Mesenchymal Stem Cell delivery of TRAIL can eliminate Metastatic Cancer

Cell Culture

Tissue culture reagents were purchased from Invitrogen (Paisley, UK) unless otherwise stated. All cells were obtained from Cancer Research UK, London Research Institute (CRUK, London, UK) and were cultured in DMEM and 10% fetal bovine serum (FBS), unless otherwise stated. Human adult mesenchymal stem cells were purchased from Tulane University (New Orleans, USA) and cultured in αMEM with 16% FBS. Adipogenic and osteogenic differentiation of MSCs was performed, as previously described. (14, 15).

TRAIL lentivirus construction and transfection of MSCs

A lentiviral plasmid (pRRL-cPPT-hPGK-mcs-WPRE) into which the Tet-on system elements had been introduced (16) (a kind gift from O. Danos, UCL, London, UK), was used as a backbone for the incorporation of TRAIL DNA. The existing reporter gene, MuSEAP was excised using the Mlu1 and EcoRV restriction sites. The IRES-eGFP sequence (from pENTR1A) was amplified and restriction sites introduced by PCR and then inserted into the plasmid in place of MuSEAP. Subsequently, human TRAIL (amino acids 1-281) (RZPD, Berlin, Germany) was similarly amplified and restriction sites introduced by PCR and then cloned into the lentivirus plasmid, via Mlu1 and BstB1 restriction sites, next to the IRES-eGFP. The plasmid constructs were confirmed by DNA sequence analysis (Cogenics, Essex, UK).

The lentivirus was produced by transfecting 293T cells with 15mls of Optimem plus 10mls of a solution made by mixing 3.6ml polyethylenimine (PEI) (Sigma Aldrich) and 56.4ml Optimem, to 600μg TRAIL plasmid, 450μg of the packaging construct pCMV-dR8.74, 150μg of a plasmid producing the VSV-G envelope, pMD.G2, and 60mls Optimem (both pCMV-dR8.74 and pMD.G2 were a kind gift from A. Thrasher, UCL, London, UK). The lentivirus was concentrated by ultracentrifugation at 18000 rpm (SW28 rotor, Optima LE80K Ultracentrifuge, Beckman) at 4°C and stored at −80°C before use.

Human mesenchymal stem cells were transduced with a multiplicity of infection (MOI) of 10 virus particles for each cell and 4μg/ml of polybrene (Sigma Aldrich, Poole, UK).

Human TRAIL expression was verified by ELISA (R&D Systems, Abingdon, UK) as per manufacturer's instructions and Western blot.

Western blots

Supernatants were centrifuged, and cells lysed in RIPA buffer (PBS, 1% Igepal Ca-630, 0.5% Sodium deoxycholate, 0.1% sodium dodecyl sulphate (Sigma)) supplemented with complete protease inhibitor cocktail (Complete-mini; Roche Diagnostics Ltd, Burgess Hill, UK). Samples were denatured, resolved with a 10% SDS-polyacrylamide gel, and transferred using a semi-dry transfer method (NovaBlot; Pharmacia LKB, Sweden).

Blots were then incubated with anti-TRAIL (1μg/ml H257 rabbit polyclonal antibody (17) (Santa Cruz Biotechnology, Inc., California, USA)) or anti-actin (0.24μg/ml, Sigma) antibodies and detected using enhanced chemiluminescence (GE Healthcare, Bucks, UK) as per manufacturer's instructions.

Co-culture experiments

Human MSCs permanently transduced with the full length TRAIL plasmid (MSCFLT) were plated with target cells. A total of 100000 cells were plated in each 6-well plate. The following day, doxycycline, or other active agents were added to the cocultures and left for 48 hours. Staining of target cells was performed with the fluorescent dye, DiI, as per manufacturer's instructions (Invitrogen, Paisley, UK).

