Surveillance of spontaneous breast cancer metastasis by TRAIL-expressing CD34+ cells in a xenograft model

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), delivered as a membrane-bound molecule expressed on the surface of adenovirus-transduced CD34⁺ cells (CD34-TRAIL⁺), was analyzed for its apoptotic activity in vitro on 12 breast cancer cell lines representing estrogen receptor-positive, HER2⁺ and triple-negative (TN) subtypes and for its effect on tumor growth, vascularization, necrosis, and lung metastasis incidence in NOD/SCID mice xenografted with the TN breast cancer line MDA-MB-231. Mesenchymal TN cell lines, which are the richest in putative tumor stem cells among the different breast cancer cell subtypes, were the most susceptible to apoptosis induced by CD34-TRAIL⁺ cells. Indeed, tumor cell ??stemness??, assessed based on the proportion of CD44⁺/CD24^?/low cells, was significantly correlated with susceptibility to TRAIL. Moreover, in vitro cytotoxicity experiments showed that CD34-TRAIL⁺ cells selectively targeted CD44⁺/CD24^?/low cells. Although in vivo treatment with CD34-TRAIL⁺ cells did not lead to tumor growth inhibition, treated mice revealed significantly larger areas of necrosis associated with damage of tumor vasculature than did control mice. Moreover, lungs from MDA-MD-231 tumor-bearing mice were completely free of metastases at 12?days after the last injection of CD34-TRAIL⁺ cells, whereas metastases were present in all control mouse lungs. An anti-metastatic effect of CD34-TRAIL⁺ cells was also observed in a model of experimental lung metastases. The correlation between in vitro susceptibility to membrane-bound TRAIL and tumor stem cell content, together with CD34-TRAIL⁺ cell-induced inhibition of the metastatic process, points to the selective targeting of cancer stem cells by CD34-armed cells and the potential value of such cells in eradicating tumor stem cells before the onset of overt metastases.     

Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy

Introduction

The phenotypic and functional differences between cells that initiate human breast tumors (cancer stem cells) and those that comprise the tumor bulk are difficult to study using only primary tumor tissue. We embarked on this study hypothesizing that breast cancer cell lines would contain analogous hierarchical differentiation programs to those found in primary breast tumors.

Methods

Eight human breast cell lines (human mammary epithelial cells, and MCF10A, MCF7, SUM149, SUM159, SUM1315 and MDA.MB.231 cells) were analyzed using flow cytometry for CD44, CD24, and epithelial-specific antigen (ESA) expression. Limiting dilution orthotopic injections were used to evaluate tumor initiation, while serial colony-forming unit, reconstitution and tumorsphere assays were performed to assess self-renewal and differentiation. Pulse-chase bromodeoxyuridine (5-bromo-2-deoxyuridine [BrdU]) labeling was used to examine cell cycle and label-retention of cancer stem cells. Cells were treated with paclitaxol and 5-fluorouracil to test selective resistance to chemotherapy, and gene expression profile after chemotherapy were examined.

Results

The percentage of CD44⁺/CD24^- cells within cell lines does not correlate with tumorigenicity, but as few as 100 cells can form tumors when sorted for CD44⁺/CD24^-/low/ESA⁺. Furthermore, CD44⁺/CD24^-/ESA⁺ cells can self-renew, reconstitute the parental cell line, retain BrdU label, and preferentially survive chemotherapy.

Conclusion

These data validate the use of cancer cell lines as models for the development and testing of novel therapeutics aimed at eradicating cancer stem cells.     

Microarray-based analysis of microRNA expression in breast cancer stem cells

Background

This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs.

Methods

We isolated ESA⁺CD44⁺CD24^-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs.

Results

The ESA⁺CD44⁺CD24^-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA⁺CD44⁺CD24^-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA⁺CD44⁺CD24^-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes.

Conclusions

We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

Distinct breast cancer stem/progenitor cell populations require either HIF1α or loss of PHD3 to expand under hypoxic conditions

The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor⁺), high mammosphere formation capacity and CD44⁺CD24^−/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44⁺CD24^−/low cell expansion. We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor⁺ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44⁺CD24^−/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.     

