Abstract
Purpose
Circulating tumor cells (CTCs) have been proved to be responsible for tumor metastasis and resistant to anticancer therapies. This study aims to isolate and characterize circulating tumor cells from human gastric cancer patients, and investigate characteristic differences between gastric CTCs and gastric cancer cell lines.
Methods
We analyzed 31 cases of gastric cancer patients using anti-CD45 antibody-conjugated magnetic microbeads negative separation, combined with fluorescence activated cell sorter CD44 positive screening. Abilities of tumor formation, metastasis, invasion, migration, irradiation and drug sensitivity of CTCs and gastric cancer cell lines were detected and compared.
Results
Of all the 31 patients, CD44+/CD45−CTCs were isolated in 14 patients, of which 3 cases were stage IIA, 2 cases stage IIB, 2 cases stage IIIC and 7 cases stage IV. The malignant behavior was demonstrated by both clonogenetic assay and tumor xenograft in nude mice. Compared with human gastric cancer cell lines, the migration and invasion abilities of CTCs increased to 3.21–12.6-fold and 2.3–6.7-fold, respectively (all p values <0.05). In addition, the metastatic potential of CTCs is much higher in vivo than that of the control. Furthermore, CTCs were found to be relatively sensitive to FU, cisplatin and paclitaxel, but relatively resistant to irradiation, oxaliplatin, cetuximab and trastuzumab.
Conclusions
CD44+/CD45− gastric CTCs were isolated and found to exhibit stronger malignant behavior when compared with human gastric cancer cell lines. Furthermore, CTCs cultured in vitro have potential implications in drug sensitivity screening for the future anticancer treatments.
Electronic supplementary material
The online version of this article (doi:10.1007/s00432-014-1814-0) contains supplementary material, which is available to authorized users.
Keywords: Circulating tumor cells, Biological characteristics, Gastric neoplasm
Introduction
Gastric cancer is one of the most common malignancies in the world, especially in east Asian countries such as China, Korea and Japan. It has been documented that gastric carcinogenesis is a multistep and multifactorial process following several sequential stages: chronic gastritis, atrophy, intestinal metaplasia and dysplasia. The main risk factors for development of gastric cancer are Helicobacter pylori infection, high salt intake, low intake of fruit and vegetables, smoking and genetic mutations (Ladeiras-Lopes et al. 2008; Fitzgerald et al. 2010; Cogliano et al. 2011; Tsugane 2005). Fifty percent of patients with gastric cancer are in advanced stages at the time of diagnosis in Western Countries and in China. The late diagnosis of gastric cancer leads to a poor prognosis of 5-year survival rates ranging from 20 to 25 % in the Western world, compared to about 60 % in Japan (Kamangar et al. 2006). So survival is dependent on the stage at which the tumor is diagnosed. For example, the survival is about 8–12 months in patients with distant metastasis who underwent treatment (Leong 2005). Unfortunately, gastric cancer has a high rate of liver metastatic. It is reported that liver metastasis is present in 4–11 % of gastric adenocarcinomas (Okano et al. 2002; Sakamoto et al. 2003; Koga et al. 2007; Cheon et al. 2008; Ueda et al. 2009).
Metastasis, the process whereby cancer cells spread from their primary site of origin and grow in adjacent or distant sites, is the primary cause of death in cancer patients. Circulating tumor cells (CTCs) are cells that leave the primary tumor and circulate in the periphery blood. It is accepted that CTCs play an important role in the initiation of tumor metastasis. Previous researches (Cristofanilli et al. 2004; de Bono et al. 2008; Cohen et al. 2009; Krebs et al. 2011; Thorsteinsson and Jess 2011; Tanaka et al. 2009) have focused on CTCs detection technology, prognosis, drug resistance and phenotypic characterization of the cancers, including breast, prostate, colorectal and lung cancer. However, fewer studies on gastric CTCs isolation and their characteristics have been reported, although, most of advances or in the prognostic value of CTCs in patients via the Cell Search System. The clinical significance of CTCs is as follows: The presence of ≥5 CTCs per 7.5 ml of whole blood in patients with metastatic breast cancer (MBC) before treatment was proved to be an independent predictor of progression-free survival (PFS) and overall survival (OS) (Cristofanilli et al. 2004). This prediction of OS was also shown that unfavorable posttreatment CTCs counts (>5 CTCs per 7.5 ml of whole blood) also predicted shorter OS at different time point (de Bono et al. 2008) in castration-resistant prostate cancer (CRPC). Interestingly, some studies have documented that CTCs counts predicted OS better than PSA decrement algorithms at all time points. In addition to OS, CTCs correlated inversely with PFS, and even more with OS, after treatment or at baseline in patients with metastatic colorectal cancer and lung cancer (Cohen et al. 2009; Krebs et al. 2011; Thorsteinsson and Jess 2011). Furthermore, other studies have shown CTCs to be a useful surrogate marker of distant metastasis in primary lung cancer (Krebs et al. 2011; Tanaka et al. 2009) and gastric cancer (Hiraiwa et al. 2008).
