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Cancer Research 68, 4810, June 15, 2008. doi: 10.1158/0008-5472.CAN-08-0160
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Neo-Organoid of Marrow Mesenchymal Stromal Cells Secreting Interleukin-12 for Breast Cancer Therapy

Nicoletta Eliopoulos1, Moïra Francois1, Marie-Noëlle Boivin1, Daniel Martineau3 and Jacques Galipeau1,2

1 Lady Davis Institute for Medical Research and 2 Division of Hematology/Oncology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada; and 3 Department of Veterinary Medicine, Université de Montréal, St-Hyacinthe, Quebec, Canada

Requests for reprints: Jacques Galipeau, Lady Davis Institute for Medical Research, 3755 Cote St-Catherine Road, Montreal, Quebec, Canada H3T1E2. Phone: 514-340-8260; Fax: 514-340-7502; E-mail: jgalipea{at}lab.jgh.mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Bone marrow–derived mesenchymal stromal cells (MSCs), beneficial for regenerative medicine applications due to their wide differentiation capabilities, also hold promise as cellular vehicles for the delivery of therapeutic plasma-soluble gene products due to their ease of handling, expansion, and genetic engineering. We hypothesized that MSCs, gene enhanced to express interleukin-12 (IL-12) and then embedded in a matrix, may act as an anticancer neo-organoid when delivered s.c. in autologous/syngeneic hosts. We performed such experiments in mice and noted that primary murine MSCs retrovirally engineered to secrete murine IL-12 can significantly interfere with growth of 4T1 breast cancer cells in vivo, with a more substantial anticancer action achieved when these cells are embedded in a matrix. Plasma of mice that received the IL-12 MSC-containing neo-organoids showed increased levels of IL-12 and IFN-{gamma}. Histopathologic analysis revealed less tumor cells in implants of 4T1 cells with IL-12 MSCs, and the presence of necrotic tumor islets and necrotic capillaries, suggesting antiangiogenesis. We also showed that the anticancer effect exerted by the IL-12 MSCs is immune mediated because it is absent in immunodeficient mice, is not due to systemic IL-12 delivery, and also occurs in a B16 melanoma model. This study therefore establishes the feasibility of using gene-enhanced MSCs in a cell-based neo-organoid approach for cancer treatment. [Cancer Res 2008;68(12):4810–8]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Bone marrow–derived mesenchymal stromal cells (MSCs) have been increasingly used clinically for regeneration of damaged tissues due to the innate potential of these progenitor cells to turn into a variety of differentiated cell types (1–5). The differentiated progeny of these primary cells includes osteocytes, chondrocytes, astrocytes, adipocytes, neurons, skeletal myoblasts, and cardiomyocytes. Studies have shown the reparative actions of MSCs on bone, cartilage, brain, spinal cord, heart, and other tissues (5–10). For instance, cell therapy with MSCs has aided in the treatment of osteogenesis imperfecta, myocardial infarction, as well as bone marrow recovery when used with hematopoietic stem cells after myeloablation (5, 11–15). Clinical trials involving MSCs are presently ongoing worldwide and comprise phase I/II studies in India for steroid refractory graft-versus-host disease, in Israel for distal tibia bone fractures, in Denmark for severe chronic myocardial ischemia, and in Japan for periodontitis (5).

Besides their favorable implications for regenerative medicine due to their cellular plasticity, MSCs have also been explored following gene enhancement for the secretion of therapeutically beneficial plasma-soluble gene products. For instance, reports have revealed the efficacy of MSCs gene modified to overexpress epidermal growth factor receptor, factor VIII, factor IX, BMP2, BMP4, COL1A1, phenylalanine hydroxylase, and other corrective proteins (16–22). The abundance of MSCs in humans together with the facility with which these cells can be isolated, expanded, and genetically engineered have rendered MSCs a desired autologous cell type for cell and gene therapy purposes.

We have previously described the use of MSCs, gene modified to secrete erythropoietin (Epo), firstly for proof-of-concept studies and subsequently for the treatment of anemia of end-stage renal disease (23–25). More specifically, we noted a pharmacologically relevant effect from Epo gene-engineered MSCs (Epo MSCs), that is, a considerable hematocrit increase, after i.p. or s.c. implantation in normal nonmyeloablated immune-competent mice, with a more significant and durable outcome when these cells were embedded within a matrix before their s.c. administration. The matrices used to form with the MSCs "neo-organoids" comprised the mouse-compatible material Matrigel as well as the human-compatible, Food and Drug Administration (FDA)-approved, bovine collagen–based substance Contigen (23, 24). We thereafter applied our neo-organoid approach in a mouse model of anemia induced by experimental chronic renal failure, and showed that our Epo MSCs embedded in Contigen and implanted s.c. engendered a rise in plasma Epo levels, a cell dose-dependent elevation in hematocrit, and an improvement in exercise capacity which we hypothesize was linked to the cardioprotective action of Epo (25). Moreover, we observed that murine MSCs, although possessing an immunosuppressive ability, were rejected in allogeneic mice and thus not immunoprivileged, signifying that use of MSCs for long-term in vivo action should be limited to autologous settings (26).

The sum of previous studies, ours and others, supported the notion that a cell and gene therapy strategy with genetically engineered autologous MSCs embedded in a matrix and acting as a neo-organoid for therapeutic protein delivery would not only be feasible but also promising for breast cancer treatment. In our earlier research, we revealed the value of interleukin (IL)-2–engineered MSCs for immunotherapy of melanoma (27). We now here report the effectiveness of IL-12 gene-modified MSCs against breast cancer as well as melanoma. In brief, our results show that murine MSCs can be retrovirally engineered to secrete considerable levels of murine IL-12 (mIL-12), embedded in collagen-based matrices to then generate s.c. neo-organoids delivering IL-12 locally, which can significantly interfere with cancer growth in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Generation of retroviral constructs and of retrovirus-producing cells. The eukaryotic expression plasmid pNGVL12 coding for mIL-12 was obtained from National Gene Vector Laboratories. A fragment comprising the IL-12 p35 and IL-12 p40 genes separated by an internal ribosomal entry site (IRES) was retrieved by restriction enzyme digest of pNGVL12 with SalI and NotI and ligated into our retroviral plasmid control construct pEmpty-Vector (24) cut with XhoI and NotI. The resulting mIL-12 retrovector was then cotransfected with the neomycin resistance gene-containing plasmid pEGFPC1 (Clontech) into GP+E86 retrovirus packaging cells. Stable GP+E86-IL-12 transfectants were used to generate retroparticles for gene transfer. Replication-free control vector retroparticles were likewise generated.

