骨髓来源细胞在肺癌发生及进展中作用的实验研究
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摘要
背景和目的
肺癌是世界发病率最高的恶性肿瘤之一，且其发病率迅速上升，并已成为癌症致病死的主要原因之一。研究肺癌的发生和进展机制，对于提高其早期诊断率，减低其发病率和死亡率具有重要意义。
目前有关肺癌的起源仍不清楚，不少的学者认为，由于干细胞存活时间较长，有更多的突变机会，因此更可能出现异常分化形成肿瘤干细胞，而肿瘤干细胞因其具有的多向分化潜能和自我更新能力使其成为肿瘤的种子。那么这些干细胞又来源何处？一般认为在肺和呼吸系统内存在一些固有的干细胞，在组织受损时参与受损组织的修复。但在组织受损严重时，固有干细胞数量得不到有效的补充，必需依靠外源性干细胞参与组织修复。已有报道BMDCs在肺损伤早期具有向损伤和炎症组织特异性趋化和修复肺损伤的潜能，在这个过程中BMDCs的生长调控和分化取决与组织的微环境，提示BMDCs可能是肺内干细胞的另一来源。在诱癌因素的作用下，具有干细胞特性的BMDCs也可能发生异常分化和增殖，进而参与肺癌的发生。目前对BMDCs可以成为实体癌起源的观点仍未达成一致，BMDCs在肺癌发生中的作用更是不清楚。
BMDCs具有向损伤和炎症组织特异性趋化的特点，而肿瘤的内环境与损伤组织有不少相似之处，从机体的角度也可以说肿瘤本身就是一个“难以愈合的损伤”。因此，已经形成的肿瘤仍可能继续释放趋化因子吸引BMDCs，持续参与肿瘤生长的微环境。既然BMDCs能在不同环境中分泌多种活性因子，那么他们在肺癌中分泌哪些细胞因子，又具体发挥了哪些作用，仍然不清楚。
目前利用BMDCs，尤其是BMSCs，作为组织损伤保护或基因治疗的细胞载体被认为是组织修复和抗癌领域大有希望的治疗策略。因此对BMDCs在参与组织异常转化和肿瘤进展中的风险评估显得尤为必要。
在本课题中，我们首选建立了多种肺癌诱导动物模型和活体标记BMDCs的骨髓嵌合模型，并最终选定在GFP骨髓嵌合小鼠体内利用NDEA诱导肺癌形成作为最适宜的研究对象，探讨BMDCs参与肺上皮细胞修复和肺上皮转化（上皮化生、不典型增生和癌）过程中的角色。对已经形成的肿瘤，我们进一步探究BMDCs对肺癌增殖、转移等进展的具体作用。这些结果可能对肺癌发生和进展研究提供新的思路，为BMSCs作为临床治疗应用前的评估提供了重要的实验依据。
研究内容
一、诱导肺癌和骨髓嵌合鼠模型的建立及评价
分别建立支气管灌注致癌物碘油诱导SD和Wistar大鼠肺癌模型，乌拉坦诱导KM小鼠肺肿瘤模型，N-亚硝基二乙胺诱导FVB小鼠肺肿瘤模型，苯并（α）芷诱导FVB小鼠肺肿瘤模型。比较各模型的成瘤时间、成瘤率、成瘤类型和各病变阶段转化时间。建立GFP-FVB/N骨髓嵌合FVB/N小鼠和性别错配骨髓嵌合Wistar大鼠模型，评估其骨髓移植后的骨髓重建、造血恢复、存活率和嵌合的骨髓细胞标记表达。选择最适宜作为进一步研究对象的动物模型。
二、BMDCs参与肺癌形成的实验研究
利用免疫组化和免疫荧光染色分析BMDCs在肺上皮损伤、化生、不典型增生到癌组织的定植和上皮分化属性，排除假阳性，进一步检测鳞癌中BMDCs的增殖活性和鳞癌标志。
三、BMDCs中BMSCs在肺癌进展中作用的实验研究
首先在裸鼠活体实验中观察BMSCs是否向肿瘤特异性趋化及对肿瘤生长、转移的影响。体外实验进一步探究，BMSCs具体通过哪些方式影响了肿瘤的生长、转移，并初步筛选起作用的主要影响因子。
主要结果和结论
1. NDEA诱导GFP-FVB/N骨髓嵌合FVB/N小鼠肺癌形成是最适宜作为研究对象的动物模型。
GFP-FVB骨髓嵌合小鼠和Y-X Wistar骨髓嵌合大鼠均能度过极期，恢复造血功能，14天存活率达100％，并能长期存活至诱癌结束时间点，供体来源骨髓也能稳定嵌合。在此基础上NDEA诱导FVB小鼠和支气管灌注致癌物碘油诱导Wistar大鼠均能诱导稳定的、均一的、高比例的和可重复性的肺癌模型。从模型的可操作性和检测方法上考虑，NDEA诱导GFP-FVB/N骨髓嵌合FVB/N小鼠肺癌形成是最适宜作为研究对象的动物模型。
