TRANSLATIONAL AND CLINICAL RESEARCH Tumor Tropism of Intravenously Injected Human-Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Their Gene Therapy Application in a Metastatic Breast Cancer Model JING YANG,a DANG HOANG LAM,a,b SALLY SALLEE GOH,a ESTHER XINGWEI LEE,b YING ZHAO,b FELIX CHANG TAY,a CAN CHEN,a SHOUHUI DU,a GHAYATHRI BALASUNDARAM,a,b MOHAMMAD SHAHBAZI,a,b CHEE KIAN THAM,c WAI HOE NG,d HAN CHONG TOH,c SHU WANGa,b Department of Biological Sciences, National University of Singapore, Singapore; bInstitute of Bioengineering and Nanotechnology, Singapore; cDepartment of Medical Oncology, National Cancer Centre Singapore, Singapore; d Department of Neurosurgery, National Neuroscience Institute, Singapore a
Key Words. Pluripotent stem cells • Neural stem cell • In vivo tracking • Gene therapy • Breast cancer
ABSTRACT Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive humaninduced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the
cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumorbearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. STEM CELLS 2012;30:1021–1029
Disclosure of potential conflicts of interest is found at the end of this article.
INTRODUCTION Recent years have seen the development of neural stem cell (NSC)-based cancer therapeutics [1–3]. These cells display intrinsic tropism for sites of brain injury and can spread through the existing migratory pathways as well as nontypical routes using cytokines, chemokines, and/or growth factors released from the injury sites as candidate migration stimulatory signals [4–6]. As a common feature of tumor development, tumor cells misregulate the expression of chemokines, growth factors, and extracellular matrix proteins. Tumor growth will further cause damage to nearby normal tissues and is considered as a nonhealing wound [7]. Hence, there might be common cell trafficking regulators between tissue injury and tumor development that attract NSCs [4–6]. While the exact tumor-homing mechanism of NSCs has yet to be fully elucidated, it is possible that these stem cells may
migrate toward either intracranial or extracranial tumors when the growth of the tumors causes tissue damage and secrets the cell trafficking regulators. Mesenchymal stem cells (MSCs) also display the ability to home to sites of injury and can be recruited into tumors, thus they are attractive as vehicles for anticancer drugs. However, it has been noted that MSCs that reach tumor stroma may contribute to tumor growth by promoting angiogenesis, creating a niche to support survival of cancer stem cells, suppressing immune responses against tumor cells, and/or promoting cancer metastasis [8–10]. Thus, there is a need to develop new cellular vehicles with tumor-homing property yet without tumor growth-promoting effects. In this regard, NSCs appear promising. To realize the full potential of the strategy of using NSCs as cancer therapeutics, it is desirable to have a reliable and stable supply of human NSCs. Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS)
Author contributions: J.Y. and D.H.L.: collection and assembly of data, data analysis and interpretation, and manuscript writing; S.S.G., E.X.L., Y.Z., F.C.T., C.C., S.D., G.B., and M.S.: collection and assembly of data and data analysis and interpretation; C.K.T., W.H.N., and H.C.T.: conception and design and financial support; S.W.: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, and final approval of manuscript. J.Y. and D.H.L. contributed equally to this article. Correspondence: Shu Wang, Ph.D., Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore. Telephone: 65-6874-7712; Fax: 65-6779-2486;; e-mail:
[email protected] Received July 1, 2011; accepted for publication December C AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/ 22, 2011; first published online in STEM CELLS EXPRESS February 6, 2012. V stem.1051
STEM CELLS 2012;30:1021–1029 www.StemCells.com
Neural Stem Cells and Breast Cancer Gene Therapy
1022
cells, can be expanded indefinitely in culture and have the potential to generate all types of cells in vitro in virtually unlimited numbers. Hence, these cells are attractive cell sources to derive differentiated cells, including NSCs [11, 12]. Human iPS cells appear more attractive for clinical applications since these cells can be relatively easily generated through reprogramming of differentiated somatic cells with transcription factors, a procedure that circumvents the bioethical controversies associated with the derivation of human ES cells from human embryos [12–14]. Also, it is assumed that transplantation of the differentiated progeny of iPS cells reprogrammed from the patient’s own cells reduces the likelihood of immune rejection. However, differentiation of human iPS cells results in a reduced and more variable yield of neural progeny when compared with human ES cells [15]. Herein, we report that tumor tropic NSCs can be derived from human iPS cells and used to attenuate tumor growth in both immunodeficient and immunocompetent mice.
