Lentiviral vectors (Genechem Chemical Technology Co., Ltd., Shanghai, China) were used to transfect the RFP gene into human SU3 glioma stem/progenitor cells (self-built in the lab) (18), and SU3 cells were obtained with high expression of RFP (SU3-RFP) screened by puromycin. SU3-RFP cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco-Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA), or in DMEM/F12 (Gibco-Invitrogen) containing 20 ng/ml recombinant human basic fibroblast growth factor (PeproTech, Princeton, NJ, USA) and recombinant human epidermal growth factor (PeproTech). SU3-RFP (150 μl) containing 1×10⁶ cells was injected with a micro-syringe directly into the abdominal cavity of the NC-C57BL/6J-GFP nude mice, which were ~6 weeks old and expressing GFP (19). The mice were then bred in an IVC isolation device (Fengshi Laboratory Animal Equipment Co., Ltd., Suzhou, China) according to specific pathogen-free level management requirements. Approximately one month later, when the abdominal circumference of the mice was observed to have increased from the in vivo imaging system (Carestream Health, Rochester, NY, USA), the mice were sacrificed, the ascites were obtained, and an abdominal anatomical procedure was performed to obtain the solid invasive-growing tumors. Cryosectioning was also performed on the peritoneal tumors for observation with a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University.

Ascites were obtained from the tumor-bearing mice and red blood cells were removed. Solid tumor tissue that had invaded into the liver and gastrointestinal wall was obtained and digested with trypsin into a single cell suspension, then the above targets were subcultured and amplified in DMEM (Gibco) containing 10% fetal bovine serum (Hyclone). Then flow cytometry (Beckman Coulter, Miami, FL, USA) was used to separate GFP⁺ cells for continuous cultivation. The limiting dilution method and the capillary method were performed for the monoclonal cell lines. Once the amplification identified cells of single-cell origin, the cells were frozen in liquid nitrogen for future use. Among these, the cell lines originating from the solid tumor on the gastrointestinal wall underwent further study and were named SU3-induced host celiac tumor cells (SU3-ihCTCs).

DMEM medium containing 10% fetal bovine serum was used for the cultivation of SU3-ihCTCs, then the cell growth was observed with an inverted fluorescence microscope (Carl Zeiss). After developing the SU3-ihCTCs on slides, hematoxylin and eosin (H&E) staining was performed and the cell morphology was observed. A total of 1×10³ cells (100 μl) were added onto a 96-well plate, and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to draw the cell growth curve. To determine the colony formation rate the cells in the logarithmic growth phase were digested with 0.25% trypsin, seeded in six-well plates with 100 cells per well and incubated overnight at 37°C and with 5% CO₂, then the number of adherent cells was calculated. After culturing for a further 6–8 days, cells were fixed with methanol for 10 min and stained with crystal violet for 20 min, then a microscope was used to determine colony counts (a colony was defined as ≥50 cells grown together) for the final calculation of the clone formation rate: clone formation rate = number of colonies/number of seeded cells × 100. This was performed three times for each well.

The cellular DNA content of SU3-ihCTCs in the logarithmic growth phase was detected by flow cytometry. Following the method of Seabright (20), cell chromosome G-banding analysis was performed. A DNeasy blood and tissue kit (Qiagen GmbH, Hilden, Germany) was used to extract the cell or tissue DNA, and the cell species was identified using the method reported by Parodi et al (21). The primers used for polymerase chain reaction (PCR) amplification of the human-specific h-cox1 gene were 5′-TTCGGCGCATGAGCTGGAGTCC and 5′-TAT GCGGGGAAACGCCATATCG, with a PCR product of 228 bp. The primers for the mouse-specific m-cox1 gene were 5′-ATTACAGCCGTACTGCTCCTAT and 5′-CCCAAAGAA TCAGAACAGATGC, with an amplified product of 150 bp. Western blot analysis was performed to detect GFP expression. RIPA cell disruption buffer (Millipore, Billerica, USA) was added to the collected SU3-ihCTCs and SU3-RFP cells and mouse spleen tissue to extract the total protein, and proteins were quantified using the bicinchoninic acid method. Total protein (50 μg) was isolated by 12% SDS-PAGE electrophoresis and transferred to a PVDF membrane, then reacted with rabbit anti-GFP antibody (1:2,000; Abcam, Hong Kong, China) and mouse anti-GAPDH antibody (1:1,000; Sigma, St. Louis, MO, USA), respectively. Following reaction with the corresponding horseradish peroxidase-conjugated secondary antibody, chemiluminescence detection and X-ray film developing were performed. Immunocytochemistry was performed to detect the expression of macrophage-specific marker protein CD68 with rat anti-mouse CD68 monoclonal antibody (1:200; Abcam).

