Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer

Abstract

Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSC) home to and incorporate within tumor tissue. We hypothesized that MSCs engineered to produce and deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumor cells, would home to and kill cancer cells in a lung metastatic cancer model. Human MSCs were transduced with TRAIL and the IRES-eGFP reporter gene under the control of a tetracycline promoter using a lentiviral vector. Transduced and activated MSCs caused lung (A549), breast (MDAMB231), squamous (H357), and cervical (Hela) cancer cell apoptosis and death in coculture experiments. Subcutaneous xenograft experiments confirmed that directly delivered TRAIL-expressing MSCs were able to significantly reduce tumor growth [0.12 cm(3) (0.04-0.21) versus 0.66 cm(3) (0.21-1.11); P < 0.001]. We then found, using a pulmonary metastasis model, systemically delivered MSCs localized to lung metastases and the controlled local delivery of TRAIL completely cleared the metastatic disease in 38% of mice compared with 0% of controls (P < 0.05). This is the first study to show a significant reduction in metastatic tumor burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumors and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumor resection.

Figure 1. MSC characterisation, transduction and gene expression

(A) MSCs, differentiation to adipocytes (Oil-Red-O) and osteoblasts (Alizarin Red S) (×4 mag), and colony forming ability. (B) Representative flow cytometry for GFP expression in MSCs transduced with the TRAIL-GFP lentivirus (MSCFLTs) and activated with doxycycline, and timescale of GFP expression with addition or removal of doxycycline. (C) TRAIL ELISA of MSCFLTs. (D) Western blots of MSCFLTs showing maximum expression of TRAIL after two days of doxycycline treatment (10μg protein loaded) and loss of expression one day after doxycycline removal (5μg protein loaded) with actin loading controls.

Figure 2. TRAIL-expressing MSCs cause cancer cell apoptosis in vitro

(A) Representative flow cytometry plots demonstrating an increase in death and apoptosis when Hela cells were co-cultured with doxycycline (dox) treated MSCFLTs compared to no doxycycline (nd), untreated controls or doxycycline treated normal MSCs. (B) Phase microscopy (×5 mag) demonstrating an increase in cell death (rounded and floating cells) when MSCFLTs were activated by doxycycline in co-culture with Hela and MDAMB231 cells. (C) Flow cytometry results from triplicate apoptosis assays showing an increase in death and apoptosis of total cells in cancer cell and MSCFLT co-cultures after doxycycline treatment. (D) Flow cytometry of co-culture experiments demonstrated DiI positive cancer cells were responsible for dead and apoptotic populations. Triplicate experiments shown on bar graph. (***p<0.001 **p<0.01).

Figure 3. TRAIL-expressing MSC-induced apoptosis occurs at low MSCFLT to cancer cell ratios via the extrinsic apoptotic pathway

(A) Results from flow cytometry apoptosis assays showing an increase in death and apoptosis of Hela cells when MSCFLTs activated with doxycycline (dox) were co-cultured with Hela cells even at low 1:16 ratios, compared to untreated controls (nd). (B) Induced cell death and apoptosis is higher using the MSCFLTs than with recombinant TRAIL (rhTRL) and can be partially blocked with blocking antibody (Ab). (C) Cell death and apoptosis is reduced when using Hela cells expressing dominant negative Fas-associated death domain (dnFADD) in comparison with those transduced with an empty vector in addition to when zVADfmk (zvad), a pan-caspase inhibitor, is used compared to the control (con). (***p<0.001 **p<0.01 *p<0.05).

Figure 4. MSCs migrate to some cancer cells in vitro and preferentially engraft in vivo

(A) Transwell migration studies demonstrated an increased number (per microscopic field) of MSCs migrating through the transwell membrane toward MDAMB231 cells, but not towards other (A549, Hela) cancer cell types compared to control 293T cells or medium alone (***p<0.001). (B) H&Es showing representative lung metastases post intravenous injection of MDAMB231 cells into NOD/SCID mice at day 10, 20, and 30 and a macroscopic picture at day 30. Scale bar represents 20μm. (C) DiI-labelled MSCFLTs (red) injected intravenously at day 10 and shown to localise to lung metastases on fluorescent microscopy with DAPI nuclear counterstain with H&E contiguous sections from day 30 harvested lungs ( ×10 mag scale bar represents 20μm and ×4 mag scale bar represents 60μm).

Figure 5. TRAIL-expressing MSCs reduce the growth of subcutaneous tumors

(A) Doxycycline (dox) treatment of subcutaneous tumors composed of mixed MDAMB231 cells and MSCFLT cells from day 0 led to a decreased size and weight (*p<0.05, **p<0.01, ***p<0.001) of the tumors compared to no doxycycline treated mice (nd). There was no reduction in tumor growth if TRAIL was activated at day 25. (B) Macroscopic appearance of the tumors. (C) TUNEL staining (green) demonstrated areas of tumor apoptosis localised with areas of MSCFLTs (DiI - red) within the tumors in mice treated with doxycycline from day 0 (DAPI nuclear counterstain - blue). Scale bar represents 25μm (D) Ex vivo single cell digestion and culture showed DiI positive (red) cells with MSC morphology that co-stained with vimentin (green) (DAPI – blue). Scale bar represents 3μm.

Figure 6. TRAIL expressing MSCs reduce the growth of lung metastases

(A) 2 million MDAMB231 cells were injected intravenously at day 0 followed by delivery or not of MSCFLT cells at days 7, 14, 21 and 28 with or without doxycycline (dox – doxycycline; nd – no dox). (B) Representative histology of lung lobes in the three experimental groups. Metastases remained, but were reduced, after injection of MSCFLT without activation of the TRAIL construct, while TRAIL activation of MSCFLTs eliminated metastatses in 3 of 8 mice (p=0.03). (C) Reduction in lung weight and metastases number per lung area with the use of MSCFLTs both with and without doxycycline treatment. There was a further significant reduction between activated MSCFLTs compared to inactivated (***p<0.001 **p<0.01 *p<0.05). (D) Immunohistochemistry demonstrating cells expressing GFP (scale bar represents 5μm) and TRAIL (scale bar represents 10μm) and consecutive immunofluorescence sections showing these cells also express DiI. There is an increase in TRAIL mRNA from lung digests treated with MSCFLT and doxycycline (*p<0.05).