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Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis in an orthotopic oral squamous cell carcinoma model
Oral Oncology (2008) 44, 1118– 1126
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Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis in an orthotopic oral squamous cell carcinoma model In-Sik Chung a, Young-Ik Son b, Ye Jeung Ko b, Chung-Hwan Baek b, Jae Keun Cho b, Han-Sin Jeong b,* a Department of Genetic Engineering and Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Republic of Korea b Department of Otorhinolaryngology-Head and Neck Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea
Received 18 December 2007; received in revised form 9 January 2008; accepted 9 January 2008 Available online 16 May 2008
KEYWORDS Tumstatin; Angiogenesis inhibitors; Oral cancer; Squamous cell carcinomas; Lymph node metastasis
Summary Tumstatin – non-collagenous (NC1) domain of the alpha 3 chain of type IV collagen – is a potent inhibitor of tumor angiogenesis. Successful tumor inhibition has been reported in glioma, bronchopulmonary cancer and melanoma experimental model. In this study, the effects of tumstatin, in vitro and in vivo, were investigated in an oral cancer model. Recombinant human tumstatin proteins were obtained by the transformation of Tn 5B1-4 cells, transfected with a plasmid containing tumstatin cDNA using the lipofection method, as previously described. Tumstatin inhibited the proliferation of human umbilical vascular endothelial cells in a dose dependent manner in a proliferation assay. For the in vivo analysis, we established an orthotopic oral squamous cell carcinoma (AT-84 cells) animal (C3H/He) model. In this animal model, the in vivo inhibitory effects of tumstatin on the tumor growth and on the metastasis of tumors were demonstrated. However, the tumors did not show complete remission. Immunostaining of the tumor microvessels (CD-31/PECAM) revealed that the density of tumor microvessels was significantly decreased in the tumstatin treated primary tumors. The results demonstrated that tumstatin delayed the tumor growth and the metastasis of oral squamous cell carcinomas. However, tumstatin alone failed to achieve tumor regression. Therefore, tumstatin might have an adjuvant role in the treatment of oral cancers, in combination with the conventional therapy.
ª 2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +82 2 3410 3579; fax: +82 2 3410 6987. E-mail address: [email protected] (H.-S. Jeong). 1368-8375/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2008.01.017
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis
Introduction Angiogenesis is not only known for embryonic development, wound healing and tissue regeneration, but also for its important role in tumor growth and metastasis.1 When a tumor grows larger than 1–2 mm in diameter, angiogenesis within the tumor is essential for its growth and survival.1,2 Since the diffusion distance of oxygen from the veins is limited to 150 lm, if angiogenesis does not accompany tumor growth, apoptosis will occur due to the interception of oxygen and other nutrients needed for tumor growth.3–5 It is thought that angiogenesis is maintained through a delicate balance between growth factors and inhibition factors1,6; therefore, the pathological angiogenesis that is required for tumor growth occurs as a result of an increase in growth factors for angiogenesis and a decrease in inhibition factors.1,2,7–9 Recently, substances have been identified that inhibit the growth of vascular endothelium cells and angiogenesis required for the growth of tumors.2,10 One of the strategies to treat tumor is the inhibition of vascular endothelium cells within the tumor using these substances. The vascular endothelium cells can be more susceptible to treatment than the tumor cells because they are genetically more stable and less resistant to anticancer therapies.2,3,6,10 Currently, there are over 20 angiogenesis inhibiting substances that are under clinical investigation for cancer treatment. Among these, one type is a low-molecular weight substance or an antibody that inhibits precursors (i.e. vascular endothelial growth factor, VEGF)11; the other type is composed of substances that already exist within the blood (i.e. thrombospondin-1 and angiostatin) or within the tissue (i.e. endostatin).12–19 To date, several agents have been tested in the oral cancer model in vivo. For example, anti-VEGF gene therapy and angiostatin gene therapy, producing angiogenesis inhibitors or blocking agents, showed promising results in this model.20–22 Recently, endostatin was reported to inhibit lymph node metastasis in an oral squamous cell carcinoma model.23 However, these trials targeting angiogenesis in oral cancer models did not achieve the complete tumor regression, but only the inhibition of growth or metastasis. Thus, a more potent anti-angiogenic agent should be required to gain the acceptable oncological results in oral cancer. One of the new substances identified is tumstatin.24,25 Tumstatin is a C-terminal globular non-collagenous (NC1) domain of the type 4 collagen alpha 3 chain.24,25 Type 4 collagen is a polymer of alpha 3 chains of the alpha 6 chains that are genetically distinct. An alpha chain consists of three spiral structures that are built with 1400 amino acids and a non-collagenous (NC1) section that is composed of 230 amino acids. The NC1 section is known to inhibit angiogenesis and tumor growth.25,26 Tumstatin inhibits angiogenesis and tumor growth more than other similar substances within tissue.26,27 Therefore, it is a candidate for the development of potential anticancer therapy.27 Although there are some data on the use of endostatin for the treatment of head and neck tumors showing its efficacy for inhibiting tumor growth,23,28–30 there is no research on tumstatin. Tumstatin has its greater potential for inhibiting angiogenesis and tumor growth in head and
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neck cancer models, as suggested by investigations on experimental tumor models of malignant melanoma, bronchopulmonary carcinoma and malignant glioma.10,31–34 Therefore, we evaluated the inhibition potential of tumstatin on angiogenesis and tumor growth in an oral cancer model.
Materials and method Preparation of purified tumstatin Recombinant human tumstatin proteins were obtained by the transformation of Tn 5B1-4 cells, transfected with the plasmid containing tumstasin cDNA using the lipofection method, as previously described.35,36 Purification of tumstatin protein was confirmed by Western blot analysis and silver staining after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).
