[Clinical Assessment of Chemosensitivity Test in Xeno-free Culture of Autologous 
Malignant Effusion Cells from Patients with Advanced Lung Adenocarcinoma].

BACKGROUND: A great individual differences to chemotherapeutic effects existed in the patient with advanced lung cancer. How to choose the optimum regimens to achieve the individuation and maximum effect of chemotherapy for lung cancer is worth exploring. The study was designed to examine the effect of ex vitro chemo-sensitivity assay in xeno-free culture of autologous malignant effusion cells from patients with advanced lung adenocarcinoma. METHODS: The 50 treatment-naive patients with lung adenocarcinoma complicated with malignant pleural or pericardial effusions were enrolled. Effusions of all cases had been controlled by closed drainage and 300 mL-500 mL of which were retained under sterile condition from 25 cases (Chemo-sensitivity group). Primary malignant effusion cells were isolated from autologous effusions of the patients. Then, xeno-free culture (average 11 days) were intervened with 8 chemotherapeutic drugs commonly used in clinical practice and were determined by CCK-8 assay. Optimum regimens were selected for chemotherapy based on the results of chemosensitivity test. As a contrast, chemotherapy regimens for the other 25 patients (Control group) were on the basis of physician's clinical experience. RESULTS: After four cycles of chemotherapy, in Chemo-sensitivity group, 17 (68.0%) cases were determined for partial response (PR), 5 (20.0%) cases for stable disease (SD), and the objective response rate (ORR) was 68.0%, the disease control rate (DCR) was 88.0%. Meanwhile, in Control group, 9 (36.0%) cases were determined for PR, 7 (28.0%) cases for SD, and, the ORR was 36.0%, the DCR was 64.0%. There were significant differences between the two groups in ORR and DCR (P<0.05). To the end of follow-up, there were 21 cases of death in Chemo-sensitivity group as well as 22 cases in Control group. The mean progression-free survival (PFS) in Chemo-sensitivity group and Control group respectively were 10.0 months and 5.8 months, and the mean overall survival (OS) in the two groups were 30.2 months and 21.2 months respectively. There were also significant differences between the two groups in PFS and OS (P<0.05). Furthermore, the adverse reactions in both groups were mild and controllable. CONCLUSIONS: Xeno-free culture of autologous malignant effusion cells from patients with advanced lung adenocarcinoma and ex vitro chemo-sensitivity assay are beneficial to the rational choices of chemotherapeutic agents used in patients with lung adenocarcinoma complicated with malignant effusions, which is a worthy trial in personalized cell culture for individualized cancer therapy and further studies.

Everolimus and zoledronic acid--a potential synergistic treatment for lung adenocarcinoma bone metastasis.

Non-small-cell lung cancer (NSCLC) frequently metastasizes to bone. It is known that zoledronic acid is cytostatic to tumors, and everolimus, the inhibitor for mammalian target of the rapamycin, could inhibit many types of cancer. Herein, we evaluated the effect of zoledronic acid alone and in combination with everolimus on treating lung adenocarcinoma bone metastasis in vitro and in vivo. Mice treated with zoledronic acid in combination with everolimus had more apoptotic lung cancer cells and more cells were arrested in the G1/G0 phase. The phosphorylation of p70S6K was inhibited in the combination treatment group. Lung cancer cell invasion was also significantly inhibited in the group with combination treatment in vitro. Bone nuclear scans revealed more metastatic lesions in controls compared with those in the combination treatment group. Bone scans and radiographic images indicated that combination therapy significantly reduced bone metastasis. The moderate survival rate suggested that the drug combination was synergistic, which can delay NSCLC bone metastasis and prolong survival in vivo.

Characterization of the attenuation of breast cancer bone metastasis in mice by zoledronic acid using (99m)Tc bone scintigraphy.

Metastatic breast cancer often metastasizes to bone. The purposes of the study were (1) to evaluate the use of (99m)Tc-MDP bone scintigraphy for detection of metastatic bone lesions, and (2) to determine the efficacy of zoledronic acid in mice with breast cancer bone metastasis. All tumor-bearing mice were analyzed with radionuclide bone scintigraphy, X-ray, and histological analysis. The metastatic bone tissue was also harvested and analyzed by western blotting and real-time qPCR. Interestingly, zoledronic acid significantly decreased both the tumor burden and the incidence of bone metastasis in mice. In addition, histomorphometric, stereological, and molecular biology analyses demonstrated that zoledronic acid may function to inhibit breast cancer cell growth in the bone microenvironment and regulate the function of osteoblasts and osteoclasts in tumor-bearing mice. Finally, the attenuation of breast cancer bone metastasis using zoledronic acid can be accurately characterized by (99m)Tc bone scintigraphy in mice.

