Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma
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- Stoff-Khalili, M.A., Rivera, A.A., Mathis, J.M. et al. Breast Cancer Res Treat (2007) 105: 157. doi:10.1007/s10549-006-9449-8
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Alternative and complementary therapeutic strategies need to be developed for metastatic breast cancer. Virotherapy is a novel therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. However, the success of virotherapy has been limited due to inefficient virus delivery to the tumor site. The present study addresses the utility of human mesenchymal stem cells (hMSCs) as intermediate carriers for conditionally replicating adenoviruses (CRAds) to target metastatic breast cancer in vivo.
HMSC were transduced with CRAds. We used a SCID mouse xenograft model to examine the effects of systemically injected CRAd loaded hMSC or CRAd alone on the growth of MDA-MB-231 derived pulmonary metastases (experimental metastases model) in vivo and on overall survival.
Intravenous injection of CRAd loaded hMSCs into mice with established MDA-MB-231 pulmonary metastatic disease homed to the tumor site and led to extended mouse survival compared to mice treated with CRAd alone.
Injected hMSCs transduced with CRAds suppressed the growth of pulmonary metastases, presumably through viral amplification in the hMSCs. Thus, hMSCs may be an effective platform for the targeted delivery of CRAds to distant cancer sites such as metastatic breast cancer.
KeywordsBreast cancerCell vehicleCRAdsMetastasesStem cellsVirotherapy
In the United States, breast cancer remains the most common malignancy in women. In some women, breast cancer is a local disease without spread. Such early breast cancers are usually diagnosed by screening mammography and are highly curable with local or regional treatment alone. However, most women with primary cancer have subclinical metastases, and in a high percentage of those treated with apparently curative surgery, distant metastases ultimately develop. The clinical course of metastatic breast cancer is variable. Chemotherapy, hormonal therapy, radiotherapy, and limited surgery are all used in the treatment of women with metastatic breast cancer, although the overwhelming majority of these women will die of their disease. In view, of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed.
In this regard, virotherapy is an exciting therapeutic approach for the treatment of cancer in which the replicating virus itself is the anticancer agent. Among various viruses, the adenovirus-based vector has emerged as a leading candidate for in vivo cancer virotherapy. Conditionally replicative adenovirus based agents (CRAds) have been designed to replicate in tumor cells whereby the virus can self-amplify and spread in the tumor from an initial infection of only a few cells. However, highly effective use of CRAd agents in tumors clinically has been heretofore hindered by three main factors: (1) low viral infectivity, (2) suboptimal replicative specificity and (3) inefficient viral agent delivery to the tumor site . With respect to breast cancer, transduction efficacy by adenovirus serotype 5 (Ad5) is often suboptimal due to the highly variable and often low expression pattern of the primary adenovirus receptor, coxsackie adenovirus receptor (CAR) [4, 9]. To circumvent this, genetic alterations of the virus fiber protein that utilizes CAR-independent entry pathways have been identified, thus bypassing CAR deficiency on cancer cells and enhancing tumor transduction. In parallel, strategies have been developed to enhance the transcription selectivity of current vector systems toward tumor cells by using tumor specific promoter (TSP) that limit ectopic expression in non-tumor cells and decrease treatment-associated toxicities. However, efficient virus delivery to the tumor site is a central mandate of virotherapy.
In this regard, cell carriers exhibiting endogenous tumor homing activity have been recently exploited to chaperone virus delivery to the tumor site. Although the utility of cells as vehicles for toxic genes, anti-angiogenic molecules, and immunostimulatory genes has been suggested in several studies, there have been only limited studies whereby cells have been exploited as carriers for virotherapeutic agents. In this regard, we have proposed human mesenchymal stromal cells (hMSCs) as carriers of oncolytic viruses in vitro . MSCs are bone marrow-derived non-hematopoietic precursor cells that when systemically administered, home to the tumor, preferentially survive and proliferate in the presence of malignant cells and become incorporated into the tumor architecture as stromal fibroblasts . In this regard, it has been recently shown that systemically administered hMSCs home to breast cancer metastasis of the lung .
