A First-Generation Multi-Functional Cytokine for Simultaneous Optical Tracking and Tumor Therapy

Creating new molecules that simultaneously enhance tumor cell killing and permit diagnostic tracking is vital to overcoming the limitations rendering current therapeutic regimens for terminal cancers ineffective. Accordingly, we investigated the efficacy of an innovative new multi-functional targeted anti-cancer molecule, SM7L, using models of the lethal brain tumor Glioblastoma multiforme (GBM). Designed using predictive computer modeling, SM7L incorporates the therapeutic activity of the promising anti-tumor cytokine MDA-7/IL-24, an enhanced secretory domain, and diagnostic domain for non-invasive tracking. In vitro assays revealed the diagnostic domain of SM7L produced robust photon emission, while the therapeutic domain showed marked anti-tumor efficacy and significant modulation of p38MAPK and ERK pathways. In vivo, the unique multi-functional nature of SM7L allowed simultaneous real-time monitoring of both SM7L delivery and anti-tumor efficacy. Utilizing engineered stem cells as novel delivery vehicles for SM7L therapy (SC-SM7L), we demonstrate that SC-SM7L significantly improved pharmacokinetics and attenuated progression of established peripheral and intracranial human GBM xenografts. Furthermore, SC-SM7L anti-tumor efficacy was augmented in vitro and in vivo by concurrent activation of caspase-mediated apoptosis induced by adjuvant SC-mediated S-TRAIL delivery. Collectively, these studies define a promising new approach to treating highly aggressive cancers, including GBM, using the optimized therapeutic molecule SM7L

Tumor Therapy Mediated by Lentiviral Expression of shBcl-2 and S-TRAIL1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells and, in combination with other agents, could enhance tumor therapy. We explored the combined therapeutic effects of a secretable form of (S) TRAIL-induced apoptosis and the downregulation of Bcl-2 in human gliomas. We constructed a lentiviral delivery system: 1) for the expression of short hairpin (sh) RNA to downregulate Bcl-2 and for the expression of S-TRAIL to induce apoptosis in glioma cells; and 2) to follow delivery in vitro and the fate of tumors in real time in vivo. We demonstrate that lentiviral-mediated simultaneous downregulation of Bcl-2 and S-TRAIL-induced apoptosis leads to an increased expression of activated caspase-3 and caspase-7, thus resulting in accelerated S-TRAIL-mediated apoptosis in glioma cells in vitro. Using a highly malignant human glioma model expressing EGFRvIII and firefly luciferase, we show that the combined effect of Bcl-2 downregulation and S-TRAIL-induced apoptosis results in complete eradication of gliomas compared to S-TRAIL monotherapy. These results show that simultaneous triggering of TRAIL-mediated death receptor pathway and downregulation of Bcl-2 by shRNA leads to enhanced eradication of gliomas and serves as a template in developing and monitoring combination therapies for the treatment of drug-resistant cancers

mNSC-SM7L prolongs SM7L delivery and attenuates GBM progression.

<p>(A) Representative FLuc bioluminescent images and summary data of mice implanted intracranially with mNSC transduced with LV-GFP-RLuc (n = 6) or SM7L (n = 6) and U87-Fluc GBM cells. FLuc bioluminescence imaging was performed from day 1 to day 35 to monitor tumor progression. On day 29, Rluc imaging was performed to determine stem cell volume and GLuc imaging was performed to confirm SM7L secretion. (B) Immunohistochemistry with antibodies against Ki67 and summary data performed on sections from brains treated with control and mNSC-SM7L 29 days post-implantation. Representative images show brain sections containing mNSC (green) or Ki67 staining (red). In all panels, *p<0.05 vs. control, and mean and SD are reported.</p

Predictive computer modeling of wild-type MDA-7/IL-24 and optimized SM7L.

<p>Predictive space-filling models of IL-20α/β receptor (A), MDA-7/IL-24 monomer (B), MDA-7/IL-24 dimer (C), and dimerized MDA-7/IL-24 binding to IL-20α/β receptor (D). (E) Representative models of wild-type MDA-7/IL-24 and the modifications to optimize the molecule. These include modification of the secretion sequence (in red) and C-terminal diagnostic fusion (yellow). (F-H) Space-filling models demonstrating the predicted structure of the modified MDA-7/IL-24 fusion protein SM7L. (F) Representative computer model of SM7L monomer containing MDA-7/IL-24 (green) and C-terminal luciferase fusion (pink). (G) Computer model showing a SM7L dimer. (H) Space filling model of SM7L dimmer interacting with IL-20α/β receptor.</p

mNSC-SM7L improves the pharmacokinetics of delivery and enhances anti-GBM efficacy <i>in vivo</i>