Novel esophageal squamous cell carcinoma bone metastatic clone isolated by scintigraphy, X ray and micro PET/CT.

AIM: To establish a Chinese esophageal squamous cell carcinoma (ESCC) cell line with high bone metastasis potency using (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) micro-pinhole scintigraphy, X ray and micro-positron emission tomography/computed tomography (PET/CT) for exploring the mechanism of occurrence and development in esophageal cancer. METHODS: The cells came from a BALB/c nu/nu immunodeficient mouse, and oncogenic tumor tissue was from a surgical specimen from a 61-year-old male patient with ESCC. The cell growth curve was mapped and analysis of chromosome karyotype was performed. Approximately 1 × 106 oncogenic cells were injected into the left cardiac ventricle of immunodeficient mice. The bone metastatic lesions of tumor-bearing mice were detected by (99m)Tc-MDP scintigraphy, micro-PET/CT and X-ray, and were resected from the mice under deep anesthesia. The bone metastatic cells in the lesions were used for culture and for repeated intracardiac inoculation. This in vivo/in vitro experimental metastasis study was repeated for four cycles. All of the suspicious bone sites were confirmed by pathology. Real-time polymerase chain reaction was used to compare the gene expression in the parental cells and in the bone metastatic clone. RESULTS: The surgical specimen was implanted subcutaneously in immunodeficient mice and the tumorigenesis rate was 100%. First-passage oncogenic cells were named CEK-Sq-1. The chromosome karyotype analysis of the cell line was hypotriploid. The bone metastasis rate went from 20% with the first-passage oncogenic cells via intracardiac inoculation to 90% after four cycles. The established bone metastasis clone named CEK-Sq-1BM had a high potential to metastasize in bone, including mandible, humerus, thoracic and lumbar vertebrae, scapula and femur. The bone metastasis lesions were successfully detected by micro-pinhole bone scintigraphy, micro-PET/CT, and X-ray. The sensitivity, specificity and accuracy of the micro-pinhole scintigraphy, X-ray, and micro-PET/CT imaging examinations were: 89.66%/32%/80%, 88.2%/100%/89.2%, and 88.75%/77.5%/87.5%, respectively. Some gene expression difference was found between parental and bone metastasis cells. CONCLUSION: This newly established Chinese ESCC cell line and animal model may provide a useful tool for the study of the pathogenesis and development of esophageal carcinoma.

[An experimental study on the Chinese lung adenocarcinoma cell clone CPA-Yang1-BR with brain metastasis potency in nude mice and in vivo imaging research].

BACKGROUND AND OBJECTIVE: Lung cancer is the leading cause of cancer-related death in men and women. It is also the most common cause of brain metastases. A brain metastasis model is difficult to be established because of the presence of the blood-brain barrier (BBB) and the lack of optimal methods for detecting brain metastasis in nude mice. Thus, the establishment of a Chinese lung adenocarcinoma cell line and its animal model with brain metastasis potency and in vivo research is of great significance. METHODS: CPA-Yang1 cells were obtained from a patient with human lung adenocarcinoma by lentiviral vector-mediated transfection of green fluorescence protein. Intracardiac inoculation of the cells was performed in nude mice, and brain metastatic lesions were detected using micro ¹8F FDG-PET/CT scanners, small animal in vivo imaging system for fluorescence, radionuclide and X ray fused imaging, magnetic resonance imaging (MRI) with sense body detection, and resection. The samples were divided into two parts for cell culture and histological diagnosis. The process was repeated in vivo and in vitro for four cycles to obtain a novel cell clone, CPA-Yang1-BR. RESULTS: A novel cell clone, CPA-Yang1-BR, was obtained with a brain metastatic rate of 50%. The use of MRI for the detection of brain metastases has obvious advantages. CONCLUSIONS: An experimental Chinese lung adenocarcinoma cell clone (CPA-Yang1-BR) and its animal model with brain metastasis potency in nude mice were established. MRI with sense body or micro MRI may be used as a sensitive, accurate, and noninvasive method to detect experimental brain metastases in intact live immunodeficient mice. The results of this study may serve as a technical platform for brain metastases from lung adenocarcinoma.