Isolation, culture, and gene modification of marrow MSCs. A female BALB/c mouse (Charles River, Laprairie Co.) weighing 15 to 20 g was sacrificed, femurs and tibias were isolated, and whole bone marrow was retrieved by flushing these bones with complete medium (Dulbecco's Modified Eagle's Medium, supplemented with 10% heat-inactivated fetal bovine serum and 50 units/ml penicillin, 50 µg/ml streptomycin; Wisent Technologies). All of these bone marrow cells were then cultured for 5 d at 37°C with 5% CO2, after which the nonadherent hematopoietic cells were discarded and the adherent MSCs were cultured for four to five passages in complete medium before gene modification.

IL-12 gene-modified BALB/c MSCs were generated by transduction of MSCs twice per day for 2 consecutive days and once more on the third day for each of 2 successive weeks. For each round of transduction, 0.45-µm filtered retroviral supernatant from GP+E86-IL-12 virus producers was placed on 60% to 70% confluent MSCs in the presence of 6 µg/mL Lipofectamine reagent (Invitrogen/Life Technologies). The ensuing polyclonal population of IL-12 gene-modified BALB/c MSCs was plated at limiting dilution for the generation of monoclonal populations of IL-12 MSCs. Control vector retroparticles served to likewise generate control MSCs. Supernatants were collected and ELISA specific for mIL-12 p70 (R&D Systems) showed the in vitro secretion of >60 ng of IL-12 per 106 cells per 24 h for the IL-12 MSCs used in this study.

Most experiments were conducted with BALB/c-derived MSCs since testing on the growth of isogenic 4T1 breast cancer cells. To evaluate MSCs from a different mouse strain and on another tumor type, we likewise generated MSCs from a 15 to 20 g C57BL/6 mouse and using our above-described IL-12 retroparticles. As control C57BL/6 MSCs, we used those we had previously produced (23).

In vitro characterization of IL-12 MSCs. IL-12 gene-modified BALB/c MSCs were incubated with CD31-biotin,390; CD34-biotin,RAM34; CD44-PE,IM7; CD45-PE,30-F11; CD73-PE,TY/23; CD80-biotin,16-10A1; CD86-biotin,PO3; CD117-PE,2B8; Kd-biotin,SF1-1.1; and I-Ad-PE,AMS-32.1 (BD PharMingen) and B7H1-PE,M1H5 and CD105-biotin,MJ7/18 (1:50 dilution; eBioscience) for 1 h at 4°C in the dark and washed with 3% fetal bovine serum (FBS) in PBS. Cells incubated with the biotin-conjugated antibodies were stained with the secondary antibodies (streptavidin-PE, dilution 1:400) for 30 min. Isotypic control analyses were conducted in parallel. Cells were washed with 3% FBS in PBS, fixed with 1% paraformaldehyde, and analyzed using a FACSCalibur flow cytometer with data analysis conducted with CellQuest Pro software (BD Immunocytometry Systems).

To ascertain their in vitro differentiation ability, IL-12 BALB/c MSCs were cultured, when ~70% confluent, in medium inductive of osteogenic or adipogenic differentiation for 1 mo and stained with Alizarin Red S or Oil Red O solutions, respectively, as previously described (25, 28, 29).

In vivo experiments assessing IL-12 MSCs on 4T1 breast cancer cell growth. 4T1 murine breast cancer cells purchased from the American Type Culture Collection and cultured in complete medium were first implanted s.c. in the right flank of isogenic BALB/c mice at 2.5 x 104 per mouse mixed with IL-12 or control BALB/c MSCs at 1 x 106 per mouse in RPMI 1640 (Wisent). In other experiments, 4T1 cells and MSCs were injected s.c. at 2.5 x 104 per mouse and 1 x 106 per mouse, respectively, after mixing with Matrigel (500 µL Matrigel per mouse; Becton Dickinson). Furthermore, most experiments here reported involved firstly the s.c. administration of the 4T1 cells (2.5 x 104 per mouse) on their own in RPMI 1640 (100 µL/mouse), the site of injection marked, and on the following day at the same site, the IL-12 or control MSCs (106 per mouse) mixed in Matrigel (500 µL Matrigel per mouse). A later study was similarly conducted with the difference that the gene-modified MSCs were administered after mixing with the human-compatible collagen-based matrix Contigen (200–300 µL Contigen per mouse; C.R. Bard, Inc.).

Tumor volume was measured over time using calipers, and percentage of tumor-free mice was determined over time. The mathematical formula used to derive tumor volume was (length x width2) ÷ 2. Peripheral blood samples were collected from the saphenous vein using heparinized microhematocrit tubes (Fisher Scientific), centrifuged, and plasma was used to measure ELISA concentrations of mIL-12 p70 and mouse IFN-{gamma} (R&D Systems).

Implant analysis. For histologic analysis of tumor cell–bearing Matrigel implants, mice were implanted s.c. with 4T1 cells, at 2.5 x 104 per mouse, mixed in Matrigel with or without IL-12 or control MSCs at 1 x 106 per mouse. Mice were sacrificed 13 d after implantation, and implants were retrieved, fixed with 10% formalin, and paraffin embedded. Sections of 5 µm along the longest axis of implants were prepared, stained with H&E, visualized with a Nikon Eclipse 50 microscope, and digitally photographed using a Nikon DS Fi1 camera and DS U2 software. Neutrophils present among tumor cells were counted in five randomly chosen fields at high power (630x). Necrotic capillaries present within the acellular central core of implants containing IL-12 MSCs were also counted.

