The effect of hyperbaric oxygen on growth of human squamous cell carcinoma xenografts.

Hyperbaric oxygen is an important adjunct to the treatment of patients with head and neck cancer with existing or recurrent wound healing problems. Anecdotal clinical observations and a recent study of chemically induced oral cancer in hamsters have raised concern that hyperbaric oxygen therapy may accelerate tumor growth in such patients. This study evaluated the effect of hyperbaric oxygen therapy on the growth of human squamous cell carcinoma xenografts in a proved animal model. Fresh tumor specimens from three patients with head and neck squamous cell carcinoma of varying degrees of differentiation were first subcutaneously transplanted into a nude mouse host. Growing xenografts were then transplanted into one of three mouse groups. Half of the mice in each group were given hyperbaric oxygen therapy. The transplant volume as an index of tumor growth was measured in controls and mice given hyperbaric oxygen therapy six times during the 3-week course. Xenograft growth was almost linear in all mice. No statistical difference in overall group mean growth rates was observed in mice given hyperbaric oxygen or control mice regardless of the degree of tumor differentiation. Xenograft tissue from all mice was microscopically examined for tumor mitotic indices and degree of differentiation. This study suggests that hyperbaric oxygen therapy has no effect on established tumor xenograft growth.     

Radiolabeled antibody therapy for squamous cell carcinoma of the head and neck.

Immunohistochemical staining for ferritin was performed on 11 human head and neck squamous cell carcinomas transplanted in nude mouse xenografts. Seven tumors were found to be positive. Using ferritin as a tumor antigen target, escalating doses of yttrium-90-labeled antiferritin antibodies were injected intravascularly into nude mice that were transplanted with a ferritin-positive human squamous cell carcinoma. Forty-five days after injection, the mean treated tumor size was 25.6% of control in the 100-microCi group, and 20.6% of control in the 200-microCi group. Ninety days after injection the mean tumor size was 27.5% that of control in the 100-microCi group and 31.7% in the 200-microCi group. Higher doses of radiation (300 and 400 microCi per mouse) caused death of most of the animals due to radiation toxicity. Presensitization of the animal, before antibody injection, with a bolus intraperitoneal injection of 7.5 mg of cisplatin per kilogram of body weight, resulted in further reduction in tumor size when compared with antibody alone or cisplatin alone. This study demonstrates that radioimmunotherapy with selected doses of yttrium-90-labeled antiferritin antibodies is effective against human head and neck squamous cell carcinoma xenografts in nude mice.     

Reovirus oncolysis in human head and neck squamous carcinoma cells

OBJECTIVE: To determine whether reovirus, a double-standed RNA virus is effective on the growth of a human head and neck squamous cell carcinoma cell line. DESIGNS: In vitro cell proliferation assay, KB cells, a human oral floor squamous cell carcinoma cell line, were treated with reovirus and the number of cells was quantitated by an assay, using trypan blue staining. In vivo tumor growth assay, KB cells were injected subcutaneously into athymic nude mice, which were given an intratumoral injection of reovirus to a maximum four times in every week. The tumor size was measured once a week. Simultaneously, apoptosis and necrosis of KB cells were investigated, using technique of immunohistochemistry. RESULTS: In vitro, the multiplication of the KB cell was inhibited depending on the concentration of reovirus. In vivo, athymic nude mice bearing KB tumors were injected with the virus intratumorally, and the tumor growth was suppressed proportionally depending on the injection time of reovirus. Necrosis was recognized extensively in the pathological specimen. On the other hand, apoptosis-inducing effect was not obvious in these specimen. CONCLUSIONS: Reovirus suppressed tumor growth of KB cells in vivo as well as in vitro. The possibility that reovirus could become the means of treatment for head and neck carcinoma, was suggested with further work.     