However, CTCs were observed in the peripheral blood of cancer patients at rare concentrations, just one tumor cell can be detected in 107–108 normal peripheral blood cells (Witzig et al. 2002). The cause of low sensitivity of the detection methods in the above researches was because not due to those detection methods were just based on the epithelial marker EpCaM, which missed the mesenchymal cells and the cells lost the epithelial elements. Biological characteristics of CTCs are of great interest in order to understand how these cells can travel in the circulation and metastasize. Therefore, the aim of this study was to find another method instead of EpCaM that can also isolate the CTCs from gastric cancer patients, to demonstrate that cells isolated by this method are circulating tumor cells and to compare the biological behaviors of CTCs from human gastric cancer patients with human gastric cancer cell lines.
Materials and methods
Sample collection
Two milliliters of blood was collected from the antecubital veins of 31 gastric cancer patients before receiving the next cycle of chemotherapy at the Department of Medical Oncology, West China Hospital, Sichuan University. And two milliliters of blood was collected from the antecubital veins of three healthy persons in the laboratory. The collected peripheral blood was kept at 4 °C before isolation. The time from collection to isolation was no more than 1 h. Written informed consent in Chinese was obtained from all patients who provided samples in this study, with prior approval from the clinical trial and biomedical institutional ethics committees of West China Hospital of Sichuan University. All clinical investigations had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
Detection of CTCs
Anti-CD45-conjugated magnetic microbeads (Miltenyi Biotec) negative sorting combined with FACS CD44 positive screening was used to isolate CTCs from peripheral blood of 31 gastric adenocarcinoma patients.
Cells were centrifuged with 2,000 rpm for 5 min, lysed of red blood cells by addition of 0.83 % ammonium chloride and then washed twice with phosphate buffered saline [pH = 7.4]. For magnetic separation, cells were labeled for 1–3 h with anti-CD45-conjugated magnetic microbeads (Miltenyi Biotec, Germany) negative sorting according to the manufactures instruction. For FACS analysis, the cells were incubated on ice for 30 min with the anti-CD44 antibody conjugated with FITC (BD Biosciences) and anti-CD45 conjugated with APC (BD Biosciences). This incubation must be done in the dark. The cells were then washed with PBS and centrifuged for 5 min at 1,000 rpm. The cells must be kept on ice until scheduled time for analysis. Samples were sterilely analyzed and sorted with a FACS Aria (BD Biosciences).
Primary culture of CTCs
The separated CD44+CD45− cells were cultured in 12-well plates with 1 ml Dulbecco’s modified Eagle’s medium containing 10 % FBS, 1 % insulin/transferrin/sodium selenite, EGF (20 ng/ml), 1 % nonessential amino acid, at 37 °C and 5 % CO2. Every 3 days, 0.5 ml of above cell culture medium was added to the 12-well plates, and medium was replaced every 7 days. About 2–3 weeks, the cells were clearly seen through microscope. For culture expansion, cells were digested with trypsin for 3 min, when the cells reached to 80–90 % of cell culture dish, and then were transferred into 100 mm petri dish.
CTCs from four gastric adenocarcinoma patients, named CTC-105, CTC-141, CTC-1 and CTC-12, were randomly obtained for the biological study. In order to make the culture control, in the biological study, cells were cultured and expanded in DMEM supplemented with 10 % FBS, just like cell lines.
Cell lines and reagents
Moderately to poorly differentiated, CD44 high-expressing human gastric adenocarcinoma cell line SGC-7901 and MKN-45 (controls) were obtained from the Cell Bank of Chinese Academy of Sciences in Beijing. CTCs and SGC-7901, MKN-45 were cultured in DMEM (Gibco) supplemented with 10 % fetal bovine serum (FBS) at 37 °C, in 5 % CO2 and 90 % humidity (Zhang et al. 2010). CD44 was detected in CTCs and gastric cancer cell lines, and no significance was found between each group (Supplement1). Solutions of 5-fluorouracil (5-Fu; Tianjin Tianyao Pharmaceuticals, Tianjin China); cisplatin, oxaliplatin (CDDP, O-HP, Qilu Pharmaceutical, Jinan, Shandong, China); paclitaxel (PTX, Xiang Fu Pharmaceutical, He Nan, China), cetuximab (Merck Co, Germany) and trastuzumab (herceptin, Roche, Switzerland) were freshly prepared in sterilized water or normal saline before each experiment.
