Human Embryonic Stem Cell-derived Endothelial Cells As Delivery System For Specific-Targeted Treatment Of Metastatic Breast Cancer

Breast cancer is one of the most prevalent cancers among women all over the world.Up to now, the primary breast cancer can be cured by mastectomy, and metastasiscauses nearly90%of the mortality among breast cancer patients. Lung is one of themost common sites of breast cancer metastasis. Multiple bilateral lung metastases areunsuitable for surgery and are usually treated with chemotherapy. However, mostchemotherapy treatments kill not only cancer cells but also normal cells, and this maycause side effects such as alopecis and nausea. If we can find a cellular vehicle whichbrings drugs or cytotoxic genes to tumor sites specifically and does not affect normaltissue, it will highly improve the efficiency of treatment and reduce the side effects.Tumor angiogenesis plays an important role in tumor growth and metastasis. Recentreports revealed that endothelial progenitor cells (EPCs) derived from bone marrowparticipate in tumor angiogenesis. Besides autologous EPCs, exogenous EPCs canalso aggregate to tumor sites via intravenous injection. Their potential in cancertherapy as cellular vehicles has been demonstrated by several studies. However, thesource of adult EPCs was limited and hard to obtain, which hampers their clinicalapplication. Thus, it is urgent for researchers to find alternative cellular deliverysystems for targeted treatment of cancer.For their ability for self-renewal and pluripotency, human embryonic stem cells(hESCs) hold tremendous potential in the field of cellular therapy as it candifferentiate into almost all cell types. There were evidences showing that hESCs atdifferent stages of differentiation can be induced into endothelial cells, and we intendto test whether human embryonic stem cell-derived endothelial cells (hESC-ECs) can also be used as delivery system for specific-targeted treatment of metastatic breastcancer.We harvested sprouting embryoid bodies from human embryonic stem cell line H9following previous protocols, and thereafter sorted hESC-ECs with the markers CD31and CD144by FACS. First, we collected hESC-ECs conditioned medium (EC-CM)to test the paracrine effects of hESC-ECs on tumor cells. After treated with EC-CM,human breast cancer MDA-MB-231proliferated slower and exhibited a higherapoptosis rate in the culture condition with low level of serum, and cells in G1phaseincreased while cells in S phase and G2/M phase decreased. The migration ability ofMDA-MB-231cells was also impaired after treated with EC-CM, and themesenchymal markers α-SMA and fibronectin were down-regulated while theepithelial marker ZO-1was up-regulated and translocated to cell surface. We alsofound that the expression of β-catenin in MDA-MB-231cells was down-regulatedfollowing EC-CM treatment.Next, breast cancer pulmonary metastasis model in NOD/SCID mice was establishedby injecting1.5×10~6MDA-MB-231cells intravenously.21days later, we injected1.0×10~6hESC-ECs labeled with Green Fluorescent Protein (GFP)-Firefly Luciferase(Fluc)(EC-BF cells) intravenously to mice bearing tumor, and observed thedistribution of injected EC-BF cells via bioluminescence imaging. We observed thatEC-BF cells can specifically aggregate to metastases, and stayed for more than72hours.Furthermore, Renilla Luciferase-Red Fluorescent Protein-HSV-truncated thymidinekinase (RL-RFP-HSV-ttk) fragment was cloned into lentiviral expression vector pLV-EF1α-MCS-IRES-Bsd, and we obtained hESC-ECs expressingRL-RFP-HSV-ttk triple fusion protein (EC-TF) by transducing hESC-ECs withpLV-EF1α-TF-Bsd. To verify the efficiency of HSV-ttk/GCV system in vitro, theprodrug of HSV-ttk, ganciclovir (GCV) was added into the culture medium of EC-TFcells, and we observed that EC-TF cells and the co-cultured MDA-MB-231cellscould be killed by GCV and bystander effects respectively.At last, the potential of EC-TF cells in treating metastatic breast cancer was evaluatedvia bioluminescence imaging of MDA-MB-231tumor labeled with GFP-FL. It wasobserved that intravenously injected EC-TF cells can significantly suppress tumorgrowth, and this effect was enhanced when pro-drug ganciclovir (GCV) was given.Taken together, our data demonstrated that hESC-ECs can suppress the Wnt/β-cateninsignaling pathway in MDA-MB-231cells via paracrine effects, thereby suppress theproliferation and metastasis of MDA-MB-231cells. And intravenously injectedhESC-ECs can specifically aggregates in pulmonary metastases. Moreover,RL-RFP-HSV-ttk triple fusion protein can be stably expressed by hESC-ECs, andGCV can kill the EC-TF cells efficiently. Last, when given GCV, intravenous injectedEC-TF cells can significantly suppress the tumor growth of MDA-MB-231pulmonary metastases via bystander effects.