The nude mouse xenograft system: a model for photodetection and photodynamic therapy in head and neck squamous cell carcinoma

Mechanisms for hematoporphyrin derivative (HPD) localization in malignant tissue and in photodynamic therapy (PDT) have not been established. The authors' experience with human cancer xenografts in nude mice as a tumor system for the experimental study of these mechanisms is described. Human mucosal head and neck squamous cell carcinoma was successfully transplanted to nude mice in 29 per cent of trials and serially passed through multiple generations in three tumor lines. Time required for progressive growth averaged 13.9 days, and 300 mm3 tumors were obtained in four weeks. Take rates per passage varied, in part because of infection, and exponential growth rates varied among the three lines. However, within a single line exponential growth rates were similar. Average growth constants for the period of exponential growth were 0.14, 0.13, and 0.09 for the three lines. Fluorescent microscopy of excised xenografts revealed HPD fluorescence principally in connective tissue cells. Minimal fluorescence was seen in malignant epithelial cells. Spectrophotometric measures of HPD uptake in homogenized tissue showed highest values in liver. Tumor HPD levels were higher than those for either skin or muscle. The authors conclude that this tumor system can be used effectively to study HPD and PDT in head and neck cancer.     

小干扰RNA对喉鳞状细胞癌裸鼠移植瘤增殖细胞核抗原及p53蛋白表达的影响

目的：应用小干扰RNA（siRNA）技术抑制裸鼠喉癌皮下移植瘤人端粒酶逆转录酶（hTERT）基因表达,探讨移植瘤中增殖细胞核抗原（PCNA）及p53蛋白表达的变化。方法：根据hTERT cDNA序列构建表达短发夹RNA（shRNA）的、靶向hTERT mRNA的真核表达质粒pshRNA,其载体质粒含荧光素报告基因。建立人喉鳞状细胞癌Hep-2细胞株裸鼠皮下接种模型,将pshRNA转染入荷瘤裸鼠瘤体内,观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤体内的表达;以免疫组织化学SP法检测PCNA及p53蛋白在肿瘤内的表达。结果：所有裸鼠均接种成功,5d后可见皮下肿瘤形成,14d左右肿瘤直径达5～7mm。pshRNA及空质粒载体转染入瘤体后,共聚焦显微镜下见癌组织中有绿色荧光表达。病理学检查发现：pshRNA组肿瘤生长受到抑制,细胞分裂少见,可见大量癌细胞坏死。与注射生理盐水的对照组比较,转染质粒完毕后7d抑瘤率为76．50％,P〈0．01。pshRNA治疗后瘤体内PCNA蛋白表达显著下调（P〈0．05）,p53蛋白表达显著上调（P〈0．05）。结论：靶向hTERT mRNA的shRNA可显著抑制人喉癌裸鼠移植瘤的生长,其机制可能是诱导PCNA下调,促进p53蛋白表达所致。     

An in vivo model for squamous cell carcinoma of the head and neck

In recent years, the successful grafting of human tumors into immune deprived animals has spurred the hopes of developing a reliable in vivo model for evaluation of human cancer. The nude mouse has created new perspectives in many different areas of cancer research, and has proved to be the most useful recipient of any animal system for tumor heterotransplantation. This study determined which of several squamous cell carcinoma (SCC) cell lines would reproducibly grow as heterotransplants in nude mice, and allowed an opportunity to determine whether characteristics of the tumor changed as a result of propagation in tissue culture. Sixteen (76%) of the 21 SCC lines heterotransplanted in nude mice remained as viable tumor nodules. Nine (43%) of these lines reproducibly produced progressively enlarging tumor masses. Five (24%) SCC lines failed to grow as heterotransplants. The average lag phase between implantation and the beginning of growth for all SCC lines tested was approximately 7 weeks and emphasizes the need for prolonged observation of nude mice following SCC heterotransplantation. The rate and pattern of growth of SCC in nude mice appears to be related specifically to the tumorigenic factors of a given cell line. The concurrent presence of other SCC or non-SCC tumors in a given mouse do not appear to influence the growth of a specific cell line. In addition, specific host factors that might vary from one mouse to another did not alter the growth patterns of SCC. Histological examination of the SCC lines successfully heterotransplanted in nude mice showed similar histological appearances to the original tumor in the human host.     