Animals
Nude mice, 4–6 weeks old, were maintained in SPF (specific-pathogen free) at 28 °C, with humidity of 40–60 %, under 12 h light–dark cycles. Sixteen mice were randomized to CTC-105, CTC-141, SGC-7901 or MKN-45 groups with four mice per group. The mice were killed after ether anesthesia. All efforts were made to minimize suffering. All experimental procedures with animals were approved by the Experimental Animal Management Committee of Sichuan University. Animal handling and all procedures on animals were carried out strictly according to the guidelines of the Animal Care and Use Committee of Sichuan University and the current Chinese Animal Ethics Committee Guidelines of the Animal Facility for protection of Animals.
Transplantation and tracking of cancer cells by magnetic resonance imaging exponentially growing cells were labeled with a concentration of 7 μmol/l ultrasmall superparamagnetic ironoxide (USPIO) (obtained from material college of Sichuan University, China) 24 h before injection. CTCs or gastric cancer cells, 2 × 106, were injected subcutaneously into the flanks of nude mice. The tumors and surrounding regions were scanned by serial 7 Tesla MRI instrument (Bruker, Germany) on the 4th, 11th and 22nd day. After 4 weeks, tumor tissues, lungs and liver were collected, fixed in 10 % neutral buffered formalin solution (Sigma, USA), paraffin embedded and subjected to hematoxylin and eosin staining analysis.
Immunohistochemical staining
The primary tumor tissues were pathologically analyzed by immunohistochemical staining, and paraffin sections were subjected to antigen retrieval for 30 min at 95 °C and then dewaxed in xylene and rehydrated with distilled water. The slides were subsequently incubated with the following antibodies overnight at 4 °C: E-cadherin, N-cadherin and vimentin (1:100, Epitomocs, USA). The reaction was performed using ABC systems (DakoCytomation) and DAB substrate chromogen (DakoCytomation) followed by hematoxylin counterstaining.
Population doubling times and growth curves
Cells were seeded in a 96-well plate (1,000 cells/well). Three wells were taken as triplicates each day, and CCK8 was added in for 2–4 h. The ratio of 490 to 630 nm absorption was measured using a UV spectrophotometer. This procedure was repeated for 5 consecutive days. Growth curves were drawn to calculate population doubling time according to the Patterson formula (Liu et al. 2010). Cells in exponential growth phase doubling time = T × lg2/lg(Nt/N0), where T is the needed time, when cell number is from N0 to Nt. The mean ± SD from at least 3 independent experiments containing 3 replicates each was obtained. Data were fit to a linear quadratic model for cell growth curves using the version of GraphPad Prism software 5 (la Jolla, Ca).
Migration and invasion assays
BD cell culture inserts (Becton–Dickinson, Franklin Lakes, NJ) precoated with Matrigel (Becton–Dickinson, Franklin Lakes, NJ) were put in 24-well plates for cell invasion assays. BD cell culture inserts un-precoated with Matrigel were put in a 24-well plate for cell migration assays.
Briefly, 1 × 104 cells per well were suspended in 0.25 ml of serum-free medium and added to the upper chamber. Medium (700 μl) supplemented with 10 % FBS was plated in the bottom of the well. The assay was carried out for 24 h in a humidified incubator. The cells that traversed the membrane pores and spread to the lower surface of the incubator were stained with hematoxylin for visualization. The average number of cells was counted per high view (40×) from 15 high-power fields using an optical microscope. The data were obtained from three independent experiments.
In vitro drug sensitivity assays
Cells were treated with various concentrations of 5-FU, CDDP, oxaliplatin (O-HP), paclitaxel (PTX), cetuximab or trastuzumab for 48 h. CCK8 assays were used to analyze the sensitivity to these drugs. The percentage of live cells was calculated according to the following formula: R(%) = A1/A2 × 100 (R is the percentage of live cells; A1 is relative absorbance value of cells that had been treated with drugs of various concentrations; A2 is relative absorbance value of control cells without any drug treatment). Cells treated with cetuximab or trastuzumab were cultured in DMEM containing 2 % FBS.
Cell irradiation and clonogenetic assays in vitro
Cell irradiation and anchorage-independent growth as a characteristic of in vitro tumor formation were assessed by clonogenetic assay. Briefly, 2 × 106 CTCs were seeded in 10 cm plates 24 h before irradiation. Radiation with various doses was administered with a dose rate of 1.743 Gy/min, using a 160 kV, 25 mA X-ray source. After irradiation, 2,000 cells were seeded in the 6 cm plates, and 14 days later, the colonies were stained with crystal violet. Only colonies of 50 or more cells were counted. Three replicates per dose were studied. The clonogenetic rate was calculated: R(%) = (colony number/total number of culture cells) ×100. The cell survival rate (%) = [(colony number/seeded cells) in irradiated group]/[colony number/seeded cells) in nonirradiated group] ×100.
