Establishment of human esophageal cancer xenograft model in immunocompetent mice and explorations of related immunological changes

[Background] To combine the primary cells of human esophageal cancer with a new type of three dimensional (3D) microcarrier 6, and then to inoculate the complex subcutaneously into immunocompetent mice. To establish a new animal xenograft tumor model of human esophageal cancer, and to explore the changes in the immune indicators of mice during tumor formation. [Methods] of and on microcarrier 6-human cell rapid simple peak the cells fast a of necrotic the to an Capillaries IHC p40 CK 5/6


[Background]
To combine the primary cells of human esophageal cancer with a new type of three dimensional (3D) microcarrier 6, and then to inoculate the complex subcutaneously into immunocompetent mice. To establish a new animal xenograft tumor model of human esophageal cancer, and to explore the changes in the immune indicators of mice during tumor formation. [Methods] 1. Isolate and extract the primary cells of human esophageal squamous cell carcinoma (SCC); mix them well with the 3D microcarriers and fully incubate them. Then, inoculate the complex into the armpits of immunocompetent mice, and record the tumor formation rate and the pathological characteristics of xenograft tumors. 2. Isolate cells in the blood, bone marrow, and spleen of the experimental mice and the control mice, and detect changes in CD3 + , CD4 + , CD8 + , myeloid-derived suppressor cells (MDSCs), and dendritic cells (DCs) by flow cytometry.

[Results]
The microcarrier 6-based model subcutaneously transplanted primary cells of human esophageal cancer, which further successfully grew into xenograft tumors in immunocompetent mice; the tumor formation rate was 80%. The hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) characteristics indicated consistencies with the human esophageal cancer cells. The flow cytometry analysis showed that CD3 + and CD4 + cells in the peripheral blood and bone marrow of the tumorformed mice were significantly reduced (P < 0.05). The cell counts of MDSCs and DCs in the blood, bone marrow, and spleen were elevated as compared with the control group, and with the MDSCs increased the most dramatically and statistically significant increase (P < 0.05).

[Conclusion]
The new type of 3D microcarriers were combined with human esophageal SCC cells; this model could be used to successfully construct an immunocompetent mouse xenograft model of human esophageal cancer. We further found that during tumor formation, the tumor cells may inhibit cellular immunity by regulating MDSCs, leading to tumor immunity escape and promoting tumor development. 4 Background Esophageal cancer is a malignant tumor that occurs in the basal cells of esophageal mucosa. In the early stage, esophageal cancer has non-specific symptoms; therefore, most patients are diagnosed in the middle and advanced stages of esophageal cancer. Hence, the prognosis is poor in most cases.
Currently, the mechanisms of occurrence and the development of esophageal cancer has not been elaborated extensively in research studies; moreover, the tumor immunology of esophageal cancer is not understood clearly till date. Presently, animal models that completely mimic human diseases are established to dynamically reveal the mechanism of tumorigenesis at the overall level; moreover, these animal models are further used in devising strategies for the prevention and treatment of diseases [1][2]. In this study, a cell culture was prepared by using primary cells of human esophageal squamous cell carcinoma (SCC); these cells were cultured on the microcarrier 6 to establish an animal model of subcutaneous xenograft tumor in immunocompetent mice. During the formation of tumor, researchers detected changes in the immune cells of the blood, bone marrow, and spleen of mice.
The modeling method was used to establish an immunocompetent mouse model of esophageal cancer and to elucidate the internal connections between the occurrence and development of esophageal cancer and the body's main immune cells, thereby providing a new pathway for the immune mechanism study of tumor formation.

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.

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 × 10 7 /ml, and microcarrier: 3 00 µg/ml), and then it was placed in a 5% CO 2 incubator for 24 h. The temperature of the incubator was maintained at 37 °C.

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 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.

Flow Cytometry
To perform flow cytometry, the fluorescence-conjugated antibodies against the myeloid-derived

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.
2 Results 2.1 General conditions of mice after inoculation Within 2-3 days after inoculation, the condition of the mice was as follows: reduced appetite, decreased activity, and poor mental state; however, not a single mouse died during this process.

