Endothelial cells nurture stem cell-like characteristics of osteosarcoma cells in a perivascular niche by CXCL12/CXCR4 pathway

CAO Ya; LIU Yang; WANG Yang; GUO Qiaonan; LYU Yangfan; ZHAO Guosheng

doi:10.16016/j.2097-0927.202505024

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Endothelial cells nurture stem cell-like characteristics of osteosarcoma cells in a perivascular niche by CXCL12/CXCR4 pathway

Basic Medicine | 更新时间：2025-07-14

- Endothelial cells nurture stem cell-like characteristics of osteosarcoma cells in a perivascular niche by CXCL12/CXCR4 pathway
- Journal of Army Medical University Vol. 47, Issue 13, Pages: 1463-1474(2025)
- 作者机构：
  
  1.陆军军医大学（第三军医大学）第二附属医院病理科，重庆
  2.重庆医科大学附属第二医院脊柱外科，重庆
- 作者简介：
  
  ZHAO Guosheng， E-mail： zhao_gs123@126.com
- 基金信息：
  
  the National Natural Science Foundation for Young Scholar of China(82303465);the General Project of Natural Science Foundation of Chongqing(CSTB2022NSCQ-MSX0103);the Regional Special Support Program of China Postdoctoral Science Foundation(2023MD734132)
- DOI：10.16016/j.2097-0927.202505024
  CLC： R322.12;R730.23;R738.1
- Received：25 May 2025，
  
  Revised：03 June 2025，
  
  Published：15 July 2025
- 稿件说明：
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曹亚，刘扬，汪洋，等.血管周围微环境中内皮细胞通过CXCL12/CXCR4信号通路促进骨肉瘤细胞干性表型［J］.陆军军医大学学报，2025，47（13）：1463-1474.DOI：10.16016/j.2097-0927.202505024.

CAO Y，LIU Y，WANG Y，et al.Endothelial cells nurture stem cell-like characteristics of osteosarcoma cells in a perivascular niche by CXCL12/CXCR4 pathway［J］.J Army Med Univ，2025，47（13）：1463-1474.DOI：10.16016/j.2097-0927.202505024.
曹亚，刘扬，汪洋，等.血管周围微环境中内皮细胞通过CXCL12/CXCR4信号通路促进骨肉瘤细胞干性表型［J］.陆军军医大学学报，2025，47（13）：1463-1474.DOI：10.16016/j.2097-0927.202505024. DOI：

CAO Y，LIU Y，WANG Y，et al.Endothelial cells nurture stem cell-like characteristics of osteosarcoma cells in a perivascular niche by CXCL12/CXCR4 pathway［J］.J Army Med Univ，2025，47（13）：1463-1474.DOI：10.16016/j.2097-0927.202505024. DOI：

摘要

目的

探究体内外血管周围微环境中血管内皮细胞对骨肉瘤细胞干性表型的影响，并初步分析其可能的分子机制。

方法

构建SV40T转化人脐静脉内皮细胞（human umbilical vein endothelial cells-T1， HUVEC-T1）与人骨肉瘤细胞系143B骨肉瘤干细胞（osteosarcoma stem cells， OSCs）体外共培养模型。体外实验分为单独培养组和共培养组，利用极限稀释克隆成球实验检测2组OSCs自我更新能力差异，利用流式细胞技术和Western blot检测2组干性标记物CD133和干性转录因子SOX2、NANOG的表达差异。采用14只雌性裸鼠（体质量18~20 g，4~6周龄）按随机数字表法分为2组（

=7），分别构建皮下移植瘤模型：注射OSCs悬液（对照组），注射OSCs+HUVEC-T1混合悬液（实验组），比较2组肿瘤体积及质量的差异。通过免疫荧光验证内皮细胞与OSCs的体内毗邻关系，通过免疫组化比较2组微血管密度（microvessel density，MVD）与CD133表达差异。通过RNA-seq筛选内皮细胞影响OSCs干性表型的潜在信号通路，利用RT-qPCR、Western blot验证测序结果。利用外源性蛋白、免疫荧光、Western blot、克隆成球等功能实验初步验证该通路调控OSCs表型的作用。