Based upon these findings, we hypothesized that hMSCs could be used as a targeting strategy for CRAds in the treatment of breast cancer metastasis of the lung. Our results, demonstrating that systemic administration of hMSC carriers can target CRAds to metastatic disease, constitute a novel therapeutic paradigm for breast cancer that couples cell therapy with virotherapy.
Materials and methods
The CRAd Ad5/3.CXCR4 was constructed as follows: the plasmid pBSKCAT/CXCR4, which contains a 279 bp sequence from the human CXCR4 promoter (−191 to +88), was a kind gift of Dr. Nelson L. Michael . The CXCR4 promoter sequence (NCBI Accession Number AY728138, from 1780 to 2059 bp) containing the 279 bp CXCR4 promoter and the simian virus 40 (SV40) polyadenylation (poly A) signal was cloned by PCR into the NotI/XhoI site of pScsE1 plasmid [12, 17] (a kind gift from Dr. Dirk Nettelbeck, Department of Dermatology, University Medical Center-Erlangen, Erlangen, Germany), resulting in pScsE1CXCR4 that contained the E1A gene downstream of the CXCR4 gene promoter. The Ad vector, pAdback 5/3 was a kind gift from Dirk Nettenbeck and contains both the E3 gene and a capsid modified F5/3 [12, 17]. After cleavage with PmeI, the shuttle vector, pScsE1CXCR4, was recombined with pAdback5/3 to generate the CRAd Ad5/3.CXCR4, where the human Ad knob serotype 5 is replaced by human Ad knob serotype 3. The Ad vector, pVK503c, was a kind gift from Dr. V. Krasnykh (M.D. Anderson, Houston, TX), and contains both the E3 gene and a capsid modified RGD4C . After cleavage with PmeI, the shuttle vector, pScsE1CXCR4, was recombined with Cla I linearized pVK503c to generate the CRAd Ad5RGD.CXCR4 with a fiber protein incorporating an RGD-motif in the HI-loop of Ad5 fiber. The recombinant plasmids were linearized with PacI and transfected into 293 cells using superfect reagent (Qiagen; Valencia, CA) to generate the Ad5/3.CXCR4 and Ad5R6D.CXCR4 adenoviruses. The other replication competent adenoviruses used in this study were: wild type Ad5wt with unmodified fiber , Ad5/3wt with a chimeric fiber having the knob of Ad5 fiber replaced by knob of Ad3 fiber [12, 17], Ad5.RGDwt with a fiber protein incorporating an RGD-motif in the HI-loop of Ad5 fiber. Ad.CXCR4Luc  was used for replication negative control. The adenoviruses were propagated in the A549 cells (a lung cancer cell line in which the CXCR4 gene is overexpressed), and purified by double CsCl density gradient centrifugation, followed by dialysis against phosphate buffered saline (PBS) containing 10% glycerol. The concentration of total viral particle numbers (PN) was determined by measuring absorption at 260 nm. Infectious PNs were determined by measuring the concentration of viral hexon protein-positive 293 cells after a 48-h infection period, using an Adeno-X Rapid Titer Kit (Clontech; Mountain View, CA).
Cell line and cell culture
MDA-MB-231 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC) and cultured as described. In brief, the cells were maintained in DME/F-12 medium (Life Technologies, Inc., Grand Island, NY), containing 10% fetal bovine serum (FBS: Gemini Bio-Products, Woodland, Ca), and 1% antibiotic–antimycotic solution (penicillin–streptomycin–fungizone, Sigma Chemicals Co., St. Louis, MO). The cells were maintained in T-175 flasks at 37°C and 5% humidified CO2, and were sub-cultured using 1% trypsin–EDTA (Gibco BRL, Life Technologies).