[Establishment of a novel chinese human lung adenocarcinoma cell line CPA-Yang3 and its real bone metastasis clone CPA-Yang3BM in immunodeficient mice].

BACKGROUND AND OBJECTIVE: The recurrence and metastasis of lung cancer is a tough problem worldwide. The aim of this study is to establish a novel Chinese lung adenocarcinoma cell line and its real bone-seeking clone sub-line for exploring the molecular mechanism of lung cancer metastasis. METHODS: The cells came from the pleural effusion of a sixty-five years old female patient with lung adenocarcinoma and supraclavicular lymph node metastases. The gene expression was detected by real-time quantitative PCR. Intracardiac injection of the cells into nude mice was performed and in vivo imaging was obtained by bone scintigraphy and conventional radiography. Bone metastases were determined on bone scintigraphy and then the lesions were resected under deep anesthesia for bone metastasis cancer cell culture. The process was repeated for four cycles to obtain a real bone-seeking clone. RESULTS: The tumorigenesis rate started at 4th passage in immunodeficient mice via subcutaneously and as well as later passages. Approximately 1×106 cancer cells were injected into left cardiac ventricle of immunodeficient mice resulted bone metastasis sites were successfully revealed by bone scintigraphy and pathological diagnosis, the mandible (100%), scapula (33%), humerus (50%), vertebral column (50%), femur (66.7%) and accompanied invasion with other organs, the adrenal gland (17%), pulmonary (33%), liver (50%), submaxillary gland (33%) in the mice after inoculation two-three weeks. The chromosome karyotype analysis of the cells was subdiploid. Quantitative real-time PCR was used to examined and compared with SPC-A-1 lung adenocarcinoma, ESM1, VEGF-C, IL-6, IL-8, AR, SVIL, FN1 genes were overexpress. The novel cell was named CPA-Yang3. The femur metastasis cell was repeated in vivo-in vitro-in vivo with three cycles and harvested a real bone metastasis clone. It was named CPA-Yang3BM. CONCLUSIONS: Tne characteristics of novel strain CPAYang3 is a highly metastasis cell line of Chinese lung adenocarcinoma and CPA-Yang3BM is a real bone-seeking clone.

Establishment of an experimental human lung adenocarcinoma cell line SPC-A-1BM with high bone metastases potency by (99m)Tc-MDP bone scintigraphy.

BACKGROUND: Bone metastasis is one of the most common clinical phenomena of late stage lung cancer. A major impediment to understanding the pathogenesis of bone metastasis has been the lack of an appropriate animal and cell model. This study aims to establish human lung adenocarcinoma cell line with highly bone metastases potency with (99m)Tc-MDP bone scintigraphy. METHODS: The human lung adenocarcinoma cancer cells SPC-A-1 were injected into the left cardiac ventricle of NIH-Beige-Nude-XID (NIH-BNX) immunodeficient mice. The metastatic lesions of tumor-bearing mice were imaged with (99m)Tc-MDP bone scintigraphy on a Siemens multi-single photon emission computed tomography. Pinhole images were acquired on a GZ-B conventional gamma camera with a self-designed pinhole collimator. The mice with bone metastasis were sacrificed under deep anesthesia, and the lesions were resected. Bone metastatic cancer cells in the resected lesions were subjected for culture and then reinoculated into the NIH-BNX mice through left cardiac ventricle. The process was repeated for eight cycles to obtain a novel cell subline SPC-A-1BM. Real-time polymerase chain reaction (PCR) was used to compare the gene expression differences in the parental and SPC-A-1BM cells. RESULTS: The bone metastasis sites were successfully revealed by bone scintigraphy. The established bone metastasis cell line SPC-A-1BM had a high potential to metastasize in bone, including mandible, humerus, thoracic vertebra, lumbar, femur, patella, ilium and cartilage rib. The expression level of vascular endothelial growth factor gene family, Bcl-2 and cell adhesion-related genes ECM1, ESM1, AF1Q, SERPINE2 and FN1 were examined. Gene expression difference was found between parental and bone-seeking metastasis cell SPC-A-1BM, which indicates SPC-A-1BM has metastatic capacity vs. its parental cells. CONCLUSION: SPC-A-1BM is a bone-seeking metastasis human lung adenocarcinoma cell line. Bone scintigraphy may be used as an accurate, sensitive, noninvasive tool to detect experimental bone metastases in intact live NIH-BNX mice.