<p>. (A–B) Representative images and summary graph showing <i>in vivo</i> imaging of multiple events in stem cell delivery of SM7L. mNSC-SM7L were implanted around established U87Fluc-mCherry tumors in subcutaneous skin flap window chambers. Intravital microscopy was used to visualize stem cell (green) and tumor (red) volumes at cellular resolution 1 and 5 days post-implantation (A). Simultaneously, Fluc and GLuc imaging was used to monitor changes in tumor volume (Fluc) and SM7L (GLuc) secretion respectively (A). Fluorescent intensities and BLI photon emission was then quantified to reveal stem cell volume, tumor volumes, and SM7L levels (B). In the panels, tumors = Fluc, red; stem cells = green; SM7L secretion = GLuc. (C–H) Summary of experiments to visualize differences in pharmacokinetics of stem cell-delivered and intravenous (IV) injection of SM7L. (C) Representative Fluc (tumor) and GLuc (SM7L) images and summary data showing SM7L levels delivered by engineered stem cells implanted around established U87-Fluc tumors at 0 and 24 h post-implantation (n = 3). (D) Ex vivo analysis of stem cell-delivered SM7L biodistribution performed by GLuc bioluminescent imaging of multiple excised tissues. (E) Summary graph of Fluc imaging performed on days 0, 2, 7, and 10 showing mNSC-SM7L inhibition of tumor progression compared to tumors treated with control stem cells. (F–H) <i>In vivo</i> imaging of conditioned medium containing SM7L injected by IV infusion. Following implantation of FLuc-positive tumors (F), a bolus of media containing SM7L was injected IV followed immediately by coelenterazine. Images were captured at 5–50 mins and 24 h post-injection (F, n = 3). Representative Fluc (tumor) and GLuc (SM7L) images and summary graphs revealing levels (F) and distribution in excised tissue (G) are shown. (H) Fluc imaging was performed on days 1, 3, 7 and 10 post-injection to determine tumor volumes.</p

Proposed signaling changes following combined SM7L/S-TRAIL therapy.

<p>Schematic simplified overview of pathways proposed to mediate the anti-tumor effects of MDA-7/IL-24 and TRAIL combination therapy. MDA-7/IL-24 primarily induces cytostatic effects through alterations in p38MAPK and ERK signaling. TRAIL induces apoptosis via activation of the caspase cascade. Elevation in these pathways were observed following combination treatment. Speculative points of cross-talk are labeled in grey. MDA-7/IL-24 up-regulates the levels of TRAIL in certain cell lines and down-regulates the TRAIL inhibitor FLIP. TRAIL has been reported to downregulate ERK in GBM cell lines leading to increased caspase activation.</p

Combined targeting of cytostatic and cytotoxic pathways with stem cell delivered SM7L and S-TRAIL improves anti-GBM efficacy <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Summary graph of the viability of U251 and U87 GBM cells pre-treated with radiation (4 Gy) and SM7L or SM7L followed by concentrated S-TRAIL. U251 and U87 GBM cells were incubated with SM7L or irradiated with 4 Gy., and treated or untreated cells were incubated with S-TRAIL (200 ng) overnight, and cell viability was determined by luciferase-based assay. (B) Summary data and representative images demonstrating the effects on cell viability of U251 and U87 GBM cells co-cultured with stem cell secreting SM7L or SM7L/S-TRAIL. Control or SM7L mNSC were overlayed on Fluc-positive GBM cells. Four days later, mNSC-STRAIL were seeded onto a subset of mNSC-SM7L and cell viability was determined by luciferase assay 24 h later. (C) Caspase 3/7 activation assay performed on U87 and U251 cells treated with conditioned media containing SM7L, S-TRAIL, SM7L/S-TRAIL, or control. (D-E) Western blot and summary graph performed on U87 and U251 cells treated with conditioned media containing SM7L and/or S-TRAIL. Cell lysates were collected and immunoblotted with antibodies against cleaved PARP, total and cleaved caspase-8 (D) as well as total and phosphorylated p38MAPK or ERK (E). (F) Representative images and summary data demonstrating the effects of stem cell-delivered SM7L or S-TRAIL monotherapy or SM7L/S-TRAIL concomitant therapy delivered by dual secreting stem cells on the progression intracranial U87-Fluc human GBMs. mNSC-GFP (control, n = 10), mNSC-SM7L (n = 10), mNSC-S-TRAIL (n = 8), or double secreting mNSC-SM7L/S-TRAIL (n = 8) were implanted together with U87-Fluc GBM cells in the frontal lobe of mice. Serial Fluc bioluminescence imaging was performed on days 0 and 29 to monitor therapeutic efficacy. (G) Representative photomicrographs and summary graph of coronal brain sections stained with H&E and corresponding fluorescence immunostaining showing mNSC distribution (GFP) or the levels caspase-3 (red, summary graph) in mice treated with mNSC-GFP (control), mNSC-SM7L, mNSC-S-TRAIL, or double secreting mNSC-SM7L/S-TRAIL. In all panels, *p<0.05 vs. control; §p<0.05 vs. SM7L, and experiments were performed in triplicate with mean and SD reported.</p