Effects of tumstatin on human umbilical vascular endothelial cells Human umbilical vascular endothelial cells (HUVECs), purchased from Clonetics, Cambrex Bio Science Inc., Walkersville, MD, USA, were cultured in the EGM-MV BulletKit (5% fetal bovine serum in endothelial basic medium, 12 lg/ml bovine brain extract, 1 lg/ml hydrocortisone, 1 ll/ml GA1000, human endothelial growth factor, hEGF) containing 30 lg/ml vitrogen. The cells were maintained in endothelial basic medium containing 0.1% fetal bovine serum for 24 h before the experiments. First, cells were washed with RPMI-1640 after incubation at 37 C temperature for 30 min in 96 well plates, in which 0.1% gelatin of 0.1 ml per each well had been coated previously. Then, HUVECs were seeded in a density of 4000 cells/0.1 ml (0.3 · 106 cells per plate) into the plate with the medium. After a 24-h culture, variable concentrations of tumstatin protein (0.2, 0.3, 0.5, 0.75, 1.0, 1.5, 2.0 lg/ml) were applied to the HUVECs. The degree of HUVEC proliferation was analyzed for 72 h. Cells were treated with 0.1% serum for 24 h, then stimulated by 10 ng/ml EGF for 20 h. They were cultured for 4 h after the addition of 1 lCi/ml [3H] thymidine per well, and washed with phosphate buffered saline (PBS) and fixed with 100% methanol in 4 C temperature for 15 min. Thereafter, 10% cold trichloroacetic acid was applied to cells in 4 C temperature for 15 min. The cells were dissolved in 0.1 N NaOH of 200 ll at room temperature for 30 min, after being washed three times with water. Then, the incorporation of [3H] thymidine was recorded in the scintillation solution (A600). A control group was treated with only normal saline.
Orthotopic squamous cell carcinoma (AT-84 cells) animal model of the floor of the mouth37,38 The tumor cell line AT-84 is a squamous cell carcinoma, spontaneously arising from oral mucosa and syngenic to C3H/HeJ mice (which were kindly provided by Dr. E.J. Shillitoe, State University of New York, Upstate Medical University). The mycoplasma free tumor cells were maintained in a humid incubator with 5% CO2 at 37 C in RPMI-1640 medium
1120 containing 10% heat-inactivated fetal bovine serum, 2 mmol/L L-gutamine, 0.1 mmol/L non-essential amino acids, 10 mmol/L N-2-HEPES buffer, 100 IU/ml penicillin and 100 lg/ml streptomycin. All of the cell culture media and the reagents were purchased from Life Technologies (Rochville, MD, USA). The cell number was calculated by performing trypan blue dye staining after detaching the adherent cells from the culture flask by gentle trypsinization. Four- to six-week-old female C3H/HeJ mice were purchased from BioLink Korea (ChungBuk, Korea). The mice were acclimated for at least 2 weeks before the experiments. The mice were provided with water and food ad libitum, and they were quarantined in a specific pathogen free environment with a 12-h light and 12-h dark photoperiod in the animal care facility, accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) international. The animal care and experiments were performed following the guidelines of AAALAC international. To establish an orthotopic oral squamous cell carcinoma animal model, 1 · 106 AT-84 cells in 0.1 ml, which was 10 times the amount of the minimum tumorigenic dose, were injected into the anterior sub-mucosa of the floor of the mouth trans-cutaneously, as previously described.37 After the tumor inoculation, primary tumor growth or formation was evaluated for 5 weeks. Every week, a group of tumor injected animals (20 animals per week, total N = 100 mice) was sacrificed. The primary tumor specimen in the floor of the mouth with the adjacent mandible, the first echelon submandibular lymph nodes, the deep cervical lymph nodes and the lung were collected to evaluate the formation of tumors and the tumor invasion into the mandible, lymph node metastasis and lung metastasis with hematoxylin and eosin staining.
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Effects of tumstatin on AT-48 tumor growth in an established animal model To evaluate the effects of tumstatin on AT-84 tumor growth, purified tumstatin protein of 50 lg/ml was injected at the same site (tumor injection site) every other day three times (days 3, 5, and 7) on day 3 after tumor inoculation (AT-84 cells, 1 · 106 cells/0.1 ml) into the floor of the mouth of C3H/HeJ mice. Then, the primary tumor growth or the formation, mandible invasion, lymph node metastasis and lung metastasis were evaluated in the same manner as described above (10 animals at 2 weeks, 10 at 3 weeks, 40 at 4 weeks, 40 at 5 weeks, and total N = 100 mice).
Evaluation of the microvessel density in the tumors The primary tumor specimens (at 5 weeks) from the floor of the mouth of animals were analyzed for microvessel density. First, the tumor specimens were fixed with acetone for 10 min. Then, to inhibit the endogenous peroxidase activity, they were incubated with methanol containing 1% hydrogen peroxide for 20 min, then cultured with 10% normal goat serum for 20 min. For these specimens, the immunostaining with CD-31/PECAM-1 (Dako, Glostrup, Denmark, 1:200 dilutions) was performed at 4 C temperature for 24 h as a primary staining. Subsequently, the specimens were washed with PBS, and treated for 1 h with secondary antibody (avidin-biotin-peroxidase complex, Vector Lab., Burlingame, CA, USA). Again, they were subjected to react with 3,30 -diaminobenzidine (DAB), and stained with hematoxylin as a counterstaining.
