1.1 Experiment animals
A total of 40 C57BL/6 male mice that were eight weeks old, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.(license number: SCXK 20140007, Shandong Province, China) These mice were raised at the specific pathogen-free (SPF)-level in the Experimental Animal Center of the Affiliated Hospital of Jining Medical College, Jining City, Shandong Province, China. All the animal experiments were conducted in compliance with the relevant regulations of the Animal Ethics Committee of the Affiliated Hospital of Jining Medical College
1.2 Esophageal Cancer Specimens
The surgical specimens of esophageal cancer were obtained from the Department of Thoracic Surgery, Affiliated Hospital of Jining Medical College, Jining City, Shandong Province, China. Prior to the study, all participants signed informed consent letters. Furthermore, the study protocol was approved by the Clinical Study Evaluation Committee of the Affiliated Hospital of Jining Medical College. This study was conducted in compliance with the relevant regulations of the Ethics Committee of Affiliated Hospital of Jining Medical College. The general patient information was presented in Table 1.
Table 1
General information of the esophageal cancer patients.
Patient | Gender | Age | Preoperative Imaging Evaluation for Metastasis | Specimen Size | Postoperative Pathology | Postoperative Lymph Nodes Examination for Metastasis |
1 | Male | 53 | None | 5.0 × 4.5 × 1.7 cm | Well-differentiated medullary SCC | Local paracardia lymph node metastasis |
2 | Female | 64 | Swelling of mediastinal lymph node, metastasis not rule out | 3.0 × 3.0 × 2.5 cm | Poorly differentiated SCC | Paraesophageal lymph node metastasis |
3 | Female | 56 | None | 2.7 × 2.4 × 1.5 cm | Well-differentiated SCC | None |
4 | Male | 68 | Local mediastinal lymph node metastasis | 5.4 × 3.5 × 1.4 cm | Moderately-differentiated medullary SCC | Subcarinal lymph node metastasis |
1.3 Microcarrier
Microcarrier 6 was purchased from Elyon Biotechnologies LLC. (Gaithersburg, MD, USA). This is a new type of microcarrier ; it is composed of organic composite polymers showing positive polymerization; moreover, it has a multi-layered pore structure. The microcarrier 6 has advantages of low immunogenicity, good biocompatibility, and metabolizable structure. Thus, it provides a stable microenvironment for cell growth.
1.4 Main Reagents
Dulbecco’s modified Eagle medium (DMEM), collagenase B, fetal bovine serum, ammonium chloride-potassium (ACK) lysing buffer, phosphate-buffered saline (PBS), and penicillin were purchased from Gibco, (Thermo Fisher Scientific, Waltham, MA,USA). The mouse monoclonal antibodies against human p40 and Cytokeratin (CK) 5/6 were purchased from Wuxi Dong Yuan Biotech, Inc(Jiang Su Province,China),
1.5 Isolation and extraction of primary cells of human esophageal cancer
The specimens of esophageal cancer were surgically extracted and placed in a sterile solution of physiological saline. After half an hour, they were sent to the laboratory for treatment. First, the superficial blood and debris were removed from the specimens by rinsing them thrice in serum-free DMEM. Then, they were cut mechanically with a tissue scissor. Thereafter, the specimens were placed in 0.05% collagenase B and digested in an incubator at 37 °C. The tissue digestion process was checked every 30 min to 1 h, and samples were repeatedly pipetted from the medium. After one hour, serum-free DMEM was added to dilute the medium and to mix the tissue samples by pipetting. After centrifuging the medium at 400 r/min for 10 min, the supernatant was removed and the precipitate was added to collagenase B for further digestion. The removed supernatant was then added to DMEM to terminate the digestion. Then, it was filtered with a 70 µm filter and centrifuged at 1200 r/min for 8 min. Thereafter, the supernatant was discarded, and the precipitate was added to ACK lysing buffer. After 5 min, DMEM was added to dilute the solution, and the sample was then centrifuged at 1000 r/min for 8 min to extract the first batch of esophageal cancer cells. Furthermore, the above steps were repeated to extract the remaining batches of esophageal cancer cells with a digestion time of 2 h and 3 h, respectively.
1.6 Establishment Of Three-dimensional (3D) Cell Culture Model
The microcarrier 6 was soaked in 75% alcohol for 24 h, and then rinsed three times with 1 × PBS, followed by incubation in DMEM for 24 h. The microcarrier was modified using 100 ng/mL stromal cell derived factor-1α (SDF-1α) and 100 ng/mL vascular endothelial growth factor (VEGF), with an incubation time of 3 h. The extracted esophageal cancer cells were placed in DMEM, which contained 10% fetal bovine serum and 1% penicillin, and the extract was then pipetted into a single-cell suspension. The microcarrier was added to the single-cell suspension and the ratio of cell to carrier volume was maintained at about 3:1 (cell count of the suspension: ~ 2 × 107/ml, and microcarrier: ~ 300 µg/ml), and then it was placed in a 5% CO2 incubator for 24 h. The temperature of the incubator was maintained at 37 °C.
1.7 Preparation Of Animal Model And The Observation Indicators
For the patient-derived xenotransplantation (PDX) group, the esophageal cancer cell-microcarrier complexes were inoculated into the right armpits of immunocompetent mice, with a dose of 100 µl/mouse; the control group was inoculated with an equal amount of microcarrier only. A total of four experiments were performed with five mice in each group. After inoculation, we observed the mental state, activity, and diet of the experimental mice; moreover, the time of local tumor occurrence and the tumor volume were recorded too. After the formation of tumor, the long diameter (a) and short diameter (b) of the tumor were measured every day, and the tumor volume was calculated according to the equation V = 1/2 × a × b^2. Fourteen days later, the PDX mice were euthanized by cervical dislocation (CD). The tumor tissues were extracted completely, and the tumor volume, texture, and the degree of necrosis were recorded. The tumor tissue was fixed with 10% neutral formalin, and it was then sent to the pathology department for hematoxylin-eosin (HE) staining and immunohistochemical (IHC) staining.
1.8 Flow Cytometry
To perform flow cytometry, the fluorescence-conjugated antibodies against the myeloid-derived suppressor cells (MDSCs, defined as CD11b+Gr-1+ cells), dendritic cells (DCs, defined as CD11c+ cells), and CD3, CD4, and CD8 markers were purchased from eBioscience (San Diego, CA, USA), and they were used at a dilution of 1:100. After euthanizing the mice, we prepared the blood, liver, and spleen cell suspensions. The cell phenotype staining samples were rinsed twice with PBS, which contained 1% fetal bovine serum and 0.1% NaN3. Standard procedures were used to incubate cells with the following anti-mouse antibodies at 4 °C for 30 minutes: CD11b-fluorescein isothiocyanate (FITC), Gr-1-algaphyrin (APC), CD3-FITC, and CD8a-phycocyanin protein (PE), CD4-APC, CD11c-PE, Ly6G-APC, and Ly6c-PE. After being washed twice with PBS, the cells were analyzed with FACSCalibur (Becton Dickinson, San Jose, CA, USA). The isotype control was performed for each antibody.
1.9 Statistical Analysis
The SPSS 13.0 software (IBM, Armonk, New York, USA) was used for statistical analysis. The measurement data was presented as x ± s, and the independent sample t test was used for the comparison of between-groups. P < 0.05 was considered as statistically significant.