Efficacy for lung metastasis induced by the allogeneic bEnd3 vaccine in mice

ABSTRACT

Background: The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice.

Methods: Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3⁺ and CD8⁺ T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice.

Results: The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates.

Conclusions: Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.

Introduction

Lung cancer ranks highest across the spectrum of human cancers in terms of morbidity and mortality. In China alone, mortality associated with lung cancer increased by more than 110% from the 1970s to the 1990s. Surgical resection still remains the main treatment for lung cancer, with chemotherapy and radiotherapy playing secondary roles. Due to extensive metastasis however, lung cancer treatments often fail, resulting in the consistently high rates of mortality. Therefore, the strategy of immunotherapy has been gaining attention as the next generation tumor treatment following the traditional surgery, chemotherapy and radiotherapy. It is a form of biotherapy combining immunology, molecular biology and genetic engineering that inhibits tumor growth by mobilizing or enhancing the body's innate anti-tumor mechanisms. Immunotherapy avoids several limitations of the other tumor treatment methods and has shown promising results in clinical studies.

Cancer biotherapy is a targeted therapy that only attacks the tumor cells and not the healthy cells. Presently, most of the commercially available tumor targeted drugs are monoclonal antibody preparations that are specific against tumor cells and tissues; some of the anti-lung cancer targeted drugs include Iressa, Tarceva, Veenat, Sutent and Sorafenib Nexavar. Although these drugs inhibit the growth of lung cancer cells and prolong patients' survival to some degree, a large number of evidence-based studies have found that their therapeutic effect is less than satisfactory.^1-5 Bevacizumab, a humanized monoclonal antibody acting directly on the vascular endothelial growth factor (VEGF), has 93% human immunoglobulin G skeleton and 7% murine-binding domain, with a half-life of 20 days. Recent phase II/III clinical data^1,3 indicate that its effect was not ideal. Moreover, the incidence of adverse effects, such as hypertension, proteinuria, bleeding, thrombus, and so on, reached 10%. Further, the long-term use of monoclonal antibodies (mAbs) is associated with an increased immunogenicity of the mAbs, and their clinical application is limited as they are expensive. Active immunotherapy specifically targeting antigens on tumor vascular endothelial cells has been proven to be effective in terms of long-time remission of cancer, lower-dosing frequency, and fewer side effects. Recently, vaccines were developed using xenogeneic vascular endothelial cell proliferation–specific proteins and DNA molecules as well as allogeneic proteins and DNA molecules, but their potency was limited due to their action on single targets and the hypersensitive response they elicited. Therefore, developing immunotherapy that would target multiple cancer cells and have minimal side effects is imperative.^6,7

In this study, a vaccine was prepared through the in vitro culture of murine brain microvascular endothelial cells (bEnd.3) derived from microvessels, and mice were immunized to observe the antitumor effect of this vaccine on lung metastases. Human umbilical vein endothelial cells (HUVECs), derived from the vein endothelium, have the potential of differentiation and proliferation and the characteristics of new blood vessel endothelial cells. A number of studies have demonstrated the tumor antiangiogenetic effects of xenogeneic HUVEC vaccine. Wei et al.⁸ used paraformaldehyde to fix the HUVEC and prepare the xenogeneic vaccine to immunize mice, which induced active immune responses against Meth A fibrosarcoma, MA782/5S breast cancer, and H22 liver cancer. Okaji et al.⁹ used glutaraldehyde to prepare a vaccine to immunize mice, which also induced an active immune response against lung metastasis of colon cancer. Chen et al.¹⁰ used live HUVEC to immunize mice directly, which induced a specific immune response against mouse myeloma and lung cancer. Therefore, we used xenogeneic HUVECs and allogeneic murine NIH3T3 fibroblasts as controls to observe tumor anti-angiogenetic effect of vascular endothelial cells from two different sources.

Results

Identification of lung metastasis in the cervical cancer U14 model and metastatic nodule count

The tumor-bearing mice exhibited lethargy, polypnea, cyanosis of lips, and decreased appetite 15 days after being injected with the U14 cervical cancer cell line. The autopsy showed cancerous nodules of different sizes on the lung surfaces (Fig. 1A), which were confirmed as cervical cancer metastasis by histopathology (Fig. 1B). Mice used in this study had one left and four right lung lobes to make the metastatic nodule count comparable across the groups; only the nodules in the left lung were counted. Left lung metastasis was significantly less in the bEnd.3 and HUVEC vaccine groups compared with the NIH3T3 vaccine and PBS groups, for both the prevention and treatment arms, based on the number of metastatic nodules. In mice immunized with bEnd.3 or HUVEC vaccines, left lung metastasis was higher in the treatment group compared with the prevention group.

Figure 1.

Lung metastasis model of cervical cancer U14. A: Gross anatomy of lung metastasis model of cervical cancer U14; B: Lung metastasis model of cervical cancer U14 under the microscope. (HE × 40) The arrow points to the cancer nodules.