Figure 1 Inhibition of endothelial cell (HUVEC) proliferation by purified tumstatin purified recombinant tumstatin was added to bovine capillary endothelial cells in a 72-h proliferation assay (tumstatin concentration P1.0 lg/ml: P < 0.05). N/S: normal saline as controls, T: tumstatin, 0.2–2.0:0.2–2.0 lg/ml.
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis First, the immunostained specimens were observed by ·40 magnification under a light microscope to the densest area of microvessel formation in the tumor tissues. Microvessels were defined as the blood vessels without muscle layers in the vessel walls, which were stained with CD-31/ PECAM-1. Around the area identified (the densest area in each tumor sample), using ·200 magnification five fields were selected to calculate the number of microvessels, and the mean value for these five fields was recorded as the microvessel density for each tumor. To determine the microvessel density for each group, two investigators evaluated three or more tumors for each group independently and then the mean values were regarded as the representative microvessel density for each group. The relative percents were also calculated with the reference standard of the number of microvessels in the control group (without any treatment) set as 100%.
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Statistical analysis The Wilcoxon rank sum test was used to analyze the significance of the differences in cell proliferation and tumor size. The occurence of metastases was compared between the groups by the chi-square test. The differences in the microvessel density were analyzed by the t-test. The Standard SPSS (version 11.0, Statistical Package for Social Science, Chicago, USA) was used for the statistical analyzes.
Results Purified tumstatin inhibited the proliferation of HUVECs in a dose dependent manner When variable concentrations (0.2–2.0 lg/ml) of purified tumstatin were applied to the HUVECs, the proliferation
Figure 2 Establishment of an orthotopic squamous cell carcinoma animal model of the floor of the mouth. (a) At 4 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the mass protruded from the floor of the mouth into the submental and submandibular area, and then the visible mass was identified grossly. (b) The tumor was fixed to the mandible bone. The enlargement of the adjacent submandibular lymph nodes (sentinel lymph nodes) was noted (white arrow). (c) The histopathological analysis of the enlarged submandibular lymph nodes revealed the replacement of normal lymph node structure with cancer cells (white arrow). (d) The tumor cells invaded into the mandible (white arrow: tumor cell nests in the mandible). (e, f) Subsequent metastases to the lung were noted in 15% of the animals (white arrow: metastatic nodules in the lung parenchyma).
1122 of HUVECs was inhibited in a dose dependent manner as the concentrations increased up to 1.0 lg/ml (Fig. 1). With the concentration over 1.0 lg/ml, the inhibitory effect of the purified tumstatin protein remained at a constant level. Compared to the control group (normal saline treated group), the proliferation of the HUVECs was significantly reduced when the concentration of tumstatin was over 1.0 lg/ml (P < 0.05 at 72 h).
Establishment of a syngenic orthotopic oral (the floor of the mouth) squamous cell carcinoma (AT-84 cells) animal (C3H/HeJ) model Most of the animals did not lose more than 25% of their baseline body weights until 5 weeks after tumor inoculation, because the tumor in the floor of the mouth did not severely affect the oral intake. Therefore, we evaluated the occurrence of metastases and the change in tumor growth up to 5 weeks. The histopathological analysis confirmed tumor invasion to the mandible bone, lymph node metastasis to the submandibular lymph nodes and lung metastasis (Fig. 2a–f). As a result, from 3 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the primary tumors consistently formed visible masses (70% of animals at 3 weeks, 80% at 4 weeks, and 90% at 5 weeks). The maximum diameter of the primary tumors was 1.0 ± 0.6 mm at 2 weeks, 3.1 ± 0.5 mm at 3 weeks, 6.3 ± 1.5 mm at 4 weeks, and 6.5 ± 2.2 mm at 5 weeks. Around 20–30% of the tumors in the floor of the mouth showed invasion of the mandible and lymphatic metastasis into the submandibular lymph nodes (the first echelon lymph nodes) (Fig. 3). The mandible invasion showed distinct pathological findings, such as cortical erosion and the presence of tumor cells in the marrow space. In addition, lung metastases were detected in 15% (3 of 20) of the animals with tumors in the floor of their mouth at 5
I.-S. Chung et al. weeks. However, lymphatic metastasis to the deep cervical lymph nodes (the secondary echelon lymph nodes) was not found in any of the animals.
Peritumor injections of tumstatin: inhibition of tumor growth and delay of lymph node metastasis without regression of tumors For comparison, the mean size (6.3 ± 1.5 mm) of the tumors at 30 days after the inoculation of tumor cells was set at 100%. When compared to the control groups, the size of the primary tumor in tumstatin treated animals was reduced to 60% compared to the control groups (3.8 ± 0.3 mm) at 30 days (P < 0.05) (Fig. 4a). However, the occurrence of tumor formation in the floor of the mouth was not different in comparisons between the control group and the tumstatin treated group (Fig. 4b). In the tumstatin treated group, primary tumors were found in 10% of the animals at 2 weeks, 60% at 3 weeks, 70% at 4 weeks, and 90% at 5 weeks. Invasion into the mandible bone showed similar frequency in the two groups. In addition, metastases to the submandibular lymph nodes were noted in only 10% of the tumstatin treated group at 5 weeks. These findings significantly differed from those of the control group (15% at 4 weeks and 30% at 5 weeks) (P < 0.05). The development of lung metastasis was reduced to 5% (2 of 40) of the tumstatin treated group at 5 weeks; however, this difference failed to reach statistical significance (15% in the control group at 5 weeks).
Tumstatin reduced the microvessel density in the tumor tissues Tumor specimens in the control group (without any treatment) showed an average microvessel number of 7.75 ± 2.7 (mean ± SD) (100%) (Fig. 5a–d). For the group
Figure 3 From 3 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the primary tumors consistently formed visible masses. About 20–30% of the tumors showed invasion of the mandible and lymphatic metastasis into the submandibular lymph nodes.
