To study the role of secreted anti-angiogenic TSP-1 on human endothelial cells in vitro and its effect on glioma angiogenesis and progression in mouse models of glioma tumors, we created different versions of secretable anti-angiogenic TSP-1 (aaTSP-1) encoding the regions of 3TSRs of TSP-1 (a.a. 412–499) where most of the current clinically used anti-angiogenic TSP-1 peptides have originated. The aaTSP-1 plasmid constructs consisting of N-terminal human Flt3 signal sequence (Ss) or the entire extracellular domain of human Flt3 (HF) and also their His6- and FLAG (Fg) tagged variants to quantify secretion of aaTSP-1 are diagrammed in . These constructs were packaged into lentivirus (LV) virions and used to transduce human NSC (hNSC) in culture. hNSC were efficiently transduced with aaTSP-1 LVs as revealed by GFP fluorescence () and by flow cytometry () and expressed aaTSP-1, as shown by dot blot analysis on culture medium (). In order to quantify secreted aaTSP-1 in the culture medium, we developed an ELISA-assay based on Ni2+ -coated wells and biotinylated anti-FLAG antibody. A higher concentration of Ss-aaTSP-1 (240 ng/mL/106 cells) compared to HF-aaTSP-1 (160 ng/mL/106 cells) protein was secreted in the culture medium () of hNSC transduced with different versions of His6- and FLAG- tagged aaTSP-1 LVs. Furthermore, hNSC continuously secreted aaTSP-1 over a 15 day period () and the expression of aaTSP-1 in hNSC did not alter their growth rate ().
Characterization of aaTSP-1 lentiviral vectors
To study the anti-angiogenic effects of different variants of aaTSP-1 in vitro
, we first measured endothelial cell branch point formation and tubule length, the two most widely used surrogates to estimate angiogenesis in vitro
, on human microvascular endothelial cells (HMVECs) incubated with conditioned medium from transduced hNSC. HMVECs incubated with Ss-aaTSP-1 containing conditioned medium showed a significant reduction in the formation of branching points () and also tubule length (supplementary Fig. 1A
), as compared to the HF-aaTSP-1 or control conditioned medium 18 hrs post incubation. There was no influence of N-terminal His and C-terminal FLAG tags on the anti-angiogenic potential of aaTSP-1 (). We then tested conditioned medium from hNSC-Ss-aaTSP1 on human brain microvascular endothelial cells (HBMVECs). hNSC-Ss-aaTSP1 significantly inhibited the formation of branching points in HBMVECs (; supplementary Fig. 1B and 1C
). Glioma cells have been reported to secrete factors that promote angiogenesis (Hanahan & Weinberg, 2000
; Papetti & Herman, 2002
). In order to study the potency of aaTSP1 on endothelial cells in presence of glioma cells, we performed additional co-culture experiments of HMVECs and HBMVECs with either established human glioma cells expressing DsRed2 (Gli36-EGFRvIII-DsRed2) or CD133 positive and negative primary human glioma cells. The conditioned medium from hNSC-Ss-aaTSP-1 caused a reduction in branching points in both HMVECs () and HBMVECs (). HMVECs co-cultured with either CD133+ or CD133− primary glioma cells, and incubated in conditioned medium from hNSC-Ss-aaTSP-1 showed a significant reduction in average branching points (). Finally, similar studies performed with a known VEGFR2 kinase inhibitor (cell permeable indolin-2-one class of RTK), showed a hNSC-Ss-aaTSP-1 comparable effect on HMVECs ().These results reveal that neural stem cell secreted aaTSP-1 has anti-angiogenic effect on both HMVECs and HBMVECs in vitro
and its efficacy is optimized by fusing it to the N-terminal human Flt3 signal sequence (Ss) rather than to the entire extracellular domain of human Flt3 (HF). Furthermore, Ss-aaTSP-1 results in the considerable reduction in the angiogenic potential of endothelial cells when co-cultured with glioma cells and this effect is comparable to the known ant-angiogenic inhibitor used in pre-clinical settings.
aaTSP-1 has anti-angiogenic effect on human endothelial cells
In order to follow the secretion of Ss-aaTSP-1 (from here on named aaTSP-1) in vitro
and in vivo
, we created a C-terminal Gaussia
luciferase (Gluc) fusion of aaTSP-1 (). Gluc is a naturally secreted bioluminescent protein that has been proven to have enhanced brightness and stability compared to other luciferases and, unlike firefly luciferase (Fluc), does not require ATP for its activity (Verhaegent & Christopoulos, 2002
). Branch point formation assays on HMVECs and HBMEVCs revealed that aaTSP-1-Gluc had considerably reduced potency as compared to aa-TSP-1 (data not shown). Gluc bioluminescence imaging on hNSC transduced with LV-aaTSP1-Gluc () showed that hNSC secreted aaTSP-1-Gluc in the culture medium (). In order to simultaneously follow the fate of hNSC and secretion of aaTSP-1, hNSC were co-transduced with LV-aaTSP-1-Gluc and LV-GFP-Fluc and both hNSC and culture medium were imaged using two different substrates, coelenterazine and D-luciferin respectively. Linear correlations between 1) aaTSP1-Gluc activity and culture medium volume; and 2) Fluc activity and the number of hNSC secreting aaTSP-1 were shown by dual in vitro
bioluminescence imaging (). When a mix of glioma cells and hNSC co-expressing GFP-Fluc and aaTSP-1-Gluc was implanted subcutaneously in mice, dual bioluminescence imaging showed that aaTSP-1 expression could be measured in vivo
and that the expression of aaTSP-1 in vivo
was stable over time (). In order to simultaneously follow tumor volumes, secretion of aaTSP-1 and localization of tumor cells and hNSC at a cellular resolution, we implanted hNSC-aaTSP-1-Gluc cells into mice with established Gli36-EGFRvIII-FD tumors, 2 mm from the site of implantation, in subcutaneous window chambers. Dual bioluminescence imaging revealed that the tumor volumes () and aaTSP-1 secretion () could be sequentially detected, and subsequent intravital microscopy on the same mice revealed the homing of NSC secreting aaTSP-1-Gluc to tumor cells () at a cellular resolution in real-time. These results reveal that that tumor volumes, anti-angiogenic agents and the delivery vehicles can be monitored longitudinally in real-time in vivo
and further demonstrate that aaTSP-1 secretion from NSC is stable in vivo
and engineered hNSC home to tumors.