Enhancement of cyclophosphamide sensitivity in squamous cell carcinoma transfected with cytochrome P-450 2B1

PURPOSE: Cyclophosphamide (CPA) has been shown to be quite effective and safe in the treatment of leukemia, but it had shown almost no efficacy in the treatment of solid tumors such as head and neck cancers. The purpose of this study was to determine whether head and neck squamous cell carcinomas can be made more sensitive to the cytotoxic effect of CPA by shifting the balance from the degradative to the activation pathway. MATERIALS AND METHODS: Human SCC-25 squamous carcinoma cells were transfected by adenoviral vector containing P-450 2B1 gene and were identified for P-450 2B1 expression by Northern blot analysis. In vitro cytotoxic assay after CPA exposure was performed using these cells. Human KB oral base squamous cell carcinoma cells were injected subcutaneously to the backs of nude mice. Then Ad.CMV-P-450 2B1 was injected intratumorally. At 48 hours after the injection of Ad.CMV-P-450 2B1, CPA was injected into the peritoneal cavity of the animals and the volume of the tumor was determined with the passage of time. All the animal procedures were performed under the guidance of the committee in our animal care facility. RESULTS: The in vitro experiments found that transduction of the P-450 2B1 gene caused a nearly 30-fold increase in the sensitivity of the host cell line to CPA on the basis of the 50% inhibitory concentration. The in vivo experiments, which were conducted in nude mice, found that the combination of P-450 2B1 gene transduction and administration of CPA showed an inhibitory effect on tumor growth. CONCLUSIONS: The P-450 2B1- cyclophosphamide system may be useful approach for gene therapy of squamous cell carcinomas of head and neck.     

Fosfomycin does not inhibit the tumoricidal efficacy of cisplatinum.

OBJECTIVES: This study investigates the effect of fosfomycin on the tumoricidal efficacy of cisplatinum. STUDY DESIGN: Prospective study utilizing the FaDu squamous cell carcinoma cell line and a nude mouse tumor xenograft model. METHODS: Tumor cell growth was assessed in vitro in the presence of cisplatinum and/or fosfomycin utilizing the MTT assay. An optimal cisplatinum dose and dosing schedule was established in a nude mouse tumor xenograft model of squamous cell carcinoma. Using this model, fosfomycin was tested at three dosages and tumor growth monitored over 4 weeks. RESULTS: Mice treated with cisplatinum and fosfomycin had smaller tumors than those treated with cisplatinum alone (P<.01). CONCLUSIONS: This study is the first demonstration that fosfomycin does not inhibit the tumoricidal efficacy of cisplatinum in vivo. This suggests that fosfomycin may be useful in preventing cisplatinum-induced ototoxicity and nephrotoxicity in humans without altering the tumor response rate.     

The biology of tumor invasion, angiogenesis and lymph node metastasis

It is now well established that the development of cervical metastases, in particular those with extranodal extension of tumor, negatively impacts both regional control and survival of patients with laryngeal carcinoma. This chapter will begin with an introduction of the important molecular events associated with the transition of the squamous epithelium of the upper aerodigestive tract to metastatic squamous cell carcinoma. We will then review the critical cellular events identified as the tumor progresses from an in situ to invasive and finally a metastatic head and neck squamous cell carcinoma. Finally we will review data from our own and other laboratories which are studying the process of new blood vessel growth (angiogenesis) induced by tumor-derived growth factors. As we develop a better understanding of the cellular and molecular mechanisms of metastasis in head and neck squamous cell carcinoma, new therapies effective at preventing the development of secondary tumors can be realized ultimately increasing the patient's survival.     

Enhancement of Cisplatin sensitivity in squamous cell carcinoma of the head and neck transfected with a survivin antisense gene

OBJECTIVE: To study a new method for treating squamous cell carcinoma of the head and neck using a survivin antisense gene. DESIGN: An adenoviral vector encoding surviving antisense was used for in vitro and in vivo experiments. KB cells were treated with pAd.CMV[cytomegalovirus]-antisurvivin. Western blot analysis, in vitro cytotoxic assay, and in vivo experiment were performed. SETTING: In vitro and in vivo study of head and neck cancer cell line KB. SUBJECTS: Male, 5-week-old BALB/c nude mice. MAIN OUTCOME MEASURES: Expression of survivin was assessed using Western blot analysis. The effect of antisurvivin to KB cells was measured by cytotoxic assay (in vitro) and tumor volume (in vivo). RESULTS: In the in vitro experiments, transduction of the survivin antisense gene caused a nearly 12-fold increase in the sensitivity of KB cells to cisplatin, as reflected by the 50% inhibitory concentration. In in vivo experiments in nude mice, tumor growth was more inhibited by the combination of cisplatin and survivin antisense gene transduction compared with either alone. CONCLUSION: These findings suggest that survivin targeting with adenoviral antisense vectors might be used for selective therapy of squamous cell carcinomas of the head and neck.     