Western blot analysis
Protein was extracted by Protein lysates (BioTeke Corporation), and concentrations were determined using the Protein dotMetric kit (geno Technology, Inc., St. louis, MO). Samples containing equal amounts of protein (20 μg/well) were loaded on 12 % Bio-Rad gels. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (amersham Pharmacia Biotech, Piscataway, nJ) using a wet transfer apparatus (Bio-Rad). Primary antibodies against anti-CD44 mouse antibody and anti-HER2 rabbit antibody were obtained from Cell Signaling Technologies, USA. Anti-HER1 mouse antibody was obtained from Santa Cruz, USA. Immunodetection was performed by enhanced immunofluorescence-conjugated secondary antibody. Western blots were quantified and normalized using GAPDH as an internal standard for protein loading.
Statistical analysis
All quantitative data were presented as mean values ± SD from three independent experiments. One-way analysis of variance (ANOVA) was used to analyze differences among groups. And the LSD multiple comparison test was used to identify differences among means of two different groups. Test level of α = 0.05, and P values less than 0.05 were considered statistically significant.
Results
Isolation and identification of gastric CTCs
Thirty-one patients were included, 20 patients were in stage IIA-IIIC and 11 patients were in stage IV. Characteristics of patients were described in Table 1.
Table 1.
Characteristics of gastric cancer patients included in this study
| Basic characteristics | Number (cases) |
|---|---|
| Sex | |
| Male | 21 |
| Female | 10 |
| Age | |
| 62 (35–78) year | |
| Clinical pathology stage | |
| Stage IIA | 4 |
| Stage IIB | 3 |
| Stage IIIA | 3 |
| Stage IIIB | 3 |
| Stage IIIC | 7 |
| Stage IV | 11 |
| Surgery | |
| No | 4 |
| Yes | 27 |
| Differentiation | |
| Median | 6 |
| Low | 20 |
| Unknown | 5 |
| Location | |
| Antrum | 13 |
| Gastric angle | 3 |
| Cardia | 4 |
| Gastric body | 6 |
| Fundus | 3 |
| Lesser curvature | 2 |
| Total | 31 |
After CD45-negative immune beads sorting combined with FACS CD44-positive screening (Fig. 1a), the ration of the number of CD44+/CD45− cells and the ration of the number of CD44−/CD45− cells were different in patients. The result was described in Table 2. After primary cell culture, among all the 31 patients, CD44+/CD45− CTCs were positively isolated in 14 patients, of which 3 were stage IIA, 2 stage IIB, 2 stage IIIC, 2 stage IV. The isolation rate of CTCs was 45.2 %, in which the isolation rate of CTCs in stage IIA–IIIC was 35 %, and stage IV was 63.6 %. After 3 weeks, continuous culture in DMEM supplemented with 10 % fetal bovine serum, 1 % insulin/transferrin/sodium selenite, EGF (20 ng/ml), 1 % nonessential amino acid, cells grew adherently in culture dish, fusiform cells were seen in morphology (Fig. 1b), and HE staining indicated that nucleus was large and deeply stained (Fig. 1c). Such cells can be expanded culture in vitro just like cell lines.
Fig. 1.
Double stain of CD44/CD45 analyzed by flow cytometry and cell morphology of gastric circulating tumor cells. a An example showed that CTCs were sorted by FACS, and the CD44 conjugated with FITC and CD45 conjugated with APC antibodies were sorted; b cell morphology through microscope (20× original magnification); c HE staining Transwell chamber membrane was as background. The micropore diameter was 8 μm
Table 2.
The ration of the number of CD44+/CD45− cells and the ration of the number of CD44−/CD45− cells sorted by FACS
| Sample no. | Ration (%) | |
|---|---|---|
| CD44+/CD45− | CD44−/CD45− | |
| 1 | 85.2 | 13.1 |
| 2 | 78.1 | 13.7 |
| 3 | 82.5 | 12.8 |
| 4 | 72.3 | 27.5 |
| 5 | 83.1 | 15.6 |
| 6 | 83.2 | 15.7 |
| 7 | 78.1 | 13.7 |
| 8 | 80.5 | 16.8 |
| 9 | 74.3 | 27.5 |
| 10 | 81.1 | 15.6 |
| 11 | 85.2 | 13.1 |
| 12 | 74.1 | 18.7 |
| 13 | 80.2 | 19.8 |
| 14 | 80.1 | 13.1 |
| 15 | 82.5 | 12.7 |
| 16 | 75.3 | 22.8 |
| 17 | 63.1 | 27.5 |
| 18 | 78.2 | 15.6 |
| 19 | 74.1 | 23.1 |
| 20 | 78.5 | 13.7 |
| 21 | 78.2 | 12.8 |
| 22 | 78.1 | 15.5 |
| 23 | 82.5 | 15.6 |
| 24 | 82.3 | 13.1 |
| 25 | 83.1 | 13.7 |
| 26 | 65.2 | 27.1 |
| 27 | 78.1 | 13.7 |
| 28 | 80.5 | 12.8 |
| 29 | 75.3 | 22.5 |
| 30 | 76.1 | 18.6 |
| 31 | 75.2 | 23.1 |
| Healthy control1 | 3.7 | 78.4 |
| Healthy control2 | 12.3 | 83.1 |
| Healthy control3 | 6.9 | 87 |
Transplantation and tracking of cancer cells by magnetic resonance imaging