Apoptosis and cell death was assessed by flow cytometry (FACSCalibur or LSRII machines (Becton Dickenson, Oxford, UK)). This was performed on floating and adherent cells from cocultures using Annexin V-647 antibody (Invitrogen, Paisley, UK) and 5μg/ml Propidium Iodide (PI) (Sigma). DAPI (2μg/ml) (Sigma) was used instead of PI when cancer cells were stained with DiI. Annexin V⁻/PI⁻ cells were judged to be non-apoptotic. Annexin V⁺/PI⁻ cells were considered to be apoptotic. Annexin V⁺/PI⁺ cells were recorded as being dead.

The following compounds were used in co-culture experiments; the pancaspase inhibitor zVADfmk (1μg/ml) (Sigma), a soluble recombinant TRAIL (200ng/ml) (R & D Systems, Abingdon, UK) and a neutralising TRAIL antibody (250ng/ml) (R & D Systems).

Production of dominant negative FADD cancer cells

Phoenix packaging cells were transiently transfected with retroviral vector for the dominant negative FADD/GFP construct (dnFADD) (18) or empty vector and the virus containing supernatant was added to infect AM12 packaging cells as previously described (19). The AM12 cell supernatant was added to Hela cells (30% confluence) with 5μg/ml polybrene and the cells were selected with 1μg/ml puromycin (20).

Cell migration assay

An in vitro cell migration assay was performed to determine the tropism of MSCs for tumor cells according to previously described methods (4, 7, 21). 150000 cancer cells or 293T cells were plated in 800μl medium on the bottom well of a transwell plate (8μm pore membrane; Becton Dickenson) for 24 hours before 40000 MSCs in 300μl of the same medium were added to the upper well. The MSCs were allowed to migrate across the membrane for 24 hours at 37°C. The cells attached to the upper side of the membrane were removed, and the migrated cells on the lower side fixed, stained using a Rapid Romanowsky staining kit (Raymond Lamb, Eastbourne, UK), and counted (5 fields/well, triplicate wells) at ×10 magnification (Olympus BX40, Watford, UK).

Xenograft cancer models

All animal studies were performed in accordance with British Home Office procedural and ethical guidelines. Six-week old NOD/SCID mice (Harlan, Bicester, UK) were kept in filter cages.

Subcutaneous tumors were obtained by the injection of two million MDAMB231 cells in 200μl PBS, subcutaneously into the left flank with a 29G needle. Tumors were measured every 3-5 days with callipers, and the volume calculated as 4/3πr³, where r denoted the radius. Metastatic lung tumors were produced by the intravenous delivery of two million MDAMB231 in 200μl PBS into the lateral tail vein.

In vivo use of TRAIL-expressing mesenchymal stem cells (MSCFLTs)

In the subcutaneous models, MSCFLTs, labelled with CM-DiI (Invitrogen) as per manufacturer's instructions, were either delivered concurrently with the cancer cells; 2 million MDAMB231 with 0.75 million MSCFLT in 200μl PBS, or after tumors had become established; 1 million MSCFLTs were injected into established subcutaneous tumors. In metastatic models, 0.75 million MSCFLT cells were suspended in 200μl PBS and injected into the lateral tail vein at 7, 14, 21 and 28 days after the cancer models had been set up (5). To activate the TRAIL constructs in the transduced MSCFLTs, mice were fed water containing 2mg/ml doxycycline and 3% sucrose, with controls fed water with 3% sucrose only.

Mice were sacrificed by CO₂ asphyxiation followed by exsanguination. Subcutaneous tumors were removed and weighed before they were split for digestion with 0.1% collagenase A (Roche Diagnostics Ltd., Burgess Hill, UK) into a single cell suspension as per manufacturer's protocol, or fixed in 4% paraformaldehyde for histology. The lungs were excised and weighed before they were insufflated with a fixed 20 cm pressure of 4% paraformaldehyde and then bathed in 4% paraformaldehyde for histology. In some lungs, the left lobe was removed and snap frozen in liquid nitrogen before insufflation.