Anthrapyrazoles and Interstitial Laser Phototherapy for Experimental Treatment of Squamous Cell Carcinoma

Interstitial laser therapy (ILT) is an effective palliative treatment for advanced head and neck cancer, but recurrence often is seen at the margin. The objective of the current study was to test combined drug and laser therapy as an experimental approach for improved treatment of human squamous cell carcinoma (SCCA). Human SCCA tumor transplants were grown in nude mice and injected with the photosensitive anthrapyrazole CI-941 before ILT. Intralesional drug injections alone at levels ranging from 60 to 1200 μg/gm of tumor induced a growth delay at the higher doses, but recurrence was seen in all 35 tumors tested. SCCA tumor transplants injected with 240 μg/gm CI-941 followed after 4 hours by ILT with the KTP532 laser led to a complete response rate of 72% (21/29) compared with 45% (13/29) for ILT alone. Laser chemotherapy was a significant improvement compared with ILT when partial and complete responses were combined (P < 0.03). The results provide preclinical evidence that laser chemotherapy may become a useful minimally invasive treatment for advanced squamous cell carcinoma of the head and neck.     

Animal Models of Cancer in the Head and Neck Region

Animal models that resemble the cancers of the head and neck region are of paramount importance in studying the carcinogenesis of these diseases. Although several methods for modeling cancer in the head and neck are available, none are fully satisfactory. Subcutaneous xenograft models of cancer in nude mice are often used in preclinical studies. However, these models are problematic in several aspects as they lack the specific interactions that exist between the tumor cells and their native environment. Establishment of tumors at the orthotopic sites restore these distinct patterns of interactions between the tumor and the host organs that are lost or altered when the tumors are established in ectopic sites. With regard to the transgenic model of cancer in the head and neck region, it should be kept in mind that the transgene used to drive the malignant transformation may not be representative of the carcinogenic process found in human tumors. Low penetrance of tumor formation also translates into high cost and time commitment in performing studies with transgenic models. In this review, we will discuss some of the commonly used methods for modeling cancer in the head and neck region including squamous cell carcinoma of the head and neck as well as thyroid carcinoma.     

Induction of immunological antitumor effects by adenovirus-mediated gene transfer of B7-1 in a murine squamous cell carcinoma cell line

OBJECTIVE: To evaluate the antitumor immune effects of B7-1 gene expression mediated by adenoviral vectors against squamous cell carcinoma. Transfection of the costimulatory molecule B7-1 gene into certain murine tumors increases antitumor immunity and suppresses tumor growth. DESIGN: In vitro and in vivo study. INTERVENTIONS: A murine squamous cell carcinoma cell line, KLN205, was infected with adenoviral vectors carrying either B7-1 (AdB7) or LacZ (AdCL) genes. Infected cells were injected subcutaneously into the flanks of DBA/2 mice. MAIN OUTCOME MEASURES: The growth of tumors infected with adenviral vectors was measured. RESULTS: AdB7-infected cells grew significantly slower than AdCL-infected cells in vivo, while there was no significant difference in the growth rates between the 2 groups in vitro. Moreover, significant growth suppression of rechallenged noninfected parental cells was observed in the mice immunized with AdB7-infected cells but not in those immunized with AdCL-infected cells. CONCLUSION: These results suggest that the B7-1 gene has therapeutic potential for immunotherapy against head and neck squamous cell carcinoma.     

Radioimmunotherapy with astatine-211 using chimeric monoclonal antibody U36 in head and neck squamous cell carcinoma