SU3-ihCTCs and the murine macrophage cell line RAW264.7 (Chinese Academy of Seed Cell Bank, Shanghai, China) were inoculated into the abdominal cavity and right forelimb armpit of nude mice at a concentration of 1×10⁷ cells/150 μl, and the tumorigenicity was observed. RAW264.7 belongs to the mouse macrophage cell line established by Raschke et al (22), with established tumorigenicity, and acted as a control of macrophage canceration in this experiment.

The RFP gene was stably transfected into SU3 cells through lentiviral vectors for both differentiated adherent-growing cells in serum culture conditions and suspended-growing stem progenitor cells in growth-factor-containing culture conditions. A red color was observed under the fluorescence microscope due to RFP expression (Fig. 1).

SU3-RFP cells gave rise to solid tumors (Fig. 2D and H) and malignant ascites (Fig. 2I) in the abdominal cavity of GFP nude mice. In the living tumor-bearing mice (Fig. 2G), in tumor nodules obtained following sacrifice (Fig. 2D–F) and in frozen sections of tumor tissues (Fig. 2A–C), observations revealed that the tumor tissues were composed of the red SU3-RFP fluorescent progenitor cells and the green fluorescent host cells.

The bloody ascites of tumor-bearing mice or tumor nodule tissues were cultured in serum-containing medium, and, while few demonstrated proliferative activity, green fluorescent cells were observed under the fluorescence microscope (Fig. 3A and B). In these green cells, macrophages with a fluctuating membrane edge could be observed (Fig. 3C), among which the majority revealed red fluorescence. Following the digestion of the tumor tissues, GFP⁺ host cells accounted for 3.77% of the mixed cells in flow cytometry (Fig. 3D). The GFP⁺ cells were then separated, enriched with flow cytometry, and the cells could be subcultured continuously.

Monocloning was performed on the above collected GFP⁺ cells, and unlimited proliferative capacity was confirmed under in vitro cultivation. The progenitor cells exhibited green fluorescence during subcultivation. SU3-ihCTCs were pleomorphic, and cell-cell contact inhibition disappeared (Fig. 4A). The growth incubation period and doubling time of SU3-ihCTCs were shorter than those of SU3 and SU3-RFP cells; the cells quickly entered the logarithmic growth phase, exhibiting rapid proliferation in the logarithmic growth phase and a short cell growth cycle (Fig. 4D). The clone formation rates of SU3-ihCTCs and SU3 cells were 57.0±4.4 and 7.3±1.0%, respectively, revealing the capacity of proliferation and formation of a single SU3-ihCTC to be stronger than that of SU3 cells, with low population dependence (Fig. 4E). SU3-ihCTCs highly expressed macrophage-specific marker protein CD68 in the same way as mouse macrophage cell line RAW264.7 (Fig. 4B and C).

High cellular DNA synthesis and chromosome changes are major features of TCs. Measured with flow cytometry, the DNA synthesis capacity of SU3-ihCTCs was 2.528 times higher than that of normal diploid mouse lymphocytes, from which it can be assumed that the chromosome number of SU3-ihCTCs was 2.528 times higher than that of normal mouse lymphocytes, and thus that SU3-ihCTCs exceed pentaploidy (2.528×2; Fig. 5C). G-banding calculation was used to calculate the number of chromosomes per cell. In the experiment, the cell chromosome splitting phase was randomly extracted, and the chromosome number was counted to be 92.70±7.15 (n=10) (Fig. 5D). PCR amplification was performed on mouse- and human-specific primers, respectively. Only the mouse cox1 gene was amplified from SU3-ihCTCs (150 bp), and GFP expression was detected using western blot analysis, while the cells revealed features including telocentric chromosomes (Fig. 5C). The above results proved that SU3-ihCTCs were host-derived TCs.

In intraperitoneally or subcutaneously inoculated nude mice, the tumorigenicity rate of SU3-ihCTCs was 100% (5/5); ascites and tumor nodules were also formed when SU3-ihCTCs were inoculated into the abdominal cavity (Fig. 6A). Since SU3-ihCTCs were deliberately inoculated in no-fluorescence nude mice, the grown TCs, either ascites or solid tumors, expressed GFP (Fig. 6B); therefore, it could be determined that the TCs were daughter cells of SU3-ihCTCs. The peritoneal solid tumors exhibited clear characteristics of pleomorphism following H&E staining, with a dense arrangement (Fig. 6C). RAW264.7 cells were also inoculated into the abdominal cavity and subcutaneous part of the GFP nude mice, with tumorigenicity rates of 5/5 and 6/6 (Fig. 6D). As the cells were not transfected with fluorescence protein, there was a clear distinction between the GFP⁻ RAW264.7 daughter cells and the GFP⁺ host cells in cultures of the bloody ascites (Fig. 6E). The peritoneal tumor nodules appeared mainly as densely arranged small round cells, and fat vacuoles were also observed (Fig. 6F).