Med Oncol (2014) 31:777 DOI 10.1007/s12032-013-0777-3
ORIGINAL PAPER
Bevacizumab improves the antitumor efficacy of adoptive cytokine-induced killer cells therapy in non-small cell lung cancer models Leilei Tao • Guichun Huang • Shujing Shi Longbang Chen
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Received: 13 August 2013 / Accepted: 16 November 2013 / Published online: 24 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Cytokine-induced killer cells (CIK cells) are a heterogeneous population of cells generated from peripheral blood mononuclear cells, which share phenotypic and functional properties with both natural killer and T cells. CIK cells therapy, as an adoptive immunotherapy with strong antitumor activity in vitro, represents a promising approach for the treatment of a broad array of malignant tumors. However, clinical trials in CIK cells therapy did not show more noticeable improvement as anticipated in cure rates or long-term survival. Possible explanations are that abnormal tumor vasculature and hypoxic microenvironment may highly limit the therapeutic benefits of CIK cells therapy. We hypothesized that antiangiogenesis therapy could enhance the antitumor efficacy of CIK cells by normalizing tumor vasculature and modulating tumor hypoxic microenvironment. In this study, we combined bevacizumab and adoptive CIK cells therapy in the treatment of lung adenocarcinoma bearing murine models. Flow cytometry,
intravital microscopy and immunohistochemistry were applied to detect tumor vasculature and hypoxic microenvironment as well as the infiltration of CIK cells. The results indicated that bevacizumab-combined adoptive CIK cells had synergistic inhibition effects on the growth of lung adenocarcinoma. Hypoxia significantly inhibited the infiltration of CIK cells into tumor tissue. Bevacizumab could normalize tumor vasculature and decrease tumor hypoxic area. Furthermore, combination therapy enhanced more CIK cells infiltrated into tumor compared with other treatment. Bevacizumab improves antitumor efficacy of CIK cells transfer therapy in non-small cell lung cancer (NSCLC). The study provides a reasonable and beneficial strategy that combined antiangiogenesis therapy with CIK cells therapy for patients of advanced stage non-small cell lung cancer.
Leilei Tao and Guichun Huang have contributed equally to this work.
Abbreviations NSCLC Non-small cell lung cancer CIK cell Cytokine-induced killer cell PBMC Peripheral blood mononuclear cells VEGF Vascular endothelial growth factor
Electronic supplementary material The online version of this article (doi:10.1007/s12032-013-0777-3) contains supplementary material, which is available to authorized users. L. Tao G. Huang S. Shi L. Chen (&) Medical Oncology Department of Jinling Hospital, Medical School of Nanjing University, 305 ZhongShan Eastern Road, Nanjing 210002, People’s Republic of China e-mail: [email protected] L. Tao e-mail: [email protected] G. Huang e-mail: [email protected] S. Shi e-mail: [email protected]
Keywords Antiangiogenesis Bevacizumab Vascular normalization Hypoxia microenvironment Adoptive CIK cells therapy
Background Lung cancer is one of the most common malignancies and leading cause of cancer-related death around the world [1]. Approximately 80 % of all lung cancer cases are non-small cell lung cancer (NSCLC). Despite development of cancer treatment and introduction of new drugs, advanced lung
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cancer remains associated with poor prognosis. Recently, adoptive immunotherapy as a new approach to treat solid tumors holds great potential compared with other traditional treatments [2]. Adoptive cytokine-induced killer cells (CIKs) transfer, as one type of adoptive immunotherapy, showed promising antitumor effect on various malignant tumors, including NSCLC [3]. Cytokine-induced killer (CIK) cells are heterogeneous in vitro-expanded T lymphocytes which along with T-NK phenotype and MHC-unrestricted antitumor ability. These biological features of CIK cells make them appealing for adoptive immunotherapy and have displayed encouraging results in tumor therapy [2]. However, the effectiveness of single cancer immunotherapy in solid tumor remains quite low in clinical trials. Thus, it is necessary to find an effective antitumor strategy aimed at enhancing more CIK cells infiltrated into tumors and increasing their intratumoral activity for patients [4]. Evidences have been provided that abnormal tumor blood vessels and tumor microenvironment play a crucial role in impeding lymphocyte cell infiltration into tumor tissue and inhibiting the activity of these immune cells [5]. Hypoxia is a common feature in a wild range of solid tumors, including non-small cell lung cancer. Hypoxia, as a crucial role, induces both angiogenesis and immunosuppression in the complex and highly dynamic tumor microenvironment. Hypoxia-induced abnormal neo-angiogenesis causes increased resistance to adoptive immune cell therapy in tumor [6]. Recent studies indicated antiangiogenesis agents could transiently normalize tumor vasculature, and its hypoxia microenvironment thus could significantly influence the response of tumor to traditional chemotherapy [7]. Bevacizumab (AvastinÒ; Genentech, Inc. South San Francisco, CA, USA) is a humanized anti-VEGF monoclonal IgG1 antibody (molecular weight, 149 kDa). Accumulated studies showed bevacizumab that targeted VEGF could result in a dramatic suppression of tumor growth, normalization of tumor blood vessel, and reduced tumor hypoxia area. Clinical trials with bevacizumab in a variety of malignancies indicated that conventional chemotherapy or irradiation in combination with bevacizumab held great potential advantages in cure rates or long-term survival of cancer patients [8, 9]. We hypothesized that bevacizumab could also enhance efficacy of CIK cell immunotherapy by normalizing tumor vessels and modulating hypoxic microenvironment of NSCLC. Our study aimed to determine whether bevacizumab could improve the antitumor effect of adoptive CIK cells and to illustrate possible mechanism that antiangiogenesis agents release the full potential of adoptive CIK cells therapy. The results of our research revealed novel mechanisms of bevacizumab promoting efficacy of adoptive CIK cells transfer
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therapy in NSCLC. This strategy provided a rational basis for bevacizumab in combination with immunotherapy and could be easily translated into clinical setting in curing NSCLC patients.