HE staining of metastatic lung cancer

Hematoxylin and eosin staining of metastatic lung cancer

A microscopic examination of hematoxylin and eosin (HE)-stained lungs of the PBS and NIH3T3 vaccine groups (both treatment and prevention arms) showed that the tumor cells were arranged as sheets or nests (parts indicated by arrow in Fig. 2C, D, G, and H), developed along the blood vessel section, or in a papillary pattern at the edges. The tumor cells displayed denser arrangement, morphological heterogeneity of cells and nuclei, and abnormal karyokinesis with asymmetry and multipolarity. The lungs of the bEnd.3 and HUVEC vaccine groups had relatively fewer tumor cells, but the tumor cell infiltration (parts indicated by an arrow) partly developed into tumor basement tissue (Fig. 2A, B, E, and F).

Figure 2.

HE staining of Metastatic cancer tissue in the prevention group and treatment group (× 400). A: bEnd.3 of prevention group; B: HUVEC of prevention group; C: NIH3T3 of prevention group; D: PBS of prevention group; E: bEnd.3 of treatment group; F: HUVEC of treatment group; G: NIH3T3 of treatment group; H: PBS of treatment group After mice in control group died, one mouse in prevention group and treatment group was sacrificed respectively. Their tumors were taken out and photograghed. The complete tumor tissues were excised and stained with HE. The black arrow means cancer cell infiltration.

Survival time observation

As shown in Figure 3, in the prevention group, the median survival time of the PBS and NIH3T3 vaccine groups was 19 and 18 days respectively, and the 95% confidence interval (CI) of median survival time of the NIH3T3 vaccine group was 16.303–19.697. The median survival time of the bEnd.3 and HUVEC vaccine groups was 34 and 31 days respectively, and the 95% CI of the two groups was 30.080–37.920 and 29040–32.960, respectively. Statistically significant differences were found between the PBS and NIH3T3 vaccine groups and the bEnd.3 and HUVEC vaccine groups in the prevention group, (P < 0.05). In the treatment group, the median survival time of both PBS and NIH3T3 vaccine groups was 19 days, and the 95% CI of the NIH3T3 vaccine group was 18.151–19.849. The survival time of the bEnd.3 and HUVEC vaccine groups was 26 and 23 days, respectively, and the 95% CI of the bEnd.3 vaccine group was 24.303–27.697. The median survival times of the bEnd.3 and HUVEC vaccine groups in the prevention group were longer than those of the treatment group (P < 0.05).

Figure 3.

Survival curve of mice. A: Survival curve of mice in prevention group; B: Survival curve of mice in treatment group. 1: bEnd.3 vaccine group; 2: HUVEC vaccine group; 3: NIH3T3 vaccine group; 4: PBS vaccine group Mice in the prevention group (n = 6) were immunized once a week for five weeks by subcutaneous injection. One week after the 5th immunization, U14 cells were injected into these mice via the tail vein. Mice in the treatment group (n = 6) were first injected with the U14 cells and then immunized with vaccine on days 1, 3, 5, 7, 9 and 11 after tumor cell injection. Survival time of each group was observed.

Detection of spleen T lymphocyte activity in immunized mice

• (1)In vitro detection of spleen CTL killing activity in the prevention group by CFSE and PI double staining

As depicted in Fig. 4A, the killing activities of bEnd.3-Ts against the bEnd.3 target cells and HUVEC-Ts against HUVEC target cells were stronger than those of PBS-Ts against both target cell types (P < 0.05 for both). The killing activities of bEnd.3-Ts and HUVEC-Ts against U14 cells were clearly weaker than those against bEnd.3 and HUVEC target cells, respectively (P>0.05). Finally, the killing activity of bEnd.3-Ts against bEnd.3 was weaker than that of HUVEC-Ts against HUVECs.

• (2)Expression of CD3 and CD8 on the surface of spleen T lymphocytes in the prevention group

Figure 4.

T lymphocytes activity detection in immunized mice. A: Flow-cytometric analysis of CFSE and PI labeled target cells to detect the Cytotoxicities of CTLs; 1: killing activity of bEnd.3-Ts against bEnd.3; 2: killing activity of bEnd.3-Ts against U14; 3: killing activity of NIH3T3-Ts against bEnd.3; 4: killing activity of PBS-Ts against bEnd.3; 5: killing activity of HUVEC-Ts against HUVEC; 6: killing activity of HUVEC-Ts against U14; 7: killing activity of NIH3T3-Ts against HUVEC; 8: killing activity of PBS-Ts against HUVEC. Histogram A: The radio of different groups. B: CD3 and CD8 expression in the surface of T lymphocyte derived from spleen of mice immunized; 9: bEnd.3-Ts; 10: HUVEC-Ts; 11: NIH3T3-Ts; 12: PBS-Ts. Histogram B: The radio of different groups; * represents P<0.5. All specimens were analyzed by FACSCalibur software, and data were obtained and analyzed by CELLQuest. The killing activity of spleen T lymphocyte CTLs in vitro and the expression of CD3 and CD8 on the surface of splenic T lymphocyte were detected by CFSE and PI double staining.

