Contribution Of BMDCs In Lung Carcinogenesis And Cancer Progress

Background and ObjectiveLung cancer is is one of the highest incidence of malignancy in the world，and hasbecome one of the major causes of death due to cancer. It is important to explore themechanism of carcinogenesis and progression for improving the early diagnosis and reducingthe mortality of lung cancer.The origin of lung cancer remains unclear. Many scholars believe that stem cells aremore likely to become cancer stem cells responsible for tumorigenesis, due to longer survivaltime with more chance of mutation. Then where do the stem cells come from? Generally,there are some intrinsic stem cells in the respiratory system responsible for tissue repair. Butwhen the damage is serious enough and intrinsic stem cells can not be an effectivesupplement, exogenous stem cells would participate in tissue repair. It has been reported thatBMDCs had potential of chemotaxis to the injury or inflammation, and contributed to therepair of injury at an early stage of lung injury, which suggestted that BMDCs may be anothersource of lung stem cells. It is reasonable to suppose that BMDCs with the characteristics ofstem cells in a continuous effect of cancer-inducing factors also might undergo aberrantdifferentiation and abnormal proliferation and involved in carcinogenesis. No agreement hasbeen reached as to BMDCs have a role in carcinogenesis of solid organs. The truecontribution of BMDCs in lung carcinogenesis remains to be determined.BMDCs exhibit specific chemotaxis towards the injury or inflammation. From anotherhand, it could be said that tumor is a kind of damage difficult to heal. Therefore, tumors couldcontinue to release chemokines and attract BMDCs to tumor microenvironment. BMDCshave ability to produce a variety of soluble factors in different environments.We also want toknow which are they produced in tumor microenvironment and what role do they play?The concept of using BMDCs, especially BMSCs, as gene vectors for targeted tumortherapy and supplement for tissue repair holds promise. However, as an important component of the tumor microenvironment, BMSCs play critical roles in regulating tumor progression.Understanding the function of BMSCs in tumor progression is necessary before BMSCs canbe used for clinical applications.In this study, the potential role of BMDCs in lung carcinogenesis was explored using anin vivo model of squamous cell carcinoma (SCC) induced by N-nitrosodiethylamine (NDEA).Green fluorescent protein (GFP)-labeled BMDCs from male donor mice were transplanted infemale recipient mice that were subsequently exposed to NDEA to identify BMDC-derivedlung epithelial cells and evaluate their participation in the various stages of lung epithelialtransformation (i.e., metaplasia, dysplasia, and carcinoma). We further explore the role ofBMDCs for lung cancer cell proliferation, migration and invasion. These results might havesignificant potential implications for both clinical cancer therapy and may provide insight intolung cancer formation and progress.Research contentsPart I. Establishment and assessment of carcinogen-induced lung cancer model andbone marrow chimeric modelSD and Wistar rats were treated with carcinogenic iodized oil by intra-trachea instillationto make lung cancer model. KM mice were treated with Urethane by intraperitoneal injection.FVB/NJ mice were treated with NDEA by Subcutaneous injection.And we tried to develop alung cancer model by B(α)P gavage. The average time to tumorigenesis, rates of tumorigenesis,tumor pathology and histologic type were compared. Bone marrow reconstitution, peripheralWBC counts, Survival rate and marker for BMDCs were analysed in bone marrow chimericmodel.An ideal model was confirmed.Part II. Study of BMDCs’ contribution to lung cancer formation in vivoEngraftment of BMDCs in lung tissue was determined using immunohistochemistry andFISH to detect GFP expression and fluorescence in situ hybridization to Y chromosomes.Epithelial differentiation, proliferative activity and a marker of SCC cells were detected withimmunefluorescent. False-positive recognition was analyzed combined with CD45+immunefluorescent staining.Part III. Study of BMDCs’ contribution to lung cancer progressIn vivo, we traced specific chemotaxis of BMSCs towards lung