Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Constitutively activated nuclear factor- b, but not induced nf- b, leads to trail resistance by up-regulation of x-linked inhibitor of apoptosis protein in human cancer cells

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappa B (NF-kappa B) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-kappa B in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-kappa B via adenoviral expression of the I kappa B-alpha super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-kappa B, pointing to a possible role of preactivated NF-kappa B in protection front TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-kappa B and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-kappa B activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-kappa B and/or XIAP only in tumor cells with constitutively activated NF-kappa B

Role of CX3CR1 receptor in monocyte/macrophage driven neovascularization.

Monocyte/macrophages are implicated in initiation of angiogenesis, tissue/organ perfusion and atherosclerosis biology. We recently showed that chemokine receptor CX(3)CR1 is an essential regulator of monocyte/macrophage derived smooth muscle cell differentiation in the vessel wall after injury. Here we hypothesised the contribution of CX(3)CR1- CX(3)CL1 interaction to in vivo neovascularization and studied the functional consequences of genetic and pharmacologic targeting of CX(3)CR1 in formation, maturation and maintenance of microvascular integrity. Cells functionally deficient in CX(3)CR1 lacked matrix tunnelling and tubulation capacity in a 3D Matrigel assay. These morphogenic and cytokinetic responses were driven by CX(3)CL1-CX(3)CR1 interaction and totally abrogated by a Rho antagonist. To evaluate the role of CX(3)CR1 system in vivo, Matrigel plugs were implanted in competent CX(3)CR1(+/gfp) and functionally deficient CX(3)CR1(gfp/gfp) mice. Leaky microvessels (MV) were formed in the Matrigel implanted in CX(3)CR1(gfp/gfp) but not in CX(3)CR1(+/gfp) mice. In experimental plaque neovascularization immature MV phenotype was observed in CX(3)CR1(gfp/gfp) mice, lacking CX(3)CR1 positive smooth muscle-like cells, extracellular collagen and basement membrane (BM) laminin compared to competent CX(3)CR1(+/gfp) mice. This was associated with increased extravasation of platelets into the intima of CX(3)CR1(gfp/gfp) but not functionally competent CX(3)CR1 mice. Pharmacologic targeting using CX(3)CR1 receptor antagonist in wild type mice resulted in formation of plaque MV with poor BM coverage and a leaky phenotype. Our data indicate a hitherto unrecognised role for functional CX(3)CR1 in Matrigel and experimental plaque neovascularization in vivo, which may buttress MV collectively in favour of a more stable non-leaky phenotype

Pharmacologic inhibition of CX₃CR1 results in formation of leaky microvessels within experimental plaque.

<p>A, Drug (F1) study protocol: Carotid artery was ligated in the C57BL6J mice (wt mice) and animals were treated with a selective CX₃CR1 inhibitor (F1) (from second week post carotid ligation for another two weeks). B, Representative cross sectional images of carotid artery from C57BL6J mice treated with saline or a selective CX₃CR1 inhibitor (F1) and stained for laminin (B; Basement membrane; Red), or CD42b (D; Platelets; Red), CX₃CR1 (GFP; Green) and DAPI (Nucleus; Blue). B & C, Significantly greater number of CX₃CR1 positive microvessels were covered by basement membrane laminin in saline treated C57BL6J mice compared to F1 treated mice. D & E, Increased platelet CD42b staining was observed in the neointimal interstitial space in the F1 treated mice compared to saline treated mice (Scale bar: 50 µm). In addition the leaky microvessel phenotype in mice treated with F1 was confirmed by presence of intravenously administered (tail vein) 2–2.5 µm diameter microspheres (red spheres) in the neointimal lesion (F & G). IgG control staining for isotype-matched antibodies shown. Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.01.</p

CX₃CR1 deficient cells lack in vitro tunnelling capacity in solid matrix and form incomplete 3D tube-like structures compared to CX₃CR1 competent cells.

<p>CX₃CR1 cells isolated from bone marrow of CX₃CR1^+/gfp or CX₃CR1^gfp/gfp mice were sandwiched between two DQ-red fortified (20 µg/ml; substrate for proteases) Matrigel layers with or without 10 ng/ml CX₃CL1 gradient and were cultured for 5 days. A & B, CX₃CR1 cells remodelled into tube-like structures (tubulation) especially under CX₃CL1 gradient (Scale bar: 10 µm). However these tubular structures were incompletely formed by CX₃CR1 deficient cells. Furthermore, the CX₃CR1 deficient cells lacked tunneling capacity (dotted lines-protease activation red) in the Matrigel (C & D) and produced significantly less laminin following CX₃CL1 stimulation (E & F). Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.</p

CX₃CR1 positive cells contribute to formation of experimental plaque angiogenesis.