The bEnd.3-Ts and HUVEC-Ts groups had more double-positive CD3⁺ CD8⁺ cells compared with the NIH3T3-Ts and PBS-Ts groups (P < 0.05). Furthermore, a significant difference was found between the expression intensities of the bEnd.3-Ts and HUVEC-Ts groups (Fig. 4B).

Detection of antibodies in the antisera of immunized mice

• (1)Measurement of antibody titer using enzyme-linked immunosorbent assay

In each group, 80% of the mice had antibodies after immunization, and those lacking any antibodies (20%) were excluded from further experiments. As illustrated in Figure 5, both the bEnd.3 and HUVEC vaccine groups had an average antibody titer of 1:6400.

• (2)Detection of specific response of the antiserum by enzyme-linked immunosorbent assay

Figure 5.

Antibody titer of mice immunized by ELISA. bEnd.3s: antibody produced by mice immunized by bEnd.3 vaccine; HUVECs: antibody produced by mice immunized by HUVEC vaccine; NIH3T3s: antibody produced by mice immunized by NIH3T3 vaccine. PBS was negative control. One week after immunization, blood of mice in each group were collected from the tail vein to prepare the serum, and the titer of the antiserum was detected by ELISA.

The immune response of the antisera from different groups to the various target cells was measured using enzyme-linked immunosorbent assay (Table 1). The bEnd.3X had a specific immune response toward the bEnd.3 membrane protein but did not react with the U14 membrane protein. Similarly, a strong specific immune response existed between HUVECX and HUVEC membrane proteins. The U14X reacted with the bEnd.3, HUVEC and U14 membrane proteins. In contrast, both NIH3T3X and PBSX showed negative responses to the bEnd.3, HUVEC, and U14 membrane proteins.

• (3)Influence of antiserum on target cell proliferation

Table 1.

Specific immune response of serum from mice immunized n = 4, ± X¯s.

	Anti-sera of different groups
Membrane protein (3μg/mL)	bEnd.3X	HUVECX	U14X	NIH3T3X	PBSX
bEnd.3	+	/	++	−	−
HUVEC	/	+++	++	−	−
U14	−	+	++	−	−

Note: P/N < 2.1 was marked as “−”; P/N ≥ 2.1 was “+”; P/N ≥ 4.2 was “++”; P/N ≥ 6.3 was “+++”; P/N ≥ 8.4 was “++++”.

The bEnd.3X and U14X inhibited the in vitro proliferation of bEnd.3 target cells, with bEnd.3X causing more significant inhibition (P < 0.05), while both HUVECX and U14X could inhibit HUVECs (P < 0.05). The U14 target cells could only be inhibited by U14X and not by bEnd.3X or HUVECX (P>0.05). Finally, neither NIH3T3X nor PBSX had any proliferative effect on the three target cells (P>0.05).

• (4)Immunohistological detection of specific antiserum response in immunized mice

Positive immune responses were seen between bEnd.3X (1:10) and bEnd.3 (Fig. 6A) and between HUVECX (1:10) and HUVEC (Fig. 6E). Weak positive responses existed among U14X (1:10), bEnd.3 (Fig. 6B), HUVEC (Fig. 6F) target cells, as well as among NIH3T3X, bEnd.3 (Fig. 6C), and HUVEC (Fig. 6G). PBSX (1:10) did not show any response to bEnd.3 (Fig. 6D) or HUVEC (Fig. 6H).

• (5)Preliminary detection of antibodies against vascular endothelial cell proliferation antigens

Figure 6.

Detection of antiserum by immunocytochemistry. A: bEnd.3X antiserum immunized bEnd.3 cells; B: U14X antiserum immunized bEnd.3 cells; C: NIH3T3X antiserum immunized bEnd.3 cells; D: PBSX antiserum immunized bEnd.3 cells; E: HUVECX antiserum immunized HUVEC cells; F: U14X antiserum immunized HUVEC cells; G: NIH3T3X antiserum immunized HUVEC cells; H: PBSX antiserum immunized HUVEC cells.

The bEnd.3X detected two specific bands at 180 and 220 kD in the bEnd.3 lysate and two bands around 180 and 150 kD in the U14 lysate. HUVECX detected two specific bands at 130 and 220 kD in the HUVEC lysate and two bands around 220 and 180 kD in the U14 lysate. The NIH3T3X and PBSX did not detect any band at 220, 180, and 130 kD with any of the target cell lysates (Fig. 7).

Figure 7.

Detection of vascular endothelial cell proliferation related antibody in antiserum by immunoblot A: 1: bEnd.3X antiserum immunized bEnd.3 cells; 2: bEnd.3X antiserum immunized U14 cells; B: 1: HUVECX antiserum immunized U14 cells; 2: HUVECX antiserum immunized HUVEC cells. BALB/c mouse bEnd.3X and HUVECX were used as first antibody, and mouse bEnd.3 membrane protein and human HUVEC membrane protein were extracted for Western blot analysis.