Materials and methods Tumor cell line Human lung adenocarcinoma cell line (A549 cells) is preserved in our laboratory and cultured in RPMI-1640 (Gibaco, USA) supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. Cells were cultured in a humidified atmosphere of 5 % CO2 at 37 °C. Generation of CIK cells Human peripheral blood samples were obtained from healthy donors with permission consent. The study was approved by the Ethic Committee of Jinling Hospital. Peripheral blood mononuclear cells (PBMC) were separated from human peripheral blood through density gradient centrifugation using Ficoll-Hypaque (Beijing Chemical Reagents Company, China). Cells were then resuspended in RPMI-1640 medium (10 % FCS, 100 U/ml penicillin and 100 lg/ml streptomycin), with the 1,000 U/ml interferon-c (Peprotech, USA) added on the first day. After 24 h, 500 U/ml Interleukin-2 (Peprotech, USA) and 50 ng/ml anti-CD3 antibody (eBioscience, USA) were added. Cells were incubated in a humidified atmosphere of 5 % CO2 at 37 °C. Thereafter, RPMI-1640 supplemented with interleukin-2 (300 U/ml) was applied every other day for 2 weeks and maintained at a final density of 2–3 9 106 cells/ml. Animal models Female BALB/c nude mice were purchased from Academy of Military Medical Science (Beijing, China) and maintained under controlled temperature and humidity, and a 12-h light–dark cycle, with sterile food and water ad libitum. All mice used in our study were 6–8 week old. Animal experiments were performed according to the guidelines of the Institute Animal Care and Use Committee of Jingling Hospital. Human lung adenocarcinoma xenograft models were established by subcutaneous inoculation of 100 ll A549 cells (1 9 107/ml) into the right flank of BALB/c nude mice. Subcutaneous tumor volumes were measured daily by caliper, and tumor volumes were calculated by the formula: tumor volume = 0.5 9 length 9 width2. The animal experiments were repeated twice.
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Treatment protocol Treatment was started when tumor volumes grew to approximately 250 mm3. The mice were randomly divided into 4 groups with 5–6 mice in each group. The detailed groups were as follows: (1) Group NS, treated with normal saline, as control group; (2) Group CIK, treated with CIK cells alone; (3) Group BEV, treated with bevacizumab alone; (4) Group BEV ? CIK, treated with bevacizumab followed by transfusion of CIK cells. The first day when treatment started was designated d1. The dose of bevacizumab was intravenous injection of 5 mg/kg at d1, intravenous transfusion of CIK cells at d3 (1 9 107cells per dose in a total volume of 100 ll). The inhibition rate of the tumor volume (TIR) was determined according to the formula: TIR = 1-(mean tumor volume in the treated group/mean tumor volume in control group). The formula (q = EA ? B/[EA ? (1 - EA) EB]) [10]was used to assess the synergism of two therapies. EA, the inhibition rate of bevacizumab therapy, which is defined as EA EB, the inhibition rate of adoptive CIK cells immunotherapy EA ? B, the inhibition rate of combination therapy q \ 0.85 means the two therapies are antagonism. q [ 1.15 means the two therapies are synergism. 0.85 B q B 1.15 means the two therapies are additive. Flow cytometry Human CIK cells were transfused into mice 3 days after treated with bevacizumab. CIK cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes Biotec. Final concentration of 5 lmol/l in PBS) and cultured at 37 °C for 5 min. Cold RPMI-1640 with 10 % FCS was added to quench the labeling reaction, and cells were washed twice with PBS to remove excess CFSE. Then CIK cells were transferred into mice. 24 h later, tumor samples were harvested, and tumor single cell suspensions were prepared from group CIK and group BEV ? CIK. Red Blood Cells were removed in Erythrocytes Lysis Buffer [155 mmol/l NH4Cl, 10 mmol/l KHCO3, and 0.1 mmol/l EDTA (pH 7.4)] at room temperature for 10 min. The amount of CFSE-labeled CIK cells in each sample was counted by FACS-Calibur and CellQuest software (BD Biosciences, San Jose, CA, USA). Intravital microscopy Intravital microscopy examination was in accordance with our previous reports [11]. In brief, mice bearing A549 tumors were anesthetized with pentobarbital sodium (50 mg/kg body weight) by intraperitoneal administration
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and then fixed on the board. Body temperature was maintained about 37 °C throughout the experiment. The skin around tumor was removed to make an observation window. The exposed tumor tissue was kept moist with normal saline during the entire surgical procedure. Subsequently, a thin sterile contact lens was covered on the upper surface of tumor to provide visual area of the tumor vascular in the experiment. The upper surface enables a large number of venules and capillaries to be monitored under intravital microscope. The dye Evans blue (Sigma-Aldrich, USA) which bounded tightly to albumin (EBA) was used to monitor vascular permeability. Evans blue (20 mg/kg) was injected via tail vein to each mouse. The distribution course of Evans blue was recorded for about 15 min. The structure of blood vessel showed by Evans blue was analyzed in the recorded video. Tumor hypoxia detection To further study the modulation of tumor hypoxia by bevacizumab, the hypoxyprobeTM-1 kit (Natural Pharmacia International, Inc, Burlington, MA, USA) constituted of 100 mg solid pimonidazole HCl (hypoxyprobeTM-1) and 1.0 ml mouse IgG1 monoclonal antibody (Mab1), was used to detect hypoxia area in tumor tissue. Pimonidazole distributes to all tissues, but it forms adducts with thiol-containing proteins only in those cells with oxygen concentration less than 14 micromolar-equivalent to a partial pressure pO2 = 10 mmHg at 37 °C. Mice were given pimonidazole (60 mg/kg) intraperitoneally 4 h before being killed. Dissected tumor tissues were fixed in 10 % formalin, embedded in paraffin and examined by immunohistochemistry with a mouse monoclonal antibody. Images of each tumor section were digitally acquired on Olympus BX-60 light microscope at 40 9 magnification. Positive staining was analyzed using NIH image analysis software, Image J, (National Institutes of Health, free link to: http://rsb.info.nih. gov/ij/) and calculating hypoxia area of the non-necrotic tumor tissue. Immunohistochemistry Formalin-fixed, paraffin-embedded tumor samples were used for immunohistochemical study. CD3 was a specific marker to enumerating T lymphocytes. Rabbit anti-human CD3 antibodies (Abcam, Cambridge, MA, USA) were used to detect human CIK cells. Continuous section slides were made and stained for hypoxia and tumor-infiltrated CIK cells, respectively. Images of each section were viewed and digitally acquired on Olympus BX-60 light microscope. Images were saved for further determination of CD3? T lymphocytes cell density and the relationship between hypoxia area and the infiltration of CD3? T cells.
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p = 0.027 \ 0.05). The combination therapy had significant antitumor activity compared with other groups. EA = 0.44, EB = 0.16, EA ? B = 0.66 and q = 1.25, which confirms the assessment of synergism effect (Fig. 1). Similar results were observed in the repeated experiments. Bevacizumab promotes tumor vessel normalization
Fig. 1 Antitumor effect of bevacizumab and adoptive CIK cells therapy on tumor growth. Female BALB/c nude mice were injected s.c. with A549 lung cancer cells and when tumor volume reached *250 mm3, mice were divided into four groups (5–6 mice in each group) randomly and received respective protocol according to the treatment schema. CIK cells therapy alone did not significantly suppress tumor growth compared with NS group (p = 0.185 [ 0.05). Bevacizumab could inhibit tumor growth compared with CIK therapy (p = 0.027 \ 0.05). Significant differences of tumor suppression effect were achieved in combination therapy compared with group of bevacizumab alone. The result showed that bevacizumab combined with CIK cells therapy exerts synergistic antitumor effect in the experiment (EA = 0.44, EB = 0.16, EA ? B = 0.66 and q = 1.25 [ 1.15, which confirms the assessment of synergism effect)
Statistical analysis Data were expressed as mean ± standard error (SE). Statistical significance was determined by one-way ANOVA, LSD and unpaired t tests. Differences were considered significant when p was \0.05. Statistical analysis was conducted using SPSS software v16.0 (SPSS Inc., Chicago, IL, USA).
