonula occludens ZO-1 and ZO-2 decreased in OCUM-12/Hypo cells, and that of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, matrix metalloproteinase-1 (MMP-1), and MMP-2 were increased in OCUM-12/Hypo cells.

Conclusion: OCUM-12 and OCUM-12/Hypo may be useful for the elucidation of disease progression associated with scirrhous gastric cancer in the setting of chronic hypoxia.

Figure 1

Upper gastrointestinal series (A) and gastro-fibrscopy (B) showed diffusely infiltrating carcinomas in which ulceration is usually not a marked feature (arrows). Histology of the primary tumour and the peritoneal metastatic tumour showed poorly differentiated adenocarcinoma (C). Primary gastric tumour accompanied by fibrosis. Immunostaining of hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9) showed that hypoxic lesions were present in both the primary tumour and the peritoneal metastatic tumour (C).

Figure 2

Morphology of OCUM-12 and OCUM-12/Hypo cells (A). OCUM-12 cells showed a round shape, whereas OCUM-12/Hypo cells were spindle-shaped and formed loose cell aggregates. G-banded karyotypes of OCUM-12 and OCUM-12/Hypo (B). The representative karyotype of OCUM-12 was XX, −X or −Y, add (1)(p36), del[1] (p32), add (4)(p11), +add (2)(q33), add(2)(q33), +3[2], del(3)(q26), +5, der(5)add(5)(p15), add(5)(q31), +6, del(6)(q13), del(6), +7, +8, +9, add(9)(p11), add(9), +10, +11, der(11)t(11;17)(p11;q11), der(11)t(11;17) × 2, −13, add(13)(q32), +14, add(15)(q24), add(16)(q11), −17, −18, +19, add(19)(p13) × 2, +20, −21, −22, +mar1, +mar2. The representative karyotype of OCUM-12/Hypo. XXY, −1, −2, der(2) add (2)(p23) add(2)(q33) × 2, −4, +5, del(5)(q31q33) × 2, del(6)(q13) × 2, +7, add(7)(p22), −8, del(9)(p13), −10, +11, add(11)(p11.2) × 2, −13, −13, add(13)(q34), +14, add(15)(p11.2), −16, −17, −18, add(19)(p13), +20, −21, −22, +5–7 mar. Arrows, breakpoints present. (C) Loss of heterozygosity analysis electropherograms using microsatellite markers. The primary gastric tumour, corresponding normal sample, OCUM-12, and OCUM-12/Hypo cells are shown by comparing the height and position of peaks. The primary tumour, OCUM-12, and OCUM-12/Hypo cells had LOH at the p53 locus and DCC/DPC4 locus, whereas no band shift was detected. The same size alleles at D18S35, D3S1611, and D5S346 probes were found in normal epithelium, primary tumour, OCUM-12, and OCUM-12/Hypo cells, which shows that the OCUM-12 and OCUM-12/Hypo cell lines are derived from the same patient. p53 sequencing analysis showed C>T transition (arrow) at codon 273 in exon 8 (D).

Figure 3

Orthotopic implantation model with OCUM-12 cells and OCUM-12/Hypo cells. (A) An orthotopic tumour in the stomach (arrows) was found 7 weeks after inoculation in both cell lines. Orthotopic implantation model with OCUM-12/Hypo cells yielded lymph node diseases (arrowheads) and peritoneal metastatic nodules (asterisks) 7 weeks after inoculation, whereas implantation with OCUM-12 cells developed few metastases. (B) Histologic findings of inoculated OCUM-12 and OCUM-12/Hypo tumours. Orthotopic tumours with OCUM-12 and OCUM-12/Hypo showed extensive fibrosis with the occasional presence of poorly differentiated adenocarcinoma cells. Masson's trichrome staining showed blue multiple fibrosis in the orthotopic tumours. Pimonidazole, HIF-1α, and CA9 staining were heterogeneously positive in OCUM-12 and OCUM-12/Hypo tumours (C).

Figure 4

The motility of OCUM-12 cells and OCUM-12/Hypo cells in vitro. (A) Migration activity. Representative phase-contrast images of the wound-healing assay show the migrating cells in the wound margin. The number of migrating OCUM-12/Hypo cells was significantly (P<0.01) greater than that of OCUM-12 cells. Dotted line, wound margin. (B) Invasion activity. Representative phase-contrast images of the invasion assay show a higher number of invading OCUM-12/Hypo cells in comparison to OCUM-12 cells.

Figure 5

mRNA expression and Gelatinolytic activity. (A) mRNA expression. The mRNA expression levels of vimentin, Snail-1, Slug/Snail-2, Twist, ZEB-1, ZEB-2, MMP-1, and MMP-2 were higher in OCUM-12/Hypo cells in comparison to the OCUM-12 cells. The expression levels of E-cadherin, ZO1, and ZO2 in OCUM-12/Hypo cells were lower than those in OCUM-12 cells, whereas that of cytokeratin 19 was not different. (B) Gelatinolytic activity. pro-MMP-2 (68 kDa) and active MMP-2 (62 kDa) were detected in OCUM-12/Hypo cells, but not in OCUM-12 cells.

Figure 6

Apoptosis induction by hypoxia. (A) The typical examples of flow cytometric analysis. (B) In OCUM-12 cells, hypoxia induced apoptosis at a rate of 24.7%, whereas apoptosis rate at 20% O₂ was 2.1%. In contrast, the apoptosis rate of OCUM-12/Hypo was 5.6% at hypoxia. The result represents the mean of three independent experiments and the bars show the s.d.