Targeting highly expressed extracellular HSP90 in breast cancer stem cells inhibits tumor growth in vitro and in vivo

Breast cancer stem cells (BCSC) have been identified in breast carcinoma as CD44⁺/CD24^?/low cells, which display tumorigenic activity and have the ability to self-renew, differentiate and metastasize. Previous studies showed that extracellular HSP90 (eHSP90) participates in the invasion and metastatic processes of various cancers including breast cancer. Here, we show for the first time that eHSP90 is over-expressed in mammosphere cultures that are derived from the MDA-MB-231, MDA-MB-453 and MCF-7 breast cancer cell lines. These mammospheres are highly enriched in cells of the CD44⁺/CD24^?/low BCSC phenotype and additionally show high expression of the BCSC markers CD49f and Sox2. Thus our results indicate that eHSP90 represents a potential novel BCSC marker. Moreover, we present evidence that eHSP90 is functionally involved in BCSC activity in vitro and in vivo. Selective neutralization of eHSP90, using the monoclonal antibody mAb 4C5, has the capacity to inhibit stem cell activity in vitro because the formation of mammosphere-derived colonies is dramatically reduced in its presence. In vivo, the treatment of mice with mAb4C5 using a prophylactic protocol, significantly inhibited the primary growth of MDA-MB-231 and mammosphere-derived tumors. More importantly, administration of this antibody in a therapeutic protocol caused a statistically significant regression of established tumors derived from MDA-MB-231 originating mammospheres. Tumor regression was even greater when mAb 4C5 was administered in combination with paclitaxel. Overall, our findings implicate eHSP90 as a potential novel BCSC biomarker. Moreover they show that eHSP90 participates in BCSC-derived primary tumor growth. Finally, we provide additional support for the possible therapeutic value of mAb4C5 in the treatment of breast cancer.     

Lung-Derived Factors Mediate Breast Cancer Cell Migration through CD44 Receptor-Ligand Interactions in a Novel Ex Vivo System for Analysis of Organ-Specific Soluble Proteins

Breast cancer preferentially metastasizes to lung, lymph node, liver, bone, and brain. However, it is unclear whether properties of cancer cells, properties of organ microenvironments, or a combination of both is responsible for this observed organ tropism. We hypothesized that breast cancer cells exhibit distinctive migration/growth patterns in organ microenvironments that mirror common clinical sites of breast cancer metastasis and that receptor-ligand interactions between breast cancer cells and soluble organ-derived factors mediate this behavior. Using an ex vivo model system composed of organ-conditioned media (CM), human breast cancer cells (MDA-MB-231, MDA-MB-468, SUM149, and SUM159) displayed cell line-specific and organ-specific patterns of migration/proliferation that corresponded to their in vivo metastatic behavior. Notably, exposure to lung-CM increased migration of all cell lines and increased proliferation in two of four lines (P < .05). Several cluster of differentiation (CD) 44 ligands including osteopontin (OPN) and L-selectin (SELL) were identified in lung-CM by protein arrays. Immunodepletion of SELL decreased migration of MDA-MB-231 cells, whereas depletion of OPN decreased both migration and proliferation. Pretreatment of cells with a CD44-blocking antibody abrogated migration effects (P < .05). “Stemlike” breast cancer cells with high aldehyde dehydrogenase and CD44 (ALDH^hiCD44⁺) responded in a distinct chemotactic manner toward organ-CM, preferentially migrating toward lung-CM through CD44 receptor-ligand interactions (P < .05). In contrast, organ-specific changes in migration were not observed for ALDH^lowCD44^- cells. Our data suggest that interactions between CD44⁺ breast cancer cells and soluble factors present in the lung microenvironment may play an important role in determining organotropic metastatic behavior.     

Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer

The CD44⁺/CD24^−/low and ALDH1⁺ cell phenotypes are associated with stemness and enhanced tumorigenic potential in breast cancer. We assessed the expression of CD44, CD24 and ALDH1 on tumor cells circulating in the peripheral blood (CTCs) of patients with metastatic breast cancer using triple-marker immunofluorescence microscopy. Among a total of 1439 CTCs identified in 20 (66.7%) out of 30 patients, 35.2% had the stem-like/tumorigenic phenotype CD44⁺/CD24^−/low, whereas 17.7% of the CTCs analyzed in seven patients, were ALDH1^high/CD24^−/low. In conclusion, we report the existence of a subpopulation of CTCs with putative stem cell progenitor phenotypes in patients with metastatic breast cancer.     

G-Protein-Coupled Estrogen Receptor Enhances the Stemness of Triple-Negative Breast Cancer Cells and Promotes Malignant Characteristics

G-protein coupled estrogen receptor (GPER) is a transmembrane receptor that mediates non-genomic effects of estrogen. This study aimed to investigate the role of GPER in the stemness formation and malignancies in triple negative breast cancer (TNBC) cells. Spheroids of MDA-MB-468 cells were induced by mammosphere culture, and the proportion of the CD44⁺ /CD24^−/low stem cell subpopulation was detected. Malignant characteristics, expression of GPER and stemness-related markers, and tumorigenesis in a xenograft assay were compared between the mammospheres and adherent cultured cells. The impacts of 17β-estradiol (E2) and the GPER-specific antagonist G15 were studied in in vitro assays. The proportion of the CD44⁺ /CD24^−/low subpopulation was increased in the mammospheres of MDA-MB-468 cells, which also showed higher expression of GPER and stemness-related markers than adherent cultured cells. The abilities of spherical colonies to proliferate, invade, and form colonies in soft agar were enhanced. Spherical cells exhibited stronger tumorigenesis ability than adherent cells in the xenograft assay. E2 treatment enhanced tumorigenicity of both adherent and spherical cells. Spherical cells treated with E2 had stronger proliferation, invasion, and colony formation abilities than other groups. Pretreatment with G15 effectively blocked the stimulation by E2. In conclusion, the expression of GPER in TNBC cells is positively related to stemness and malignant features.     

miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5

Effective therapeutic targets for triple-negative breast cancer (TNBC), a special type of breast cancer (BC) with rapid metastasis and poor prognosis, are lacking, especially for patients with chemotherapy resistance. Decitabine (DCA) is a Food and Drug Administration-approved DNA methyltransferase inhibitor that has been proven effective for the treatment of tumors. However, its antitumor effect in cancer cells is limited by multidrug resistance. Cancer stem cells (CSCs), which are thought to act as seeds during tumor formation, regulate tumorigenesis, metastasis, and drug resistance through complex signaling. Our previous study found that miR-155 is upregulated in BC, but whether and how miR-155 regulates DCA resistance is unclear. In this study, we demonstrated that miR-155 was upregulated in CD24⁻CD44⁺ BC stem cells (BCSCs). In addition, the overexpression of miR-155 increased the number of CD24⁻CD44⁺ CSCs, DCA resistance and tumor clone formation in MDA-231 and BT-549 BC cells, and knockdown of miR-155 inhibited DCA resistance and stemness in BCSCs in vitro. Moreover, miR-155 induced stemness and DCA resistance by inhibiting the direct target gene tetraspanin-5 (TSPAN5). We further confirmed that overexpression of TSPAN5 abrogated the effect of miR-155 in promoting stemness and DCA resistance in BC cells. Our data show that miR-155 increases stemness and DCA resistance in BC cells by targeting TSPAN5. These data provide a therapeutic strategy and mechanistic basis for future possible clinical applications targeting the miR-155/TSPAN5 signaling axis in the treatment of TNBC.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.     

沉默CD44表达对乳腺癌细胞MDA-MB-231增殖与侵袭的影响

目的:探索乳腺癌细胞MDA-MB-231及MCF-7中CD44分子的表达水平差异及沉默CD44对乳腺癌细胞MDA-MB-231增殖、侵袭和迁移的影响。方法:利用qRT-PCR及Western blot技术检测细胞中CD44基因表达水平;设计并合成CD44的siRNA片段(CD44-siRNA)转染乳腺癌细胞,利用qRT-PCR、Western blot技术检测细胞中CD44基因表达水平的变化;MTT检测MDA-MB-231细胞增殖;Transwell侵袭实验检测MDA-MB-231细胞的迁移与侵袭能力变化。结果:CD44在侵袭性乳腺癌细胞MDA-MB-231中的表达高于非侵袭性乳腺癌细胞MCF-7,CD44-siRNA下调了 MDA-MB-231细胞中CD44 mRNA与蛋白水平的表达,并抑制了细胞的增殖和侵袭转移能力。结论:CD44-siRNA能够下调CD44的表达,并有效抑制乳腺癌细胞MDA-MB-231的增殖及其侵袭迁移力。     