Human mesenchymal stem cells and labeling
Human mesenchymal stem cells were obtained from the Tulane Center for Gene Therapy (Tulane University Health Sciences Center, New Orleans, LA, USA) and cultured according to the protocol provided. Carbocyanine dye (CellTracker CM-Dil; Molecular Probes Inc, Eugene, Ore) was used to label the human mesenchymal stem cells according to the manufacturer’s standard protocol as previously described .
In vitro cytotoxicity assay of CRAds
For determination of virus-mediated cytotoxicity, 1 × 104 MDA-MB-231 cells were seeded in 48-well plates and infected with adenoviruses at MOI of 1–1000 or were mock-infected . To visualize cell killing, cells were fixed and stained with 1% crystal violet in 70% ethanol for 20 min followed by washing with tap water to remove excess dye at day 3, 5, 7 and 9. The plates were dried and images were captured with a Kodak DC260 digital camera (Eastman Kodak, Rochester, NY, USA).
In vitro cytotoxicity assay of human mesenchymal stem cells loaded with CRAd
About 3 × 105 hMSCs were plated in 6 well plates and infected with Ad5/3.CXCR4 at MOI of 1–1000 in 2% media. After 18 h the Ad5/3.CXCR4 infected hMSCs, now called hMSC-Ad5/3.CXCR4, were washed three times with PBS and trypsinized. Next, 5 × 104 MDA-MB-231 breast cancer cells plated in 48 well plates were co-cultured with 5 × 104 hMSCs carrying Ad5/3.CXCR4 at different MOIs. To visualize cell killing, cells were fixed and stained with 1% crystal violet in 70% ethanol for 20 min followed by washing with tap water to remove excess dye at day 3, 5, 7, and 9. The plates were dried and images were captured with a Kodak DC260 digital camera (Eastman Kodak, Rochester, NY, USA).
Quantitating virus replication
Purification of the DNA and quantitative real-time PCR for E4 was performed as previously described . About 1.5 × 105 cells were seeded per well in a six-well plate. The next day cells were infected with the indicated viruses at different MOIs or mock infected and growth medium was collected at the indicated time points. Negative controls without templates were performed for each reaction series, and an internal control (human GAPDH) was used to normalize the copy number for the E4 genes. Comparison of replication rates of different treatment groups were performed with a Student’s t-test.
Mouse xenograft model for experimental metastatic breast cancer to the lung
Female C.B-17 SCID mice (6-weeks old) were obtained from Charles River Laboratories, Inc (Wilmington, MA, USA). Mice were used according to approved institutional protocols. The mouse xenograft model for metastatic breast cancer to the lung and the route of hMSC injection was performed as described . Mice were injected intravenously in the lateral tail vein with 2 × 106 MB-MDA-231 suspended in 200 μl of PBS . In preliminary experiments, we determined that all mice injected with 2 × 106 MB-MDA-231 developed macroscopic tumor nodules in their lungs at 14 days after tumor cell injection (data not shown). Fourteen days later, treatment was started. The following preparations were made prior intravenous injection: 106 hMSCs were infected with Ad5/3.CXCR4 at MOI of 1000 in 2% media. After 18 h the Ad5/3.CXCR4 infected hMSCs, now called hMSC-Ad5/3.CXCR4, were washed three times with PBS and trypsinized. Then hMSC-Ad5/3.CXCR4 and hMSCs alone were labeled with dye (CellTracker CM-Dil; Molecular Probes Inc., Eugene, Ore) at 37°C in complete media for 30 min followed by three washes with PBS and resuspended in 100 ul of PBS. Then, at day 14, mice were we injected intravenously in the lateral tail vein with either 106 hMSCs which had been infected with Ad5/3.CXCR4 of MOI 1000 (n = 8), hHMSC alone (n = 8) or Ad5/3.CXCR4 of MOI of 1000 alone (n = 8) suspended in 100 μL PBS. Non-treated MDA-MB-231 tumor bearing mice (n = 8) and healthy mice (n = 8) served as a control.