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis
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Figure 4 The inhibitory effect of primary tumor growth by tumstatin in an orthotopic oral squamous cell carcinoma animal model. (a) The size of the primary tumor formation according to the time sequence was evaluated in the tumstatin (50 lg/ml) treated group (n) compared with the two control groups; tumstatin-free media treated group (r) and normal saline treated group (m). (b) The primary tumor and the mandible invasion were evaluated in the tumstatin (50 lg/ml) treated group (Tumor-eTumstatin Tx, Mandible invasion-Tumstatin Tx) and the control groups (Tumor-Control, Mandible invasion-Control). The rate of lymphatic metastases into the submandibular lymph nodes (LN mets-Tumstatin Tx) in the tumstatin treated group was compared with the control groups (LN mets-Control).
treated only with normal saline, the microvessel density was 8.15 ± 1.7 per high power field (105.2%), and for the group treated with tumstatin free media, it was 7.35 ± 2.0 (94.8%). However, the tumor specimens treated with tumstatin (50 lg/ml) had a microvessel density of 5.25 ± 2.0 per high power field (67.7%), which was significantly lower than the control groups (P < 0.05) (Fig. 5e).
Discussion The results of this study demonstrated the inhibitory effects of tumstatin, a potent endogenous inhibitor of angiogenesis,
on oral tumor growth. This is the first study to investigate the effects of tumstatin on oral cancer. The animal model used in these oral cancer experiments had the characteristics of orthotopic syngenic tumors (AT-84 cells) in an immunocompetant host (C3H/HeJ).37,38 Thus, the results from this study are more clinically relevant. Tumstatin has been reported to be the most potent inhibitor of angiogenesis among the currently known endogenous inhibitors.27 It inhibits tumor angiogenesis through aVb3 integrin signaling on tumor associated vascular endothelium, and then induces apoptosis.25,26 In our study, tumstatin inhibited the proliferation of HUVECs in vitro
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Figure 5 Immunostaining of microvessels in the tumor specimen (CD-31). (a, b) Tumor specimen treated without any treatment (control group). (c, d) Tumor treated with tumstatin (concentration 50 lg/ml). (a, c) ·40, (b) ·100, (d) ·200 high power field. T: tumor, B: bone, M: muscle, black arrow: CD-31 stained microvessel. (e) Blood vessel density was determined by counting the vessels with CD-31 (+) vascular endothelial cells per ·200 high power fields. Blood vessel density in tumors treated with tumstatin was compared to the control groups. In the tumstatin treated group, the blood vessel density was significantly lower than that in the control groups: A–C (P < 0.05). ((A) control group without any intervention, (B) control group treated with normal saline 1 ml, (C) control group treated with tumstatin-negative media, and (D) tumstatin concentration 50 lg/ml).
and reduced the microvessel density in tumor tissues in vivo, which is consistent with the previous results.25,26 Tumstatin is thought to inhibit tumor cells directly, through the same signaling pathway, aVb3 integrin.34,39–41 As the tumor grows, the expression of aVb3 integrin increases, tumstatin works directly on tumor cells through aVb3 integrin. In other experiments, our data showed the induction of apoptosis of the tumor cells (the elevation of tumor necrosis factor-a level), possibly through aVb3 integrin (data not shown here).
To evaluate the in vivo effect of tumstatin, we first evaluated an oral tongue squamous cell carcinoma model. However, the tumor mass on the tongue frequently impaired the oral intake of the animals, which resulted in the loss of more than 30% of baseline body weight at 3–4 weeks. Thus, we could not follow the animals over sufficient time for the development of metastasis. However, the tumor in the floor of the mouth did not severely affect oral intake and we could monitor the occurrence of metastases for up to 5 weeks without the significant loss of body weight. Subse-
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis quently, these tumors invaded into the mandible bone, metastasized to the submandibular lymph nodes, and lung as the primary tumor grew, which can be a model for tumor growth in human patients. Our data showed that tumstatin delayed the primary tumor growth of oral squamous cell carcinomas and lymph node metastasis, but failed to achieve tumor regression in the animal models. These findings are consistent with the previously reported results,25,26 where tumstatin was used in relatively high concentrations (80–100 lg/ml) compared to the 50 lg/ml in our study. Therefore, the results suggest that tumstatin treatment alone is not enough to eradicate oral cancers as a single treatment modality. Considering the beneficial effects of tumstatin, tumstatin might have an important role in the adjuvant setting in combination with conventional treatment modalities or targeted therapy. The limitations of this study include the following. First, the purified tumstatin protein was obtained from the transformation of Tn 5B1-4 cells, transfected with the plasmid containing tumstatin cDNA using the lipofection method, as previously described.35,36 This method is time consuming and requires multiple steps for protein purification; therefore, high concentrations (over 100 lg/ml) of tumstatin were not available to be used in this study. For future clinical application, a high throughput method of purification is needed. Second, peritumor injection of the tumstatin protein may be associated with an error of the delivery into the exact tumor formation site. Therefore, in situ expression of tumstatin, after gene transfection into the target cells, should be performed in subsequent studies. Nevertheless, this study showed that tumstatin could inhibit the tumor growth of oral squamous cell carcinomas, similar to other cancers by reducing tumor angiogenesis. However, tumstatin alone failed to achieve tumor regression. Further study is needed to investigate the potential role of tumstatin as an adjuvant therapy for oral cancers, in combination with the conventional therapy.
Conflict of interest statement None declared.
Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (Ministry of Education and Human Resources Development, MOEHRD) (KRF2007-313-E00263).