In vivo testing of IL-12 MSCs contralateral to 4T1 breast cancer cell implantation. BALB/c mice were implanted s.c. in the right flank with 4T1 cells at 2.5 x 104 per mouse in RPMI 1640. The following day, two of the three groups of 4T1-implanted mice received by s.c. injection in the opposite side (i.e., left flank) IL-12 or control BALB/c MSCs, at 1 x 106 per mouse, mixed in Matrigel. Tumor volumes were measured for over 1 mo.

In vivo experiments in nonobese diabetic-severe combined immunodeficient mice. Female nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice, obtained from The Jackson Laboratory, were implanted firstly with 4T1 cells at 2.5 x 104 per mouse, s.c. in the right flank, and the following day at the same site, with IL-12 or control BALB/c, MSCs mixed in Matrigel at the cell density of 1 x 106 per mouse. One group of mice received only 4T1 cells. Tumor measurements were conducted for over 1 mo.

In vivo testing of MSCs in the B16 melanoma model. B16 murine melanoma cancer cells previously used in our laboratory (27) were cultured in complete medium and implanted s.c. in the right flank of isogenic C57BL/6 mice at 105 per mouse in RPMI 1640 (100 µL/mouse). The next day, IL-12 or control C57BL/6 MSCs, polyclonal populations, were mixed at 5 x 105 per mouse in Contigen and administered s.c. in the same area where the B16 cells had been implanted (200–300 µL Contigen per mouse). Tumor volume was measured over time and percentage of tumor-free mice was determined for over 2 mo. Animals were handled under the guidelines promulgated by the Canadian Council on Animal Care.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
In vitro characterization of IL-12 gene-modified MSCs. To genetically engineer MSCs to render them capable of secreting significant levels of IL-12, we first generated the mIL-12 retrovector, a construct containing the two subunits of mIL-12, specifically p35 and p40, separated by an IRES, which we then used to produce replication-free IL-12–containing retroparticles. We likewise generated the control retrovector (i.e., not expressing IL-12) and then replication-free control retroparticles. Replication-free retroparticles were used for the gene modification of MSCs. Using MSCs isolated from BALB/c and C57BL/6 mice, we created polyclonal and monoclonal preparations of IL-12 gene-modified murine MSCs secreting >60 ng of mIL-12 per 106 cells per 24 h in vitro by ELISA on cell supernatants. More precisely, mIL-12 secretion by the IL-12 MSCs mainly used was 75 ± 3.7 ng of mIL-12 per 106 cells per 24 h (n = 5, mean ± SE). Control MSCs were found not to secrete detectable levels of mIL-12 (<2.5 pg/mL).

To determine if the IL-12 BALB/c MSCs to be tested in our 4T1 breast cancer model express cell surface antigens typically present on MSC preparations, we characterized our cells by flow cytometry analysis and found them, as shown in Fig. 1A , to be CD44+, CD73+, CD117+, CD105–, CD31–, CD45–, MHC class I+ (i.e., Kd+), and MHC class II– (I-Ad–; Fig. 1A) and we also show that they possess mesenchymal plasticity in vitro (Fig. 1B).


Figure 1
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  		Figure 1. Characterization of IL-12 MSCs. A, the BALB/c mouse-derived MSCs, gene modified to secrete mIL-12 and used in this study, were analyzed by flow cytometry analysis for the expression of cell surface antigens CD31, CD34, CD44, CD45, CD73, CD80, CD86, CD105, CD117, Kd (MHC class I), I-Ad (MHC class II), and B7H1. B, for analysis of their mesenchymal differentiation ability, IL-12–secreting MSCs, undifferentiated and confluent (left), were cultured, when ~70% confluent, in conditions inductive of osteogenic or adipogenic differentiation, respectively. Middle, following osteogenic differentiation, calcium in the mineralized extracellular matrix was shown by Alizarin Red S staining; right, following adipogenic differentiation, lipid droplets were evident by light microscopy as well as by their staining with Oil Red O. Photographs were taken under light microscopy using a Contax167MT camera (Kyocera) with a 400 ISO film attached to an Axiovert25 Zeiss microscope (Carl Zeiss).

 
Effect of IL-12 MSCs on 4T1 breast cancer cell growth. To learn if the IL-12 secreted in vivo by IL-12 gene-modified BALB/c MSCs can engender a beneficial effect against 4T1 cells in vivo, we implanted isogenic BALB/c mice with 4T1 cells s.c. in the presence or absence of IL-12 MSCs or control MSCs. We observed a significant effect against breast cancer progression in recipients of IL-12 MSCs compared with the other groups of mice (P < 0.01, log-rank test; Fig. 2A ). At 3 weeks after implantation, there were 43% of mice tumor-free among those that had received IL-12 MSCs, in contrast to 0% of control mice that had been implanted with 4T1 cells alone or with control MSCs. By 5 weeks after implantation, there were still 14% of tumor-free mice in the IL-12 group (Fig. 2A).


Figure 2
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  		Figure 2. Percentage of tumor-free BALB/c mice after implantation with 4T1 breast cancer cells and gene-modified MSCs with/without Matrigel, and plasma IL-12 p70 and IFN-{gamma} levels. A, 4T1 cells (2.5 x 104 per mouse) alone or mixed with IL-12–secreting or control MSCs (1 x 106 per mouse) were injected s.c., without a matrix, in syngeneic BALB/c mice, and percentage of tumor-free mice was determined over time (n = 7). B, BALB/c mice were implanted s.c. with 4T1 cells (2.5 x 104 per mouse) and the next day, at the same site, with IL-12 MSCs or control MSCs (1 x 106 per mouse) mixed in Matrigel. Percentage of tumor-free mice was determined over time (n = 6). C, plasma collected from peripheral blood from BALB/c mice 1 wk following implantation with 4T1 cells with/without subsequent day IL-12 MSCs or control MSCs in Matrigel was used in mouse-specific IL-12 (left) and IFN-{gamma} (right) ELISAs to determine IL-12 and IFN-{gamma} levels. Columns, mean; bars, SE.