Determination of effect of CRAd loaded hMSCs on MB-MDA-231 tumor weight in mouse lung
Fourteen days after MB-MDA-231 tumor cell injection (as described above), the mice obtained treatment intravenously in the lateral tail vein with either 106 hMSCs which had been infected with Ad5/3.CXCR4 of MOI 1000 (n = 4), MSC alone (n = 4) or Ad5/3.CXCR4 of MOI of 1000 alone (n = 4) suspended in 100 μl PBS. Mice injected with MB-MDA-231 tumor cells alone (n = 4) and healthy mice with no tumor cell injection (n = 4) served as controls. Mice were sacrificed by asphyxiation with CO2 30 days after tumor cell injection. We measured the weight of whole lungs in all groups of mice and used whole lung weight as a surrogate endpoint of MD-MBA-231 tumor burden in the lung and to assess the effect of hMSCs, Ad5/3.CXCR4 on tumor growth . All mice were followed daily until euthanasia. None of the mice had to be sacrificed because of excessive bleeding, open wound infection, moribund status, or prostration with weight loss of more than 25% of initial body weight.
Determination of effect of CRAd loaded hMSCs on survival in mice bearing metastatic breast cancer
Fourteen days after MD-MBA-231 tumor cell injection (as described above), the mice obtained treatment intravenously in the lateral tail vein either 106 hMSCs which had been infected with Ad5/3.CXCR4 of MOI 1000 (n = 10), hMSC alone (n = 10) or Ad5/3.CXCR4 of MOI of 1000 alone (n = 10) suspended in 100 μl PBS. Mice injected with MB-MDA-231 tumor cells alone (n = 10) and healthy mice with no tumor cell injection (n = 10) served as controls. All mice were followed daily until day 150. None of the mice had to be sacrificed because of excessive bleeding, open wound infection, moribund status, or cachexia.
MDA-MB-231 lung metastases
To assess the effect of transient transfection of hMSC-Ad5/3.CXCR4 on MDA-MB-231 lung metastasis, lungs were harvested 30 days later, fixed in 10% neutral-buffered Formalin, longitudinally trisected, paraffin embedded, and three 5–7 μm thickness sections were cut at 200 μm intervals from each embedded block. Tissue sections were stained with hematoxylin and eosin, examined for the presence of tumor nodules by a pathologist unaware of the treatments.
Tissue processing and imaging studies
Lungs from each group of mice were harvested 30 days after treatment, fixed in 10% neutral-buffered formalin, longitudinally trisected, paraffin embedded, and three 5–7 μm thickness sections were cut at 200 μm intervals from each embedded block. Tissue sections were stained with hematoxylin and eosin, examined for the presence of tumor nodules by a pathologist. In addition, lungs for each group of mice were embedded in Tissue TEK OTC compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −80°C.
GFP labeled hMSCs were identified in the lung as following: Frozen tissue sections were air dried, fixed in 4% paraformaldehyde for 1 h, permeabilized in PBS with 0.2% Triton X-100, washed 5 min in PBS and blocked in 25% goat serum for 30 min. The sections were washed in PBS three times for 5 min, mounted with VectaShield mounting medium with 4′,6-diamidino-2-phenylindole (H-1200; Vector Laboratories) and then analyzed by fluorescence microcopy. Adenoviral hexon was identified in the lung as following: Frozen tissue sections were air dried, fixed in 4% paraformaldehyde for 1 h, permeabilized in PBS with 0.2% Triton X-100, washed 5 min in PBS and blocked in 25% goat serum for 30 min. Then the tissue sections were treated overnight with goat anti-hexon antibody (Chemicon) at 4°C. The sections were washed in PBS three times for 5 min. The tissue sections were treated with Alexa 534 labeled anti-goat secondary antibody (Alexa 534, Molecular Probes, Invitrogen) for 1 h, washed three times in PBS. All sections were mounted with VectaShield mounting medium with 4′,6-diamidino-2-phenylindole (H-1200; Vector Laboratories). The images were captured with either a Texas Red or a FITC filter in an Olympus AX70 fluorescence microscope equipped with a Zeiss Axiocam camera (Carl Zeiss, Oberkochem, Germany). Individual images were processed and merged using Adobe Photoshop 5.5 application software.