Discussion

Recently, angiogenesis-targeted immunotherapy of tumor has made great progress, especially, the antitumor immunotherapy with vascular endothelial cell vaccine which has become one of the hot spots. However, whether the effect of immunotherapy with vascular endothelial cell vaccine is better than that of tumor cell vaccine remains to be further studied. In this study, mouse brain microvascular endothelial cells bEnd.3, were cultured in vitro and prepared into a vaccine. Then mice were immunized with this vaccine to observe its antitumor effect against lung metastases. Vaccines made of mouse brain microvascular endothelial cells bEnd.3 are allogeneic for vaccine receptor mice, while HUVEC is xenogenic. bEnd.3 expresses not only intrinsic markers of endothelial cell but also genes related to endothelial cell proliferation, such as vascular endothelial growth factor receptor (VEGFR)-II and endoglin, consistent with the experimental design. HUVEC was chosen as a control because it originates from the large vein and possesses the potential to differentiate and proliferate and has the characteristics of new vascular endothelial cells. Wei et al. have confirmed the presence of anti-VEGFR-II and integrin αv antibody in immunized mice antiserum. The NIH3T3 fibroblast was taken as a control because it does not express antigen related to vascular endothelial proliferation. Moreover, it derives from mouse and is allogeneic with bEnd.3 cell, but is not a kind of vascular endothelial cell.

All aforementioned results indicated that both allogeneic bEnd.3 and xenogeneic HUVEC vaccines can inhibit lung metastasis of cervical cancer U14 and prolong the survival time. However, the inhibitory effect in the prevention group was not as good as that in the treatment group, thereby validating the conclusion drawn by Wei et al. and Okaji et al. Further experiments are needed to determine the cause behind the better antitumor activity of the treatment and not the prevention group, as well as to explore the optimal ratio of vaccine dose and tumor cell load.

The T-cell antigen receptor on the surface of T lymphocyte binds to the antigenic peptide major histocompatibility complex (MHC) I/II molecule complex on the surface of dendritic cell molecules, and double signals jointly induce the mixed lymphocyte reaction. The MHC I molecule captures alloantigen-activated cytotoxicity mediated by CD8+ T lymphocyte, and the MHC II molecule captures xenogeneic antigens activating CD4+ T lymphocyte subsets, thus mediating the cellular immune effect.¹¹

Allogeneic vaccine mostly induces cytotoxicity mediated by the CD8+ T lymphocyte, and the T-cell subset elimination test has proved that the CD4+ T lymphocyte subset is necessary for antitumor immunity. The elimination of CD4+ lymphocyte stops the cytotoxicity mediated by CD8+ T lymphocyte, which further indicates the importance of CD4+ T lymphocyte in allogeneic immunity.¹² A study by Ankathatti Munegowda, M et al.¹³ showed that the expression of CD8+ on the surface of T lymphocytes and the activation of CTL are vital in antitumor immunity. The detection of splenic T lymphocyte activity after immunization also exhibited consistent results that both two kinds of vascular endothelial cells induced CTLs and have a specific killing effect on target cells.

The proportion of CD3+CD8+ cells in splenic T lymphocytes in the two vascular endothelial cell vaccine groups increased, indicating that the vaccine induced CTLs that target vascular endothelial cells.

Antibody titer is an index that measures the antibody level in serum. Various methods are available that measure the antibody titer of mice, such as agar gel immunodiffusion test and ELISA test.¹⁴ The ELISA method possesses the features of high detection sensitivity, simplicity, and accuracy; moreover, the false-positive and false-negative results could be avoided by setting duplicated wells and repeating tests.¹⁵ In this study, the detection of antibody specificity indicated that the U14 tumor cells in mice had the same gene expression as the vascular endothelial cells, and the vascular endothelial cell vaccine was not only targeted against tumor vascular endothelial cells but also against tumor cells, consistent with the conclusions made by Xiang et al.¹⁶