 
Effect of Matrigel-embedded IL-12 MSCs on 4T1 breast cancer cell growth. To reveal the potential of a clinically translatable anticancer approach where genetically engineered MSCs would be implanted embedded in a matrix as a neo-organoid at the tumor site, we mixed the IL-12 BALB/c MSCs in Matrigel and injected these at the site were 4T1 tumor cells had been implanted on their own, and without any matrix, 1 day earlier. In mice implanted s.c. with 4T1 cells and the following day with Matrigel-embedded IL-12 MSCs in the same area, 100% were tumor-free at 20 days after implantation versus 0% of mice implanted with 4T1 cells only or with next day Matrigel-embedded control MSCs (P < 0.001, log-rank test). Furthermore, 67% of IL-12 MSC-implanted mice remained tumor-free for >55 days (Fig. 2B).

To determine IL-12 plasma levels, mIL-12 p70–specific ELISA was conducted on plasma samples collected 1 week after implantation and revealed concentrations of 362 ± 61.3 pg/mL in IL-12 MSC recipients versus 27.3 ± 2.60 pg/mL in control MSC-implanted mice (P < 0.01, Student's t test; Fig. 2C, left). To establish if increased IL-12 plasma levels led to elevated IFN-{gamma} plasma concentrations, mIFN-{gamma}–specific ELISA was performed and revealed IFN-{gamma} levels of 452 ± 113 pg/mL in these IL-12 mice, in contrast to no detectable levels (i.e., <2 pg/mL) in control MSC recipients (P < 0.05, Student's t test; Fig. 2C, right). However, plasma concentrations of both IL-12 and IFN-{gamma} declined over time following implantation of IL-12 MSCs, as we determined a drop of ~35% in IL-12 and ~95% in IFN-{gamma} at 3 weeks after implantation, more precisely to 233 ± 46.1 pg/mL and 24 pg/mL, respectively (data not shown).

Histologic analysis of implants of 4T1 cells with/without IL-12 or control MSCs. To analyze and compare the histology of implants of tumor cells in the presence or absence of gene-modified MSCs, implants were retrieved 2 weeks after implantation. Matrigel implants from mice implanted with 4T1 cells and IL-12 MSCs contained much less tumor cells (Fig. 3C ) than Matrigel implants from mice implanted with 4T1 cells alone (Fig. 3A) or 4T1 cells with control MSCs (Fig. 3B). Tumors were subdivided in large ill-defined lobules by thin delicate fibrovascular bands, and within lobules, tumor cells formed a solid sheet where few capillaries were interspersed. Lobules contained small and few areas of lytic necrosis where cell debris were mixed with few neutrophils. Tumor cells were poorly differentiated: they were closely and disorderly packed (they did not form glandular acini), and their nuclei were large, vesicular, and highly pleomorphic and often contained multiple amphophilic nucleoli. The chromatin was coarsely clumped. The cytoplasm was scarce and cellular outline was indistinct. We noted that there were no striking quantitative or qualitative changes between the IL-12 group and both control groups regarding inflammation. A quantitative analysis of neutrophils revealed the presence of 3.3 ± 2.2, 1.2 ± 0.5, and 0.7 ± 0.3 neutrophils (mean ± SE) per high-power field in implants retrieved from mice 2 weeks after implantation with 4T1 cells in Matrigel, 4T1 cells and control MSCs in Matrigel, and 4T1 cells and IL-12 MSCs in Matrigel, respectively. There were no significant differences between the three different groups. Furthermore, lymphocytes were noted to be even scarcer than neutrophils.


Figure 3
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  		Figure 3. Histologic analysis of retrieved Matrigel implants. Implants of Matrigel with 4T1 cells alone (A), 4T1 cells mixed with control MSCs (B), or 4T1 cells mixed with IL-12 MSCs (C) were retrieved from BALB/c recipient mice 13 d after implantation, processed, sectioned, H&E stained, and analyzed by a veterinary pathologist. Shown are representative sections from four implants per group of mice. A, top, implant of 4T1 cells occupied by a solid sheet of tumor cells; middle, interface between dense population of tumor cells (white asterisk) and Matrigel matrix. The matrix is infiltrated by long viable capillaries (long arrows) and islets of viable tumor cells (arrowheads). Bar, 500 µm. Bottom, above boxed region at higher magnification. The matrix is infiltrated by long viable capillaries (long arrow) and islets of viable tumor cells (white asterisk). Arrowhead, few neutrophils are present. Bar, 200 µm. B, top, implant of 4T1 cells and control MSCs occupied by a solid sheet of tumor cells; middle, interface between tumor cells and Matrigel. The matrix is infiltrated by long viable capillaries (long arrow) and numerous randomly distributed islets of viable tumor cells (short arrows). White asterisks, a dense population of tumor cells rims the Matrigel implant. Bar, 500 µm. Bottom, above boxed region at higher magnification. The matrix is infiltrated by long viable capillaries (long arrow) and islets of viable tumor cells (white asterisk). Short arrows, few neutrophils are present. Bar, 200 µm. C, top, in implant of 4T1 cells and IL-12 MSCs, a thin peripheral rim of tumor cells occasionally thickened; middle, interface between tumor cells and Matrigel. The matrix contains a long necrotic branching capillary (upper three short arrows) originating from the dense population of tumor cells that rims the implant. Long arrow, junction between viable (left of the arrow) and branching necrotic (right of the arrow) segments of the capillary. Lower two short arrows, numerous islets of necrotic tumor cells are randomly distributed within Matrigel. Bar, 500 µm. Bottom, above boxed region at higher magnification. It shows an abrupt demarcation between viable and necrotic capillary segments (long arrow), a viable segment of the capillary on the left of the axis of the long arrow, and necrotic segments on the right. The capillary wall of the necrotic segments contains hyaline eosinophilic material (fibrinoid necrosis), and endothelial cells are vacuolated and have pyknotic nuclei (short arrows). Bar, 200 µm.