<p>Plaque angiogenesis was created by ligation of carotid artery at its bifurcation for 4 weeks in CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice. Perfusion fixed carotid arteries were isolated and OCT embedded. 5 µm cross sections were stained with smooth muscle marker (Calponin; Red) and confocal images were acquired. A, Representative bright field cross section image of carotid artery from CX₃CR1^+/gfp mice stained with DAPI (Nucleus; Blue). Red blood cells (RBCs) (arrow heads) were observed in the microvessels indicating these microvessels were functional. B, Mice competent for CX₃CR1 function had a higher proportion of MV containing RBCs C, Representative cross section image of carotid artery from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice were stained with calponin (Red) and DAPI (nucleus; blue). CX₃CR1 positive cells (GFP positive; Green) integrated into microvascular wall and were also present in perivascular region and co-expressed smooth muscle marker (Calponin; Red) (Scale bar: 10 µm). In the CX₃CR1 functionally deficient (CX₃CR1^gfp/gfp) mice the number of vascular and perivascular cells (GFP positive; Green) (D) and co-expressing smooth muscle marker (E) were significantly reduced. Data is expressed as mean ± SEM of 20 carotid artery cross sections/mice (n = 4 independently performed experiments). F, Microvessels in the plaque stained with CX₃CR1 (Green) and DAPI (nucleus; blue) were 3D reconstructed using IMARIS software to depict signet ring structures and their tubular architecture. G, The number of signet-ring cells was significantly reduced in CX₃CR1^gfp/gfp mice. H, Schematic representing the three major phenotypes of CX₃CR1 cells associated with microvascular structures in the plaque. * denotes p<0.05.</p

Functional deficiency of CX₃CR1 results in leaky microvessel phenotype in experimental plaque.

<p>Representative cross sectional images of carotid artery plaques from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice stained with CD42b (Platelets; Red) (A) or laminin (Basement membrane; Red) (C) and DAPI (Nucleus; Blue). B, Significantly increased staining for platelet CD42b was observed in the neointimal interstitial space in CX₃CR1^gfp/gfp mice compared to competent CX₃CR1^+/gfp mice (Scale bar: 50 µm). D, A significantly greater number of CX₃CR1 positive microvessels were covered by basement membrane laminin in CX₃CR1^+/gfp mice compared to CX₃CR1^gfp/gfp mice (insets in panel C show GFP and laminin co-staining present in CX₃CR1^+/gfp but not CX₃CR1^gfp/gfp mice). Data is represented as mean ± SEM of 15 plaque sections/mice (n = 4 independently performed experiments); * denotes p<0.05.</p

Rho activation is essential for tubulation of CX₃CL1 stimulated CX₃CR1 cells.

<p>CX₃CR1 cells isolated from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice were treated with 100 nM CX₃CL1 and 30 minutes post probed for expression of activated RhoA. A, Representative immuno blot showing active RhoA protein expression. B, Active RhoA expression within 30 mins post CX₃CL1 stimulation was significantly higher in CX₃CR1 cells isolated from CX₃CR1^+/gfp compared to CX₃CR1^gfp/gfp mice. C, CX₃CR1 cells isolated from bone marrow of CX₃CR1^+/gfp mice were sandwiched between two Matrigel layers with 10 ng/ml CX₃CL1 gradient and were cultured for 5 days in absence or presence of Rho inhibitor (Y27632; 10 µM, Scale bar: 10 µm). C & D, Inhibition of Rho using Y27632 (10 µM) prevented the formation of tube-like structures by CX₃CR1 cells isolated from CX₃CR1^+/gfp mice. Data is represented as mean ± SEM of 4 independently performed experiments;* denotes p<0.01.</p

CX₃CR1 positive cells contribute to formation of microvessels (MV) in Matrigel and deficiency of CX₃CR1 results in leaky MV phenotype.

<p>500 µl of Matrigel subcutaneously implanted in CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice for 4 weeks. A, Representative sections of Matrigel isolated from CX₃CR1^+/gfp and CX₃CR1^gfp/gfp mice, intravitally stained with lectin (Rhodamine <i>Griffonia Simplicifolia</i>; Red) to show functional MV and DAPI (Nucleus; Blue) show presence of CX₃CR1 (GFP) positive MV. B, Deficiency of CX₃CR1 restricted CX₃CR1 cells to mostly the perivascular region of functional MV within Matrigel. Moreover, the number of integrated vascular associated CX3CR1 positive MV were significantly higher in Matrigels isolated from CX₃CR1^+/gfp mice compared to Matrigels isolated from CX₃CR1^gfp/gfp mice. Data is expressed as mean ± SEM of 25 Matrigel sections/mice (n = 3 independently performed experiments). C, Three dimensional reconstruction of the confocal images using IMARIS indicate CX₃CR1 positive cells integrated into periluminal vascular wall in the Matrigel implanted in CX₃CR1^+/gfp but not in CX₃CR1^gfp/gfp mice where cells remained in perivascular region (Scale bar: 10 µm). Microvessels formed in Matrigels isolated from CX₃CR1^gfp/gfp mice were leaky, which was evident by increased heme content (D & E), Evan's blue dye leak (F & G), and extravasated platelets (CD42b; Red) (H & I) in the matrix compared to Matrigels implanted in CX₃CR1^+/gfp mice. Data is represented as mean ± SEM of 8 Matrigels/group and 10 Matrigel sections/mice (n = 4 independently performed experiments); * denotes p<0.05.</p