结果

成功构建HUVEC-T1与OSCs体外直接共培养模型，与对照组比较，共培养组中OSCs自我更新能力显著增强，干性标志物CD133阳性比例升高［（8.20±1.64）%

（4.32±1.34）%，

0.05］，CD117、SOX2、NANOG表达增加（

0.05），成球数量（

0.05）及SOX2/NANOG蛋白表达增加。实验组移植瘤体积［（643.10±413.50）

（247.90±93.66） mm³，

0.05］和质量［（0.52±0.27）

（0.24±0.10） g，

0.05］均高于对照组，对应其MVD［（22.57±11.84）

（11.43±5.38），

0.05］与CD133表达也增加（

0.05）。免疫荧光证实CD31标记的内皮细胞与CD133标记的OSCs在移植瘤内存在毗邻关系。RNA-seq与功能实验发现CXCR4在共培养的OSCs中表达升高，CXCL12在共培养的内皮细胞中高表达，而外源性的CXCL12能提高OSCs的成球能力和干性标记物SOX2、NANOG表达（

0.05）。

结论

血管周围微环境中内皮细胞可能通过CXCL12/CXCR4通路促进骨肉瘤细胞的干性表型维持。

Abstract

Objective‍　‍To investigate the effect of endothelial cells in the perivascular niche on the stem cell-like characteristics of osteosarcoma cells and primarily explore the possible molecular mechanism. Methods‍　‍A co-culture model was established

in vitro

using SV40T- human umbilical vein endothelial cells (HUVEC-T1) and osteosarcoma stem cells (OSCs) derived from the human osteosarcoma cell line 143B.

Thus

there were 2 groups of cells

OSCs and co-cultured OSCs. The self-renewal capacity of OSCs between the 2 groups was assessed using a limiting dilution forming sphere assay. Flow cytometry and Western blotting were used to detect the expression of stem cell marker CD133 and stemness transcription factors SOX2 and NANOG. Fourteen female nude mice (4~6 weeks old

weighing 18~20 g) were randomly and equally divided into 2 groups of subcutaneous xenograft models: the control group (OSCs suspension) and the experimental group (OSCs+HUVEC-T1 mixed suspension). The tumor volume and mass were compared between the 2 xenograft groups. Immunofluorescence (IF) staining was used to verify the spatial proxim

ity between endothelial cells and OSCs

in vivo

while immunohistochemistry was employed to compare microvessel density (MVD) and CD133 expression level between the 2 groups. RNA-seq was performed to identify potential signaling pathways of endothelial cells affecting the stemness of OSCs. PCR and Western blotting were applied to confirm the RNA-seq findings. Exogenous protein treatment

IF staining

Western blotting and sphere formation assay were utilized to preliminarily validate the role of the identified pathway in regulating the stemness phenotype of OSCs. Results‍　‍The

in vitro

co-culture model of HUVEC-T1 and OSCs was successfully established. Compared with the control group

the co-culture group exhibited significantly enhanced self-renewal ability of OSCs

laeger proportion of the stemness marker CD133

[(8.20±1.64)%

(4.32±1.34)%

0.05

]

enhanced expression of CD117

SOX2 and NANOG (

0.05)

along with more sphere formation (

0.05) and elevated SOX2/NANOG protein levels. The xenograft mice from the experimental group showed larger tumor volume (643.10±413.50

247.90±93.66 mm³

0.05) and heavier tumor weight (0.52±0.27

0.24±0.10 g

0.05) when compared the control group

correspondingly showing increased MVD (22.57±11.84

11.43±5.38

0.05) and elevated CD133 expression (

0.05). IF staining confirmed the adjacency of CD31-labeled endothelial cells and CD133-labeled OSCs

in vivo

. RNA-seq and functional experiments demonstrated that CXCR4 was highly expressed in co-cultured OSCs

CXCL12 was highly expressed in co-cultured endothelial cells

and exogenous CXCL12 promoted the sphere formation and expression levels of SOX2 and NANOG of OSCs (

0.05). Conclusion‍　‍Endothelial cells within the perivascular nic

he may promote the stemness phenotype of osteosarcoma cells

via

the CXCL12/CXCR4 pathway.

