Establishment And Biologic Activity Of A Highly Brain Metastatic Nonsmall Cell Lung Cancer Cell Sub-clone

BACKGROUND Primary lung cancer is one of the commonest malignant tumors threatening the health of human being. Its incidence rate and mortality rate are both very high. The mortality of lung cancer is often ascribed to the distant organ metastases, most patients with lung cancer were staged into advanced disease on diagnosis because of systemic metastases. About 25-30% patients with lung cancer suffere from brain metastases. Brain metastatic lesions develop rapidly and generate many obvious symptoms. Brain metastases becomes a fatal factor because of the resistance to drugs which make it hard to be cured. So brain metastases is a serious problem demanded to be solved. Unfortunately, the mechanisms of brain metastases are still unclear. The studies are always focus on earlier detection, chemical therapy, surgery and observation of survival. The basic research of brain metastases of lung cancer is very weak. So we are trying to select a sub-clone from a common lung adencarcinoma cell line and compare its biologic activity to its mother-line, aiming to reveal some mechanisms of brain metastases of lung cancer.OBJECTIVE The purpose of this study was to select a sub-clone with increased potential of brain metastases through repeated injection of human lung cancer cells into the tail vein of nude mice, and to establish an animal model of brain metastatases. Furthermore, In order to reveal some mechanisms of brain metastases in lung cancer, the biologic activities were compared among these cell lines: selected sub-clone with increased potential of brain metastases, PC14 which metastasize to brain occasionaly and A549 which never metastasize to brain. In vitro cell attachment, migration and invasion assays with ECM were performed. The expressions of VEGF and MMP-9 between these cell lines were also measured and compared by immunohistochemical staining. The different will reveal some mechanisims of brain metastases of lung cancer.METHODS A lung adencarcinoma cell line, PC14, was selected for establishment of a highly brain metastatic sub-clone. The tumor cells were injected into the tail vein of nude mice. Five weeks later, the injected mice were sacrificed and the brain metastatic neoplasms were primarily cultured. Then collect the primarily cultured tumor cells and injected them into nude mice again. Four or five cycles were carried till a highly brain metastatic subclone was selected. Then compared the biologic activity between these cell lines: the selected highly brain metastatic sub-clone, PC14 which occasionaly metastasize to brain and A549 which never metastasize to brain. 1) In vitro cell adherent capability was carried on the cultured surface coated with extracellular matrix gel and measured by MTT assay. 2) The cell migratory capability was evaluated by number of cells which migrate from millicell inner chamber to outer chamber through an 8μm microporous membrane, the number of cells was measured by MTT assay. 3) The millicell chamber microporous membrane was coated with extracellular matrix gel, then the microporous membrane would have the similar construction as ECM and base membrane, the cell invasive capability was evaluated by the number of cells which invade from millicell inner chamber to outside chamber through ECM and base membrane, the number of cells was also measured by MTT assay. VEGF and MMP-9 expression of these cell lines was measured and compared by LsAB immunohistochemical staining.RESULTS After four cycles, a sub-clone with enhanced capability of brain metastases compared with PC14, named as PC14/B, was established, which could induce about 4/5 brain metastases through tail vein injection in nude mice. PC14/B has similar characters of distant organ metasetsese as PC14 except brain. The factors of adhesion of PC14/B, PC14 and A549 cell lines were 0.24±0.03, 0.09±0.03 and 0.12±0.03 respectively(P=0.004),PC14/B has an enhanced adherent capability than the other two(P value was 0.001 and 0.011 respectively). The cells migrating through microporous membrane were measured by MTT assay, the ODs were 0.51±0.12,0.32±0.13 and 0.19±0.06(P=0.007),PC14/B has an enhanced migratory capability than the other two(P value was 0.034 and 0.002 respectively). The Cells invading from millicell inner chamber to outside chamber through ECM and base membrane were measured by MTT assay, the ODs were 0.69±0.17,0.38±0.06 and 0.27±0.06(P=0.001),PC14/B has an increased invasive capability than the other two(P value was 0.005 and <0.001 respectively). But no difference had been observed in attachment and migration experiments between PC14 and A549, the former has a higher invasive capability than the later. VEGF and MMP-9 expressed higher in PC14/B and A549 than PC14.CONCLUSIONS Repeated intravenous injection of lung cancer cells maybe a possible method to establish a highly brain metastatic subclone and an in vivo model of experimental brain metastasis in nude mice. The results indicate that VEGF and MMP-9 enhanced the adherent, migratory and invasive capabilities in higher brain metastases subclone cells. They maybe promote brain metastases in lung cancer, but they are not enough to induce brain metastases.