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Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis in an orthotopic oral squamous cell carcinoma model In-Sik Chung a, Young-Ik Son b, Ye Jeung Ko b, Chung-Hwan Baek b, Jae Keun Cho b, Han-Sin Jeong b,* a Department of Genetic Engineering and Plant Metabolism Research Center, Kyung Hee University, Suwon 449-701, Republic of Korea b Department of Otorhinolaryngology-Head and Neck Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea
Received 18 December 2007; received in revised form 9 January 2008; accepted 9 January 2008 Available online 16 May 2008
KEYWORDS Tumstatin; Angiogenesis inhibitors; Oral cancer; Squamous cell carcinomas; Lymph node metastasis
Summary Tumstatin – non-collagenous (NC1) domain of the alpha 3 chain of type IV collagen – is a potent inhibitor of tumor angiogenesis. Successful tumor inhibition has been reported in glioma, bronchopulmonary cancer and melanoma experimental model. In this study, the effects of tumstatin, in vitro and in vivo, were investigated in an oral cancer model. Recombinant human tumstatin proteins were obtained by the transformation of Tn 5B1-4 cells, transfected with a plasmid containing tumstatin cDNA using the lipofection method, as previously described. Tumstatin inhibited the proliferation of human umbilical vascular endothelial cells in a dose dependent manner in a proliferation assay. For the in vivo analysis, we established an orthotopic oral squamous cell carcinoma (AT-84 cells) animal (C3H/He) model. In this animal model, the in vivo inhibitory effects of tumstatin on the tumor growth and on the metastasis of tumors were demonstrated. However, the tumors did not show complete remission. Immunostaining of the tumor microvessels (CD-31/PECAM) revealed that the density of tumor microvessels was significantly decreased in the tumstatin treated primary tumors. The results demonstrated that tumstatin delayed the tumor growth and the metastasis of oral squamous cell carcinomas. However, tumstatin alone failed to achieve tumor regression. Therefore, tumstatin might have an adjuvant role in the treatment of oral cancers, in combination with the conventional therapy.
ª 2008 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +82 2 3410 3579; fax: +82 2 3410 6987. E-mail address: [email protected] (H.-S. Jeong). 1368-8375/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2008.01.017
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis
Introduction Angiogenesis is not only known for embryonic development, wound healing and tissue regeneration, but also for its important role in tumor growth and metastasis.1 When a tumor grows larger than 1–2 mm in diameter, angiogenesis within the tumor is essential for its growth and survival.1,2 Since the diffusion distance of oxygen from the veins is limited to 150 lm, if angiogenesis does not accompany tumor growth, apoptosis will occur due to the interception of oxygen and other nutrients needed for tumor growth.3–5 It is thought that angiogenesis is maintained through a delicate balance between growth factors and inhibition factors1,6; therefore, the pathological angiogenesis that is required for tumor growth occurs as a result of an increase in growth factors for angiogenesis and a decrease in inhibition factors.1,2,7–9 Recently, substances have been identified that inhibit the growth of vascular endothelium cells and angiogenesis required for the growth of tumors.2,10 One of the strategies to treat tumor is the inhibition of vascular endothelium cells within the tumor using these substances. The vascular endothelium cells can be more susceptible to treatment than the tumor cells because they are genetically more stable and less resistant to anticancer therapies.2,3,6,10 Currently, there are over 20 angiogenesis inhibiting substances that are under clinical investigation for cancer treatment. Among these, one type is a low-molecular weight substance or an antibody that inhibits precursors (i.e. vascular endothelial growth factor, VEGF)11; the other type is composed of substances that already exist within the blood (i.e. thrombospondin-1 and angiostatin) or within the tissue (i.e. endostatin).12–19 To date, several agents have been tested in the oral cancer model in vivo. For example, anti-VEGF gene therapy and angiostatin gene therapy, producing angiogenesis inhibitors or blocking agents, showed promising results in this model.20–22 Recently, endostatin was reported to inhibit lymph node metastasis in an oral squamous cell carcinoma model.23 However, these trials targeting angiogenesis in oral cancer models did not achieve the complete tumor regression, but only the inhibition of growth or metastasis. Thus, a more potent anti-angiogenic agent should be required to gain the acceptable oncological results in oral cancer. One of the new substances identified is tumstatin.24,25 Tumstatin is a C-terminal globular non-collagenous (NC1) domain of the type 4 collagen alpha 3 chain.24,25 Type 4 collagen is a polymer of alpha 3 chains of the alpha 6 chains that are genetically distinct. An alpha chain consists of three spiral structures that are built with 1400 amino acids and a non-collagenous (NC1) section that is composed of 230 amino acids. The NC1 section is known to inhibit angiogenesis and tumor growth.25,26 Tumstatin inhibits angiogenesis and tumor growth more than other similar substances within tissue.26,27 Therefore, it is a candidate for the development of potential anticancer therapy.27 Although there are some data on the use of endostatin for the treatment of head and neck tumors showing its efficacy for inhibiting tumor growth,23,28–30 there is no research on tumstatin. Tumstatin has its greater potential for inhibiting angiogenesis and tumor growth in head and
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neck cancer models, as suggested by investigations on experimental tumor models of malignant melanoma, bronchopulmonary carcinoma and malignant glioma.10,31–34 Therefore, we evaluated the inhibition potential of tumstatin on angiogenesis and tumor growth in an oral cancer model.
Materials and method Preparation of purified tumstatin Recombinant human tumstatin proteins were obtained by the transformation of Tn 5B1-4 cells, transfected with the plasmid containing tumstasin cDNA using the lipofection method, as previously described.35,36 Purification of tumstatin protein was confirmed by Western blot analysis and silver staining after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).