2. BMDCs可分化为多种肺上皮细胞，促进肺修复。
GFP-FVB/N小鼠骨髓移植后一月大量供体来源的GFP阳性BMDCs在肺内定植，部分定植的BMDCs参与构成肺泡腔，有的胞体伸展呈梭形，提示为Ⅰ型肺泡上皮细胞。免疫荧光双标显示，支气管腔内细胞表达GFP/PCK双阳性，提示供体来源的BMDCs分化为PCK阳性的支气管上皮细胞，参与放射性损伤后支气管上皮修复。肺泡内可见GFP/SPC双阳性细胞，供体来源的BMDCs可分化为Ⅱ型肺泡上皮细胞，进而补充Ⅰ型肺泡上皮细胞，从而参与放射性损伤后的肺泡修复。
3.若损伤因素持续存在，BMDCs也会直接形成鳞状化生、不典型增生、甚至鳞癌细胞，并参与肺鳞癌的形成和生长。
BMDCs在支气管上皮鳞状化生、不典型增生和鳞状细胞癌中的表达率逐渐升高，尤其是在癌组织内其比例明显升高和成群表达。免疫荧光双标显示，这些病变中的GFP阳性细胞多数也表达上皮细胞标记PCK阳性，癌巢中的GFP阳性细胞同时也表达鳞癌细胞标记P63。利用Y染色标记作为另一种独立的BMDCs标记检测BMDCs细胞的上皮细胞属性也证实了上述结果。提示BMDCs参与了肺鳞癌恶性转化的全过程。此外，发现这些GFP+/PCK+细胞部分同时表达Ki67，BMDCs来源的增殖细胞占肿瘤中所有增殖细胞的9.7±3.92%，提示供体来源的BMDCs不仅具备癌细胞的上皮表型，实际也部分参与了肿瘤的生长。多数BMDCs来源细胞表达XY单套染色体，提示直接分化可能是BMDCs形成上述细胞的主要机制，但细胞融合仍不能完全排除。
4. BMSCs可能通过多途径促进肿瘤生长、迁移、侵袭，IL-6和PGE2可能是BMSCs影响肺癌细胞生长和转移的主要细胞因子。
裸鼠活体移植实验证实标记的BMSCs能够向肺癌肿瘤特异性趋化，并在活体中呈现出促肺癌细胞生长成瘤和促肺癌细胞转移的特性。进一步发现，在有BMSCs的作用时，移植瘤血管密度更高，CD133阳性的肺癌细胞增殖能力更强，肺癌干细胞克隆球的数量增多、体积增大、其内CD133阳性细胞比例升高，提示BMSCs可能通过促肿瘤干细胞增殖和微血管形成而促进肿瘤生长。另一方面，在BMSCs作用下，肺癌细胞向划痕迁移的速度更快，肺癌细胞跨膜侵袭的能力更强，肺癌细胞形态变得更长更细，细胞间连接明显减少，E－cadherin蛋白表达明显降低，提示下调肺癌细胞E－cadherin、促进肺癌细胞的迁移、侵袭能力，可能是BMSCs促进肺癌转移的作用方式。单用BMSCs条件介质代替BMSCs共培养也能获得上述相同的结论，而BMSCs条件介质中含明显高浓度的IL-6，在作用肺癌细胞后促进肺癌细胞进一步分泌IL-6，PGE2在BMSCs与肺癌细胞相互作用后表达升高，提示旁分泌IL-6和PGE2可能是BMSCs影响肺癌细胞生长和转移的主要细胞因子。此外，BMDCs来源的CAFs也可能作为BMDCs参与肺癌进展的另一方式。
Background and Objective
Lung 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 contents
Part I. Establishment and assessment of carcinogen-induced lung cancer model andbone marrow chimeric model
SD 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 vivo
Engraftment 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 progress
In 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 conclusions
1. 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.