We used the Wilcoxon rank sum test to perform pairwise comparisons of treatment effect on lung weight between all groups. Survival was measured from the day of MDA-MB-231 cell injection until the day of 130. For the survival data, the log-rank test was used to assess differences in survival among the four treatment groups. Because this overall test showed that the difference between MSC-Ad5/3.CXCR4–treated and control mice was statistically significant (P < 0.001), pairwise log-rank tests were performed. All statistical tests were two-sided; a P value of less than 0.05 was considered statistically significant. Statistical analyses were performed by using GraphPad Prism software (GraphPad Software, San Diego, CA).
Identification of a CRAd allowing efficient loading of hMSCs and maximal oncolysis of breast cancer cells
Ad5/3.CXCR4 loaded human mesenchymal stem cells display oncolysis of breast cancer cells in vitro
At day 3 of the co-culture experiment, hMSC-Ad5/3.CXCR4 cells showed evidence of initial oncolysis in MDA-MB-231 cells at MOI of 1000, which was completed at day 7. Interestingly, between days 7 and 9 of co-culture, an increase of oncolysis of MDA-MB231 of more than 3 orders of magnitude was achieved, resulting in complete oncolysis at MOI of 1. These results were correlated with PCR assays of Ad DNA in the culture media, which showed a significant increase in viral copy number between days 7 and 9 corresponding to enhanced viral oncolysis (data not shown). In the aggregate, co-culturing of MDA-MB-231 cells with hMSC-Ad5/3.CXCR4 resulted in increased oncolysis of MDA-MB-231 cells with time, suggesting viral amplification in hMSCs. The killing of MDA-MB-231 was protracted, thus indicating a sufficient time window to manipulate hMSCs with CRAds.
MSC-Ad5/3.CXCR4 homes to breast cancer metastases in the lung in vivo
Systemically administered MSC-Ad5/3.CXCR4 reduces the growth of MDA-MB-231 cell derived lung metastases in vivo
Treatment with hMSC-Ad5/3.CXCR4 improved survival of mice bearing breast cancer metastases in the lungs
In the present study, we have exploited the utility of hMSCs as cellular carriers of CRAds for the therapy of metastatic breast cancer to the lung. We have demonstrated both in vitro and in vivo the capability of hMSCs as intermediate carriers for CRAds. Importantly, we have shown the ability of systematically administered CRAd loaded hMSCs to home to and to kill breast cancer pulmonary metastases. The exploitation of mammalian cells represents a novel approach of coupling virotherapy with cell therapy. In this regard, delivery of conditional replication HSV type 1 viruses by neural precursor cells has been endeavored for the treatment of glioma . Tumor cells have also been used as cellular carriers of replication competent parvoviruses for the treatment of hepatic cancer . Recently, the ability of mesenchymal stem cells to deliver replication competent adenoviruses to ovarian and cervical cancer cells in vitro was investigated . Herein, our present study extends this paradigm to an in vivo approach to distant metastatic cancer.
In the design of our study we investigated a combined cell vehicle therapy and virotherapy approach by exploiting a CRAd agent designed with enhanced tumor infectivity and specificity in vitro as well as anti-tumor potency in vivo. With respect to virotherapy, our CRAd was designed to address the biological requirements of breast cancer. Tumor infectivity enhancement of this CRAd was achieved by incorporation of the knob domain from the human adenovirus serotype 3 (Ad5/3) to provide CAR-independent tropism . Enhanced specificity was achieved by placing the CRAd E1A gene under the transcriptional control of the tumor selective promoter CXCR4. In this regard, the CXCR4 gene promoter revealed a superior “breast cancer—on/liver-off” profile in a preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system . Furthermore, recent evidence points to the SDF-1α-CXCR4 complex as having an important role in progression to metastasis in several tumor contexts [13, 24]. This CRAd combining transductional and transcriptional targeting strategies has been recently shown to exhibit superior infectivity and tumor selective replication in breast cancer (Stoff-Khalili et al., unpublished observations).