 
However, an antiangiogenic effect was determined based on the following observations. Besides the smaller implant size and apparent lower number of tumor cells in implants from IL-12 MSC recipient mice, the striking feature of this group was the presence of "ghost" necrotic capillaries in the acellular central part of the implants (Fig. 3C). Quantitative analysis revealed counts of 13 ± 2.4 necrotic capillaries (mean ± SE) within the acellular central core of implants retrieved from recipient mice 2 weeks following implantation with 4T1 cells and IL-12 MSCs in Matrigel. In the other two groups, necrotic capillaries were very rarely seen (less than 2) because these implants, in contrast to those with IL-12 MSCs, were almost entirely populated by tumor cells.

In addition, between these necrotic capillaries, rare islets of tumor cells, also necrotic (ghost like), were present. This type of necrosis, coagulation necrosis, strongly suggests ischemia as a causal factor.

Assessing if effect of IL-12 MSCs on 4T1 tumor growth is local or systemic. To establish if the slowing of 4T1 tumor progression observed in mice that also received IL-12–secreting gene-modified MSCs is due to a local or systemic action of the IL-12, we conducted experiments where Matrigel-embedded MSCs were injected s.c. in the flank contralateral to where 4T1 cells were injected the previous day. As revealed in Fig. 4 , there was no slowing of tumor growth when IL-12 MSCs were not administered in the same flank and location as 4T1 cancer cells. Tumor growth in mice implanted with 4T1 cells alone or with contralateral injection of control MSCs or IL-12 MSCs in Matrigel was similar (P > 0.1, Student's t test). At day 29 after implantation, tumor volumes in these groups of mice were 639 ± 125, 739 ± 236, and 894 ± 301 µL, respectively (Fig. 4).


Figure 4
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  		Figure 4. Effect of gene-modified MSCs on tumor growth in BALB/c mice 1 d following injection of 4T1 breast cancer cells in contralateral flank. BALB/c mice were implanted s.c. in the right flank with 4T1 cells (2.5 x 104 per mouse) and the next day, at the contralateral site (i.e., left flank), with IL-12 MSCs or control MSCs (1 x 106 per mouse) mixed in Matrigel. Tumor volume was measured over time. Points, mean (n = 7–8); bars, SE.

 
Effect of IL-12 MSCs on 4T1 cancer growth in immunodeficient mice. Aiming to determine the importance of the immune response in the anticancer effect exerted by the IL-12 MSCs, we similarly tested these cells on 4T1 tumor cell growth in immunodeficient mice. We therefore implanted NOD-SCID mice with 4T1 cells and the next day with Matrigel-embedded gene-modified BALB/c MSCs at the same injection site. We noted that no beneficial effect arose from the IL-12 MSCs because 4T1 tumor growth was not significantly different than that in the other two groups of mice (P > 0.05, Student's t test; Fig. 5 ).


Figure 5
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  		Figure 5. Tumor growth in NOD-SCID mice following implantation with 4T1 breast cancer cells and Matrigel-embedded gene-modified MSCs. NOD-SCID mice were injected s.c. with 4T1 cells (2.5 x 104 per mouse) and the next day, at the same site, with Matrigel-embedded IL-12 MSCs or control MSCs (1 x 106 per mouse). Tumor volume was determined over time. Points, mean (n = 6–8); bars, SE.

 
Effect of IL-12 MSCs on 4T1 tumor growth when embedded in a human-compatible matrix. As the matrix material for embedding MSCs, we first used Matrigel, a mouse-compatible basement membrane derived from EHS mouse sarcoma. We then tested if an anticancer effect can also be achieved with MSCs embedded in a human-compatible matrix. Thus, we carried out experiments where MSCs were embedded in the human-compatible, FDA-approved, bovine collagen–based matrix Contigen and remarked a beneficial effect of IL-12 MSCs on 4T1 tumor progression in BALB/c mice. IL-12 MSCs in Contigen injected 1 day following 4T1 cells at the same site led to 88% of recipient mice being tumor-free at day 23 when mice in both other groups all had tumors (P < 0.0005, log-rank test; Fig. 6A ). Thirty-eight percent of the Contigen-embedded IL-12 MSC recipients did not develop any tumor up to day 56, after which this group comprised 13% tumor-free mice until day 70.


Figure 6
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  		Figure 6. Percentage of tumor-free mice following implantation with 4T1 or B16 cancer cells and gene-modified MSCs embedded in human-compatible matrix Contigen. A, BALB/c mice were implanted s.c. with 4T1 cells (2.5 x 104 per mouse) and the next day, at the same site, with IL-12 MSCs or control MSCs (1 x 106 per mouse) mixed in Contigen. Percentage of tumor-free mice was determined over time (n = 7–8). B, C57BL/6 mice were injected s.c. with B16 cells (105 per mouse) and the next day, at the same site, with syngeneic IL-12 MSCs or control MSCs (5 x 105 per mouse) embedded in Contigen. Percentage of tumor-free mice was determined over time (n = 7–8).