Immunohistochemistry

Fixed specimens were embedded in paraffin and cut into 3μm sections for Haematoxylin and Eosin (H&E) staining. GFP antibody (Rabbit polyclonal: Invitrogen) and TRAIL antibody (Rabbit monoclonal: Santa Cruz, Heidelberg, Germany) were used as primary antibodies and detected with biotinylated secondaries and diaminobenzidine (DAB; Vector Laboratories, California, USA). Fluorescent microscopy was used to detect DiI positive cells with DAPI counterstain, and secondary fluorescent antibodies against primary vimentin antibodies (sc7557, Santa Cruz). TUNEL staining was performed according to the manufacturer's instructions (Roche Diagnostics, Burgess Hill, UK). Microscopy was performed using light (Olympus BX40, Watford, UK), fluorescent (Carl Zeiss Ltd., Axioskop 2, Welwyn Garden City, UK) or confocal microscopes (Bio-Rad MRC 1024, Hercules, California, USA).

Quantitative Real-Time RTPCR

RNA was extracted from the snap frozen left lung with TRIzol (Invitrogen) and treated with DNAse (Ambion, Texas, USA). cDNA was synthesized using a RT-PCR kit, according to manufacturer's instructions (Roche Diagnostics). Real-time PCR amplification was performed with Light Thermocycler (Roche Diagnostics) using SYBR Green qPCR kit (Invitrogen) with primers for human TRAIL and the control 18s.

Metastases Quantification

The number and size of tumor nodules were assessed in H&E sections using similar methodology to that previously described (22). Photomicrographs of representative sections of the entire lung were taken at ×4 magnification. This created a complete picture of all lobes of the lungs of the mice. Image analysis software (SimplePCI High Performance Imaging Software, Hamamatsu Photonics, Herrsching, Germany) was used to trace around the metastatic deposits and the lung sections and then calculate lung and metastasis area and number of metastases.

Statistics

Statistical analysis was performed using GraphPad Prism v4 (GraphPad Software, California, USA). Multiple groups were analysed by Anova or Kruskal-Wallis tests. Single group data was assessed using Students t-test. All in vitro experiments were performed in triplicate unless specified.

Characterisation and TRAIL transduction of Mesenchymal stem cells

Fully characterised MSCs were purchased from Tulane University and were shown to differentiate into fat and bone in differentiation assays and have the expected colony forming efficiency (48% ± 2.83 (SD)) (Figure 1A). Mesenchymal stem cells were stably transduced with our lentivirus expressing TRAIL and GFP under the control of doxycycline at different multiplicities of infection (MOI). An MOI of 10 was used for further experiments producing 82.13% ± 0.40 successful transduction (GFP expression) at day seven (Figure 1B), while limiting the number of dead cells 4.8% ± 1.83).

MSCFLTs were examined for TRAIL and GFP expression using flow cytometry, Western blots and ELISA. Flow cytometry demonstrated <0.5% GFP expression before activation with doxycycline while 48 hours after doxycycline addition to the media, this increased to 74.7% ± 2.5 (Figure 1B). Withdrawl of doxycycline, after the cells had been exposed for five days, led to a fall in GFP expression with 12.2% ± 1.0 of cells weakly positive at day seven (Figure 1B and Supplementary Figure S1).

ELISA and immunoblotting for TRAIL demonstrated it was only produced when the transgene was activated by doxycycline (Figure 1C,D). Western blots demonstrated TRAIL protein expression in MSCFLT lysates was maximal after two days of doxycycline stimulus (Figure 1D), but very little TRAIL protein remained after the doxycycline stimulus had been removed for one day, following a preceding doxycycline stimulus of five days (Figure 1D).