After isolation and expanded culture, tumor-forming ability of human gastric CTCs and human gastric cell lines SGC-7901 and MKN-45 was determined. Cells were labeled with ultrasmall superparamagnetic iron oxide (USPIO) 24 h before injection. In consistence with previous reports, cell growth was similar after USPIO labeling in vitro (Fig. 2a). Ten days after the cells were injected subcutaneously into the flanks of nude mice, nodes were visible at the injection sites. Serial 7 Tesla MRI of the tumors and surrounding regions was performed on the 4th, 11th and 22nd day. Tumors with low signal on T2 sequence and high signal on T2 star sequence were clearly visualized by MRI at various time point. Metastatic tumors formed by CTCs were found in the portal vein on the 11th day. On the contrary, no metastasis was found in SGC-7901 or MKN-45 groups until the 22nd day. Figure 2b, c shows MRI scanning sequences of tumors and micro-metastasis derived from SGC-7901 and CTCs, respectively. After 4 weeks, tumor tissues, lungs and liver were collected, stained with hematoxylin and eosin (Fig. 3a, b). Histologically, the tumor cells formed by CTCs seemed to be mesenchymal, the cells were fusiform, and so immunohistochemical analysis was performed in the tumor tissue derived from cultured CTCs; the result revealed that E-cadherin was lowly expressed, while N-cadherin and vimentin were highly expressed. The primary tumor cells generated from CTCs showed character of epithelial to mesenchymal transition (EMT). One of the results is shown in Fig. 4. HE staining of liver and lungs tissues indicated that tumors derived from CTCs with higher rate of lungs and liver metastasis than those of human gastric cancer cell lines. The tumor volumes and the number of mice with liver metastasis are depicted in Fig. 3c, tumor volume was similar between CTC-141 and SGC-7901, and CTC-105 was the smallest of the four groups. Mice number with liver metastasis was the biggest in CTC-105 group, followed by CTC-141 group, and the number of mice with liver metastasis in SGC-7901 group was 0. Figures 2 and 3 indicate that the capacity of metastasis was much greater and earlier in CTCs than that of gastric cancer cell lines.
Fig. 2.
MRI scanned of USPIO labeled CTCs and gastric cancer cell lines in vivo. A Cell growth curves of CTCs labeled with USPIO. Cell growth curves were drawn in groups of CTCs before or after labeled with USPIO, and the relative values were evaluated in 5 consecutive days. The growth curves were similar between CTCs and CTCs-USPIO+. B One of the images of tumors derived from SGC-7901. a–f Images of subcutaneous primary tumor derived from SGC-7901 scanned by 7 Tesla series MRI on the 4th, the 11th and the 22nd days, respectively. g, h No metastatic tumor embolus in the portal vein was scanned on the 22nd day. a, c, e, g were T2 sequences, and b, d, f, h were T2 star sequences. C One of the images of tumors derived from CTCs. a–f Images of subcutaneous primary tumors derived from CTCs scanned by 7 Tesla series MRI on the 4th, the 11th and the 22nd day, respectively. g, h Metastatic tumor embolus in the portal vein was scanned on the 11th day. a, c, e, g were T2 sequences, and b, d, f, h were T2 star sequences
Fig. 3.
Tumors formed by CTCs and gastric cancer cell lines in vivo. A Gross morphology of tumor and liver metastasis lesions in CTCs and SGC-7901 groups; a and c derived from SGC-7901; b and d derived from CTCs. Arrow denotes tumor (a, b) and a metastatic node (d). B Hematoxylin and eosin staining of tumor tissues, liver and lungs of the CTCs and SGC-7901 groups (20× original magnification). (a–c) An example of tumor, liver and lungs of CTCs group, respectively. (d–f) An example of tumor, liver and lungs of SGC-7901 groups, respectively. C The tumor volumes and the number of mice with liver metastasis in vivo. a Tumor volumes in various groups in vivo. b Number of mice with liver metastasis in various groups
Fig. 4.
Immunohistochemical analysis of the original human tumor generated from gastric CTCs and gastric cancer cell lines (40× original magnification). E-cadherin (a); N-cadherin (b); vimentin (c)
Growth speed was different between CTCs and gastric cancer cell lines
Next, tumor growth speed was compared between CTCs and gastric cancer cell lines. Tumor formation assays in nude mice demonstrated that the volumes of CTCs-formed tumors were not greater than that derived from gastric cell lines SGC-7901 or MKN-45 in vivo. The growth curves of CTCs and the two gastric cancer cell lines were depicted in Fig. 5a. According to the Patterson formula, the population doubling time of CTC-105,CTC-141,CTC-1,CTC-12,SGC-7901 and MKN-45 was 30 ± 2 h, 42.49 ± 5 h, 83.72 ± 6 h, 76.27 ± 3 h, 46.5 ± 2 h and 40 ± 4 h, respectively. CTC-105 grows faster than SGC-7901 and MKN-45 (p values were all <0.05). Tumor growth speed was compared between CTCs and gastric cancer cell lines. These results from the available data suggested that, consistent with gastric cell lines, different gastric CTCs showed different proliferative potential.