Pharmacokinetics of hNSC-aaTSP1
To assess the effects of aaTSP-1 delivered via engineered hNSC on gliomas in vivo, we first implanted a mix of human Gli36-EGFRvIII-FD glioma cells and either aaTSP-1 expressing or control GFP-Rluc expressing hNSC. The presence of hNSC (green) within the implanted tumors (red) was confirmed by intravital microscopy 4 days post implantation (). Serial Fluc bioluminescence imaging on mice revealed a significant reduction in glioma burden in animals bearing hNSC expressing aaTSP-1 as compared to the hNSC expressing control GFP-Rluc starting at day 6 post-implantation (). Histopathological analysis with anti-CD31 antibody on tumor sections revealed that control tumor presented significantly higher number of vascular endothelial cells than treated tumors (). In order to simultaneously follow tumor cells, NSC and tumor vasculature at a cellular resolution, we created a human malignant glioma cell line expressing a new red shifted marker, tdTomato, which is the brightest of the red shifted fluorophore variants (Gli36-EGFRvIII-tdTomato) () (1.6 times brighter than DsRed2; t0.5 for maturation: 1 hr as compared to 7 hr for DsRed2) and implanted a mix of therapeutic hNSC-aaTSP-1 or control hNSC-GFP-Rluc and Gli36-EGFRvIII-tdTomato tumor cells in subcutaneous window chambers. Intravital confocal microscopy on mice 4 days after implantation revealed that tumor cells, hNSC and vasculature could be detected simultaneously () in both aaTSP-1 treated and control GFP-Rluc treated mice. Furthermore, a significant reduction in the microvascular density within the tumor mass was seen in aaTSP-1 treated tumors ( and ) as compared to the controls at day 6 post implantation ( and ). These experiments demonstrate that anti-angiogenic effects of aaTSP-1 have a substantial influence on the growth and progression of tumor cells in subcutaneous glioma tumors. Furthermore, using different combinations of optical imaging modalities, anti-angiogenic effects of hNSC delivered aaTSP-1 can be monitored in real-time in vivo.
NSC delivered aaTSP-1 influences tumor formation in vivo
Next, we tested the effect of hNSC-aaTSP-1 in the intracranial glioma models. Two different sets of experiments were performed. In the first set of experiments, we implanted a mix of hNSC-aaTSP-1 or control hNSC-GFP-Rluc and Gli36-EGFRvIII-tdtomato tumor cells in a cranial window model. Similar to the subcutaneous window chamber model, intravital confocal microscopy on mice revealed that tumor cells, hNSC and blood vasculature could be detected simultaneously in both control GFP-Rluc () and aaTSP-1 treated () mice. A considerable reduction in glioma volumes in tumors implanted with hNSC-aaTSP-1 () as compared to the controls () was observed 4 days after implantation. Furthermore, a significant reduction in vasculature around Gli36-EGFR-vIII-tdTomato/NSC-aaTSP-1 transplanted areas as compared to the controls ().
NSC delivered aaTSP-1 has anti-proliferative effect on established intracranial gliomas
A number of previous studies have revealed that repeated systemic administration of anti-angiogenic drugs in animal models result in controlled cell proliferation but incomplete eradication of established gliomas. In the second set of experiments, we assessed the effect of stem cell delivered aaTSP-1 on established gliomas by implanting hNSC-aaTSP-1 in the close vicinity to Gli36-EGFR-vIII-FD established gliomas. Dual bioluminescence imaging revealed the progression of glioma tumors () and fate of hNSC in real time (). Glioma growth was significantly reduced at day 6, day 12 and day 16 after implantation compared to the control hNSC-GFP-Rluc implanted animals, where tumors grew more rapidly (). Kaplan-Meier survival analysis revealed a statistically significant prolongation of the survival in the hNSC-aaTSP-1 treated group as compared to control hNSC-GFP-Rluc (). Histological examination revealed prominent presence of endothelial cells in control tumors as compared to the aaTSP-1 tumors (). These results show that a single administration of hNSC-aaTSP-1 in mice with established glioma result in significant reduction glioma growth and prolonged survival.
In order to investigate the fate of transplanted hNSC-aaTSP-1 in mice, immunohistochemical analysis on brain sections from mice bearing gliomas and implanted with hNSC-aaTSP-1 12 days post-implantation was performed. A robust expression of neural stem cell marker, nestin in hNSC 12 day post-implantation was observed (). Furthermore a majority of hNSC-aaTSP-1 did not stain for the proliferation marker Ki67 (), the astrocytic marker, GFAP () and the neuronal marker, MAP-2 () where as tumor cells were stained for Ki67 () and normal mouse brain stained for both GFAP () and MAP-2 (). These results reveal that therapeutic hNSC do not proliferate and remain in an un-differentiated state in the brains of glioma bearing mice brains.
Engineered hNSC do not differentiate in mouse glioma model