Trail-expressing MSCs cause tumor cell death in vitro

MSCFLTs were co-cultured with tumor cells, and apoptotic and dead cells increased significantly when doxycycline was added. This effect was observed with lung cancer (A549) (apoptotic and dead cells increased from 3.1% ± 0.1 to 19.6% ± 0.8 (p=0.001, t-test) with the addition of doxycycline), breast cancer (MDAMB231) (15.4% ± 1.7 to 37.7% ± 6.5)(p=0.001, t-test), squamous cell cancer (H357) (12.5% ± 0.3 to 34.5% ± 1.0)(p=0.001, t-test) and cervical cancer (Hela) (6.7% ± 1.0 to 36.1% ± 3.5)(p=0.0001, t-test) cells (Figure 2A-C). These apoptosed and dead cells were shown to come specifically from the cancer cell population by labelling the cancer cell populations with the fluorescent dye (DiI). 50.7% ± 2.7 of the Hela cell population were dead or apoptotic compared to only 8.3% ± 4.6 of the MSCFLT population (p<0.0001, t-test) (Figure 2D).

Co-culture experiments were repeated with decreasing numbers of MSCFLTs. A significant increase in apoptosis and death of the cancer cells was achieved at all concentrations of MSCFLT used (p<0.001 at ratio MSCFLT:Hela cells of 1:16, Anova). There was a cell ratio-dependent effect (Figure 3A). This experiment under-estimates the true killing capacity of the MSCFLT cells as the seeding ratios do not account for any proliferation of cancer cells compared to the MSCFLTs before the addition of doxycycline.

Trail-expressing MSCs kill cancer cells by TRAIL induction of the extrinsic apoptosis pathway

A TRAIL antibody with some ability to neutralise the bioactivity of TRAIL was added to the MSCFLT and Hela cell co-cultures. The antibody was used at a dose to give maximal neutralising effect (250ng/ml) (23). The amount of MSCFLT-induced death and apoptosis was significantly reduced with this antibody (38.03% ± 0.49 compared to 50.67% ± 3.8)( p<0.001, Anova) however not back to baseline (11.48% ± 1.49) (Figure 3B). We found our doxycycline-induced MSCFLTs were potent inducers of death and apoptosis of Hela cells, with a larger proportion of apoptotic and dead cells in comparison to maximal doses (200ng/ml) of recombinant soluble TRAIL (50.67% ± 3.8 compared to 27.50% ± 0.70)(p<0.001, Anova) (Figure 3B).

The caspases are a family of closely related enzymes crucial to apoptosis. zVADfmk is a cell permeable, pan-caspase inhibitor. Application of this compound to the 1:1 co-cultured MSCFLTs and Hela cells caused a 51.6% reduction in death and apoptosis (26.5% ± 0.7 compared to a DMSO treated control 54.8% ± 3.0)( p=0.003, t-test) confirming the importance of caspases in the mechanism of TRAIL-induced cell death (Figure 3C).

To confirm the mechanism of MSCFLT induced cancer cell death is via the extrinsic pathway, Hela cancer cells were retrovirally transduced with a dominant negative Fas-associated death domain (dnFADD) construct or an empty vector (18). The dnFADD consists of the death domain that binds to the TRAIL receptor, but not the domain responsible for caspase 8 recruitment and the triggering of apoptosis. We observed a significant reduction (p=0.018, t-test) in death and apoptosis of Hela cells transduced with dnFADD after co-culture with activated MSCFLT cells (Figure 3C). This data presents strong evidence the MSCFLT cells induce apoptosis via the extrinsic pathway.

MSCs migrate towards some tumor cells

In vitro transwell experiments demonstrated the ability of MSCs to home towards tumors. A549, MDAMB231, and Hela cancer cell cultures were used to attract MSCs, with benign fibroblast 293T cells and medium alone used as controls. There was significantly increased migration to the MDAMB231 cells; 250.4 ± 0.8 cells/field compared to 98.6 ± 9.3 cells to 293T and 57.6 ± 7.6 towards the medium (p<0.001, Anova). There was not a significantly increased migration towards the A549 (92.0 ± 7.1) and Hela (81.0 ± 2.1) cells (Figure 4A).