CD24 enhances DNA damage-induced apoptosis by modulating NF-κB signaling in CD44-expressing breast cancer cells

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.     

Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

Background

In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44⁺/CD24^- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44⁺/CD24^- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?

Methods

Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.

Results

MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.

Conclusions

MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

     

CD44 targets Na+/H+ exchanger 1 to mediate MDA-MB-231 cells' metastasis via the regulation of ERK1/2

Background:

CD44, a transmembrane glycoprotein expressed in a variety of cells and tissues, has been implicated in tumour metastasis. But the molecular mechanisms of CD44-mediated tumour cell metastasis remain to be elucidated.

Methods:

The downregulation of CD44 was determined by immunofluorescence. Moreover, the motility of breast cancer cells was detected by wound-healing and transwell experiments. Then the spontaneous metastasis of CD44-silenced MDA-MB-231 cells was tested by histology with BALB/c nude mice.

Results:

A positive correlation between CD44 and Na⁺/H⁺ exchanger isoform 1 (NHE1) was found in two breast cancer cells. CD44 downregulation could inhibit the metastasis of MDA-MB-231 cells and the expressions of Na⁺/H⁺ exchanger 1. Moreover, CD44 overexpression upregulated the metastasis of MCF-7 cells, but the elevated metastatic ability was then inhibited by Cariporide. Interestingly, during these processes only the p-ERK1/2 was suppressed by CD44 downregulation and the expression of matrix metalloproteinases and metastatic capacity of MDA-MB-231 cells were greatly inhibited by the MEK1 inhibitor PD98059, which even had a synergistic effect with Cariporide. Furthermore, CD44 downregulation inhibits breast tumour outgrowth and spontaneous lung metastasis.

Conclusions:

Taken together, this work indicates that CD44 regulates the metastasis of breast cancer cells through regulating NHE1 expression, which could be used as a novel strategy for breast cancer therapy.     

P2Y2R activation by nucleotides released from the highly metastatic breast cancer cell contributes to pre-metastatic niche formation by mediating lysyl oxidase secretion,collagen crosslinking,and monocyte recruitment

Tumor microenvironmental hypoxia induces hypoxia inducible factor-1α (HIF-1α) overexpression, leading to the release of lysyl oxidase (LOX), which crosslinks collagen at distant sites to facilitate environmental changes that allow cancer cells to easily metastasize. Our previous study showed that activation of the P2Y₂ receptor (P2Y₂R) by ATP released from MDA-MB-231 cells increased MDA-MB-231 cell invasion through endothelial cells. Therefore, in this study, we investigated the role of P2Y₂R in breast cancer cell metastasis to distant sites. ATP or UTP released from hypoxia-treated MDA-MB-231 cells induced HIF-1α expression and LOX secretion by the activation of P2Y₂R, and this phenomenon was significantly reduced in P2Y₂R-depleted MDA-MB-231 cells. Furthermore, P2Y₂R-mediated LOX release induced collagen crosslinking in an in vitro model. Finally, nude mice injected with MDA-MB-231 cells showed high levels of LOX secretion, crosslinked collagen and CD11b⁺ BMDC recruitment in the lung; however, mice that were injected with P2Y₂R-depleted MDA-MB-231 cells did not exhibit these changes. These results demonstrate that P2Y₂R plays an important role in activation of the HIF-1α–LOX axis, the induction of collagen crosslinking and the recruitment of CD11b⁺ BMDCs. Furthermore, P2Y₂R activation by nucleotides recruits THP-1 monocytes, resulting in primary tumor progression and pre-metastatic niche formation.