关键词

Keywords

references

WANG Y ， HU Q ， CAO Y ， et al . FOSL1 promotes stem cell-like characteristics and anoikis resistance to facilitate tumorigenesis and metastasis in osteosarcoma by targeting SOX2 ［J］. Int J Mol Med ， 2024 ， 54 （ 5 ）： 94 . DOI： 10.3892/ijmm.2024.5418 http://dx.doi.org/10.3892/ijmm.2024.5418 .

LIU B ， MA W ， JHA R K ， et al . Cancer stem cells in osteosarcoma： recent progress and perspective ［J］. Acta Oncol ， 2011 ， 50 （ 8 ）： 1142 - 1150 . DOI： 10.3109/0284186x.2011.584553 http://dx.doi.org/10.3109/0284186x.2011.584553 .

YAN G N ， LV Y F ， GUO Q N . Advances in osteosarcoma stem cell research and opportunities for novel therapeutic targets ［J］. Cancer Lett ， 2016 ， 370 （ 2 ）： 268 - 274 . DOI： 10.1016/j.canlet.2015.11.003 http://dx.doi.org/10.1016/j.canlet.2015.11.003 .

PRAGER B C ， XIE Q ， BAO S D ， et al . Cancer stem cells： the architects of the tumor ecosystem ［J］. Cell Stem Cell ， 2019 ， 24 （ 1 ）： 41 - 53 . DOI： 10.1016/j.stem.2018.12.009 http://dx.doi.org/10.1016/j.stem.2018.12.009 .

CALABRESE C ， POPPLETON H ， KOCAK M ， et al . A perivascular niche for brain tumor stem cells ［J］. Cancer Cell ， 2007 ， 11 （ 1 ）： 69 - 82 . DOI： 10.1016/j.ccr.2006.11.020 http://dx.doi.org/10.1016/j.ccr.2006.11.020 .

ZHU T S ， COSTELLO M A ， TALSMA C E ， et al . Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells ［J］. Cancer Res ， 2011 ， 71 （ 18 ）： 6061 - 6072 . DOI： 10.1158/0008-5472.can-10-4269 http://dx.doi.org/10.1158/0008-5472.can-10-4269 .

YAN G N ， YANG L ， LV Y F ， et al . Endothelial cells promote stem-like phenotype of glioma cells through activating the Hedgehog pathway ［J］. J Pathol ， 2014 ， 234 （ 1 ）： 11 - 22 . DOI： 10.1002/path.4349 http://dx.doi.org/10.1002/path.4349 .

LU J ， YE X C ， FAN F ， et al . Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1 ［J］. Cancer Cell ， 2013 ， 23 （ 2 ）： 171 - 185 . DOI： 10.1016/j.ccr.2012.12.021 http://dx.doi.org/10.1016/j.ccr.2012.12.021 .

JIANG H M ， ZHOU C ， ZHANG Z ， et al . Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1 ［J］. Nat Commun ， 2020 ， 11 ： 5129 . DOI： 10.1038/s41467-020-18860-4 http://dx.doi.org/10.1038/s41467-020-18860-4 .

ZHANG Q ， XIA Y ， WANG L ， et al . Targeted anti-angiogenesis therapy for advanced osteosarcoma ［J］. Front Oncol ， 2024 ， 14 ： 1413213 . DOI： 10.3389/fonc.2024. 1413213 http://dx.doi.org/10.3389/fonc.2024.1413213 .

XU E J ， HUANG Z ， ZHU K P ， et al . PDGFRB promotes dedifferentiation and pulmonary metastasis through rearrangement of cytoskeleton under hypoxic microenvironment in osteosarcoma ［J］. Cell Signal ， 2025 ， 125 ： 111501 . DOI： 10.1016/j.cellsig.2024.111501 http://dx.doi.org/10.1016/j.cellsig.2024.111501 .

YANG C ， TIAN Y ， ZHAO F ， et al . Bone microenvironment and osteosarcoma metastasis ［J］. Int J Mol Sci ， 2020 ， 21 （ 19 ）： E6985 . DOI： 10.3390/ijms21196985 http://dx.doi.org/10.3390/ijms21196985 .

WEIDNER N ， FOLKMAN J ， POZZA F ， et al . Tumor angiogenesis： a new significant and independent prognostic indicator in early-stage breast carcinoma ［J］. J Natl Cancer Inst ， 1992 ， 84 （ 24 ）： 1875 - 1887 . DOI： 10.1093/jnci/84.24.1875 http://dx.doi.org/10.1093/jnci/84.24.1875 .