Results Combination of bevacizumab and CIK cells therapy significantly inhibits the growth of lung cancer in mice models To assess the inhibitory effect of the combination therapy, bevacizumab was injected via tail vein into A549 carcinomabearing BABL/C nude mice, and 3 days later, CIK cells were transfused into mice. Tumor volumes were recorded every other day throughout the experiment. The results showed that CIK cells monotherapy had no significant antitumor activity compared with NS group (968.52 ± 93.42 vs. 1,158.51 ± 81.18, p = 0.185 [ 0.05). Bevacizumab limited tumor growth compared with NS controls and reached statistical significance (647.17 ± 37.35 vs. 1,158.51 ± 81.18
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A549 tumor-bearing mice were treated with bevacizumab (5 mg/kg, i.v.). Twenty four hours later, intravital microscopy was performed to clarify tumor vessel normalization. Vascular permeability was observed indirectly by the diffusion of Evans blue-Albumin from tumor vessel into tumor parenchyma after injection of Evans blue. At the same time, the vasculature wall was visualized by Evans blue and turned red. Tumor vessels were less tortuous and less dilated in the bevacizumab group compared with NS group (Fig. 2a, b, up right). Bevacizumab augments the homing of CIK cells in the tumor To test whether the combination therapy augments CIK cells infiltration into tumor tissue, the accumulation of CD3? T lymphocyte was observed in tumor tissue by immunohistochemistry. Tumors from all experiment groups were removed, and CD3 was used as a specific marker to enumerate T lymphocytes. Only a few CIK cells infiltrated in the tumor of NS group (Fig. 3A–a). CIK cells therapy alone slightly increased the level of infiltrating CD3? T lymphocytes compared with the NS group (Fig. 3A-b). However, tumor-infiltrating CD3? T lymphocytes were significantly increased in the combination group (Fig. 3A–c). Similar results were also verified by flow cytometry. A549 lung cancer-bearing mice were infused with CFSE-labeled CIK cells 3 days after prior bevacizumab treatment. The percentage of CFSE-labeled CIK cells in tumor tissues 24 h after transfusion was analyzed by Flow cytometry. The combination therapy group had more CIK cells infiltrated than the group treated with CIK cells alone (12.9 ± 0.7 % vs. 8.33 ± 0.2 %, p \ 0.05) (Fig. 3b, c). Bevacizumab decreases hypoxic area in tumor microenvironment and hypoxia inhibits the homing of CIK cells Hypoxic areas of tumor tissue were distributed mainly on the edge of necrosis tissues and stained by hypoxic probe. Bevacizumab could normalize the structure of tumor vessel (Fig. 2a, b, up right). Bevacizumab could decrease hypoxia area, as determined by pimonidazole in A549 lung carcinoma compared with the group NS (9.23 ± 5.5 % vs.
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Fig. 2 Bevacizumab promotes vessel normalization of A549 lung carcinoma and decreases tumor hypoxic area. A549 tumor-bearing mice were treated with bevacizumab (5 mg/kg), normal NS group as control. Intravital microscopy was performed to clarify tumor vessel normalization. Meanwhile, mice were killed and tumor samples were stained by hypoxyprobe in A549 lung carcinoma. a Hypoxic area of NS group, and the tumor vascular structure captured by intravital
microscopy in NS group was showed in upper right corner; b Hypoxic area of bevacizumab group and the tumor vascular structure captured by intravital microscopy after treat with bevacizumab was showed in the upper right corner; c columns represented tumor hypoxic area in two groups. Representative sections are shown from all groups with a magnification of 9400. The green bar represents 50 lm, while the black bar means 200 lm. *p \ 0.05, indicating significant difference
Fig. 3 Accumulation of CIK cells in the tumor after CIK cells therapy and combinatorial therapy. a A549 tumor-bearing mice were treated with normal saline, CIK cells and bevacizumab ? CIK cells, respectively. Tumor tissue from 3 groups were prepared and analyzed by anti-human CD3 antibody staining. Individual fields at 9400 magnification were chosen to detect intratumoral CD3? T lymphocytes. A-a Group NS, A-b Group CIK cells alone. A-c Group BEV ? CIK. b A549 tumor-bearing mice were treated with CIK cells and bevacizumab (5 mg/kg) ? CIK cells, respectively. CFSE-labeled 1 9 107 CIK cells were transfused i.v. into mice of each group. 24 h
after CIK cells transfusion, mice were killed and single cell suspensions of tumor tissue were prepared and then analyzed by flow cytometry. Flow cytometry analysis data showing the percentages of CFSE-labeled CIK cells infiltrating into the tumor in the CIK cells group and the combination group. All experiments were repeated at least three times. c The columns represent mean ± SE of CFSElabeled CIK cells infiltrating percentages into the tumor of the hosting mice in two groups. The black bar means 200 lm. *p \ 0.05 indicating statistical significance. The experiment were repeated twice and with similar results
30.7 ± 3.7 % p \ 0.05). In the experiment, we further analyzed special relationship between tumor hypoxic area and the density of tumor-infiltrating CIK cells. Two
continuous section slides from each tumor sample were prepared and stained for hypoxia and anti-CD3 antibodies, respectively. The amount of the infiltrating CD3? CIK
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Fig. 4 Hypoxia inhibits the infiltration of CIK cells. A549 tumorbearing mice were transfused i.v. with CIK cells. Twenty four hours after CIK cells transfusion, mice were given pimonidazole, and mice were killed 4 h later. Continuous sections slides were made and tumor hypoxia and tumor-infiltrating CIK cells were analyzed, respectively. Tumor hypoxic areas were stained by monoclonal antibody (Mab1) against protein adducts of pimonidazole. Tumor-infiltrating CIK cells were stained by anti-human CD3 antibody. The figures showed that less CD3? CIK cells infiltrated in hypoxic area compared with
normoxic area, indicating impaired CIK cells recruitment to tumor tissue by hypoxia. a Representative figure of tumor hypoxic area at 9400 magnification. b Representative figure of tumor-infiltrating CD3 ? CIK cells at 9400 magnification. The black bar in the figure means 200 lm. The black arrows refer to the CD3? CIK cells. c The columns represent mean ± SE of CIK cells infiltrating number in tumor hypoxic and normoxic area under microscopy (9400). *p \ 0.05 indicating statistical significance
cells in normoxic tumor tissue was more than it in tumor hypoxic area (Fig. 4). The study showed that hypoxia impeded the recruitment of CIK cells into tumor.