In antitumor studies, the therapeutic effect is usually determined by tumor volume, histology, immunohistochemistry, ELISA, and flow cytometry.¹⁷ Recent studies found the tumor inhibitory mechanism of antitumor angiogenesis: the tumor growth in vivo can be inhibited by inducing the formation of CTLs targeting the proliferation of vascular endothelial cells, promoting the production of antibodies linked to vascular endothelial cell proliferation such as anti-VEGFR-2, anti-endoglin, anti-integrin αν antibodies, and so on, and triggering cellular and humoral immune responses.¹⁸ Antiangiogenic therapy using specific antibodies against vascular endothelial cells is currently being researched by many groups as an alternative to the traditional anticancer therapies. Tung et al.¹⁹ found that inhibition of bovine lactoferrin and VEGF could inhibit the growth of lung cancer through a negative effect on inflammation. Hiratsuka et al.²⁰ suggested that inhibition of VEGFR-1 was able to impede metastasis of lung cancer. Studies by Paauwe et al.^21,22 indicated that endoglin targeting antibody could inhibit angiogenesis and metastasis in breast and ovarian cancers. Immunotherapy targeting specific proteins in non-small-cell lung cancer^23,24 and colon cancer²⁵ are also being actively explored. Ramucirumab is a humanized monoclonal antibody blocking the synthesis of intracellular proteins in new tumor endothelial cells and downstream angiogenesis pathways. Ramucirumab has been approved for treating advanced gastric cancer, gastroesophageal junction adenocarcinoma, colorectal cancer, lung cancer.^26,27 Western blotting was performed with the positive mouse antisera as a primary antibody to identify the specific antibodies involved in this study,. Results showed that mouse bEnd.3, HUVEC cells and U14 cells expressed integrin αν gene, but VEGFR II was expressed only in mouse bEnd.3 and HUVEC cells. Wei et al.⁸ found anti-VEGFR-II and integrin αν antibody in antiserum after the mouse was immunized by HUVEC. VEGFR-II, with a molecular weight of 220 kD, is one of the most important receptors of VEGF that mediated VEGF and promoted proliferation, differentiation, and migration of endothelial cells. Integrin ανβ3, with a molecular weight of 130 kD, is a member of adhesion molecule family. Endoglin is a transmembrane protein molecule with a molecular weight of 180 kD.^28,29 Therefore, blockade of angiogenesis mediated by VEGFR-II, integrin αν and endoglin has become an important means of antiangiogenesis therapy.³⁰ The preliminary results of this study suggested that the mouse bEnd.3 antiserum might produce ant-VEGFR-II antibody but not anti-integrin αν antibody, which might be related to the nonexpression of integrin αν in the protein of bEnd.3 cells.³¹ HUVEC antiserum might produce anti-VEGFR-II antibody and anti integrin αν antibody, and both the vaccines might produce anti-endoglin antibody. The analysis showed that the band at the 180 kD might be that of endoglin.

This study found that allogeneic mouse bEnd.3 cell vaccine exhibited inhibitory potential against growth and lung metastasis of cervical cancer U14 in mice and prolonged the survival time of mice; besides these, the prevention group had a better antitumor effect than the treatment group. It worked by inducing the formation of CTLs targeting vascular endothelial cells, antibodies against the proliferation of vascular endothelial cells, and cellular and humoral immune responses of antiangiogenesis, thus inhibiting the growth of tumor.

However, this study had several limitations. The number of metastatic nodules on the lung surface was counted by the naked eye under the anatomical lens, which might have resulted in a bias of the experimental results. This issue needs attention in further studies. Moreover, not only should the metastatic nodules be counted under the anatomical lens, but also the complete tumor should be removed and weighed so as to evaluate the maximum and transverse diameters of the subcutaneous tumor and calculate the tumor volume. In this study, the reasons for the disappearance of tumor in the bEnd.3 and the HUVEC groups of the prevention group were not clear. It is possible that the tumor cells were attacked by cellular immunity after blood supply was stopped. In the whole experiment, although the antitumor results of the treatment group were always better than those of the prevention group, further experiments are needed to explore the best proportion between the optimal dose and the tumor cell load.

Compared with the exogenous supplements (such as antibodies, small-molecule compounds), active immunotherapy has a longer effect without requiring frequent repeated administration and with fewer side effects. Therefore, active immunotherapy inducing tumor antiangiogenesis has a good application prospect. Glutaraldehyde-fixed vascular endothelial cell vaccine can effectively break the immune tolerance of the organism, induce an active immune response, and achieve multitarget treatment. The treatment of a single gene or protein vaccine is more targeted with lesser side effect. Therefore, its induction of active immune response of antitumor vessel is worth further studying. In this study, the treatment group had a worse antitumor effect compared with the prevention group, but actually, the treatment of tumor is clinically more needed. Therefore, exploration of the number of cells to prepare a vaccine, the number of cells loaded by antigen, and the time and route of immunization was the main content of this study. The adoption of whole vascular endothelial cells to induce the active immune response of antitumor vessels might become a new method of biotherapy for tumors.

Materials and methods

Ethical statement: The study has been sanctioned by the animal use and care committee.

Materials

Pathogen free female BALB/c mice, aged 6∼8 weeks and weighing 16∼18 g, were purchased from the Laboratory Animal Center of Zhengzhou University (license number: SCXK HENAN 2005–0001). The bEnd.3 mouse brain microvascular endothelial cell line was kindly provided by the Institute of Genetics, Fudan University, the U14 mouse cervical cancer strain was purchased from Cancer Institute, Chinese Academy of Medical Science, NIH3T3 mouse fibroblast endothelial cell line was purchased from Wuhan Cell Collection Center and umbilical cords were provided by the Department of Gynaecology and Obstetrics, the Third Affiliated Hospital of Zhengzhou University.

Cell culture and vaccine preparation

• (1)Allogeneic bEnd.3 cell culture

  According to the American Type Culture Collection, the cells were cultured with high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). As the cell confluency reached 80%, the cells were washed twice with phosphate-buffered saline (PBS) and then dissociated with 0.05% trypsin–0.02% EDTA at 37°C for 5 min. When the cell morphology changed into rounded cells, high-glucose DMEM containing 10% FBS was added for neutralization. Centrifugation was performed at 1200 rpm for 6 min. The supernatant was discarded, and the cells were divided for bottle grouping with 1.0 × 104/cm² cells per bottle. Then, the cells were cultured with high-glucose DMEM containing 10% FBS in the cell culture incubator with 5% CO₂ at 37°C. The medium was changed every 2 days.