MARDANI A ， GHEYTANCHI E ， MOUSAVIE S H ， et al . Clinical significance of cancer stem cell markers CD133 and CXCR4 in osteosarcomas ［J］. Asian Pac J Cancer Prev ， 2020 ， 21 （ 1 ）： 67 - 73 . DOI： 10.31557/apjcp.2020.21.1.67 http://dx.doi.org/10.31557/apjcp.2020.21.1.67 .

MOUKENGUE B ， LALLIER M ， MARCHANDET L ， et al . Origin and therapies of osteosarcoma ［J］. Cancers （Basel）， 2022 ， 14 （ 14 ）： 3503 . DOI： 10.3390/cancers14143503 http://dx.doi.org/10.3390/cancers14143503 .

TU S M ， ESTECIO M R ， LIN S H ， et al . Stem cell theory of cancer： rude awakening or bad dream from cancer dormancy ［J］. Cancers （Basel）， 2022 ， 14 （ 3 ）： 655 . DOI： 10.3390/cancers14030655 http://dx.doi.org/10.3390/cancers14030655 .

VOOG J ， JONES D L . Stem cells and the niche： a dynamic Duo ［J］. Cell Stem Cell ， 2010 ， 6 （ 2 ）： 103 - 115 . DOI： 10.1016/j.stem.2010.01.011 http://dx.doi.org/10.1016/j.stem.2010.01.011 .

YANG J ， HU Y ， WANG L ， et al . Human umbilical vein endothelial cells derived-exosomes promote osteosarcoma cell stemness by activating Notch signaling pathway ［J］. Bioengineered ， 2021 ， 12 （ 2 ）： 11007 - 11017 . DOI： 10.1080/21655979.2021.2005220 http://dx.doi.org/10.1080/21655979.2021.2005220 .

ZHAO L J ， QIU Z X ， YANG Z N ， et al . Lymphatic endothelial-like cells promote glioblastoma stem cell growth through cytokine-driven cholesterol metabolism ［J］. Nat Cancer ， 2024 ， 5 （ 1 ）： 147 - 166 . DOI： 10.1038/s43018-023-00658-0 http://dx.doi.org/10.1038/s43018-023-00658-0 .

SEANO G . Targeting the perivascular niche in brain tumors ［J］. Curr Opin Oncol ， 2018 ， 30 （ 1 ）： 54 - 60 . DOI： 10.1097/cco.0000000000000417 http://dx.doi.org/10.1097/cco.0000000000000417 .

马红敏，郝翔麟，吕杨帆，等 . 内皮细胞通过抑制LGI4表达激活NOTCH 4 信号通路促进骨肉瘤细胞转移［J］. 陆军军医大学学报， 2022 ， 44 （ 20 ）： 2088 - 2097 . DOI： 10.16016/j.2097-0927.202208043 http://dx.doi.org/10.16016/j.2097-0927.202208043 .

MA H M ， HAO X L ， LYU Y F ， et al . Endothelial cells promote osteosarcoma cell metastasis by inhibiting LGI4 expression and activating NOTCH 4 signaling pathway ［J］. J Army Med Univ ， 2022 ， 44 （ 20 ）： 2088 - 2097 . DOI： 10.16016/j.2097-0927.202208043 http://dx.doi.org/10.16016/j.2097-0927.202208043 .

BENSLIMANE-AHMIM Z ， PEREIRA J ， LOKAJCZYK A ， et al . Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization ［J］. Cancer Lett ， 2017 ， 395 ： 11 - 19 . DOI： 10.1016/j.canlet.2017.02.032 http://dx.doi.org/10.1016/j.canlet.2017.02.032 .

LI B ， WANG Z ， WU H ， et al . Epigenetic regulation of CXCL12 plays a critical role in mediating tumor progression and the immune response in osteosarcoma ［J］. Cancer Res ， 2018 ， 78 （ 14 ）： 3938 - 3953 . DOI： 10.1158/0008-5472.can-17-3801 http://dx.doi.org/10.1158/0008-5472.can-17-3801 .

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