We successfully established a new gastric cancer cell line, XGC-1, in vitro using a fresh sterile specimen derived from the primary tumour of a gastric cancer patient. From the primary culture, we succeeded in freezing, thawing and subculturing cells for > 90 generations in DMEM/F12 supplemented with 10% FBS.
In vitro characteristics of XGC-1 cells
Under light microscopy, most of the cells appeared spindle-shaped and oval-shaped, a few cells were polygonal, the cell poles appeared sharp, the nuclei were obvious, a few cells were multinucleated, and the nucleolus was clearly visible (Fig. 1a). The cells aggregated and grew during culture. When the nutrient level was insufficient, the cells floated in the culture medium, and they continued to adhere to the wall in nutrient-rich conditions (Fig. 1b). H&E staining showed that the nuclei were deeply stained and the proportion of nucleoplasm was high, which is consistent with cancer cell characteristics (Fig. 1c). Transmission electron microscopy revealed that most cell nuclei increased in volume, were round or elliptical, and had a large proportion of nucleoplasm. Chromatin in the nucleus was often significant, part of the nuclear membrane was recessed, and the nucleolus was significantly enlarged and solid; the number of free ribosomes increased and there were fewer organelles, mitochondrial swelling was obvious, the crest morphology was irregular and the crest number was small, the connection between the cells was scarce, and there were no microvilli structures (Fig. 1d). The cells were passaged > 90 generations, and there was no obvious change in morphology during cell passage. The growth of the XGC-1 cell line was assayed by the MTT method. The XGC-1 cell line showed vigorous growth and a doubling time of approximately 48 h (Fig. 1e).
The cells began to divide on the third day of culture in low-melting agarose, and colonies were formed in the independent single-cell culture at 2 weeks. The cells were stacked in a circular shape, and white colonies were observed with the naked eye at 3 weeks. The colony formation rate was approximately 72.0% (Fig. 2a and b).
Karyotype of the XGC-1 cell line
The karyotype was triploid, many deletions were observed, ectopic and derived chromosomes (→) were visible, the X chromosome had an abnormal arm, the Y chromosome was lost, no normal chromosome 3 was present, the Philadelphia chromosome (Ph') was observed, and 4 chromosomes with unknown sources were detected (?). The representative karyotype was 69, X, -Y, der(X)add(X)(P?), i(X)(p10), der(1)add(1)(p?), der(1)add(1)(p?), -2, del(3)(q10), der(3)t(3;5) (p10;q10), der(3)t(3;9)(q10;p10), -4, del(4)(p10), der(4)del(4)(p15)add(4)(q?), -5, i(5)(p10)x2, del(6)(q21), +der(7)add(7)(p?), del(9)(p21), -10, -11, -13, der(13)add(13)(p?), -14, del(17)(q24), -18, del(22)(q11), +mar1, +mar2, +mar3, +mar4 (Fig. 3a and b).
Subcutaneous inoculation for 5 days resulted in a mung bean-sized tumour. One month later, the BALB/C nude mice were sacrificed (Fig. 4a). The subcutaneous tumours were removed for histological examination (Fig. 4b) and were compared with the original primary tumour. Both primary tumours and subcutaneous tumours (Fig. 4c and d) were characterized by typical gastric adenocarcinoma (H&E) features, and all were poorly differentiated. The liver, kidney, lung and other important organs showed no tumour metastasis.
PBS was used as the negative control (Fig. 5a). The results of immunohistochemical staining revealed that cytokeratin expression was positive (Fig. 5b), and cytoplasmic keratin was stained a tan colour. The membrane and nucleus were not stained, and vimentin expression was negative (Fig. 5c). Cytoplasmic vimentin staining was not observed, which was consistent with the epithelial source characteristics. Nuclear Ki-67 was stained brown (Fig. 5d). The patina and capsule were not stained, and 5 fields were randomly observed under a microscope. The average staining rate of the fields of view was approximately 75%.