Fig. 5.
Cell growth and clonogenetic ability in vitro. a Cell growth curves of CTCs and gastric cancer cell lines. Cell growth speed was calculated by CCK8 in 5 consecutive days, and exponentially, population doubling time was evaluated according to the Patterson formula. b The colony number formed by various cells. Various numbers of cells were seeded in the 6 cm plates, and 14 days later, the colonies were stained with crystal violet. Only colonies of 50 or more cells were counted. Three replicates per number were studied. c Clone forming number after irradiation in vitro and the cell survival rate. a The clone forming number in different groups with various doses were depicted in the quadrant. b Cell survival rate was drawn according to the above formula
CTCs form much more colonies than gastric cancer cell lines in vitro
Next, in vitro clonogenetic assays were used to compare colony-forming ability between CTCs and gastric cancer cell lines. Different doses of cells were seeded in the 6 cm plates, and 14 days later, the colonies were stained with crystal violet. Only colonies of 50 or more cells were counted. Data are representative of three independent experiments. The results showed that MKN-45 and SGC-7901 cells did not form colonies even cultured with 200 cells per culture dish, while CTCs formed various numbers of colonies even cultured with 30 cells per culture dish (Table 3; Fig. 5b). These results showed that CTCs form much more colonies than gastric cancer cell lines in vitro clonogenetic assays.
Table 3.
Clonogenetic rate in CTCs and gastric cancer cell lines with various number of cells
| Clonogenetic rate | Number of culture cells | |||||
|---|---|---|---|---|---|---|
| 30 | 50 | 80 | 100 | 150 | 200 | |
| SGC-7901 | 0 | 0 | 0 | 0 | 0 | 0 |
| MKN-45 | 0 | 0 | 0 | 0 | 0 | 0 |
| CTC-105 | 20 % | 12 % | 15 % | 20 % | 12 % | 10 % |
| CTC-141 | 13.3 % | 18 % | 21.25 % | 22 % | 18.6 % | 19.5 % |
| CTC-1 | 20 % | 42 % | 48.75 % | 49 % | 56.6 % | 44 % |
| CTC-12 | 26.6 % | 74 % | 56.25 % | 48 % | 40.6 % | 41.5 % |
CTCs were more tolerate to radiation than gastric cancer cell lines
In addition to chemotherapy, radiotherapy is another adjuvant treatment of gastric cancer. In order to determine whether CTCs were relatively sensitive or resistant to radiation, CTCs were irradiated with various doses of X-ray. CTCs irradiated with 6 Gy or higher failed to form colony after 14th day in culture, while SGC-7901 and MKN-45 cells irradiated with 2 Gy or higher failed to form foci after 14th day of culture. Mean numbers of colonies formed and the cell survival rate after irradiation are shown in Fig. 5c. It seemed that CTCs were more tolerant to radiation than gastric cancer cell lines.
CTCs exhibited greater migration, invasion and metastasis abilities than gastric cancer cell lines
In addition to growth, migration, invasion and metastasis abilities were detected. According to transwell assays, migration capability of CTCs is 3.21–12.1-fold of SGC-7901 and 3.48–12.8-fold of MKN-45, invasive capacity of CTCs was 2.3–5.1-fold of SGC-7901 and 3.05–6.74-fold of MKN-45. According to LSD analysis, there is no significance between SGC-7901 and MKN-45. However, significance was found between CTC-105 and SGC-7901; CTC-105 and MKN-45; CTC-141 and SGC-7901; CTC-141 and MKN-45; CTC-1 and SGC-7901; CTC-1 and MKN-45; CTC-12 and SGC-7901, CTC-12 and MKN-45 (p < 0.05 for all comparisons) (Fig. 6). In vitro and in vivo tests indicated that circulating tumor cells exhibited greater migration, invasion and metastasis abilities than gastric cancer cell lines.
Fig. 6.