• (2)
  Xenogeneic HUVEC cell culture

  • (i)
    First, 15–20 cm of undamaged umbilical cord of healthy fetuses via cesarean section was collected under sterile conditions. The blood in the umbilical cord was squeezed out before putting the umbilical cord into the saline solution, and then HUVEC was isolated and cultured for 4 h.
  • (ii)
    Blood around the outer membrane of the umbilical cord was washed with saline solution (adding penicillin–streptomycin 200 μg/mL). The saline solution was injected to one end of the umbilical vein using a 50-mL syringe to rinse the venous cavity repeatedly until the umbilical cord was pale.
  • (iii)
    The proximal portion of the umbilical vein was clipped followed by injecting 15 mL of 0.25% trypsin to the distal end. The distal end was clipped when the venous cavity was full. The umbilical cord was put into a 37°C incubator for 10–12 min after no trypsin leakage was confirmed.
  • (iv)
    The umbilical cord was taken out followed by several oscillations and blood vessel massage to make the endothelial cells fall off. The proximal umbilical vein was cut open to collect the digest solution. Then, 15 mL of saline solution was used to wash the umbilical vein again and collected together with the digest solution. The collected liquid was centrifuged at 1200 rpm for 8 min.
  • (v)
    The precipitated cells were collected and suspended in the endothelial cell medium (ECM). They were inoculated in a 25-cm² disposable culture bottle with gelatin, and cultured in an incubator containing 5% CO₂ at 37°C.
  • (vi)
    The culture medium was discarded after 24 h. The cells were washed once with PBS to remove the red blood cells and the floating cells. The rest was cultured with ECM with the medium changed every 2–3 days. The cells grew into monolayer cells within 2–3 days.
  • (vii)
    When the confluency of primary isolated HUVECs reached 80%, they were digested with 0.25% trypsin and observed under an inverted microscope. When the cell morphology changed into rounded cells but was still attached, the trypsin was discarded and the ECM medium was added. After uniform blending, the cells were passaged at a proportion of 1:2 in an incubator containing 5% CO₂ at 37°C. The medium was changed every 2–3 days.
• (3)Mouse bEnd.3 cells were conventionally cultured in-vitro as described. HUVECs were isolated from human umbilical cord and propagated in-vitro. The identity of the two cell types in culture was confirmed by immuno-histological detection of the cell surface markers vWF, CD31 and CD144, and through RT-PCR.
• (4)RT-PCR was used to detect the expression of the vascular endothelial cell proliferation related antigens VEGFR-II and integrin αν in the bEnd.3, HUVEC, NIH3T3 and U14 cell lines. After total mRNA extraction, target cDNA fragments were obtained through reverse transcription and the amplification products were detected by 2% agarose gel electrophoresis.
• (5)Vaccine preparation

After the cells of bEnd.3, HUVEC, and NIH3T3 were amplified in vitro, 0.025% glutaraldehyde was used to fix and prepare the vaccine. The final vaccine concentration was 2.5 × 10⁷ cells/mL. The cells with a confluence of 80% were washed twice with PBS and dissociated with 0.05% trypsin–0.02% EDTA at 37°C for 5 min. When the cell morphology changed into rounded cells, high-glucose DMEM containing 10% FBS was added for neutralization, followed by cell centrifugation at 2000 rpm for 6 min. The supernatant was discarded, and 1–2 mL of 0.025% glutaraldehyde was added to fix the cells at 4°C for 20 min. The cells were washed twice with PBS before counting, and 1 mL of PBS was added to 2.5 × 10⁷ cells. The mixture was placed into a 1.5 mL of Eppendorf tube followed by mixing it well, and was used for immediate injection or preserved at −80°C for further use. Especially, before vaccination, the vaccine was mixed well, and 10 μL was diluted, and the cell number and cell morphology were observed microscopically.

Preparation of mouse lung metastases model

• (1)Culture of mouse cervical cancer U14 cells: The cells were cultured with DMEM containing 10% FBS. When cells grew to 80%, they were prepared into cell suspension. Cells were passaged at a proportion of 1:3, and a passage was generally completed within 3–4 days.
• (2)Preparation of inoculated cervical cancer U14 cells: Cervical cancer U14 cells in the logarithmic growth phase were taken to prepare a cell suspension and counted and washed twice with PBS. Centrifugation was conducted at 1500 rpm for 6 min. Also, the cell density was adjusted to 3 × 10⁶ cells/0.2 mL for backup.
• (3)Subculture of mouse cervical cancer U14 cells: Mouse cervical cancer U14 cells were cultured in the ascites fluid and harvested upon reaching confluency. The harvested cells were then used to make the tumor cell fluid at the concentration of 2.0 × 10⁷ cells/mL. The mice were each inoculated with 0.2mL of the tumor cell fluid (containing 4 × 10⁶ U14 cells) via the tail vein, randomized, and observed for tumor growth. The duration of the subculture cycle was 7–9 days.
• (4)Preparation of mouse lung metastases model: Tumor cell fluid of well-grown ascites of U14 was selected to prepare tumor cell fluid with a concentration of 2.5 × 10⁷ cells/mL under a microscope; and then each mouse received tail vein injection at 5 × 10⁶ cells/0.2 mL, after which tumor growth was observed.