 
Effect of IL-12 MSCs on B16 melanoma progression. To learn whether the antitumor effect accomplished with the use of matrix-embedded IL-12 gene-modified MSCs is specific to only one mouse strain and one tumor type, we tested in the B16 melanoma model, isogenic C57BL/6 mouse-derived MSCs genetically engineered to secrete IL-12 and embedded in Contigen. Results showed a significant decline in tumor progression in C57BL/6 mice that received the Contigen-embedded IL-12–secreting C57BL/6 MSCs in contrast to control MSCs or no MSCs 1 day after administration of the B16 cells at the same injection site (P < 0.0005, log-rank test; Fig. 6B). One hundred percent of mice implanted with IL-12 MSCs were tumor-free at day 13 versus 0% of mice that received B16 cells alone or with next day control MSCs. Fifty-seven percent of IL-12 mice did not develop a tumor until almost day 50 after B16 implantation (Fig. 6B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
IL-12, produced mostly by antigen-presenting cells, is a multifunctional, heterodimeric Th1 cytokine shown to induce a cellular immune response, cancer cell apoptosis, as well as antiangiogenesis (31–33). Many studies have evaluated the anticancer effectiveness of IL-12 (34, 35). However, significant toxicity from recombinant human IL-12 administration has been noted in clinical trials in advanced cancers, limiting its clinical use (36–38). Therefore, sustained delivery of IL-12, avoiding toxic peaks seen with intermittent i.v. infusions, would be desirable. One means of achieving this and thus providing sustained and sufficient amounts of IL-12 at the tumor site is through a cell and gene therapy approach. In such a strategy tested in mice, investigators observed IL-12–mediated antiangiogenesis in SCID mice bearing human or murine tumors that were coinoculated with IL-12–secreting gene-modified 3T3 fibroblasts (39). In a phase I dose-escalation study, autologous human fibroblasts retrovirally transduced to secrete human IL-12 and injected in the peritumoral surroundings showed promise for the treatment of disseminated cancer (40). In another more recent study, autologous dendritic cells were transfected with an adenovirus encoding for human IL-12 and injected in the tumors of 17 patients with metastatic gastrointestinal tumors (41). These and other reports support the feasibility of a cell therapy platform for IL-12 delivery to overcome its dose-limiting toxicity.

Based on our previous work with MSCs (24, 25, 27), we here proposed using MSCs as part of a neo-organoid to obtain optimal amounts of IL-12 for cancer treatment. We assessed our IL-12 gene-enhanced cell therapy "neo-organoid" approach mainly against breast cancer cell growth.

We established that IL-12 gene-modified MSCs maintained their progenitor cell properties and that IL-12 did not seem to alter their phenotype, which is consistent with the absence of IL-12 receptor expression by MSCs. We then initially ascertained that IL-12 MSCs can engender an anticancer effect on their own without any matrix, as we noted a slowing of tumor progression in mice implanted with IL-12–secreting BALB/c MSCs mixed with 4T1 breast cancer cells compared with control mice (Fig. 2A).

All mice, however, in this experiment did develop tumors by day 36 (Fig. 2A). Hence, seeing as we had earlier published that the effect of a secreted transgene product can be augmented and prolonged by embedding the gene-modified MSCs in a matrix before their s.c. delivery (23, 24), we sought out to determine whether a greater and more persistent antitumor effect can be realized with matrix-embedded IL-12 MSCs. This indeed did occur. The antitumor effect exerted by the IL-12 MSCs was very markedly superior and longer lasting when these cells were delivered mixed with the matrix material Matrigel (Fig. 2B). This finding is of great interest as it indicates that a neo-organoid containing IL-12 MSCs implanted in the vicinity of already present breast cancer cells can lead to a significant slowing of cancer growth and to increased survival with the possibility of complete tumor regression. We also showed that these IL-12–secreting neo-organoids had engendered increased systemic levels of mIL-12, as concentrations were over 10-fold higher than in the control mice (Fig. 2C, left). Voest and colleagues (33) have shown that IL-12 has antiangiogenic effects that are indirect and that can inhibit tumor growth, and proposed that this antiangiogenic action may be due to IL-12–induced IFN-{gamma}. Consequently, due to the fact that IL-12 has been reported to induce an antiangiogenic effect involving IFN-{gamma} (33, 42), we also measured mouse IFN-{gamma} and noted increased plasma levels in the IL-12 MSC mice (Fig. 2C, right).

With the aim of visualizing and better describing the local in vivo action of the IL-12 MSCs on the 4T1 breast cancer cells, we carried out an implant analysis experiment where 4T1 cells were admixed with MSCs in Matrigel, injected s.c., and subsequently retrieved ~2 weeks later. Histopathology revealed substantially less tumor cells in implants with IL-12 MSCs (Fig. 3), confirming the slower tumor progression observed in these mice. With regards to inflammation, there were no striking quantitative or qualitative differences between the IL-12 group and both control groups. Very interestingly, however, IL-12 MSC mice contained not only less tumor cells in their implants but also strikingly necrotic capillaries and necrotic islets of tumor cells, suggesting ischemia perhaps caused by an antiangiogenic effect of IL-12 and more specifically blood vessel necrosis. These observations together with the elevated IFN-{gamma} plasma levels support an antiangiogenic role of the IL-12 (33, 42) delivered by our gene-modified MSCs. Additive to the antiangiogenic effect, an intact immune system is required for the antitumor effect of IL-12 because their activity is lost in immunodeficient NOD-SCID mice (Fig. 5).

Moreover, we discovered that, for our neo-organoid matrix constituent, a human-compatible substance could also be used for embedding IL-12 MSCs and similarly obtaining a considerable slowing of tumor growth (Fig. 6A). These results confirm the utility of this FDA-approved, viscous bovine collagen–based material (i.e., Contigen) that we previously tested, and published our observations of it permitting an enhanced and more durable effect from a gene product secreted by the MSCs (24). Because Matrigel is a matrix derived from a murine sarcoma cell line and comprises various mouse proteins, it is immunologically incompatible for use in humans. As we aim to eventually bring our transgenic cell therapy neo-organoid approach to the clinic, we previously tested and found that Contigen will support mouse MSCs as efficiently as Matrigel and is retrievable leading to the cessation of the effect of the transgene (24).

Furthermore, we showed that the beneficial effect of our gene-enhanced cell therapy neo-organoid strategy is not restricted to 4T1 breast cancer and not restricted to the BALB/c mouse strain. Using C57BL/6-derived MSCs genetically engineered to secrete IL-12 and embedded within the Contigen scaffold, we likewise achieved a slowing of B16 melanoma progression (Fig. 6B). Our therapeutic approach therefore holds potential against different types of cancer.