Effects of tumstatin on human umbilical vascular endothelial cells Human umbilical vascular endothelial cells (HUVECs), purchased from Clonetics, Cambrex Bio Science Inc., Walkersville, MD, USA, were cultured in the EGM-MV BulletKit (5% fetal bovine serum in endothelial basic medium, 12 lg/ml bovine brain extract, 1 lg/ml hydrocortisone, 1 ll/ml GA1000, human endothelial growth factor, hEGF) containing 30 lg/ml vitrogen. The cells were maintained in endothelial basic medium containing 0.1% fetal bovine serum for 24 h before the experiments. First, cells were washed with RPMI-1640 after incubation at 37 C temperature for 30 min in 96 well plates, in which 0.1% gelatin of 0.1 ml per each well had been coated previously. Then, HUVECs were seeded in a density of 4000 cells/0.1 ml (0.3 · 106 cells per plate) into the plate with the medium. After a 24-h culture, variable concentrations of tumstatin protein (0.2, 0.3, 0.5, 0.75, 1.0, 1.5, 2.0 lg/ml) were applied to the HUVECs. The degree of HUVEC proliferation was analyzed for 72 h. Cells were treated with 0.1% serum for 24 h, then stimulated by 10 ng/ml EGF for 20 h. They were cultured for 4 h after the addition of 1 lCi/ml [3H] thymidine per well, and washed with phosphate buffered saline (PBS) and fixed with 100% methanol in 4 C temperature for 15 min. Thereafter, 10% cold trichloroacetic acid was applied to cells in 4 C temperature for 15 min. The cells were dissolved in 0.1 N NaOH of 200 ll at room temperature for 30 min, after being washed three times with water. Then, the incorporation of [3H] thymidine was recorded in the scintillation solution (A600). A control group was treated with only normal saline.
Orthotopic squamous cell carcinoma (AT-84 cells) animal model of the floor of the mouth37,38 The tumor cell line AT-84 is a squamous cell carcinoma, spontaneously arising from oral mucosa and syngenic to C3H/HeJ mice (which were kindly provided by Dr. E.J. Shillitoe, State University of New York, Upstate Medical University). The mycoplasma free tumor cells were maintained in a humid incubator with 5% CO2 at 37 C in RPMI-1640 medium
1120 containing 10% heat-inactivated fetal bovine serum, 2 mmol/L L-gutamine, 0.1 mmol/L non-essential amino acids, 10 mmol/L N-2-HEPES buffer, 100 IU/ml penicillin and 100 lg/ml streptomycin. All of the cell culture media and the reagents were purchased from Life Technologies (Rochville, MD, USA). The cell number was calculated by performing trypan blue dye staining after detaching the adherent cells from the culture flask by gentle trypsinization. Four- to six-week-old female C3H/HeJ mice were purchased from BioLink Korea (ChungBuk, Korea). The mice were acclimated for at least 2 weeks before the experiments. The mice were provided with water and food ad libitum, and they were quarantined in a specific pathogen free environment with a 12-h light and 12-h dark photoperiod in the animal care facility, accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) international. The animal care and experiments were performed following the guidelines of AAALAC international. To establish an orthotopic oral squamous cell carcinoma animal model, 1 · 106 AT-84 cells in 0.1 ml, which was 10 times the amount of the minimum tumorigenic dose, were injected into the anterior sub-mucosa of the floor of the mouth trans-cutaneously, as previously described.37 After the tumor inoculation, primary tumor growth or formation was evaluated for 5 weeks. Every week, a group of tumor injected animals (20 animals per week, total N = 100 mice) was sacrificed. The primary tumor specimen in the floor of the mouth with the adjacent mandible, the first echelon submandibular lymph nodes, the deep cervical lymph nodes and the lung were collected to evaluate the formation of tumors and the tumor invasion into the mandible, lymph node metastasis and lung metastasis with hematoxylin and eosin staining.
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Effects of tumstatin on AT-48 tumor growth in an established animal model To evaluate the effects of tumstatin on AT-84 tumor growth, purified tumstatin protein of 50 lg/ml was injected at the same site (tumor injection site) every other day three times (days 3, 5, and 7) on day 3 after tumor inoculation (AT-84 cells, 1 · 106 cells/0.1 ml) into the floor of the mouth of C3H/HeJ mice. Then, the primary tumor growth or the formation, mandible invasion, lymph node metastasis and lung metastasis were evaluated in the same manner as described above (10 animals at 2 weeks, 10 at 3 weeks, 40 at 4 weeks, 40 at 5 weeks, and total N = 100 mice).
Evaluation of the microvessel density in the tumors The primary tumor specimens (at 5 weeks) from the floor of the mouth of animals were analyzed for microvessel density. First, the tumor specimens were fixed with acetone for 10 min. Then, to inhibit the endogenous peroxidase activity, they were incubated with methanol containing 1% hydrogen peroxide for 20 min, then cultured with 10% normal goat serum for 20 min. For these specimens, the immunostaining with CD-31/PECAM-1 (Dako, Glostrup, Denmark, 1:200 dilutions) was performed at 4 C temperature for 24 h as a primary staining. Subsequently, the specimens were washed with PBS, and treated for 1 h with secondary antibody (avidin-biotin-peroxidase complex, Vector Lab., Burlingame, CA, USA). Again, they were subjected to react with 3,30 -diaminobenzidine (DAB), and stained with hematoxylin as a counterstaining.
Figure 1 Inhibition of endothelial cell (HUVEC) proliferation by purified tumstatin purified recombinant tumstatin was added to bovine capillary endothelial cells in a 72-h proliferation assay (tumstatin concentration P1.0 lg/ml: P < 0.05). N/S: normal saline as controls, T: tumstatin, 0.2–2.0:0.2–2.0 lg/ml.