responses observed in these clinical or preclinical trials are not as well as expected. Different explanations can be depicted to justify the decreased antitumor effect of the immunotherapy. All of them take into account two major obstacles: On one hand, reduced homing of immune cells to the tumor site, and on the other, hampering of antitumor immune functions due to tumor microenvironment [4]. Accumulating evidences have been investigated that improvement of tumor microenvironment could increase the efficacy of adoptive immune therapy [15]. Different strategies can be envisaged to facilitate recruitment of effector T cells into tumors. Our previous work showed TP regimen, and recombinant human endostatin (rh-endostatin) could markedly enhance the antitumor response of adoptively transferred CIK cells in lung cancer, partially by reducing the tumor hypoxia [11, 16]. Bevacizumab is a humanized monoclonal anti-VEGF antibody that targets all isoforms of human VEGF. It is the first antiangiogenic antibody approved by Food and Drug Administration for cancer therapy, based on its efficacy in combination with standard chemotherapy in metastatic
Discussion Human CIK cells are a heterogeneous cells isolated from peripheral blood mononuclear cells after in vitro expanded by interferon-c, anti-CD3 monoclonal antibodies and interleukin-2. They present functional and phenotypic properties of T-NK cells and are also endowed with a MHC-unrestricted antitumor ability. Because of these unique characteristics of CIK cells, adoptive CIK cells transfer therapy holds great potential as a promising approach treated with malignant tumor refractory to traditional therapies. The clinical or preclinical trials of adoptive CIK cells therapy against several malignant disease including leukemia [12], renal carcinoma [13], colorectal cancer [14] and lung cancer [3] have been described in many literatures. However, the therapeutic antitumor
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colorectal cancer [17]. VEGF is an angiogenic factors overexpressed in many solid tumors. Dysfunctional tumor vessels can be a significant barrier to effective cancer therapy. The anti-VEGF treatment reduces tumor size as well as permeability of dilated and tortuous tumor vascular [18]. Targeting VEGF can effect transient ‘‘normalization’’ of the tumor vasculature in a wide range of solid tumors models with reduced tumor vessel permeability, lower tumor interstitial pressures, and, in turn, alleviated tumor hypoxia. For this reason, we hypothesized that bevacizumab could prompt CIK cells infiltration into tumor and enhance the effectiveness of adoptive CIK cells by normalizing tumor vessels and reducing hypoxic area in tumor tissue. Of all results in our study, bevacizumab in combination with CIK cells therapy showed significant antitumor ability, and seems more effective than other groups. Almost little or no inhibitory effect when single CIK cells were transferred into the mice. Considering all possible causes about this phenomenon, the number of transfused CIK cells and immunosuppressive tumor microenvironment are two important factors. In the experiment, each mouse was given 1 9 107 CIK cells that may be not a sufficient amount to exert an effective immune response in killing tumor cells. The CIK cells migrated into tumor are not fully functional under the immunosuppressive/hypoxic tumor microenvironment which caused by abnormal tumor vessels. To further approve the combination group has more CIK cells infiltrating into tumor parenchyma, immunohistochemistry and flow cytometry were used to detect hypoxic area of tumor tissue and the amount of infiltrating CIK cells, respectively. The result of flow cytometry indicated more CIK cells infiltration into tumors in combination group compared with single CIK cells transfer group. Tumor hypoxia area significantly reduced after treat with bevacizumab have been verified by immunohistochemistry. The negative effect of abnormal vascular in cancer treatment has been previously described. Aberrant vessels characterized these features including widened interendothelial junctions, increased numbers of fenestrations, vesicles and vesico-vacuolar channels, and a lack of normal basement membrane and perivascular cells. Because of the above-mentioned ultrastructural alterations in the tumor vessel wall, vascular permeability of solid tumors is generally higher than in normal tissues. The heterogeneity of tumor blood flow cause abnormal microenvironment in tumors and hinders the delivery and efficacy of CIK cells therapy [19]. Intravital microscopy was used to detect the morphology and structure of tumor vasculature. Evans blue was used to observe the permeability of tumor vascular. Evans blue could strongly bind to albumin in the blood. Its behavior reflects the transport of albumin, which is about 67 kDa with a diameter of about 7 nm. This size is similar
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to effective pore size of 6–7 nm occurring in the most normal blood vessel [20]. Aberrant tumor vessels were characterized with larger inter-endothelial junctions than normal blood vessels; there might be a little hindrance for the transvascular transport of Evans blue-albumin complex. The morphology of tumor vessels is visually more normal and less dilated than the control group. The phenomenon showed bevacizumab could normalize tumor vasculature. The normalized tumor vasculatureinduced tumor oxygenation supply increased. As a result, hypoxia area of tumor tissue could be reduced by bevacizumab. Tumor hypoxia is associated with treatment resistance, cell proliferation, and metastatic potential and a poor overall prognosis [6]. To study the relationship between tumor hypoxia and infiltration of CIK cells, we used pimonidazole and anti-human CD3 antibody staining the tumor tissue with immunohistochemistry. Pimonidazole is reductively activated in an oxygen-dependent manner and is covalently bound to thiol-containing proteins in hypoxic cells. Pimonidazole can be used for qualitative and quantitative assessment of hypoxia [21]. The density of CIK cells in the tumor was judged by the immunohistochemistry of anti-human CD3 antibodies. After analyzing the result of our experiment, we found that bevacizumab could significantly reduce hypoxic area of the tumor tissue. Less CD3? CIK cells infiltrated in hypoxic area compared with normoxic area, indicating that hypoxia significantly impaired CIK cells recruitment to tumor tissue. It also deduced that improvement in tumor hypoxia was benefic for the infiltration of CIK cells. In addition, the efficiency of tumor inhibition may be further enhanced. Thus, we concluded tumor hypoxia microenvironment may be the possible mechanism limiting cancer immunotherapy. Bevacizumab that induce vascular normalization can alleviate hypoxia and increase the efficacy of immunotherapy therapy in A549 lung carcinoma. The antiangiogenic agents could enhance more CIK cells accumulated in tumor tissue and make it play a greater role in the treatment. Although similar conclusions had been verified in other models, our findings still open new perspectives for combining antiangiogenesis and immunotherapy in cancer patients with lung cancer.
Conclusion Our study clearly showed hypoxia hinder the infiltration of CIK cells into tumor tissue. Bevacizumab could normalize tumor vascular and reduce tumor hypoxic area, and then increase intratumoral infiltration of CIK cells, thereby enhancing efficacy of antitumor ability of adoptive CIK cells transfer therapy. Thus, bevacizumab in combination with adoptive CIK cells therapy can be a rational regimen
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applied to clinical treatment, and it would be beneficial to patients with advanced stage NSCLC. Acknowledgments The work was supported by National Natural Science Foundation of China (No. 81101739) and China International Medical Foundation, CIMF-F-H001-02. Conflict of interests peting interests.