OBJECTIVES: In advanced head and neck squamous cell carcinoma (HNSCC), there is a need for an adjuvant treatment. We aim to evaluate the biodistribution and therapeutic effect of radioimmunotherapy using the alpha emitting, astatine-211-labeled, chimeric monoclonal antibody U36 (U36) on the HNSCC cell line UT-SCC7 in vivo. STUDY DESIGN: Xenograft tumors were inoculated subcutaneously in nude mice. Astatine-211-labeled U36 was injected intravenously with or without blocking of target with nonlabeled U36. METHODS: In the biodistribution experiments, radioactivity was measured in tumors and various organs at set time points. In the therapeutic experiments, two groups (with or without blocking) received therapy, and the tumor growth was compared with that of controls. In addition, one group received nonlabeled U36 only. RESULTS: The biodistribution experiments demonstrated that astatine-211-labeled U36 could target UT-SCC7 xenografts in nude mice. With time, uptake increased in tumors and decreased in normal organs. Nonlabeled U36 did not influence tumor growth. In the two therapy groups, 18 of 20 tumors responded to therapy by decreasing or stabilizing their volumes. Significant difference was seen between the treated groups and the controls (P < .05). CONCLUSION: The study illustrates the specific binding of astatine-211-labeled U36 to HNSCC and suggests radioimmunotherapy with the alpha emitting radionuclide to be a useful treatment modality.     

Antivascular therapy of oral tongue squamous cell carcinoma with PTK787

OBJECTIVES/HYPOTHESIS: Vascular endothelial growth factor (VEGF) is an important mediator in tumor vascularization, growth, and metastasis. We investigated whether blockade of the VEGF receptor (VEGF-R) signaling pathway by the tyrosine kinase inhibitor PTK787 combined with CPT-11, a semisynthetic camptothecin analogue, can inhibit the tumor growth and angiogenesis of squamous cell carcinoma of the oral tongue in an orthotopic nude mouse model. METHODS: JMAR human oral squamous cell carcinoma cells were injected into the tongues of nude mice. Seven days later, the mice were randomized to receive a placebo, daily oral PTK787, weekly CPT-11 injection, or PTK787 plus CPT-11. After 4 weeks of treatment, the mice underwent necropsy, and the tongue tumors, cervical lymph nodes, and lungs were removed for immunohistochemical analyses. RESULTS: CPT-11, PTK787, and PTK787 plus CPT-11 significantly decreased tumor volumes and prolonged survival. The combination treatment group had the most significant decrease in volume and increase in survival. PTK787 alone or in combination with CPT-11 reduced the phosphorylation of VEGF-R in tumor cells and tumor-associated endothelial cells, was associated with decreased microvessel density, a decreased proliferative index, and an increased apoptotic index. PTK787 alone or the combination therapy resulted in apoptosis of both tumor cells and tumor-associated endothelial cells. CONCLUSIONS: These results suggest that targeting VEGF-R tyrosine kinase activity can be an effective therapeutic approach in squamous cell carcinoma of the oral tongue.     

头颈鳞癌血管生成与颈淋巴结转移相关性研究

目的 :探讨头颈鳞癌血管生成与其颈淋巴结转移的关系以及血管内皮生长因子(VEGF)在头颈鳞癌血管生成中的作用。方法 :应用免疫组化SABC法检测 5 8例头颈鳞癌组织中微血管密度 (IMVD)及VEGF的表达。结果 :5 8例头颈鳞癌组织中IMVD为 2 3.93± 8.77,肿瘤分化程度 ,高与中、高与低间 ,IMVD差异有显著性意义 (均P <0 .0 5 ) ;中与低间 ,差异无显著性意义 (P >0 .0 5 )。颈淋巴结转移组IMVD(2 7.92± 9.11)明显高于非转移组 (2 0 .6 9± 7.0 8) ,其差异有显著性意义 (P <0 .0 1)。癌组织中VEGF表达与瘤内IMVD呈正相关 (rs=0 .4 87,P <0 .0 1)。结论 :瘤内IMVD可作为预测头颈鳞癌颈淋巴结转移的一个重要指标 ;VEGF可促进头颈鳞癌血管生成。     

The Role of Copper Suppression as an Antiangiogenic Strategy in Head and Neck Squamous Cell Carcinoma