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis First, the immunostained specimens were observed by ·40 magnification under a light microscope to the densest area of microvessel formation in the tumor tissues. Microvessels were defined as the blood vessels without muscle layers in the vessel walls, which were stained with CD-31/ PECAM-1. Around the area identified (the densest area in each tumor sample), using ·200 magnification five fields were selected to calculate the number of microvessels, and the mean value for these five fields was recorded as the microvessel density for each tumor. To determine the microvessel density for each group, two investigators evaluated three or more tumors for each group independently and then the mean values were regarded as the representative microvessel density for each group. The relative percents were also calculated with the reference standard of the number of microvessels in the control group (without any treatment) set as 100%.
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Statistical analysis The Wilcoxon rank sum test was used to analyze the significance of the differences in cell proliferation and tumor size. The occurence of metastases was compared between the groups by the chi-square test. The differences in the microvessel density were analyzed by the t-test. The Standard SPSS (version 11.0, Statistical Package for Social Science, Chicago, USA) was used for the statistical analyzes.
Results Purified tumstatin inhibited the proliferation of HUVECs in a dose dependent manner When variable concentrations (0.2–2.0 lg/ml) of purified tumstatin were applied to the HUVECs, the proliferation
Figure 2 Establishment of an orthotopic squamous cell carcinoma animal model of the floor of the mouth. (a) At 4 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the mass protruded from the floor of the mouth into the submental and submandibular area, and then the visible mass was identified grossly. (b) The tumor was fixed to the mandible bone. The enlargement of the adjacent submandibular lymph nodes (sentinel lymph nodes) was noted (white arrow). (c) The histopathological analysis of the enlarged submandibular lymph nodes revealed the replacement of normal lymph node structure with cancer cells (white arrow). (d) The tumor cells invaded into the mandible (white arrow: tumor cell nests in the mandible). (e, f) Subsequent metastases to the lung were noted in 15% of the animals (white arrow: metastatic nodules in the lung parenchyma).
1122 of HUVECs was inhibited in a dose dependent manner as the concentrations increased up to 1.0 lg/ml (Fig. 1). With the concentration over 1.0 lg/ml, the inhibitory effect of the purified tumstatin protein remained at a constant level. Compared to the control group (normal saline treated group), the proliferation of the HUVECs was significantly reduced when the concentration of tumstatin was over 1.0 lg/ml (P < 0.05 at 72 h).
Establishment of a syngenic orthotopic oral (the floor of the mouth) squamous cell carcinoma (AT-84 cells) animal (C3H/HeJ) model Most of the animals did not lose more than 25% of their baseline body weights until 5 weeks after tumor inoculation, because the tumor in the floor of the mouth did not severely affect the oral intake. Therefore, we evaluated the occurrence of metastases and the change in tumor growth up to 5 weeks. The histopathological analysis confirmed tumor invasion to the mandible bone, lymph node metastasis to the submandibular lymph nodes and lung metastasis (Fig. 2a–f). As a result, from 3 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the primary tumors consistently formed visible masses (70% of animals at 3 weeks, 80% at 4 weeks, and 90% at 5 weeks). The maximum diameter of the primary tumors was 1.0 ± 0.6 mm at 2 weeks, 3.1 ± 0.5 mm at 3 weeks, 6.3 ± 1.5 mm at 4 weeks, and 6.5 ± 2.2 mm at 5 weeks. Around 20–30% of the tumors in the floor of the mouth showed invasion of the mandible and lymphatic metastasis into the submandibular lymph nodes (the first echelon lymph nodes) (Fig. 3). The mandible invasion showed distinct pathological findings, such as cortical erosion and the presence of tumor cells in the marrow space. In addition, lung metastases were detected in 15% (3 of 20) of the animals with tumors in the floor of their mouth at 5
I.-S. Chung et al. weeks. However, lymphatic metastasis to the deep cervical lymph nodes (the secondary echelon lymph nodes) was not found in any of the animals.
Peritumor injections of tumstatin: inhibition of tumor growth and delay of lymph node metastasis without regression of tumors For comparison, the mean size (6.3 ± 1.5 mm) of the tumors at 30 days after the inoculation of tumor cells was set at 100%. When compared to the control groups, the size of the primary tumor in tumstatin treated animals was reduced to 60% compared to the control groups (3.8 ± 0.3 mm) at 30 days (P < 0.05) (Fig. 4a). However, the occurrence of tumor formation in the floor of the mouth was not different in comparisons between the control group and the tumstatin treated group (Fig. 4b). In the tumstatin treated group, primary tumors were found in 10% of the animals at 2 weeks, 60% at 3 weeks, 70% at 4 weeks, and 90% at 5 weeks. Invasion into the mandible bone showed similar frequency in the two groups. In addition, metastases to the submandibular lymph nodes were noted in only 10% of the tumstatin treated group at 5 weeks. These findings significantly differed from those of the control group (15% at 4 weeks and 30% at 5 weeks) (P < 0.05). The development of lung metastasis was reduced to 5% (2 of 40) of the tumstatin treated group at 5 weeks; however, this difference failed to reach statistical significance (15% in the control group at 5 weeks).
Tumstatin reduced the microvessel density in the tumor tissues Tumor specimens in the control group (without any treatment) showed an average microvessel number of 7.75 ± 2.7 (mean ± SD) (100%) (Fig. 5a–d). For the group
Figure 3 From 3 weeks after the inoculation of AT-84 cancer cells into the floor of the mouth of C3H/HeJ mice, the primary tumors consistently formed visible masses. About 20–30% of the tumors showed invasion of the mandible and lymphatic metastasis into the submandibular lymph nodes.