Invasion and migration of CTCs and gastric cancer cell lines in vitro. a Number of cells per high view that migrated through the transwell membrane. Results are presented as means of 15 high-power views (40× original magnification). A representative experiment in triplicate, along with standard errors is shown. One-way analysis of variance (ANOVA) was used to analyze differences among groups, and the LSD multiple comparison test was used to identify differences among different groups (**p < 0.01). b A representative result of transwell migration and invasion assays of CTCs and gastric cancer cell lines scanned through microscope (10× original magnification)
CTCs were relatively sensitive to paclitaxel, cisplatin and FU, but relatively resistant to oxaliplatin, cetuximab and trastuzumab
Chemotherapy has been a major strategy for the treatment of advanced gastric cancer patients; fluorouracil (FU), cisplatin, paclitaxel (PTX) and oxaliplatin are frequently used. However, they often fail after a few cycles of treatment due to multidrug resistance. Therefore, the drug sensitivity of CTCs and gastric cancer cell lines was compared. As shown in Fig. 7, the percentage of live CTCs and gastric cancer cells decreased with a dose-dependent manner when treated with different dose of 5-Fu, cisplatin and PTX. No significance was found between each groups treated with 5-Fu, PTX and cisplatin (p > 0.05 for all comparisons). The percentage of live SGC-7901 and MKN-45 cells decreased in the presence of high dose of oxaliplatin. However, the percentage of live cells in CTC-105 and CTC-141 was not decreased with increased concentrations of oxaliplatin, and CTCs were less sensitive than SGC-7901 and MKN-45. Significances were found between CTC-105 and SGC-7901 (p < 0.05), CTC-105 and MKN-45 (p < 0.05), CTC-141 and SGC-7901 (p < 0.05), CTC-141 and MKN-45 (p < 0.05). However, no significance was found between CTC-1 and SGC-7901, CTC-12 and SGC-7901, CTC-1 and MKN-45, CTC-12 and MKN-45 (p > 0.05 for all comparisons). When treated with cetuximab and trastuzumab, the percentage of live cells in groups of gastric cancer cell lines and CTCs was not decreased with increased concentrations of cetuximab and trastuzumab. No significance was found between each groups (p > 0.05 for all comparisons).
Fig. 7.
Percentage of live cells treated with various concentrations of drugs. a–f Percentage of live cells treated with various concentrations of FU, cisplatin, oxaliplatin, PTX, cetuximab and trastuzumab, respectively
Discussion
According to previously reported researches on CTCs, it has been accepted that CTCs must be epithelial-like in order to be captured. Therefore, the technology of CTCs capture relies on the expression of epithelial cell adhesion molecule (EpCAM) on the surface of epithelial cells. However, disseminated CTCs from the primary tumor have a high potential for invasion as a result of epithelial mesenchymal transition (EMT). It has been hypothesized that the circulating tumor cells (CTCs) may have a cancer stem cell-like potential and have underwent EMT, which enhance the metastatic capacity of CTCs (Witzig et al. 2002). Indeed, some evidence indicated CTCs undergone EMT as part of the process of dissociating from the original tumor mass and initiating the metastatic process (Maheswaran and Haber 2010; Aktas et al. 2009). Aktas et al. reported that a major proportion of CTCs in metastatic breast cancer patients showed EMT and characteristics of tumor stem cells (Aktas et al. 2009). Others reported similarly that the majority (>80 %) of these CTCs in patients with metastatic CRPCs and over 75 % of CTCs from women with metastatic breast cancer co-expressed epithelial genes such as EpCAM, CK and E-cadherin, as well as genes representing for mesenchymal, including vimentin, N-cadherin and O-cadherin, and the stem cell marker CD133 (He et al. 2010). There is also strong evidence that the mesenchymal properties of invasiveness and motility are required for metastases and that EMT leads to expression of cancer stem cell markers including CD44 (Armstrong et al. 2011). CTCs which have underwent EMT with low EpCAM expression were associated with high levels of vimentin, suggesting that capture of mesenchymal tumors may be challenging just using EpCAM. Other antibodies for the capture of CTCs may be required to isolate full range of them (Punnoose et al. 2010). Studies on colorectal cancer have revealed the coexistence of CD133+ CD44+ stem-like cells and CD133+ CD44 high subsets that exhibited higher invasiveness in vitro and greater metastasis to liver in vivo (Chen et al. 2011; Botchkina et al. 2009; Du et al. 2008). For these reasons, we chose the cancer stem cell-like biomarker CD44 in breast cancer, as a targeted molecular so that CTCs with mesenchymal phenotype, and cancer stem-cell like cells could be also captured. However, little is known about the biology of these cells. Detailed characterization of CTCs is necessary to provide the opportunity for their clinical use in the treatment of selected cancers.
The results of the current study using clonogenetic assays and tumor xenograft in nude mice indicated that captured cells are true circulating tumor cells but not leukocytes. Retrospective analysis of clinical and pathological data, we found that positive isolation rate of CTCs in stage IIA-IIIC was 35 %, and stage IV was 63.6 %, which indicated that the positive CTCs rate was positively correlated with clinical stage of gastric cancer patients.