肺癌是世界发病率最高的恶性肿瘤之一，且其发病率迅速上升，并已成为癌症致病死的主要原因之一。研究肺癌的发生和进展机制，对于提高其早期诊断率，减低其发病率和死亡率具有重要意义。
目前有关肺癌的起源仍不清楚，不少的学者认为，由于干细胞存活时间较长，有更多的突变机会，因此更可能出现异常分化形成肿瘤干细胞，而肿瘤干细胞因其具有的多向分化潜能和自我更新能力使其成为肿瘤的种子。那么这些干细胞又来源何处？一般认为在肺和呼吸系统内存在一些固有的干细胞，在组织受损时参与受损组织的修复。但在组织受损严重时，固有干细胞数量得不到有效的补充，必需依靠外源性干细胞参与组织修复。已有报道BMDCs在肺损伤早期具有向损伤和炎症组织特异性趋化和修复肺损伤的潜能，在这个过程中BMDCs的生长调控和分化取决与组织的微环境，提示BMDCs可能是肺内干细胞的另一来源。在诱癌因素的作用下，具有干细胞特性的BMDCs也可能发生异常分化和增殖，进而参与肺癌的发生。目前对BMDCs可以成为实体癌起源的观点仍未达成一致，BMDCs在肺癌发生中的作用更是不清楚。
BMDCs具有向损伤和炎症组织特异性趋化的特点，而肿瘤的内环境与损伤组织有不少相似之处，从机体的角度也可以说肿瘤本身就是一个“难以愈合的损伤”。因此，已经形成的肿瘤仍可能继续释放趋化因子吸引BMDCs，持续参与肿瘤生长的微环境。既然BMDCs能在不同环境中分泌多种活性因子，那么他们在肺癌中分泌哪些细胞因子，又具体发挥了哪些作用，仍然不清楚。
目前利用BMDCs，尤其是BMSCs，作为组织损伤保护或基因治疗的细胞载体被认为是组织修复和抗癌领域大有希望的治疗策略。因此对BMDCs在参与组织异常转化和肿瘤进展中的风险评估显得尤为必要。
在本课题中，我们首选建立了多种肺癌诱导动物模型和活体标记BMDCs的骨髓嵌合模型，并最终选定在GFP骨髓嵌合小鼠体内利用NDEA诱导肺癌形成作为最适宜的研究对象，探讨BMDCs参与肺上皮细胞修复和肺上皮转化（上皮化生、不典型增生和癌）过程中的角色。对已经形成的肿瘤，我们进一步探究BMDCs对肺癌增殖、转移等进展的具体作用。这些结果可能对肺癌发生和进展研究提供新的思路，为BMSCs作为临床治疗应用前的评估提供了重要的实验依据。
研究内容
一、诱导肺癌和骨髓嵌合鼠模型的建立及评价
分别建立支气管灌注致癌物碘油诱导SD和Wistar大鼠肺癌模型，乌拉坦诱导KM小鼠肺肿瘤模型，N-亚硝基二乙胺诱导FVB小鼠肺肿瘤模型，苯并（α）芷诱导FVB小鼠肺肿瘤模型。比较各模型的成瘤时间、成瘤率、成瘤类型和各病变阶段转化时间。建立GFP-FVB/N骨髓嵌合FVB/N小鼠和性别错配骨髓嵌合Wistar大鼠模型，评估其骨髓移植后的骨髓重建、造血恢复、存活率和嵌合的骨髓细胞标记表达。选择最适宜作为进一步研究对象的动物模型。
二、BMDCs参与肺癌形成的实验研究
利用免疫组化和免疫荧光染色分析BMDCs在肺上皮损伤、化生、不典型增生到癌组织的定植和上皮分化属性，排除假阳性，进一步检测鳞癌中BMDCs的增殖活性和鳞癌标志。
三、BMDCs中BMSCs在肺癌进展中作用的实验研究
首先在裸鼠活体实验中观察BMSCs是否向肿瘤特异性趋化及对肿瘤生长、转移的影响。体外实验进一步探究，BMSCs具体通过哪些方式影响了肿瘤的生长、转移，并初步筛选起作用的主要影响因子。
主要结果和结论
1. NDEA诱导GFP-FVB/N骨髓嵌合FVB/N小鼠肺癌形成是最适宜作为研究对象的动物模型。
GFP-FVB骨髓嵌合小鼠和Y-X Wistar骨髓嵌合大鼠均能度过极期，恢复造血功能，14天存活率达100％，并能长期存活至诱癌结束时间点，供体来源骨髓也能稳定嵌合。在此基础上NDEA诱导FVB小鼠和支气管灌注致癌物碘油诱导Wistar大鼠均能诱导稳定的、均一的、高比例的和可重复性的肺癌模型。从模型的可操作性和检测方法上考虑，NDEA诱导GFP-FVB/N骨髓嵌合FVB/N小鼠肺癌形成是最适宜作为研究对象的动物模型。
2. BMDCs可分化为多种肺上皮细胞，促进肺修复。