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Figure 4 The inhibitory effect of primary tumor growth by tumstatin in an orthotopic oral squamous cell carcinoma animal model. (a) The size of the primary tumor formation according to the time sequence was evaluated in the tumstatin (50 lg/ml) treated group (n) compared with the two control groups; tumstatin-free media treated group (r) and normal saline treated group (m). (b) The primary tumor and the mandible invasion were evaluated in the tumstatin (50 lg/ml) treated group (Tumor-eTumstatin Tx, Mandible invasion-Tumstatin Tx) and the control groups (Tumor-Control, Mandible invasion-Control). The rate of lymphatic metastases into the submandibular lymph nodes (LN mets-Tumstatin Tx) in the tumstatin treated group was compared with the control groups (LN mets-Control).
treated only with normal saline, the microvessel density was 8.15 ± 1.7 per high power field (105.2%), and for the group treated with tumstatin free media, it was 7.35 ± 2.0 (94.8%). However, the tumor specimens treated with tumstatin (50 lg/ml) had a microvessel density of 5.25 ± 2.0 per high power field (67.7%), which was significantly lower than the control groups (P < 0.05) (Fig. 5e).
Discussion The results of this study demonstrated the inhibitory effects of tumstatin, a potent endogenous inhibitor of angiogenesis,
on oral tumor growth. This is the first study to investigate the effects of tumstatin on oral cancer. The animal model used in these oral cancer experiments had the characteristics of orthotopic syngenic tumors (AT-84 cells) in an immunocompetant host (C3H/HeJ).37,38 Thus, the results from this study are more clinically relevant. Tumstatin has been reported to be the most potent inhibitor of angiogenesis among the currently known endogenous inhibitors.27 It inhibits tumor angiogenesis through aVb3 integrin signaling on tumor associated vascular endothelium, and then induces apoptosis.25,26 In our study, tumstatin inhibited the proliferation of HUVECs in vitro
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Figure 5 Immunostaining of microvessels in the tumor specimen (CD-31). (a, b) Tumor specimen treated without any treatment (control group). (c, d) Tumor treated with tumstatin (concentration 50 lg/ml). (a, c) ·40, (b) ·100, (d) ·200 high power field. T: tumor, B: bone, M: muscle, black arrow: CD-31 stained microvessel. (e) Blood vessel density was determined by counting the vessels with CD-31 (+) vascular endothelial cells per ·200 high power fields. Blood vessel density in tumors treated with tumstatin was compared to the control groups. In the tumstatin treated group, the blood vessel density was significantly lower than that in the control groups: A–C (P < 0.05). ((A) control group without any intervention, (B) control group treated with normal saline 1 ml, (C) control group treated with tumstatin-negative media, and (D) tumstatin concentration 50 lg/ml).
and reduced the microvessel density in tumor tissues in vivo, which is consistent with the previous results.25,26 Tumstatin is thought to inhibit tumor cells directly, through the same signaling pathway, aVb3 integrin.34,39–41 As the tumor grows, the expression of aVb3 integrin increases, tumstatin works directly on tumor cells through aVb3 integrin. In other experiments, our data showed the induction of apoptosis of the tumor cells (the elevation of tumor necrosis factor-a level), possibly through aVb3 integrin (data not shown here).
To evaluate the in vivo effect of tumstatin, we first evaluated an oral tongue squamous cell carcinoma model. However, the tumor mass on the tongue frequently impaired the oral intake of the animals, which resulted in the loss of more than 30% of baseline body weight at 3–4 weeks. Thus, we could not follow the animals over sufficient time for the development of metastasis. However, the tumor in the floor of the mouth did not severely affect oral intake and we could monitor the occurrence of metastases for up to 5 weeks without the significant loss of body weight. Subse-
Peritumor injections of purified tumstatin delay tumor growth and lymphatic metastasis quently, these tumors invaded into the mandible bone, metastasized to the submandibular lymph nodes, and lung as the primary tumor grew, which can be a model for tumor growth in human patients. Our data showed that tumstatin delayed the primary tumor growth of oral squamous cell carcinomas and lymph node metastasis, but failed to achieve tumor regression in the animal models. These findings are consistent with the previously reported results,25,26 where tumstatin was used in relatively high concentrations (80–100 lg/ml) compared to the 50 lg/ml in our study. Therefore, the results suggest that tumstatin treatment alone is not enough to eradicate oral cancers as a single treatment modality. Considering the beneficial effects of tumstatin, tumstatin might have an important role in the adjuvant setting in combination with conventional treatment modalities or targeted therapy. The limitations of this study include the following. First, the purified tumstatin protein was obtained from the transformation of Tn 5B1-4 cells, transfected with the plasmid containing tumstatin cDNA using the lipofection method, as previously described.35,36 This method is time consuming and requires multiple steps for protein purification; therefore, high concentrations (over 100 lg/ml) of tumstatin were not available to be used in this study. For future clinical application, a high throughput method of purification is needed. Second, peritumor injection of the tumstatin protein may be associated with an error of the delivery into the exact tumor formation site. Therefore, in situ expression of tumstatin, after gene transfection into the target cells, should be performed in subsequent studies. Nevertheless, this study showed that tumstatin could inhibit the tumor growth of oral squamous cell carcinomas, similar to other cancers by reducing tumor angiogenesis. However, tumstatin alone failed to achieve tumor regression. Further study is needed to investigate the potential role of tumstatin as an adjuvant therapy for oral cancers, in combination with the conventional therapy.
Conflict of interest statement None declared.
Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (Ministry of Education and Human Resources Development, MOEHRD) (KRF2007-313-E00263).
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