tumor and assessed tumor growth and metastasis when interaction with BMSCs.In vitro, we investigated theeffect of BMSCs on proliferation, migration and invasion of lung cancer cells, and screen thepotential soluble factors suggestting molecular mechanism.The main results and conclusions1. NDEA-induced lung squamous cell carcinoma in GFP bone marrow chimericFVB/NJ mice could establish an ideal model.GFP-FVB chimeric FVB/NJ mice and Y-X chimeric Wistar rat all could recover fromlethal radiation damage and survive long time enough as induced lung cancer model. Bonemarrow reconstitution was observed and stable chimerism was analysised in the recipient.The lung cancer models induced with NDEA in FVB/NJ mice and with carcinogenic iodizedoil in Wistar rat were stable, uniform, high proportion and reproducible. Considering theoperation and testing methods, NDEA-induced lung squamous cell carcinoma in GFP bonemarrow chimeric FVB/NJ mice could establish an ideal model.2. BMDCs engrafted as different types of lung epithelial cells and contribute to lungrepair.After just4weeks post-transplantation, GFP+BMDCs was observed in the lung ofrecipient.Some spindle-shaped cells of them engrafted as pneumocytes in the alveoli. PCK+/GFP+cells were found in bronchi suggestted that BMDCs engrafted as bronchial epithelialcells to contribute to radiation damage repair in in bronchi.Besides, GFP+/SPC+cells werefound in the alveoli suggestted donor derived Type II alveolar cells as Transient amplifyingcell which also could regenerate type I pneumocytes.3. BMDCs induced by carcinogen could become metaplasia, dysplasia, andsquamous cell carcinoma cells, participate in lung SCC formation and partiallycontribute to tumor growth.BMDC appeared at three stages of lung SCC progression: metaplasia, dysplasia, andcarcinoma. There was a significantly higher proportion of GFP+cells within lung SCC thanwas found in metaplasia and dysplasia. GFP+BMDCs were also observed in clusters withinseveral SCC nests. Furthermore, most GFP+cells in SCC were PCK+epithelial cells, andsome exhibited proliferative activity as determined by Ki67staining, which suggested thatsome donor BMDCs differentiated into proliferating epithelial cells. Analysis of p63 expression, a marker of SCC cells, indicated that the presence of GFP+p63+cells in inner partsof the SCC. These findings strongly suggest that BMDC-derived lung epithelial cells couldparticipate in lung SCC formation and partially contribute to tumor growth. Most of the cellscontain XY suggestting monosomy, which suggestted differentiation was possible mechanismfor donor derived cells, but cell fusion could not be ruled out completely.4. BMSCs could promote lung tumor growth, migration and invasion throughmultiple pathways,and interacted with lung cancer cells possiblely through IL-6andPGE2.In NOD mice, labeled BMSCs introduced into the tibia traffic to sites of growing lungtumor xenografts where they accelerated tumor growth and increasing tumor metastasis.Furthermore, the tumor co-injected with BMSCs have a much higher microvessel densityvalue. In CD133+cells isolated from both lung cancer cells, co-culture with BMSCs forsignificantly increased their proliferation. BMSCs also significantly increased lung cancersphere enriched in cancer stem cells(CSCs)formation and CD133+fractions in the sphere. Allthese suggestted that BMSCs contributed to tumor growth through CSCs proliferation andtumor vessel formation. On the other hand, faster migration and increased invasion of the lungcancer cells were promoted by BMSCs in scratch assays and in transmembrane invasion assay.The changed morphology, reduced intercellular junctions and decreased E-cadherin proteinwere observed in the lung cancer cells co-cultured with BMDCs. All these suggestted thatBMSCs contributed to tumor metastasis through cell migration, invasion and E-cadherinprotein. The effect of the BMSCs-conditioned medium containing the soluble factors wassimilar to the effect of co-cultured with BMDCs. IL-6levels was much higher in the cultureof BMSCs than that of lung cancer cells, and a further induction of IL-6in the co-culture.PGE2was significantly induced in the co-culture not in the single conditioned medium. IL-6and PGE2produced by BMDCs minght be the promising cytokine acting on lung cancercells.