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The authors declare that they have no com-
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ORIGINAL PAPER
Bevacizumab improves the antitumor efficacy of adoptive cytokine-induced killer cells therapy in non-small cell lung cancer models Leilei Tao • Guichun Huang • Shujing Shi Longbang Chen
•
Received: 13 August 2013 / Accepted: 16 November 2013 / Published online: 24 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Cytokine-induced killer cells (CIK cells) are a heterogeneous population of cells generated from peripheral blood mononuclear cells, which share phenotypic and functional properties with both natural killer and T cells. CIK cells therapy, as an adoptive immunotherapy with strong antitumor activity in vitro, represents a promising approach for the treatment of a broad array of malignant tumors. However, clinical trials in CIK cells therapy did not show more noticeable improvement as anticipated in cure rates or long-term survival. Possible explanations are that abnormal tumor vasculature and hypoxic microenvironment may highly limit the therapeutic benefits of CIK cells therapy. We hypothesized that antiangiogenesis therapy could enhance the antitumor efficacy of CIK cells by normalizing tumor vasculature and modulating tumor hypoxic microenvironment. In this study, we combined bevacizumab and adoptive CIK cells therapy in the treatment of lung adenocarcinoma bearing murine models. Flow cytometry,
intravital microscopy and immunohistochemistry were applied to detect tumor vasculature and hypoxic microenvironment as well as the infiltration of CIK cells. The results indicated that bevacizumab-combined adoptive CIK cells had synergistic inhibition effects on the growth of lung adenocarcinoma. Hypoxia significantly inhibited the infiltration of CIK cells into tumor tissue. Bevacizumab could normalize tumor vasculature and decrease tumor hypoxic area. Furthermore, combination therapy enhanced more CIK cells infiltrated into tumor compared with other treatment. Bevacizumab improves antitumor efficacy of CIK cells transfer therapy in non-small cell lung cancer (NSCLC). The study provides a reasonable and beneficial strategy that combined antiangiogenesis therapy with CIK cells therapy for patients of advanced stage non-small cell lung cancer.
Leilei Tao and Guichun Huang have contributed equally to this work.
Abbreviations NSCLC Non-small cell lung cancer CIK cell Cytokine-induced killer cell PBMC Peripheral blood mononuclear cells VEGF Vascular endothelial growth factor
Electronic supplementary material The online version of this article (doi:10.1007/s12032-013-0777-3) contains supplementary material, which is available to authorized users. L. Tao G. Huang S. Shi L. Chen (&) Medical Oncology Department of Jinling Hospital, Medical School of Nanjing University, 305 ZhongShan Eastern Road, Nanjing 210002, People’s Republic of China e-mail: [email protected] L. Tao e-mail: [email protected] G. Huang e-mail: [email protected] S. Shi e-mail: [email protected]
Keywords Antiangiogenesis Bevacizumab Vascular normalization Hypoxia microenvironment Adoptive CIK cells therapy
Background Lung cancer is one of the most common malignancies and leading cause of cancer-related death around the world [1]. Approximately 80 % of all lung cancer cases are non-small cell lung cancer (NSCLC). Despite development of cancer treatment and introduction of new drugs, advanced lung
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cancer remains associated with poor prognosis. Recently, adoptive immunotherapy as a new approach to treat solid tumors holds great potential compared with other traditional treatments [2]. Adoptive cytokine-induced killer cells (CIKs) transfer, as one type of adoptive immunotherapy, showed promising antitumor effect on various malignant tumors, including NSCLC [3]. Cytokine-induced killer (CIK) cells are heterogeneous in vitro-expanded T lymphocytes which along with T-NK phenotype and MHC-unrestricted antitumor ability. These biological features of CIK cells make them appealing for adoptive immunotherapy and have displayed encouraging results in tumor therapy [2]. However, the effectiveness of single cancer immunotherapy in solid tumor remains quite low in clinical trials. Thus, it is necessary to find an effective antitumor strategy aimed at enhancing more CIK cells infiltrated into tumors and increasing their intratumoral activity for patients [4]. Evidences have been provided that abnormal tumor blood vessels and tumor microenvironment play a crucial role in impeding lymphocyte cell infiltration into tumor tissue and inhibiting the activity of these immune cells [5]. Hypoxia is a common feature in a wild range of solid tumors, including non-small cell lung cancer. Hypoxia, as a crucial role, induces both angiogenesis and immunosuppression in the complex and highly dynamic tumor microenvironment. Hypoxia-induced abnormal neo-angiogenesis causes increased resistance to adoptive immune cell therapy in tumor [6]. Recent studies indicated antiangiogenesis agents could transiently normalize tumor vasculature, and its hypoxia microenvironment thus could significantly influence the response of tumor to traditional chemotherapy [7]. Bevacizumab (AvastinÒ; Genentech, Inc. South San Francisco, CA, USA) is a humanized anti-VEGF monoclonal IgG1 antibody (molecular weight, 149 kDa). Accumulated studies showed bevacizumab that targeted VEGF could result in a dramatic suppression of tumor growth, normalization of tumor blood vessel, and reduced tumor hypoxia area. Clinical trials with bevacizumab in a variety of malignancies indicated that conventional chemotherapy or irradiation in combination with bevacizumab held great potential advantages in cure rates or long-term survival of cancer patients [8, 9]. We hypothesized that bevacizumab could also enhance efficacy of CIK cell immunotherapy by normalizing tumor vessels and modulating hypoxic microenvironment of NSCLC. Our study aimed to determine whether bevacizumab could improve the antitumor effect of adoptive CIK cells and to illustrate possible mechanism that antiangiogenesis agents release the full potential of adoptive CIK cells therapy. The results of our research revealed novel mechanisms of bevacizumab promoting efficacy of adoptive CIK cells transfer
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therapy in NSCLC. This strategy provided a rational basis for bevacizumab in combination with immunotherapy and could be easily translated into clinical setting in curing NSCLC patients.