Grouping and inoculation

• (1)Mice were randomized into four groups on the basis of vaccine type: the bEnd.3, HUVEC and NIH3T3 cell vaccine groups and the phosphate buffer saline (PBS) negative control group.
• (2)Antiserum preparation by mouse tail vein blood sampling

  The mouse tail was immersed in warm water of 50°C for 1 min and taken out. Then, it was dried and disinfected by 75% alcohol. Then, 1 cm from the end of the tail was cut and gently squeezed along the tail to collect 50 μL of blood into an Eppendorf tube. The blood was placed at room temperature for 1 h and at 4°C for one night. On the second day, the upper serum was suctioned and stored at 4°C. The antiserum of each immune group was represented by bEnd.3X, HUVECX, NIH3T3X, and PBSX, and normal mouse serum was represented by NX.

• (3)Mice grouping and inoculation

The randomized mice were further divided into the prevention and treatment groups each with 10 mice. In the prevention group, mice were directly immunized once a week for five weeks with 5 × 10⁶ cells of the specific vaccine by subcutaneous injection around the axillary lymph node. After the 5th immunization, blood was collected from their tail veins and the antibody titer was measured by the enzyme-linked immunosorbent assay (ELISA); only mice that produced antibodies were used in the next experiment. One week after the 5th immunization, 5 × 10⁶ U14 cells were injected into these mice via the tail vein. In treatment group, the mice were first injected with the U14 cells (5 × 10⁶) and then immunized with the specific vaccine on days 1, 3, 5, 7, 9 and 11 after tumor cell injection.

Metastatic cancer nodule count on lung surface

After mice in the PBS negative control group died, the mice in all other groups were sacrificed and their left lungs taken out. The metastatic nodules on the lung surfaces that were visible to the naked eye were counted before fixing the lungs with 4% paraformaldehyde. Following fixation, incisions 0.2cm apart were cut along the longest cross section of left lung and 3 randomly selected sections were stained with hematoxylin-eosin (HE). The metastatic nodules were counted again under the microscope and a nodule appearing on more than one section was counted as one nodule. The final count of the metastatic nodules was the sum of the naked-eye and microscopic counts; accordingly the mean of the total left lung metastatic nodules in each group was calculated.

Observation of survival time

The survival times of mice in the prevention and treatment groups were observed and recorded starting from the day of the U14 cell injection.

Detection of T lymphocytes activity in immunized mouse

• (1)In-vitro detection of spleen cytotoxic T lymphocytes (CTLs) activity in the prevention group by CFSE and PI double staining: cultured bEnd.3 and HUVEC cells in the logarithmic phase of growth and U14 cells directly extracted from ascites were used as target cells. The cells were suspended in the suitable medium at 2 × 10⁵ cells/mL, incubated in the dark with 0.4 μl CFSE solution (at concentration of 5 mM) for 15 minutes at 37˚C and washed twice with PBS at 1500 r/min for 6 minutes each before resuspension in 2 mL medium. T lymphocytes extracted from the spleens of mice immunized with different cells were used as effector cells and correspondingly called bEnd.3-Ts, HUVEC-Ts, NIH3T3-Ts and PBS-Ts. The effector and target cells were mixed in the ratio 30:1 and incubated with 5% CO₂ at 37˚C for 4 hours. After the co-incubation, the cells were centrifuged at 1700 r/min for 8 minutes and re-suspended in 1 mL PBS with 10% FBS. Except for the bEnd.3 and HUVEC negative controls, 5 μL (PI) was added to the other tubes and the cells incubated in the dark for 10 min before being pelleted, re-suspended and analysed. The killing rate was calculated using the formula: Killing rate (%) = (CFSE and PI double positive cells/total target cells) × 100%.
• (2)Expression of CD3 and CD8 on the surface of spleen T lymphocytes: mice from the non-tumor-bearing groups (one mouse per group) were sacrificed on the 7^th day after the last immunization to extract T lymphocytes. The T cells were counted and 5 × 10⁶ cells were re-suspended in 100 μL PBS with 10% FBS. To each tube, 1 μL anti-CD3ε (with concentration of 0.5mg/ml) and 2.5 μL anti-CD8α (with concentration of 0.5mg/ml) antibodies purchased from BioLegend were added and the cells were incubated for 30 min at 4˚C in the dark. The cells were then washed twice with PBS, re-suspended in buffer and analysed by flow cytometry.