Tumor formation time, tumor volume, and tumor formation rate
The xenograft tumor began to grow in about 8-10 days, and it reached the peak value of growth in about 12-16 days. The formed tumors had a diameter of 0.6-1.0 cm. These tumors were mostly round or oval in shape; they had a tough or hard texture and a grayish-white cut surface. The rate of tumor formation was 80%.

HE Staining
The xenograft tumor tissue displayed heterogeneous cells. These cells were of various sizes, and they exhibited a disordered arrangement under a microscope. In general, most cells were oval in shape; these cells had large and varied nuclei, coarse chromatin, obvious nucleoli, and a clearly visible nuclear division. Large necrosis was observed in the center of the tumor, and capillaries were visibly distributed around the tumor.

IHC Detection
According to the IHC results, the expression of p40 and CK 5/6 cells was found to be positive. This confirms that atypical cells were actually human-derived esophageal cancer cells.

Flow Cytometry
Peripheral blood of the PDX group was compared with the control group. It was found that the PDX group showed significantly reduced expression of CD3 + and CD4 + cells (P = 0.0071 and P = 0.0325, respectively) and a slightly increased expression of CD8 + cells as compared to the control group. In the PDX group, the total MDSCs and granulocytic MDSCs (G-MDSCs) were significantly increased (P < 0.0001 for both); however, the monocytic MDSCs (M-MDSCs) were decreased in the PDX group as compared to that in the control group. The DCs of the PDX group were significantly more than that in the control group (P = 0.002). (Fig. 4-8) The bone marrow of the PDX group was compared with the control group. It was found that the PDX group had decreased CD3 + , CD4 + , and CD8 + cells as compared to those in the control group, with CD3 + cells showing the least expression and statistically significantly reduced activity (P = 0.0126), followed by CD8 + cells' statistically reduced expression (P = 0.0712). The total, G-and M-MDSCs were all increased in the PDX group as compared to those in the control group, with the total MDSCs being the highest in number (P = 0.0009). The DCs of the PDX group were slightly increased as compared with the control group, but not significantly increased statistically (p = 0.2307). (Fig. 4-8) Finally, the spleen tissue of the PDX group was compared with the control group. It was found that the PDX group had a decreased expression of CD3 + , CD4 + , and CD8 + cells as compared to the control group, with CD4 + cells' expression being the most reduced and also significantly reduced statistically gastric cancer transplant tumor model, and also achieved certain results [11].
As is well known, tumor cell immunity is closely related to the occurrence, development, and prognosis of tumors. The T lymphocytes are the most crucial function cells in the human body's immune system.. The investigation of changes in subsets and functions of T cells in the occurrence and development of tumor is helpful to reveal the mechanisms of tumorigenesis,which play a pivotal role in the occurrence and development of tumor .In this experiment, changes in the T cell subsets of CD3 + , CD4 + , and CD8 + were measured. The results indicate that in the bone marrow and spleen, the PDX group had fewer CD3 + , CD4 + , and CD8 + cells than in the control group. In the bone marrow, the CD3 + cells declined the most and the decline was statistically significant (P = 0.0126). In spleen tissues, CD4 + cells decreased most significantly (P = 0.0139), and CD3 + and CD8 + also dropped to some extent (P = 0.0525 and P = 0.0961, respectively). Compared with the control group, the CD3 + and CD4 + cells in the peripheral blood of the PDX group were reduced significantly (P = 0.0071 and P = 0.0325, respectively), but the change in CD8 + cells have a mild rise. We believe that if the sample size increases, the value will also show a downward trend. A clinical study [12] showed that in the peripheral blood of patients with esophageal cancer, CD3 + and CD4 + cells were significantly reduced before surgery; however, their expression were significantly enhanced after surgery (P < 0.05). This indicates that in esophageal cancer patients who underwent radical operation, the cell immunosuppression ability was improved significantly. This change was in complete agreement with the CD3 + and CD4 + changes in our xenograft model, suggesting a suppressed cell immune function in the esophageal cancer xenograft model during tumor formation.
In the current experimental study, MDSCs are a heterogeneous group of cells that originate from the myeloid tissue at various stages of differentiation. These cells play an essential role in tumor-related immunosuppression.Moreover, MDSCs mediate the immune escape of tumors by suppressing the effector T cells and the natural killer (NK) cells. Based on the differential expression of CD11b and the lymphocyte antigen 6 complex (locus G, Ly6g, and Gr-1) on the cell surface of mice, the MDSCs could be divided into two independent groups, namely, the CD11b + Ly6C low Ly6G + granulocytic group, and the CD11b + Ly6C + Ly6G − monocytic group. Nowadays they have become a hotspot in studies related to tumor immunity and autoimmunity because of their significant immunosuppressive functions [13][14][15]. In this study, the total MDSCs and their subtypes (G-MDSCs and M-MDSCs) were tested, and the antigen-presenting cells, DCs, were studied as well. The total MDSCs and G-MDSCs in the peripheral blood of the PDX group were found to be significantly increased as compared to those in the control group (both with P < 0.0001), and the DCs were significantly elevated in the PDX group too (P = 0.002). The total, G-and M-MDSCs in the bone marrow of the PDX group were all enhanced as compared with the control group; moreover, the total MDSCs increased particularly significantly (P = 0.0009). Furthermore, the DCs of the PDX group were slightly increased, but the increase was just not