MATERIALS
AND
METHODS
Cells A Cre-excisable polycistronic lentiviral vector containing Oct4, Klf4, Sox2, and c-Myc genes (Millipore, Bedford, MA, http://www.millipore.com) was used to generate iPS cells from human foreskin fibroblasts (Millipore, Supporting Information Figs. S1, S2; Table S1). Prior to differentiation of the generated iPS cells into NSCs, iPS cell colonies cultured on Matrigel were dissociated to single cells using Accumax (Millipore). The generated single cells were cultured in an NSC medium [16] to derive NSCs (iPS-NSCs, Supporting Information Fig. S2). After 1 month of passaging, a homogeneous cell population with characteristic bipolar NSC morphology was achieved. The doubling time of the generated iPSNSCs was approximately 3–4 days. For the passage of the iPS-NSCs, the cells were treated with accutase (Millipore) and subcultured at a split ratio of 1:2. The iPS-NSCs could be cryopreserved in the NSC medium containing 10% dimethyl sulfoxide (DMSO) and remained viable after thawing from liquid nitrogen storage.
In Vivo Imaging to Assess Cell Distribution Female-specific pathogen-free athymic nude (nu/nu) BALB/c mice, non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) IL2Rgamma-c null (NSG) mice, and immunocompetent BALB/c mice (4- to 6-week old) were used to establish orthotopic breast cancer models. 4T1-luc2, a luciferaseexpressing mouse breast cancer cell line (Caliper Life Sciences, Hopkinton, MA, http://www.caliperls.com), was inoculated into the mammary fat pad of anesthetized mice at a dose of 1 105 cells in 50 ll PBS per animal. One week later, a lipophilic, near-infrared fluorescent dye 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl indotricarbocyanine iodide (DiR, Caliper Life Sciences) was used to label iPS-NSCs. DiR has an excitation/emission spectrum in the near infrared (750/780 nm), which makes it ideal for in vivo imaging due to significantly reduced autofluorescence from animals at higher wavelengths. Cells were labeled with 5 lg/ml of DiR overnight, followed by washing with PBS three times. The labeled cells, 1 106 in 200 ll PBS per animal, were injected into mice through the tail vein. The labeled cells were also tested in mice without tumors. To evaluate in vivo NSC distribution, whole-animal imaging and ex vivo organ imaging were performed using the in vivo imaging systems (IVIS) imaging system coupled with cool charge coupled device (CCD) camera and the indocyanine green (ICG) filter (Caliper Life Sciences). Images and measurements of fluorescent signals were acquired and analyzed using Living Image 3.2 (Caliper Life Sciences), quantified as efficiency. Firefly luciferase bioluminescence signals of 4T1-luc2 cancer cells were acquired using the IVIS imaging system with an emission filter of 560 nm after intraperitoneal injection of Dluciferin (100 mg/kg, Promega, Madison, WI, http://www. promega.com). For histological examination of NSC distribution, mice were sacrificed by cardiac perfusion with PBS, followed by 4% paraformaldehyde in PBS. The organs were harvested for cryostat sectioning. Cryostat sections were observed under a near-infrared confocal microscope. The fluorescent intensity was quantified using the Image J software (U.S. National Institutes of Health, Baltimore, MD) and the mean value was shown. All handling and care of animals were carried out according to the Guidelines on the Care and Use of Animals for Scientific Purposes issued by the National Advisory Committee for Laboratory Animal Research, Singapore.
DNA Extraction and Real-Time Quantitative Polymerase Chain Reaction Baculoviral Transduction Baculoviral vectors were produced and propagated in Spodoptera frugiperda (Sf9) insect cells preadapted to Sf-900 II serum-free medium (Invitrogen, Carlsbad, CA, http://www. invitrogen.com), as described previously [16, 17]. Subsequent baculovirus preparation steps and virus titer measurements were conducted according to the Bac-to-Bac baculovirus expression system manual (Invitrogen). To examine baculoviral transduction efficiency in iPSNSCs, a recombinant baculovirus vector containing the enhanced green fluorescent protein (eGFP) gene expression cassette [17] was used. iPS-NSCs were seeded at a density of 5 105 cells per well in a six-well plate and transduced overnight with baculoviral vectors at an multiplicity of infection (MOI) of 100 in the NSC medium. Fluorescence-activated cell sorting (FACS) analysis was used to quantify the transduction efficiency of the baculoviral vector in iPS-NSCs. After accutase dissociation, the cells were collected, washed in phosphate buffered saline (PBS) twice, and analyzed with the FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, http://www.bd.com).