Materials and methods Tumor cell line Human lung adenocarcinoma cell line (A549 cells) is preserved in our laboratory and cultured in RPMI-1640 (Gibaco, USA) supplemented with 10 % fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin. Cells were cultured in a humidified atmosphere of 5 % CO2 at 37 °C. Generation of CIK cells Human peripheral blood samples were obtained from healthy donors with permission consent. The study was approved by the Ethic Committee of Jinling Hospital. Peripheral blood mononuclear cells (PBMC) were separated from human peripheral blood through density gradient centrifugation using Ficoll-Hypaque (Beijing Chemical Reagents Company, China). Cells were then resuspended in RPMI-1640 medium (10 % FCS, 100 U/ml penicillin and 100 lg/ml streptomycin), with the 1,000 U/ml interferon-c (Peprotech, USA) added on the first day. After 24 h, 500 U/ml Interleukin-2 (Peprotech, USA) and 50 ng/ml anti-CD3 antibody (eBioscience, USA) were added. Cells were incubated in a humidified atmosphere of 5 % CO2 at 37 °C. Thereafter, RPMI-1640 supplemented with interleukin-2 (300 U/ml) was applied every other day for 2 weeks and maintained at a final density of 2–3 9 106 cells/ml. Animal models Female BALB/c nude mice were purchased from Academy of Military Medical Science (Beijing, China) and maintained under controlled temperature and humidity, and a 12-h light–dark cycle, with sterile food and water ad libitum. All mice used in our study were 6–8 week old. Animal experiments were performed according to the guidelines of the Institute Animal Care and Use Committee of Jingling Hospital. Human lung adenocarcinoma xenograft models were established by subcutaneous inoculation of 100 ll A549 cells (1 9 107/ml) into the right flank of BALB/c nude mice. Subcutaneous tumor volumes were measured daily by caliper, and tumor volumes were calculated by the formula: tumor volume = 0.5 9 length 9 width2. The animal experiments were repeated twice.
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Treatment protocol Treatment was started when tumor volumes grew to approximately 250 mm3. The mice were randomly divided into 4 groups with 5–6 mice in each group. The detailed groups were as follows: (1) Group NS, treated with normal saline, as control group; (2) Group CIK, treated with CIK cells alone; (3) Group BEV, treated with bevacizumab alone; (4) Group BEV ? CIK, treated with bevacizumab followed by transfusion of CIK cells. The first day when treatment started was designated d1. The dose of bevacizumab was intravenous injection of 5 mg/kg at d1, intravenous transfusion of CIK cells at d3 (1 9 107cells per dose in a total volume of 100 ll). The inhibition rate of the tumor volume (TIR) was determined according to the formula: TIR = 1-(mean tumor volume in the treated group/mean tumor volume in control group). The formula (q = EA ? B/[EA ? (1 - EA) EB]) [10]was used to assess the synergism of two therapies. EA, the inhibition rate of bevacizumab therapy, which is defined as EA EB, the inhibition rate of adoptive CIK cells immunotherapy EA ? B, the inhibition rate of combination therapy q \ 0.85 means the two therapies are antagonism. q [ 1.15 means the two therapies are synergism. 0.85 B q B 1.15 means the two therapies are additive. Flow cytometry Human CIK cells were transfused into mice 3 days after treated with bevacizumab. CIK cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes Biotec. Final concentration of 5 lmol/l in PBS) and cultured at 37 °C for 5 min. Cold RPMI-1640 with 10 % FCS was added to quench the labeling reaction, and cells were washed twice with PBS to remove excess CFSE. Then CIK cells were transferred into mice. 24 h later, tumor samples were harvested, and tumor single cell suspensions were prepared from group CIK and group BEV ? CIK. Red Blood Cells were removed in Erythrocytes Lysis Buffer [155 mmol/l NH4Cl, 10 mmol/l KHCO3, and 0.1 mmol/l EDTA (pH 7.4)] at room temperature for 10 min. The amount of CFSE-labeled CIK cells in each sample was counted by FACS-Calibur and CellQuest software (BD Biosciences, San Jose, CA, USA). Intravital microscopy Intravital microscopy examination was in accordance with our previous reports [11]. In brief, mice bearing A549 tumors were anesthetized with pentobarbital sodium (50 mg/kg body weight) by intraperitoneal administration
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and then fixed on the board. Body temperature was maintained about 37 °C throughout the experiment. The skin around tumor was removed to make an observation window. The exposed tumor tissue was kept moist with normal saline during the entire surgical procedure. Subsequently, a thin sterile contact lens was covered on the upper surface of tumor to provide visual area of the tumor vascular in the experiment. The upper surface enables a large number of venules and capillaries to be monitored under intravital microscope. The dye Evans blue (Sigma-Aldrich, USA) which bounded tightly to albumin (EBA) was used to monitor vascular permeability. Evans blue (20 mg/kg) was injected via tail vein to each mouse. The distribution course of Evans blue was recorded for about 15 min. The structure of blood vessel showed by Evans blue was analyzed in the recorded video. Tumor hypoxia detection To further study the modulation of tumor hypoxia by bevacizumab, the hypoxyprobeTM-1 kit (Natural Pharmacia International, Inc, Burlington, MA, USA) constituted of 100 mg solid pimonidazole HCl (hypoxyprobeTM-1) and 1.0 ml mouse IgG1 monoclonal antibody (Mab1), was used to detect hypoxia area in tumor tissue. Pimonidazole distributes to all tissues, but it forms adducts with thiol-containing proteins only in those cells with oxygen concentration less than 14 micromolar-equivalent to a partial pressure pO2 = 10 mmHg at 37 °C. Mice were given pimonidazole (60 mg/kg) intraperitoneally 4 h before being killed. Dissected tumor tissues were fixed in 10 % formalin, embedded in paraffin and examined by immunohistochemistry with a mouse monoclonal antibody. Images of each tumor section were digitally acquired on Olympus BX-60 light microscope at 40 9 magnification. Positive staining was analyzed using NIH image analysis software, Image J, (National Institutes of Health, free link to: http://rsb.info.nih. gov/ij/) and calculating hypoxia area of the non-necrotic tumor tissue. Immunohistochemistry Formalin-fixed, paraffin-embedded tumor samples were used for immunohistochemical study. CD3 was a specific marker to enumerating T lymphocytes. Rabbit anti-human CD3 antibodies (Abcam, Cambridge, MA, USA) were used to detect human CIK cells. Continuous section slides were made and stained for hypoxia and tumor-infiltrated CIK cells, respectively. Images of each section were viewed and digitally acquired on Olympus BX-60 light microscope. Images were saved for further determination of CD3? T lymphocytes cell density and the relationship between hypoxia area and the infiltration of CD3? T cells.
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p = 0.027 \ 0.05). The combination therapy had significant antitumor activity compared with other groups. EA = 0.44, EB = 0.16, EA ? B = 0.66 and q = 1.25, which confirms the assessment of synergism effect (Fig. 1). Similar results were observed in the repeated experiments. Bevacizumab promotes tumor vessel normalization
Fig. 1 Antitumor effect of bevacizumab and adoptive CIK cells therapy on tumor growth. Female BALB/c nude mice were injected s.c. with A549 lung cancer cells and when tumor volume reached *250 mm3, mice were divided into four groups (5–6 mice in each group) randomly and received respective protocol according to the treatment schema. CIK cells therapy alone did not significantly suppress tumor growth compared with NS group (p = 0.185 [ 0.05). Bevacizumab could inhibit tumor growth compared with CIK therapy (p = 0.027 \ 0.05). Significant differences of tumor suppression effect were achieved in combination therapy compared with group of bevacizumab alone. The result showed that bevacizumab combined with CIK cells therapy exerts synergistic antitumor effect in the experiment (EA = 0.44, EB = 0.16, EA ? B = 0.66 and q = 1.25 [ 1.15, which confirms the assessment of synergism effect)
Statistical analysis Data were expressed as mean ± standard error (SE). Statistical significance was determined by one-way ANOVA, LSD and unpaired t tests. Differences were considered significant when p was \0.05. Statistical analysis was conducted using SPSS software v16.0 (SPSS Inc., Chicago, IL, USA).