[Establishment of a Novel Chinese Human Lung Adenocarcinoma Cell Line CPA-Yang1 which Produces Highly Bone Metastases in Immunodeficient Mice.].

BACKGROUND: Lung cancer is a common malignancy and is the major determinant of overall cancer mortality worldwidely. Approximately 70% of lung cancer patients will die from metastatic diseases. The aim of this study is to establish a Chinese lung adenocarcinoma cell line with high metastasis potency for exploring the mechanism of occurrence and development in lung cancer. METHODS: The cell came from the pericardial effusion of a fifty-year old male patient with lung adenocarcinoma and the cells in primary culture were obtained successfully. Immunodeficient mice tumorigenicity was assayed. The cell growth curve was mappinged. Analysis of chromosome karyotype was tested. Tumor marker was detected by radioimmunoassay. The gene expression was measured by real-time quantitative PCR. RESULTS: The first passage cells were planted in immunodeficient mice subcutaneously and the tumorigenesis rate was 100% as well as later passages. Under the microscope, the cell showed larger and semi-suspension, semi-adherence. Approximately 0.8*10(6) cancerous cells were injected into left cardiac ventricle or tail vein of immunodeficient mice resulted start to appear lower limb paralysis and spine swelling deformation in the mice after inoculation two-three weeks. The bone metastasis rate was 90% in the tumor bearing mice by bone scintigraphy and pathology and only pulmonary metastasis 10% at the same time. The chromosome karyotype analysis of the cells was sub-triploid. The tumor marker CEA was detected in higher secretion by radioimmunoassay in the cell culture suspension. Quantitative real-time PCR was used to examined and compared SPC-A-1 lung adenocarcinoma, VEGF-C, IL-6, IL-8, genes were overexpressed. The novel cell line was named CPA-Yang1. CONCLUSIONS: Tne novel strain CPA-Yang1 is an parental cell with characteristics of bone metastasis of Chinese lung adenocarcinoma. It has stable traits, highly metastatic ability and a good experimental model for lung cancer research.

[Establishment and Characterization of a Novel Chinese Human Lung Adenocarcinoma Cell Line CPA-Yang2 in Immunodeficient Mice.].

BACKGROUND: The recurrence, metastasis and multidrug resistance (MDR) in lung cancer are the tough problems worldwide. This study was to establish a novel chinese lung adenocarcinoma cell line with high metastasis potential for exploring the mechanism of reccurrence, development and MDR in lung cancer. METHODS: The cell came from the abdominal dropsy of a fifty-six years old female patient with lung adenocarcinoma and the tumor markers CA125, CYFRA21-1, CEA, NSE were detected to be higher secretion by radioimmunoassay in the abdominal dropsy. Tumorigenicity of immunodeficient mice was confirmed in 8th passage. The cell growth curve was mapped. Analysis of chromosome karyotype was tested. The gene expression was measured by real-time quantitative PCR. RESULTS: The tumorigenesis rate started at 8th passage in 3/10 immunodeficient mice via subcutaneously and the fully tumorigenicity was at 11th passage as well as later passages. Under the microscope, the cell showed oval-shap and adherence. The chromosome karyotype analysis of the cells was sub-triploid. Approximately 1*10(6) and 1.5*10(6) cancerous cells were injected into left cardiac ventricle and tail vein of immunodeficient mice respectively. The results showed multiorgan metastasis in the mice after three-four weeks, including mandible, scapula, humerus, vertebral column, femur, rib and brain, liver, adrenal gland, pulmonary in the mice after inoculation. The bone metastasis rate was 100% in the tumor bearing mice by bone scintigraphy and pathology. Quantitative real-time PCR was used to examined and compared with SPC-A-1 lung adenocarcinoma, ESM1, VEGF-C, IL-6, IL-8, AR genes were overexpressed. The novel cell was named CPA-Yang2 CONCLUSIONS: The characteristics of novel strain CPA-Yang2 is a highly metastasis cell line of Chinese lung adenocarcinoma. It has stable traits, highly metastasis ability and maybe is a MDR lung cancerous cell line. Of course, it's a good experimental model for lung cancer research.