Consent for publication
Not applicable

Availability of data and materials
Not applicable

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by grant from National Natural Science Foundation of China (81170395,

81570556)
Authors' contributions RRH coordinated the whole process of the experiment, responsible for collecting samples, isolating and extracting primary cancer cells, analyzing the experimental results and writing papers. He was the main first author.
FH was the main project designer, evaluated the experimental quality, participated in the modification of the paper, and was the corresponding author.
HMZ is responsible for data statistics and paper revision, and is the second author.
YZB has an important contribution to cell separation and extraction.
XYYand GYJ participated in flow cytometry detection and data analysis.
QYW participated in pathological examination and analysis.
HXW provides surgical specimens.
YFQ and HRZ participated in the modification of the paper.
All authors read and approved the final manuscript.    Changes of CD3+ in various tissues. Control (control group); PDX (tumor formation group). * P < 0.05, ** P < 0.01, *** P < 0.0001. In peripheral blood, bone marrow, and spleen, the PDX group shows decreased CD3+ cells compared with the control group; the declines in peripheral blood and bone marrow are statistically significant.

Figure 5
Changes of CD4+ in various tissues. Control (control group); PDX (tumor formation group). * P < 0.05, ** P < 0.01, *** P < 0.0001. In peripheral blood, bone marrow, and spleen, the PDX group shows decreased CD4+ cells compared with the control group; the declines in peripheral blood and spleen are statistically significant (P < 0.05). Compared with the control group, the PDX group has slightly increased CD8+ cells in peripheral blood and decreased CD8+ cells in bone marrow and spleen.

Figure 6
Changes of DCs in various tissues. Control (control group); PDX (tumor formation group). * P < 0.05, ** P < 0.01, *** P < 0.0001. In peripheral blood, bone marrow, and spleen, the PDX group shows increased DCs compared with the control group; moreover, the increases in peripheral blood and spleen are statistically significant.

Figure 7
Changes of total MDSCs in various tissues. Control (control group); PDX (tumor formation group). * P < 0.05, ** P < 0.01, *** P < 0.0001. In peripheral blood, bone marrow, and spleen, the PDX group shows statistically significant increase in MDSCs as compared with that of the control group; moreover, the increases in bone marrow and peripheral blood are the most significant (P < 0.0001).

Figure 8
Changes of G-and M-MDSCs in various tissues. Control (control group); PDX (tumor formation group). * P < 0.05, ** P < 0.01, *** P < 0.0001. The PDX group shows decreased MDSCs in peripheral blood and increased MDSCs in bone marrow and spleen as compared with those of the control group. The G-MDSCs of the PDX group in the three tissues are all increased, with the increases in peripheral blood and spleen being statistically significant.