Results Combination of bevacizumab and CIK cells therapy significantly inhibits the growth of lung cancer in mice models To assess the inhibitory effect of the combination therapy, bevacizumab was injected via tail vein into A549 carcinomabearing BABL/C nude mice, and 3 days later, CIK cells were transfused into mice. Tumor volumes were recorded every other day throughout the experiment. The results showed that CIK cells monotherapy had no significant antitumor activity compared with NS group (968.52 ± 93.42 vs. 1,158.51 ± 81.18, p = 0.185 [ 0.05). Bevacizumab limited tumor growth compared with NS controls and reached statistical significance (647.17 ± 37.35 vs. 1,158.51 ± 81.18
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A549 tumor-bearing mice were treated with bevacizumab (5 mg/kg, i.v.). Twenty four hours later, intravital microscopy was performed to clarify tumor vessel normalization. Vascular permeability was observed indirectly by the diffusion of Evans blue-Albumin from tumor vessel into tumor parenchyma after injection of Evans blue. At the same time, the vasculature wall was visualized by Evans blue and turned red. Tumor vessels were less tortuous and less dilated in the bevacizumab group compared with NS group (Fig. 2a, b, up right). Bevacizumab augments the homing of CIK cells in the tumor To test whether the combination therapy augments CIK cells infiltration into tumor tissue, the accumulation of CD3? T lymphocyte was observed in tumor tissue by immunohistochemistry. Tumors from all experiment groups were removed, and CD3 was used as a specific marker to enumerate T lymphocytes. Only a few CIK cells infiltrated in the tumor of NS group (Fig. 3A–a). CIK cells therapy alone slightly increased the level of infiltrating CD3? T lymphocytes compared with the NS group (Fig. 3A-b). However, tumor-infiltrating CD3? T lymphocytes were significantly increased in the combination group (Fig. 3A–c). Similar results were also verified by flow cytometry. A549 lung cancer-bearing mice were infused with CFSE-labeled CIK cells 3 days after prior bevacizumab treatment. The percentage of CFSE-labeled CIK cells in tumor tissues 24 h after transfusion was analyzed by Flow cytometry. The combination therapy group had more CIK cells infiltrated than the group treated with CIK cells alone (12.9 ± 0.7 % vs. 8.33 ± 0.2 %, p \ 0.05) (Fig. 3b, c). Bevacizumab decreases hypoxic area in tumor microenvironment and hypoxia inhibits the homing of CIK cells Hypoxic areas of tumor tissue were distributed mainly on the edge of necrosis tissues and stained by hypoxic probe. Bevacizumab could normalize the structure of tumor vessel (Fig. 2a, b, up right). Bevacizumab could decrease hypoxia area, as determined by pimonidazole in A549 lung carcinoma compared with the group NS (9.23 ± 5.5 % vs.
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Fig. 2 Bevacizumab promotes vessel normalization of A549 lung carcinoma and decreases tumor hypoxic area. A549 tumor-bearing mice were treated with bevacizumab (5 mg/kg), normal NS group as control. Intravital microscopy was performed to clarify tumor vessel normalization. Meanwhile, mice were killed and tumor samples were stained by hypoxyprobe in A549 lung carcinoma. a Hypoxic area of NS group, and the tumor vascular structure captured by intravital
microscopy in NS group was showed in upper right corner; b Hypoxic area of bevacizumab group and the tumor vascular structure captured by intravital microscopy after treat with bevacizumab was showed in the upper right corner; c columns represented tumor hypoxic area in two groups. Representative sections are shown from all groups with a magnification of 9400. The green bar represents 50 lm, while the black bar means 200 lm. *p \ 0.05, indicating significant difference
Fig. 3 Accumulation of CIK cells in the tumor after CIK cells therapy and combinatorial therapy. a A549 tumor-bearing mice were treated with normal saline, CIK cells and bevacizumab ? CIK cells, respectively. Tumor tissue from 3 groups were prepared and analyzed by anti-human CD3 antibody staining. Individual fields at 9400 magnification were chosen to detect intratumoral CD3? T lymphocytes. A-a Group NS, A-b Group CIK cells alone. A-c Group BEV ? CIK. b A549 tumor-bearing mice were treated with CIK cells and bevacizumab (5 mg/kg) ? CIK cells, respectively. CFSE-labeled 1 9 107 CIK cells were transfused i.v. into mice of each group. 24 h
after CIK cells transfusion, mice were killed and single cell suspensions of tumor tissue were prepared and then analyzed by flow cytometry. Flow cytometry analysis data showing the percentages of CFSE-labeled CIK cells infiltrating into the tumor in the CIK cells group and the combination group. All experiments were repeated at least three times. c The columns represent mean ± SE of CFSElabeled CIK cells infiltrating percentages into the tumor of the hosting mice in two groups. The black bar means 200 lm. *p \ 0.05 indicating statistical significance. The experiment were repeated twice and with similar results
30.7 ± 3.7 % p \ 0.05). In the experiment, we further analyzed special relationship between tumor hypoxic area and the density of tumor-infiltrating CIK cells. Two
continuous section slides from each tumor sample were prepared and stained for hypoxia and anti-CD3 antibodies, respectively. The amount of the infiltrating CD3? CIK
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Fig. 4 Hypoxia inhibits the infiltration of CIK cells. A549 tumorbearing mice were transfused i.v. with CIK cells. Twenty four hours after CIK cells transfusion, mice were given pimonidazole, and mice were killed 4 h later. Continuous sections slides were made and tumor hypoxia and tumor-infiltrating CIK cells were analyzed, respectively. Tumor hypoxic areas were stained by monoclonal antibody (Mab1) against protein adducts of pimonidazole. Tumor-infiltrating CIK cells were stained by anti-human CD3 antibody. The figures showed that less CD3? CIK cells infiltrated in hypoxic area compared with
normoxic area, indicating impaired CIK cells recruitment to tumor tissue by hypoxia. a Representative figure of tumor hypoxic area at 9400 magnification. b Representative figure of tumor-infiltrating CD3 ? CIK cells at 9400 magnification. The black bar in the figure means 200 lm. The black arrows refer to the CD3? CIK cells. c The columns represent mean ± SE of CIK cells infiltrating number in tumor hypoxic and normoxic area under microscopy (9400). *p \ 0.05 indicating statistical significance
cells in normoxic tumor tissue was more than it in tumor hypoxic area (Fig. 4). The study showed that hypoxia impeded the recruitment of CIK cells into tumor.
