1. Volume 17, Issue 7, Pages 1241-1249 (July 2009)
Autologous Bone Marrow Stromal Cells Genetically Engineered to Secrete an IGF-I Receptor Decoy Prevent the Growth of Liver Metastases 
Ni Wang, Lucia Fallavollita, Long Nguyen, Julia Burnier, Moutih Rafei, Jacques Galipeau, Shoshana Yakar, Pnina Brodt 
Molecular Therapy 
Volume 17, Issue 7, Pages (July 2009)
DOI: /mt
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Genetically engineered bone marrow stromal cells produce a soluble IGF-IR. MSCs were cultured in serum-free medium for 24 hours, the conditioned media harvested and concentrated 30-fold. The concentrated proteins were separated on a 6% SDS–polyacrylamide gel under reducing (R) or nonreducing (NR) conditions using 80 µg protein per lane. Proteins were detected with a rabbit antibody to the α subunit of human IGF-IR followed by a peroxidase-conjugated donkey anti-rabbit IgG. Shown are the results of a representative western blot of four performed. Numbers on the left denote the positions of MW markers. The positions of the bands correspond to the α subunit (R) and the truncated soluble tetramer (NR). IGF-IR, insulin-like growth factor-I receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Detection of circulating soluble IGF-IR in mice implanted with genetically engineered marrow stromal cells. Ten million MSCs were mixed with Matrigel and implanted subcutaneously into (a) syngeneic C57Bl/6 or (c) athymic mice. The mice were bled at several intervals postimplantation, the plasma separated and soluble IGF-IR levels measured using the ELISA. To avoid daily bleeding of the same mice, the animals were separated into groups that were bled twice weekly and the data for each time point pooled to generate the curve shown. Each value represents the mean (and SD) of a minimum of three (and up to 33) individual measurements performed on the indicated days. Blood samples collected from mice implanted with mock-transduced MSC (MSCGFP) or MSC producing erythropoietin (MSCEPO) were used as controls. Shown in b are representative confocal microscopy images taken of sections prepared from formalin fixed and paraffin-embedded Matrigel plugs containing the indicated cells that were removed 22 days following subcutaneous implantation. The sections were stained with a rabbit antibody to GFP followed by an Alexa Fluor 568 secondary antibody and imaged using confocal microscopy with a ×40 objective. ELISA, enzyme-linked immunosorbent assay; IGF-IR, insulin-like growth factor-I receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
The soluble IGF-IR forms a complex with circulating IGF-I. Plasma concentrations of sIGFIR-bound mouse IGF-I were semiquantified by ELISA. Pooled plasma samples obtained at each of the indicated time intervals were used for the analysis. Shown are the means (and SD) of values obtained from three different plasma pools, each derived from at least six mice. ELISA, enzyme-linked immunosorbent assay; IGF-IR, insulin-like growth factor-I receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Bone marrow stromal cells producing a soluble IGF-IR inhibit experimental hepatic metastasis of H-59 cells. (a,b) Syngeneic female C57Bl/6 or (c,d) nude mice were implanted with 107 genetically engineered MSCsIGFIR or control MSC embedded in Matrigel. (a) Nine or (b) 14-days later, the mice were inoculated via the intrasplenic/portal route with 105 H-59 cells. Mice were euthanized and liver metastases enumerated 14 days post-tumor injection. Shown are the pooled data of (a-left) two and (a-right) three experiments, each performed using a different control MSC population, as shown. Representative livers from one of the experiments included in the right panel are shown (a-bottom panel). Shown in b are the pooled results of three experiments in which H-59 cells were injected 14 days post-MSC implantation and the mice euthanized 14–16 days later. Results of in vivo imaging performed with the IVIS 100 Xenogen system on day 15 post-tumor inoculation into nude mice are shown in c and representative hematoxylin and eosin stained sections obtained from formalin fixed and paraffin-embedded livers of these mice 18 days post-tumor inoculation and acquired with a ×4 (left and center panels) or ×40 (right panels) objective are shown in d. The P values as determined by the nonparametric Mann–Whitney test were (a-left) P < 0.001, (a-right) P < 0.005, and (b) P < when MSCsIGFIR-treated mice were compared to each control group. L, liver; Le, leukocytes; T, tumor. IGF-IR, insulin-like growth factor-I receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Bone marrow stromal cells producing a soluble IGF-IR inhibit colon carcinoma metastasis. (a) Mice were inoculated with 5 × 104 MC-38 or (b) 106 KM12SM 14 days post-MSC implantation. Mice were euthanized and liver metastases enumerated (a) 18 or (b) 21 days post-tumor injection. Shown are the results of individual experiments using the indicated numbers of mice per group. Representative images of hematoxylin and eosin stained sections obtained from formalin fixed and paraffin embedded livers of KM12SM-injected nude mice (experiment depicted in b) using ×4 (left and center panels) or ×40 (right panels) objectives are shown in c. The P values were (a) P < and (b) P < 0.01 when MSCSigfir-treated mice were compared to each of the control groups. L, liver; T, tumor. IGF-IR, insulin-like growth factor-I receptor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Reduced angiogenesis, increased apoptosis and decreased proliferation in micrometastases of mice implanted with MSCsIGFIR. Mice were implanted with MSC as described in the legend to Figure 4 and 105 GFP-tagged H-59 cells were injected 14 days later. Livers were obtained on day 6 post-tumor cell inoculation and processed for immunohistochemistry as described in Materials and Methods. Microvessels within micro-metastases were detected using a rat anti-CD31 antibody followed by an Alexa Fluor 568 goat anti-rat IgG. A total of six sections derived from three different livers were analyzed per group and five randomly selected fields were analyzed per section (for a total of 30 fields). CD31+ microvessels were counted, and the number of vessels per µm determined with the aid of the Zeiss LSM Image Browser software. Shown in a are means (and SE of the means) based on 30 individual images analyzed (top, P < two-tailed t-test) and two representative images/group acquired with a ×40 objective (bottom). Results of a TUNEL assay performed on sections derived from the same livers are shown in b. Following the TUNEL assay (as described in Materials and Methods), nuclei were DAPI stained and the numbers of TUNEL+ cells per total nuclei in each field were calculated using images acquired with a ×63 objective. Shown are means (and SE) of the proportions of TUNEL+ nuclei per total nuclei seen in 12 individual images (top, P = , two-tailed t-test) and representative images showing GFP+ tumor cells (green), total nuclei (blue), and apoptotic cells (red) and the merged images for each group acquired with a ×63 objective (bottom). Proliferating cells within the micrometastases were detected using an antibody to Ki67. A total of six sections derived from three different livers were analyzed per group and two randomly selected fields were analyzed per section (for a total of 12 fields). Images for each of the 12 fields were acquired with a ×63 objective, the numbers of Ki67+ cells (red) and total GFP+ tumor cells per field were recorded and the percentages of Ki67 positive cells in each field were calculated. Shown in c are the mean proportions of Ki67+ cells recorded in 12 individual images (top, P = , two-tailed t-test) and representative merged images of green and red fluorescence acquired with a ×63 objective (bottom). DAPI, 4′,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end–labeling.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2009 The American Society of Gene Therapy Terms and Conditions