In vivo NSC distribution was also evaluated using a real-time quantitative polymerase chain reaction (qPCR) method. Mice tissues were homogenized in buffer animal tissue lysis (ATL) (Qiagen, Valencia, CA, http://www.qiagen.com), and genomic DNA was extracted with DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol. qPCR was carried out in the iQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, http://www.bio-rad.com) using the iQ SYBR Green Supermix (Bio-Rad) with 100 ng total genomic DNA in triplicate in a 20-ll reaction. A pair of primers (forward: 50 GCTCAGTTCCAGTTGCTTG-30 , reverse: 50 -GCAGTGAGCCAAGATTGCAC-30 ) that amplify a 215-bp fragment of human leukocyte antigen (HLA)-A short tandem repeat (STR)#54 [18, 19] were used to detect human DNA in 100 ng genomic DNA from mice injected with human cells. Mouse GAPDH primers (forward: 50 -GGGTGGAGCCAAACGGG TC-30 , reverse: 50 -GGAGTTGCTGTTGAAGTCGCA-30 ) that amplify a 550-bp fragment were used to verify the integrity of mouse DNA and act as an internal reference gene. The final concentration for HLA-A STR#54 primers was 250 nM each, and the final concentration for mouse GAPDH primers
Yang, Lam, Goh et al.
1023
Figure 1. In vivo imaging to demonstrate the tumor tropism of induced pluripotent stem (iPS)-NSCs. Immunodeficient NSG (A, C) and immunocompetent BALB/c (B, D) mice were inoculated with 4T1-luc2 breast cancer cells (Luc-4T1) in the mammary fat pad. At day 7 post-tumor inoculation, DiR-labeled iPS-NSCs were injected through the tail vein into the tumor-bearing mice. Imaging was performed on the indicated days. Imaging at day 0 post-NSC injection was performed 5 minutes after NSC tail vein injection. (A, B): Dual-color, whole-animal imaging with the in vivo imaging systems (IVIS) imaging system. Three mice without tumors (normal mice) and three tumor-bearing mice (4T1 mice) were imaged ventrally. DiR near-infrared fluorescence imaging was used to show the distribution of systemically injected iPS-NSCs, whereas bioluminescence imaging was used to document tumor growth in the same set of tumor-bearing mice. (C, D): iPS-NSC accumulation in the region with orthotopic 4T1 breast tumor within 2 weeks post-NSC injection in 4T1 mice. DiR signal values of the iPS-NSCs in the tumor region are expressed as a percentage of total DiR signals in an animal (mean 6 SD, n ¼ 3 mice per time point). Abbreviations: NSCs, neural stem cells; NSG, NOD/SCID IL2Rgamma-c null.
was 200 nM each. PCR cycling conditions were: initial 95 C for 3 minutes, then 95 C for 15 seconds and 60 C for 1 minute with real-time data acquisition for 40 cycles, followed by melt curve detection at 0.5 C per 10-second increment from 55 C to 95 C. The specificity of the PCR products was verified by agarose gel electrophoresis. The number of human iPS-NSCs was calculated from respective CT (cycle threshold) values using a linear equation from a human iPS-NSC genomic DNA standard curve and normalized to the mouse GAPDH reference gene.
Animal Experiments to Evaluate Therapeutic Efficacy To investigate therapeutic effects of iPS-NSC-mediated gene delivery, orthotopic breast cancer models were established as described above in both immunodeficient and immunocompetent mice. Recombinant baculovirus vectors containing the herpes simplex virus thymidine kinase (HSVtk) gene expression cassette [16] were used for transduction of iPS-NSCs. Seven days after tumor inoculation, baculovirus-transduced iPS-NSCs (1 106 in 200 ll PBS per animal) were injected into the mice through the tail vein. Animals were intraperitoneally administered with 200 ll of 5 mg/ml ganciclovir www.StemCells.com
(GCV) (50 mg/kg body weight) or PBS daily from days 8 to 28 post-tumor inoculation. Tumor growth was monitored using whole-animal imaging as described above, and animal survival rates were recorded.
Statistical Analysis All data are represented as mean 6 SD. The statistical significance of differences was determined by unpaired Student’s t test or the analysis of variance with replication followed by Fisher’s least significant difference post hoc analysis. The statistical analysis of survival data was performed using the log-rank test followed by Holm–Sidak method for pairwise multiple comparison tests. A p-value of
