| The Metastatic spread of cancer cells from a primary tumor to distant sites in the body is one of the major biological behaviors of malignant neoplasm as well as responsible for most cancer patient morbidity and mortality. It has been reported that many affairs involved in the process of tumor cell invasion and metastasis, such as lower homotypic adhension;higher heterotypic adhesion ,mobility and invasive potency;and the ability to produce proteolytic enzymes which can degrade the natural barrier surrounding neoplasm lesion by neoplasm cells or by host cells; and the ability to release lots of angiogenetic factors which play a important role in neoangio-genesis and in distant or second metastasis. It has been demonstrated that numerous molecules have changed during metastasis process, which participating in cell growth,differention, apopotosis, inflammatory respons,exocytosis, stress response, cell adhesion, energic metabolism, cell skeleton assemble and signal introduction etc. So far, we have not got a definite blueprint of the key molecules during metastasis process which will benefit us to supervise and cure neoplasm metastases. We have found before that primary neoplasm is heterogenic,only a very few of the tuomr cells released into the circulation develop into metastases. Someone else demonstrated that most cancer cells are rapidly destroyed in the circulation. Recent studies have shown that metastatic inefficiency are the post-extravasation survival and growth of cells. We now believe that every step in the metastasis process can influence tumor cells. It is the ability of self-condition and self-adaptability that decided the fate of the released tumor cells to be dormant, be destroyed, or grow into overt lesion. If a detailed comparative proteomic analysis among the tumor cells living in different steps in the whole metastasis process were carried out, maybe, we could find out some molecules playing key roles in tumor metastases, interpret the mechanism of tumor metastases, and set some targets for cure and supervise tumor metastases at the same time. Because the proteomic technique is a high-flux, high resolution and hypersensitive technique, it can be used to proceed an overall view comparative analysis of cell or tissue protein. On the basis of these considerations, we recruit two sub strains, PLA801C and PLA801D, of human giant-cell lung carcinoma cell strain PLA-801, which were reported to have different metastatic property, and proteomic strategy to carry out our research. We want to find outa series of protein cluster deeply involved in metastatic process from tumor cells living in different steps in metastatic process or from tumor cells which possess different metastatic property. In the present study, we tested the biologic behavior related to metastasis in vitro and the metastatic potency in vivo used Balb/c nude mouse of the two sub strains PLA801C and PLA801D; verified the value of the two sub strains in research about tumor metastasis mechanism; investigated the depletion magnetic separation used to purifying tumor cells from metastatic lesion; established 2-DE profiles of total proteins of tumor lesion subcutaneously transplanted to nude mouse and of the two sub strains cultured in vitro and the membrane proteins of the later. We identified two proteins separated from PLA801D membrane proteins, which were higher expressed, comparing with these from PLA801C.More detailed results are in progress. 1 Identification of the biologic behaviors related to metastasis in vitro of PLA801C and PLA801D The two strains exert a similar proliferation ability measured by growth curve, but PLA801D produced more foci in soft agar than PLA801C; In adhesive ability assay, we discovered that their homotypic adhesion potency and heterotypic adhesion potency are alike; PLA801C showed a higher motility potency and a lower invasive potency than PLA801D in cell invasion and motility assay in vitro. We concluded from above that the biologic behaviors of the two sub strains in vitro are statistic different ;metastasis is a mixed ability of poly ring-jointproperty . because that the biological parameter in vitro is well associated with metastasis potency iv vivo,and that extraorgan essay cycle time is shoter and easy to be quality controlled ,it has a great significance to carry out a proteomic analyze of the two sub strains in vitro. 2 Study the spontaneous metastatic potency in vivo of human pulmonary giant cell carcinoma cell sub strains PLA801C and PLA801D When being subcutaneously transplanted 2 ～2.5 ×106 cells to Balb/c-nu/nu nude mouse's right axillary space,the tumorigenicity of the two sub strains in nude mice were all 100%,their metastatic rates to lymph nodes and lung were 0 and 100% for PLA801C, 33.33% and 66.67% for PLA801D.The result of this assay indicated that there is a obviously different metastasis rate between the two sub strains to lung and to lymph notes .The metastatic regional difference hints that the microenvironmental selection pressure can influence the metastasis ability of tumor cells.It will reveal the molecule mechanisms of metastasis if a comparative proteomic analysis between tumor cells metastasised to diffirent region were carried out.In addition , we have got specimen for proteomic analysis in this part,which means that we have afforded material foundation for proteome analysis. 3 Investigation of depletion magnetic separation used to purifying tumor cells from metastatic lesion. To search the proteome alternations associated with the different metastatic property or with specific metastasis progress duringmetastasis process ,we will adapt comparative proteomic strategy to separate and identify the proteins which have significantly changed among PLA801C,PLA801D cultured in vitro, subcutaneous primary lesion and metastatic lesion of the two sub strains.In this study it is important to acquire pure tumor cells from small lesions to avoid interference by irrelevant proteins. The method we take to sort tumor cells must not affect the protein expressing status of tumor cells and can get a highly pure tumor cells during the sorting process. We planed to take depletion magnetic separation which is a common method used in many labs for sorting cells .We prepared comprehensive Balb/c nude mice lung single cells as antigen to immunize changchun white rabbit,and obtained polyclonal antibody, its titer reached to 1:10240. After being precipitated with saturated ammonium sulfate and proceed through ion exchange column, the prepared antibody was practically determined the optimum titer by flow cytometric analysis. The optimum titer was 736ug/100ul/106 cells, where a bright labeling of the target cells is achieved, combined with lowest nonspecific staining of non-target cells. We practiced depletion magnetic separation using the primary antibody prepared by ourselves,and the purity of sorted tumor cells from lung metastatic lesion reached to 87.86%. In this part, we laid a foundation for further study the proteins of small metastatic lesion. 4 We identified two proteins which associated whith lung cancer cell metastasis by proteomic technique We established 2-DE profiles of intracellular proteins of tumor...
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