Other preclinical studies using IL-12 MSCs in cancer treatment have also been conducted. In one latest investigation, MSCs were gene modified with an adenovirus coding for mIL-12 and then investigated for prophylaxis against tumor formation in mice (43). With all three cancer cell types examined (i.e., hepatocellular carcinoma, Lewis lung carcinoma, and B16 melanoma), the IL-12 MSCs prevented tumors from arising in most mice (43). In another recent study, human MSCs retrovirally engineered to express rat IL-12 were tested by intratumoral injection in B16F10 melanoma-bearing C57BL/6 mice and found to slow tumor growth and prolong survival (44). However, the authors suggested that the immune rejection of these xenogeneic MSCs impaired their effectiveness (44). Other than for IL-12 delivery, studies by us and others have revealed the preclinical efficacy of genetically engineered MSCs in cancer treatment. For instance, human MSCs adenovirally transduced to express human IFN-β were noted to suppress, after i.v. administration, the growth of A375SM human melanoma in immunodeficient mice (45). These cells were found to have a tropism for the tumor site where local production of the IFN-β then led to the antitumor effect. A slowing of tumor growth and increase in mouse survival also occurred when these IFN-β MSCs were mixed with the melanoma cells and administered s.c. (45). No beneficial effects were observed when the IFN-β MSCs were administered distally (i.e., on the side contralateral to the tumors; ref. 45), an observation we also make in our present study (Fig. 4). We here established that the antitumor effect seen with our IL-12 MSCs was due to local, and not systemic, IL-12 delivery because we did not observe this result when the IL-12 MSCs were implanted in the flank contralateral to the 4T1 breast cancer cells. Tumor progression was similar in all groups of mice (Fig. 4), therefore indicating the importance of the proximity of the IL-12 MSCs to the cancer cells for an anticancer effect to arise from the secreted IL-12. In a more recent investigation by Studeny and colleagues (46), IFN-β gene-modified human MSCs were noted following i.v. administration to reduce the pulmonary metastasis from established human A375SM melanoma or MDA-231 breast cancer cells, an effect the authors suggest is due to tumor site production of IFN-β. In another investigation, these IFN-β–enhanced human MSCs tested in mice with human glioma were also found to lead to increased survival following intravascular delivery or via intratumoral injection (47). This anticancer effect again was only obtained through local delivery of the IFN-β MSCs. In these reports, such as in ours, a high concentration of the transgene at the tumor site plays an important role.

Other than IL-12 and IFN-β, research using rat MSCs genetically engineered to produce human IL-2 showed decreased tumor growth and significantly increased survival time following intratumoral injection in an established rat glioma model (48). We have previously reported that mIL-2 gene-modified mouse MSCs can generate an anticancer immune response engendering a significant slowing of B16 melanoma tumor growth in recipient mice following s.c. delivery (27). We have also found that MSCs can act as antigen-presenting cells and proposed that this feature can be exploited for augmenting an immune response against a tumor (49, 50).

In conclusion, our investigation shows the potential of using MSCs gene enhanced for delivery of therapeutically relevant levels of a plasma-soluble gene product with an antineoplastic effect, in this case IL-12, as part of a cell-based neo-organoid strategy for cancer therapy. The ease of implantation and removal makes this approach safe and clinically desirable.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: U.S. Army Breast Cancer Research program award DAMD17-02-1-0447 and Canadian Institutes of Health Research operating grant MOP-15017. N. Eliopoulos is a previous recipient of a fellowship from the U.S. Army Medical Research Acquisition Activity, Fort Detrick, Frederick, MD (U.S. Army Medical Research and Material Command Breast Cancer Research program, award no. DAMD17-02-1-0447). J. Galipeau is a Fonds de Recherche en Santé du Québec Chercheur-Boursier Sénior.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/14/08. Revised 3/28/08. Accepted 4/ 4/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