The present study addresses the utility of hMSCs as intermediate carriers for CRAds to target metastatic breast cancer in vivo. The choice of hMSCs as cellular vehicles was based on their described tumor homing capacity and the observation that intravenously administered hMSCs do not engraft in healthy organs (i.e. lung, liver, spleen, kidney and muscle) [7, 14, 21]. Indeed, fluorescence microscopy of lung tissue sections showed homing of labeled hMSCs to the breast cancer metastases, while no hMSCs could be identified in healthy regions of the lung. It has been suggested that signals mediating increased turnover and proliferation of connective stromal cells in tumors may induce the homing of MSCs to this site [20, 21]. In this regard, it has been recently suggested that this process may be related to high local concentrations of paracrine growth factors such as fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor-β, or other mediators within the tumor microenvironment .
In order to exploit hMSCs as cellular carriers for CRAds, it was important to consider how to limit viral activity in the carrier cells until the hMSCs would reach the metastatic breast cancer cells in the lung. We selected the Ad5/3.CXCR4 CRAd as optimal for our strategy because this agent exhibited limited cytotoxicity in hMSCs and maximal cytotoxicity in breast cancer cells. Indeed, the identification of labeled hMSCs loaded with the CRAd at metastatic tumor sites in the lungs after systemic injection suggested a sufficient time window for the hMSCs to carry this agent to the disease. The prevention of cytotoxicity of viruses on cell carriers is an important practical issue relevant to our strategy [8, 11]. In this regard, several approaches have been designed to attenuate the cytotoxicity of viruses on cell carriers, such as mimosine treatment to avoid premature destruction of neural precursors serving as cellular carriers for conditionally replicative herpes simplex virus type 1 vectors 
We demonstrated here that hMSC based viral delivery enhanced the oncolytic effect of virotherapeutic treatment and increased the survival of tumor-bearing animals. This result validates the feasibility of using hMSCs as cellular carriers for replication competent adenoviruses. The amount of virus loaded into hMSCs was measured at the time of in vivo delivery and corresponded to the titer of CRAd injected alone. Therefore, the superior therapeutic response of metastatic breast cancer in the lung from treatment with CRAd loaded hMSCs cannot be attributed to a higher dose of the CRAd initially introduced. This response may result from the amplification of viral load in the hMSC carriers or from targeted virus delivery and release of the virus in the vicinity of tumor cells at multiple foci. Further studies are in progress to investigate our hypothesis that hMSCs may function as local factories of CRAds to explain the superior oncolysis of CRAd loaded hMSCs compared to CRAd alone in vivo. In our study, we used an immune-compromised animal model. In future studies, we will investigate if hMSCs could also serve as a “Trojan horse” for CRAd delivery by evading an antiviral immune response. As a result of the transport of CRAds inside hMSCs, CRAds may be protected not only from untargeted trapping but also from inactivation through hemaglutination and neutralization through preexisting antiviral immunity.
In conclusion, this study represents to the best of our knowledge, the first attempt to demonstrate the proof of principle that hMSCs can serve as cellular carriers to deliver CRAds to distant tumors such as breast cancer metastases in the lungs and mediate oncolysis in vivo after systemic administration. This work provides the first evidence to suggest that hMSCs as carrier cells represent a promising mode of administration of CRAds for the treatment of distant neoplastic metastatic disease.
This work was supported by Grant of the Deutsche Forschungsgemeinschaft Sto 647/1-1 (to M. A. Stoff-Khalili), by grants from the National Institutes of Health 5T32CA075930 (NIH Training Grant) and Department of Defense: W81Xwh-05-1-035 (to D. T. Curiel). R01CA93796, R01CA98543 and AR46031 (to G. P. Siegal) and from the Louisiana Gene Therapy Research Consortium, Inc. (to J. M. Mathis) and R01CA108585 (to J. T. Douglas).