responses observed in these clinical or preclinical trials are not as well as expected. Different explanations can be depicted to justify the decreased antitumor effect of the immunotherapy. All of them take into account two major obstacles: On one hand, reduced homing of immune cells to the tumor site, and on the other, hampering of antitumor immune functions due to tumor microenvironment [4]. Accumulating evidences have been investigated that improvement of tumor microenvironment could increase the efficacy of adoptive immune therapy [15]. Different strategies can be envisaged to facilitate recruitment of effector T cells into tumors. Our previous work showed TP regimen, and recombinant human endostatin (rh-endostatin) could markedly enhance the antitumor response of adoptively transferred CIK cells in lung cancer, partially by reducing the tumor hypoxia [11, 16]. Bevacizumab is a humanized monoclonal anti-VEGF antibody that targets all isoforms of human VEGF. It is the first antiangiogenic antibody approved by Food and Drug Administration for cancer therapy, based on its efficacy in combination with standard chemotherapy in metastatic
Discussion Human CIK cells are a heterogeneous cells isolated from peripheral blood mononuclear cells after in vitro expanded by interferon-c, anti-CD3 monoclonal antibodies and interleukin-2. They present functional and phenotypic properties of T-NK cells and are also endowed with a MHC-unrestricted antitumor ability. Because of these unique characteristics of CIK cells, adoptive CIK cells transfer therapy holds great potential as a promising approach treated with malignant tumor refractory to traditional therapies. The clinical or preclinical trials of adoptive CIK cells therapy against several malignant disease including leukemia [12], renal carcinoma [13], colorectal cancer [14] and lung cancer [3] have been described in many literatures. However, the therapeutic antitumor
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colorectal cancer [17]. VEGF is an angiogenic factors overexpressed in many solid tumors. Dysfunctional tumor vessels can be a significant barrier to effective cancer therapy. The anti-VEGF treatment reduces tumor size as well as permeability of dilated and tortuous tumor vascular [18]. Targeting VEGF can effect transient ‘‘normalization’’ of the tumor vasculature in a wide range of solid tumors models with reduced tumor vessel permeability, lower tumor interstitial pressures, and, in turn, alleviated tumor hypoxia. For this reason, we hypothesized that bevacizumab could prompt CIK cells infiltration into tumor and enhance the effectiveness of adoptive CIK cells by normalizing tumor vessels and reducing hypoxic area in tumor tissue. Of all results in our study, bevacizumab in combination with CIK cells therapy showed significant antitumor ability, and seems more effective than other groups. Almost little or no inhibitory effect when single CIK cells were transferred into the mice. Considering all possible causes about this phenomenon, the number of transfused CIK cells and immunosuppressive tumor microenvironment are two important factors. In the experiment, each mouse was given 1 9 107 CIK cells that may be not a sufficient amount to exert an effective immune response in killing tumor cells. The CIK cells migrated into tumor are not fully functional under the immunosuppressive/hypoxic tumor microenvironment which caused by abnormal tumor vessels. To further approve the combination group has more CIK cells infiltrating into tumor parenchyma, immunohistochemistry and flow cytometry were used to detect hypoxic area of tumor tissue and the amount of infiltrating CIK cells, respectively. The result of flow cytometry indicated more CIK cells infiltration into tumors in combination group compared with single CIK cells transfer group. Tumor hypoxia area significantly reduced after treat with bevacizumab have been verified by immunohistochemistry. The negative effect of abnormal vascular in cancer treatment has been previously described. Aberrant vessels characterized these features including widened interendothelial junctions, increased numbers of fenestrations, vesicles and vesico-vacuolar channels, and a lack of normal basement membrane and perivascular cells. Because of the above-mentioned ultrastructural alterations in the tumor vessel wall, vascular permeability of solid tumors is generally higher than in normal tissues. The heterogeneity of tumor blood flow cause abnormal microenvironment in tumors and hinders the delivery and efficacy of CIK cells therapy [19]. Intravital microscopy was used to detect the morphology and structure of tumor vasculature. Evans blue was used to observe the permeability of tumor vascular. Evans blue could strongly bind to albumin in the blood. Its behavior reflects the transport of albumin, which is about 67 kDa with a diameter of about 7 nm. This size is similar
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to effective pore size of 6–7 nm occurring in the most normal blood vessel [20]. Aberrant tumor vessels were characterized with larger inter-endothelial junctions than normal blood vessels; there might be a little hindrance for the transvascular transport of Evans blue-albumin complex. The morphology of tumor vessels is visually more normal and less dilated than the control group. The phenomenon showed bevacizumab could normalize tumor vasculature. The normalized tumor vasculatureinduced tumor oxygenation supply increased. As a result, hypoxia area of tumor tissue could be reduced by bevacizumab. Tumor hypoxia is associated with treatment resistance, cell proliferation, and metastatic potential and a poor overall prognosis [6]. To study the relationship between tumor hypoxia and infiltration of CIK cells, we used pimonidazole and anti-human CD3 antibody staining the tumor tissue with immunohistochemistry. Pimonidazole is reductively activated in an oxygen-dependent manner and is covalently bound to thiol-containing proteins in hypoxic cells. Pimonidazole can be used for qualitative and quantitative assessment of hypoxia [21]. The density of CIK cells in the tumor was judged by the immunohistochemistry of anti-human CD3 antibodies. After analyzing the result of our experiment, we found that bevacizumab could significantly reduce hypoxic area of the tumor tissue. Less CD3? CIK cells infiltrated in hypoxic area compared with normoxic area, indicating that hypoxia significantly impaired CIK cells recruitment to tumor tissue. It also deduced that improvement in tumor hypoxia was benefic for the infiltration of CIK cells. In addition, the efficiency of tumor inhibition may be further enhanced. Thus, we concluded tumor hypoxia microenvironment may be the possible mechanism limiting cancer immunotherapy. Bevacizumab that induce vascular normalization can alleviate hypoxia and increase the efficacy of immunotherapy therapy in A549 lung carcinoma. The antiangiogenic agents could enhance more CIK cells accumulated in tumor tissue and make it play a greater role in the treatment. Although similar conclusions had been verified in other models, our findings still open new perspectives for combining antiangiogenesis and immunotherapy in cancer patients with lung cancer.
Conclusion Our study clearly showed hypoxia hinder the infiltration of CIK cells into tumor tissue. Bevacizumab could normalize tumor vascular and reduce tumor hypoxic area, and then increase intratumoral infiltration of CIK cells, thereby enhancing efficacy of antitumor ability of adoptive CIK cells transfer therapy. Thus, bevacizumab in combination with adoptive CIK cells therapy can be a rational regimen
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applied to clinical treatment, and it would be beneficial to patients with advanced stage NSCLC. Acknowledgments The work was supported by National Natural Science Foundation of China (No. 81101739) and China International Medical Foundation, CIMF-F-H001-02. Conflict of interests peting interests.
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The authors declare that they have no com-
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