  1. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–4.[Abstract/Free Full Text]
  2. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7.[Abstract/Free Full Text]
  3. Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 2003;5:32–45.[Medline]
  4. Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 2004;32:414–25.[CrossRef][Medline]
  5. Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient's bedside: an update on clinical trials with mesenchymal stem cells. J Cell Physiol 2007;211:27–35.[CrossRef][Medline]
  6. Shake JG, Gruber PJ, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 2002;73:1919–25.[Abstract/Free Full Text]
  7. Diduch DR, Jordan LC, Mierisch CM, et al. Marrow stromal cells embedded in alginate for repair of osteochondral defects. Arthroscopy 2000;16:571–7.[Medline]
  8. Kinnaird T, Stabile E, Burnett MS, et al. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004;109:1543–9.[Abstract/Free Full Text]
  9. Lu D, Mahmood A, Wang L, Li Y, Lu M, Chopp M. Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome. Neuroreport 2001;12:559–63.[CrossRef][Medline]
  10. Hofstetter CP, Schwarz EJ, Hess D, et al. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A 2002;99:2199–204.[Abstract/Free Full Text]
  11. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–13.[CrossRef][Medline]
  12. Horwitz EM, Prockop DJ, Gordon PL, et al. Clinical responses to bone marrow transplantation in children with severe osteogenesis imperfecta. Blood 2001;97:1227–31.[Abstract/Free Full Text]
  13. Horwitz EM, Gordon PL, Koo WKK, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 2002;99:8932–7.[Abstract/Free Full Text]
  14. Chen SL, Fang W, Ye F, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004;94:92–5.[CrossRef][Medline]
  15. Koc ON, Gerson SL, Cooper BW, et al. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol 2000;18:307–16.[Abstract/Free Full Text]
  16. Sato H, Kuwashima N, Sakaida T, et al. Epidermal growth factor receptor-transfected bone marrow stromal cells exhibit enhanced migratory response and therapeutic potential against murine brain tumors. Cancer Gene Ther 2005;12:757–68.[CrossRef][Medline]
  17. Van Damme A, Chuah MK, Dell'accio F, et al. Bone marrow mesenchymal cells for haemophilia A gene therapy using retroviral vectors with modified long-terminal repeats. Haemophilia 2003;9:94–103.[Medline]
  18. Lu DR, Qiu XF, Zheng B, Xue JL. High expression of human clotting factor IX cDNA in the bone marrow stroma cells of a hemophilia B patient. Chin Sci Bull 1995;40:1374–7.
  19. Zhang XS, Linkhart TA, Chen ST, et al. Local ex vivo gene therapy with bone marrow stromal cells expressing human BMP4 promotes endosteal bone formation in mice. J Gene Med 2004;6:4–15.[CrossRef][Medline]
  20. Turgeman G, Pittman DD, Muller R, et al. Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy. J Gene Med 2001;3:240–51.[CrossRef][Medline]
  21. Oh HJ, Lee H, Park JW, et al. Reversal of gene expression profile in the phenylketonuria mouse model after adeno-associated virus vector-mediated gene therapy. Mol Genet Metab 2005;86:S124–32.[Medline]
  22. Pochampally RR, Horwitz EM, DiGirolamo CD, Stokes DS, Prockop DJ. Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects. Gene Ther 2005;12:1119–25.[CrossRef][Medline]
  23. Eliopoulos N, Al-Khaldi A, Crosato M, Lachapelle K, Galipeau J. A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice. Gene Ther 2003;10:478–89.[CrossRef][Medline]
  24. Eliopoulos N, Lejeune L, Martineau D, Galipeau J. Human-compatible collagen matrix for prolonged and reversible systemic delivery of erythropoietin in mice from gene-modified marrow stromal cells. Mol Ther 2004;10:741–8.[CrossRef][Medline]
  25. Eliopoulos N, Gagnon RF, Francois M, Galipeau J. Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure. J Am Soc Nephrol 2006;17:1576–84.[Abstract/Free Full Text]
  26. Eliopoulos N, Stagg J, Lejeune L, Pommey S, Galipeau J. Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice. Blood 2005;106:4057–65.[Abstract/Free Full Text]
  27. Stagg J, Lejeune L, Paquin A, Galipeau J. Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy. Hum Gene Ther 2004;15:597–608.[CrossRef][Medline]
  28. Meirelles LD, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003;123:702–11.[CrossRef][Medline]
  29. Phinney DG, Kopen G, Isaacson RL, Prockop DJ. Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 1999;72:570–85.[CrossRef][Medline]
  30. Le BK, Tammik L, Sundberg B, Haynesworth SE, Ringden O. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 2003;57:11–20.[CrossRef][Medline]
  31. Trinchieri G, Scott P. Interleukin-12: basic principles and clinical applications. Curr Top Microbiol Immunol 1999;238:57–78.[Medline]
  32. Gee MS, Koch CJ, Evans SM, et al. Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12. Cancer Res 1999;59:4882–9.[Abstract/Free Full Text]
  33. Voest EE, Kenyon BM, O'Reilly MS, Truitt G, D'Amato RJ, Folkman J. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 1995;87:581–6.[Abstract/Free Full Text]
  34. Del VM, Bajetta E, Canova S, et al. Interleukin-12: biological properties and clinical application. Clin Cancer Res 2007;13:4677–85.[Abstract/Free Full Text]
  35. Colombo MP, Trinchieri G, Colombo MP, Trinchieri G. Interleukin-12 in anti-tumor immunity and immunotherapy. Cytokine Growth Factor Rev 2002;13:155–68.[CrossRef][Medline]
  36. Car BD, Eng VM, Lipman JM, Anderson TD. The toxicology of interleukin-12: a review. Toxicol Pathol 1999;27:58–63.[Abstract/Free Full Text]
  37. Cohen J. Clinical trials: IL-12 deaths: explanation and a puzzle. Science 1995;270:908.[Medline]
  38. Atkins MB, Robertson MJ, Gordon M, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997;3:409–17.[Abstract]
  39. Duda DG, Sunamura M, Lozonschi L, et al. Direct in vitro evidence and in vivo analysis of the antiangiogenesis effects of interleukin 12. Cancer Res 2000;60:1111–6.[Abstract/Free Full Text]
  40. Kang WK, Park C, Yoon HL, et al. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. Hum Gene Ther 2001;12:671–84.[CrossRef][Medline]
  41. Mazzolini G, Alfaro C, Sangro B, et al. Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J Clin Oncol 2005;23:999–1010.[Abstract/Free Full Text]
  42. Kerbel RS, Hawley RG. Interleukin 12: newest member of the antiangiogenesis club. J Natl Cancer Inst 1995;87:557–9.[Free Full Text]
  43. Chen XC, Wang R, Zhao X, et al. Prophylaxis against carcinogenesis in three kinds of unestablished tumor models via IL12-gene-engineered MSCs. Carcinogenesis 2006;27:2434–41.[Abstract/Free Full Text]
  44. Elzaouk L, Moelling K, Pavlovic J. Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model. Exp Dermatol 2006;15:865–74.[CrossRef][Medline]
  45. Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-β delivery into tumors. Cancer Res 2002;62:3603–8.[Abstract/Free Full Text]
  46. Studeny M, Marini FC, Dembinski JL, et al. Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 2004;96:1593–603.[Abstract/Free Full Text]
  47. Nakamizo A, Marini F, Amano T, et al. Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas. Cancer Res 2005;65:3307–18.[Abstract/Free Full Text]
  48. Nakamura K, Ito Y, Kawano Y, et al. Antitumor effect of genetically engineered mesenchymal stem cells in a rat glioma model. Gene Ther 2004;11:1155–64.[CrossRef][Medline]
  49. Stagg J, Pommey S, Eliopoulos N, Galipeau J. Interferon-{gamma}-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell. Blood 2006;107:2570–7.[Abstract/Free Full Text]
  50. Pommey S, Galipeau J. The use of mesenchymal stromal cells in oncology and cell therapy. Bull Cancer 2006;93:901–7.[Medline]



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