To examine if MSCFLTs could be traced to metastatic tumors we used a lung metastasis model. Intravenously injected MDAMB231 cells produced lung metastases in all of 20 NOD/SCID mice. These tumors were visible at day ten post-injection and grew to large metastases by day 30 (Figure 4B). DiI-stained MSCFLTs were injected at day 10 and shown histologically to preferentially localise to lung metastases (Figure 4C). DiI-stained fibroblasts were not detected in the tumors or indeed the lungs ten days after injection (data not shown). This histological data shows MSCFLTs preferentially engraft and are maintained in the in vivo tumor environment compared to surrounding lung parenchyma while fibroblasts are not.

Trail-expressing MSCs reduce subcutaneous tumor growth in mice

Two million MDAMB231 cells were coinjected with 0.75 million MSCFLT cells and the TRAIL transgene activated with the addition of doxycycline at day 0, day 25, or never, with tumors harvested at day 42. A recent study has demonstrated that coculture of MSCs with MDAMB231 cells did not alter the tumour growth (24). In our study with transduced MSCs, activation at day 0 resulted in a significantly reduced tumor size (0.12 cm³ ± 0.12 vs 0.66 cm³ ± 0.49)(p<0.001, 2-way Anova) and weight (0.07 g ± 0.06 vs 0.33 g ± 0.33) (p<0.05, Kruskal-Wallis), however there was no change in the tumor growth when TRAIL was activated after the tumor had already become established at day 25 (Figure 5A,B). TUNEL staining of excised tumors demonstrated areas of apoptosis co-localised with DiI-labelled MSCFLT cells (Figure 5C). The subcutaneous tumors were excised and digested to a single cell suspension. Confocal microscopy of the ex vivo DiI-labelled cells included cells of MSC type morphology and demonstrated vimentin staining (Figure 5D). A separate experiment injected transduced MSCFLTs into established tumors. Similar to late activation of co-injected cells, the late injection of MSCFLT cells into established tumors and activation of TRAIL at this later stage was unable to alter the growth of the tumors (Supplementary Figure S2).

Systemic delivery of TRAIL-expressing MSCs reduces and can eliminate lung metastases

Intravenously injected MDAMB231 cells were used to produce lung metastases in NOD/SCID mice. MSCFLT were then used as an intravenous combined cellular and gene therapy with delivery at day 7, 14, 21 and 28 (Figure 6A). At day 35, tumor metastases were found in all mice (8/8) without MSCFLT and all mice with MSCFLT without the use of doxycycline (8/8). In the MSCFLT plus doxycycline arm, three out of eight mice were tumor free (p=0.032, Chi²) (Figure 6B). In addition to the elimination of metastases with MSCFLT plus doxycycline, the lung weight (p<0.01, Anova), which serves as a correlate for metastases load, was also significantly reduced in all of these mice. Similar results were achieved when metastases numbers per lung area was used as an endpoint (p<0.001, Anova) (Figure 6C). Three mice had to be excluded from this latter analysis as the lungs did not inflate during the fixation procedure. MSCFLT cells delivered to control mice without doxycycline were unable to clear the metastases in any mice, but did have less metastases per area (p<0.001, Anova) and a lower lung weight (p<0.05, Anova) than the untreated mice. Doxycycline treatment however produced a further significant reduction (p<0.05, Anova) (Figure 6B,C). In a subsequent experiment, TRAIL and GFP were both shown to be expressed in vivo by the MSCFLT with immunohistochemistry when mice were harvested two days after treatment with MSCFLT and doxycycline (Figure 6D) (primary antibody controls showed no staining, data not shown). TRAIL mRNA derived from the lungs of these mice was also increased (Figure 6D).