GFP-FVB/N小鼠骨髓移植后一月大量供体来源的GFP阳性BMDCs在肺内定植，部分定植的BMDCs参与构成肺泡腔，有的胞体伸展呈梭形，提示为Ⅰ型肺泡上皮细胞。免疫荧光双标显示，支气管腔内细胞表达GFP/PCK双阳性，提示供体来源的BMDCs分化为PCK阳性的支气管上皮细胞，参与放射性损伤后支气管上皮修复。肺泡内可见GFP/SPC双阳性细胞，供体来源的BMDCs可分化为Ⅱ型肺泡上皮细胞，进而补充Ⅰ型肺泡上皮细胞，从而参与放射性损伤后的肺泡修复。
3.若损伤因素持续存在，BMDCs也会直接形成鳞状化生、不典型增生、甚至鳞癌细胞，并参与肺鳞癌的形成和生长。
BMDCs在支气管上皮鳞状化生、不典型增生和鳞状细胞癌中的表达率逐渐升高，尤其是在癌组织内其比例明显升高和成群表达。免疫荧光双标显示，这些病变中的GFP阳性细胞多数也表达上皮细胞标记PCK阳性，癌巢中的GFP阳性细胞同时也表达鳞癌细胞标记P63。利用Y染色标记作为另一种独立的BMDCs标记检测BMDCs细胞的上皮细胞属性也证实了上述结果。提示BMDCs参与了肺鳞癌恶性转化的全过程。此外，发现这些GFP+/PCK+细胞部分同时表达Ki67，BMDCs来源的增殖细胞占肿瘤中所有增殖细胞的9.7±3.92%，提示供体来源的BMDCs不仅具备癌细胞的上皮表型，实际也部分参与了肿瘤的生长。多数BMDCs来源细胞表达XY单套染色体，提示直接分化可能是BMDCs形成上述细胞的主要机制，但细胞融合仍不能完全排除。
4. BMSCs可能通过多途径促进肿瘤生长、迁移、侵袭，IL-6和PGE2可能是BMSCs影响肺癌细胞生长和转移的主要细胞因子。
裸鼠活体移植实验证实标记的BMSCs能够向肺癌肿瘤特异性趋化，并在活体中呈现出促肺癌细胞生长成瘤和促肺癌细胞转移的特性。进一步发现，在有BMSCs的作用时，移植瘤血管密度更高，CD133阳性的肺癌细胞增殖能力更强，肺癌干细胞克隆球的数量增多、体积增大、其内CD133阳性细胞比例升高，提示BMSCs可能通过促肿瘤干细胞增殖和微血管形成而促进肿瘤生长。另一方面，在BMSCs作用下，肺癌细胞向划痕迁移的速度更快，肺癌细胞跨膜侵袭的能力更强，肺癌细胞形态变得更长更细，细胞间连接明显减少，E－cadherin蛋白表达明显降低，提示下调肺癌细胞E－cadherin、促进肺癌细胞的迁移、侵袭能力，可能是BMSCs促进肺癌转移的作用方式。单用BMSCs条件介质代替BMSCs共培养也能获得上述相同的结论，而BMSCs条件介质中含明显高浓度的IL-6，在作用肺癌细胞后促进肺癌细胞进一步分泌IL-6，PGE2在BMSCs与肺癌细胞相互作用后表达升高，提示旁分泌IL-6和PGE2可能是BMSCs影响肺癌细胞生长和转移的主要细胞因子。此外，BMDCs来源的CAFs也可能作为BMDCs参与肺癌进展的另一方式。
Background and Objective
Lung 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 contents
Part I. Establishment and assessment of carcinogen-induced lung cancer model andbone marrow chimeric model
SD 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 vivo
Engraftment 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 progress
In 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 conclusions
1. 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.
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