Studies on the biological characteristics of circulating tumor cells are relatively few, Kallergi et al. have reported the use of M30 staining apoptotic CTCs in breast cancer, but also analyze positive percentage of Ki67 to evaluate the proliferative ability of CTCs in different stages. They found that both CTCs and apoptotic CTCs can be detected in early and late stages of breast cancer, and in addition, in patients with metastatic breast cancer, the number of apoptosis-positive is less than that of in early stage patients, and the CTC’s proliferative ability in early stages is higher than that of in the late stage of patients (Kallergi et al. 2013).
In the current study, drug sensitivity screening assay indicated that CTCs from four patients were neither sensitive to oxaliplatin nor cetuximab and trastuzumab antibody. Those four patients underwent several cycles FOLFOX or SOX chemotherapy and had progressive disease before CTCs isolation. Western blots indicated that CTCs were moderate to low expressed of HER2, but no expression of HER1, which may explain why they were resistant to cetuximab and herceptin in vitro. Results of immunochemical staining of HER2 and EGFR in the primary tumor tissue were not obtained. HER1 and HER2 expressions in CTCs and gastric cancer cell lines were detected by Western blot (Fig. 8). This result was consistent with the result of a phase III clinical trial that advanced gastric cancer patients cannot benefit from cetuximab antibody (Lordick et al. 2013). In that study, because the median score of EGFR immunochemical staining was 0, patients did not benefit from cetuximab. Similarly, in patients with breast cancer, only those who had HER2-positive expression both in tumor tissues and CTCs derived significant benefit from anti-HER2 therapy. Conversely, up to 52 % patients who were histologically HER2-positive, yet with HER2-negative CTCs, did not benefit from anti-HER2 therapy (Liu et al. 2013). In the radiation study, compared with gastric cancer cell lines SGC-7901 and MKN-45, CTCs showed relatively tolerate to X-ray.
Fig. 8.
Expression of HER1 and HER2 in CTCs and gastric cancer cell lines detected by Western blot
Biological behavior study found that gastric circulating tumor cells had stronger colony formation ability in vitro and exhibited higher capacity of migration, invasion metastasis and much more relative resistance to irradiation, some chemodrugs and target drugs. Previous study indicated that CK+ CTCs were reduced and CK+/CD133+ CTCs were increased after chemotherapy in nonmetastatic breast cancer. So that CD133+ CTCs subsets were thought to be the cause of resistance to chemotherapy (Nadal et al. 2013). Retrospective analysis of clinical data, all CTCs were isolated from peripheral blood of patients who were undergoing adjuvant or first-line chemotherapy, indicating that in the course of treatment, a subset of CD44+/CD45− CTCs has already exhibited treatment resistant, so it may also indicate that the presence of these subset cells is the cause of clinical treatment failure. From the available results, it indicates that gastric CTCs may serve as a useful tool in exploring the potential therapeutic response and screening relatively sensitive medicine in vitro in the future.
Limitations of the present study include false-negatives of CTCs isolation due to low sensitivity of detection and lack of quantification of cells per ml. One of the most apparent limitations of our study was the small sample size, and the CTC-positive separation rate may be changed if the sample size enlarged. Because only four cases were chosen to be studied in the biological behaviors’ research, biological behaviors of CTCs from all the cases should be tested, which could tell us more precise conclusions. CTCs from more patients are needed to test the biological behaviors in the future.
Conclusions
CD44+/CD45− CTCs from gastric cancer patients have been isolated, characterized and found to exhibit clearly malignant behavior. Drug sensitivity screening for captured CTCs may provide a predictive approach in future treatment selection.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary material 1 Expression of CD44 in CTCs and gastric cancer cell lines detected by Western blot. CD44 was detected in CTCs and gastric cancer cell lines, and no significance was found between each group. (TIFF 39 kb)
Acknowledgments
We greatly appreciate the financial support from the National Basic Research Program of China (973 Program, 2011CB935800) (http://www.973.gov.cn/English/Index.aspx) and the National Natural Science Foundation (30971519) (http://www.nsfc.gov.cn/Portal0/default166.htm). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Conflict of interest
We declare that we have no conflict of interest.
Abbreviations
- CTCs
Circulating tumor cells
- FACS
Fluorescence activated cell sorter
- USPIO
Ultrasmall superparamagnetic iron oxide
- EMT
Epithelial–mesenchymal transition
- HER1
Epidermal growth factor receptor1
- HER2
Epidermal growth factor receptor2
- EpCaM
Epithelial cell adhesion molecule
Contributor Information
Ming Liu, Phone: +86-28-85423203, Email: mingliu721@aliyun.com.
Feng Bi, Phone: +86-28-85423203, Email: bifeng@medmail.com.cn, Email: bifeng@med.com.cn.
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Supplementary Materials
Supplementary material 1 Expression of CD44 in CTCs and gastric cancer cell lines detected by Western blot. CD44 was detected in CTCs and gastric cancer cell lines, and no significance was found between each group. (TIFF 39 kb)