Antisera antibody test of immunized mice

• (1)
  Membrane protein extraction and concentration detection

  • (i)
    No less than 1 × 10⁷ cells were collected and washed twice with cold PBS. Centrifugation was performed at 3000 rpm for 5 min. Cells were placed into a 50-mL centrifuge tube, and the excess PBS was removed.
  • (ii)
    Subsequently, 1 mL of protein extraction reagent A-MIX was added to the ice, and the ice water mixture was ultrasonicated for 2 min at an interval of 5 s.
  • (iii)
    The homogenate obtained after ultrasonication was directly centrifugated at 2000 rpm for 10 min at 4°C, and the precipitate was discarded.
  • (iv)
    The supernatant was transferred to a new cold Eppendorf tube and centrifugated at 13,000 rpm for 30 min at 4°C. Then, it was transferred to a new tube for cryopreservation of cytosolic protein.
  • (v)
    Then, 100 μL of cold protein extraction reagent D-MIX was added to the precipitate. After uniform blending, it was placed at 4°C for 30 min.
  • (vi)
    Centrifugation was performed at 13,000 rpm for 30 min at 4°C, and the samples were divided into upper layer and precipitate.
  • (vii)
    The supernatant finally obtained was the membrane protein extract, and 2 mL of the supernatant was collected to determine the protein content. The rest was stored at −80°C.
• (2)Antisera titer detection by ELISA: venous blood was collected from every group and stored first at room temperature for one hour and then overnight at 4˚C. The serum supernatants (the antisera) were collected and suitable labelled as bEnd.3X, HUVECX, NIH3T3X and PBSX as per the vaccine group; the serum of control mice was called NX. Plasma membrane proteins were extracted from each group and 3 mg/L membrane solution was used to coat the ELISA plates. Different concentrations of the antisera and goat anti-mouse HRP-IgG (purchased from Boster Biological Technology co. Ltd) were added onto the coated plates and incubated at 37˚C for 30 min. Following the coloration reaction, the absorbance at 450nm was measured and antibody titer in the different antisera was calculated.
• (3)Specific response detection of immunized mouse by ELLISA: Cross reaction was performed in every group. Positive antiserum with a titer of 1:100 was taken as the first antibody to observe specific response of immunized mouse antiserum. 4 wells were set for each group and the absorbance of each well at 450nm was determined. NX represented serum of normal BALB/c mouse, blank control was set to zero and P/N≥2.1 indicates a positive result.
• (4)Detecting the effect of immunized mouse antiserum on target cell proliferation by CCK experiment: bEnd.3, HUVEC and NIH3T3 cells at logarithmic phase were collected and routinely trypsinized. U14 cells were extracted from murine ascites and inoculated into 96-well plate with 1 × 10⁴ cells per well, and 4 wells were set for each group. The cells in each well were resuspended by 70 μL corresponding medium, and then 30 μL antiserum was added, maintaining the total volume of each well at 100 μL. Inhibition rate = [(target cell A₄₅₀ value _– experimental group A₄₅₀ value)]/target cell A₄₅₀ value × 100%.
• (5)The specificity of antisera was determined by immunocytochemistry as follows: mouse bEnd.3 and human HUVEC cells were inoculated into 96-well plate for overnight culture and then fixed with 40 g/L paraformaldehyde for 30 min. The immunized mouse antisera and goat anti-mouse HRP-IgG were used as the primary and secondary antibodies respectively and diaminobenzidine was used for color detection. An inverted phase contrast microscope was used to examine and photograph the degree of staining, and the Biosens Digital Imaging System⁹ was used for scanning and analyzing.
• (6)Preliminary detection of specific antibodies in the immunized mouse antisera against the vascular endothelia cell proliferation antigens by Western blot: one mouse from every group was bled retro-orbitally and the sera were collected. SDS-gel electrophoresis was performed with 50 μg protein sample from each group and the separated proteins were transferred to PVDF membrane through semi-dry electro blotting. The membranes were blocked at room temperature for three hours and incubated overnight at 4˚C with the antisera (as the primary antibody). After washing, the membranes were incubated with the secondary goat anti-mouse HRP-IgG at room temperature for one hour. The membranes were stained with the enhanced chemiluminescence (ECL) reagent and developed in a dark room. The Gel-Doc imaging system (Quantity One 1-D Analysis Software) was used for imaging.

Observation of mouse health and changes in the immune system organs

During the experiment, mice were weighed every week and their fur color, appetite, feces and activity were observed. After sacrificing the mice, the morphological changes in their livers, lungs and kidneys were observed. The thymi and spleens were also taken from each group for HE staining to detect any changes of tumor cells.

Statistical analysis

The nodules on the lung surface were expressed as (X±s).^10,11 The CTL killing and target cell proliferation experiments were repeated at least three times; their mean values were calculated and the results were analyzed with the SPSS 23.0 software and expressed as [(X±s)%]. One-way ANOVA was used for statistical analysis and the LSD method for pairwise comparison. The survival times, survival rate and mean survival times were determined by the Kaplan-Meier method with α = 0.05 as the test standard.

Funding Statement

This project was supported by Grant: W2013FZ33.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

Thanks should be given to workmates of the authors within the same department and all the researchers of Biomedical Engineering Technology and Data Mining Research Institution of Zhengzhou University for their general support.