Objective To determine whether tetrathiomolybdate (TM), a powerful chelator of copper, is capable of lowering the body stores of copper and suppressing the growth of head and neck squamous cell carcinoma in an orthotopic murine model. Study Design In vivo, murine model. Methods Twelve 8‐week‐old male C3H/HeJ mice were assigned to either a TM treatment group (n = 7) or a control group (n = 5). Serum samples were obtained from a single mouse in each group to measure the level of ceruloplasmin as a surrogate marker of total body copper on days 0, 4, and 7. Mice in both groups received a floor‐of‐mouth injection of 1.5 × 10⁵ SCC VII/SF cells. After 7 to 10 days of tumor growth the treatment group received fresh water daily, to which TM was added to achieve an oral intake of 50 mg per mouse. The control group received only fresh drinking water daily. Tumor volume measurements were obtained every other day. Microvessel density counts were assessed in the tumors by Factor VIII analysis. Results Measurable tumor growth was achieved in 100% of the mice by the tenth day. Total body copper was reduced by 28% from baseline levels in mice in the treatment group. The difference in mean tumor volume in the control group was 4.7 times greater than the TM‐treated group at the completion of treatment (3004 mm³ and 633mm³, respectively). This accounted for an overall suppression rate of 79% (P = .008; two‐tailed Student t test). In addition, microvessel density was reduced by 50% in the TM‐treated group. Conclusion In this initial study, the first of its kind in head and neck squamous cell carcinoma, we have demonstrated the ability of TM to significantly suppress both the growth of squamous cell carcinoma and tumor vascularity in this orthotopic murine model, suggesting its potential for efficacy in the treatment of this disease in humans.     

Critical remarks on the role of collagenolytic enzymes in carcinomas of the head and neck

Collagenolytic activity of eight squamous cell carcinomas of the head and neck as well as xenografts of such tumors in nude mice was investigated by two different methods. High enzyme activities seem to be present in necrotic tumor areas, whereas the germinative zones tend to show lower collagenase levels. There is experimental evidence for the assumption of a collagen-stabilizing factor (CSF). A hypothetic concept including the results of other authors is developed concerning the role of collagenase in infiltrative growth of malignant tumors. The greater part of these questions being unanswered, further studies on collagenolytic activity in malignancy will be necessary.     

表皮生长因子受体介导的多肽载体系统对人喉鳞状细胞癌的基因导入研究

目的检测一种新的以头颈肿瘤细胞表面受体为靶向的多肽基因导入系统对人喉鳞状细胞癌(简称鳞癌)的基因导入效果。方法链霉亲和素标记生物素(1abeled streptavidin biotin，LSAB)法检测喉癌组织标本和培养的Hep2喉癌细胞表皮生长因子受体(epidermal growth factor receptor，EGFR)的表达。建立人喉癌裸鼠移植瘤模型，分别构建绿色荧光蛋白标记基因和多肽载体系统、半乳糖苷酶报告基因和多肽载体系统四元复合物，分别进行报告基因和标记基因的体内外导入实验。结果EGFR在65％(15／23)喉癌组织标本呈阳性表达。Hep2细胞的EGFR亦呈阳性表达。EGFR主要位于细胞膜，部分位于细胞质内。绿色荧光蛋白基因转染Hep2后48h显微镜下可见荧光，72h达高峰，导入率大于80％(78／97)。β-半乳糖苷酶报告基因荷人喉癌裸鼠移植瘤瘤内转染12h即表达蓝染，24h蓝染程度加深，72h仍有表达。结论EGFR广泛存在于人喉癌细胞，新的多肽基因导入系统借助于EGFR可靶向性将报告或标记基因导人体内外人喉癌细胞，并得到高效表达，为头颈鳞癌的基因治疗提供了一个新的靶向高效的基因导入系统。     

Growth of xenografted head and neck cancer in nude mice pre-treated with whole body irradiation.

In an attempt to enhance the primary acceptance rate of human squamous cell carcinoma of the head and neck (HNSCC), nude mice (BALB/c) were given whole body irradiation (WBI) prior to heterotransplantation of tumour specimens. A total of 27 fresh HNSCC biopsies were transplanted, with a take rate of 15%. No difference in primary tumour acceptance rate was found between irradiated and non-irradiated mice. Only one of the accepted tumours established growth both in irradiated and non-irradiated mice. In a second experiment, three previously established tumour lines of HNSCC were re-transplanted to irradiated and non-irradiated mice. As compared with non-irradiated mice, the growth rate was lower in all tumours transplanted to irradiated mice, the difference being statistically significant in two out of the three tumour lines. The results of the study show that attempted immunosuppression by WBI of nude mice prior to tumour implantation does not improve the growth conditions of HNSCC. These findings further emphasise the complexity of the transplantation barrier against human tumour xenografts in nude mice.