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Genetically modified lentiviruses that preserve microvascular function protect against late radiation damage in normal tissues

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Science Translational Medicine  24 Jan 2018:
Vol. 10, Issue 425, eaar2041
DOI: 10.1126/scitranslmed.aar2041
  • Fig. 1 Irradiation with 50 Gy/3 fractions generates LAE characterized by SOD2 depletion and CTGF overexpression.

    (A) Representative photographs of bilateral superficial inferior epigastric artery (SIEA) flaps (edges tattooed in Indian ink) in Fischer (F344) male rats showing characteristic late adverse effect (LAE) features observed in irradiated flaps including contracture, induration of the skin, telangiectasia, and hair loss (n = 6 per group). (B) Changes in skin paddle surface area of irradiated and control SIEA flaps [±95% confidence interval (CI)]. (C) Acute and late RTOG scores including component subscores by tissue type: skin, subcutaneous (s/c), and joint. ROTG, Radiation Therapy Oncology Group. (D) Passive range-of-movement (ROM) measurement at the knee joint in irradiated and nonirradiated hindlimbs (n = 6 per group). (E) T2-weighted magnetic resonance imaging (MRI) of bilateral SIEA flaps showing an irradiated flap (L) and unirradiated control (R) and quantification of volumetric changes (mean ± 95% CI, n = 6 per group). fx, fractions. (F) Connective tissue growth factor (CTGF) enzyme-linked immunosorbent assay (ELISA) and superoxide dismutase 2 (SOD2) biochemical activity assay performed on flap tissues taken from irradiated and control flaps (n = 6 per group). (G) Western blotting of control and irradiated SIEA flaps for SOD2, phospho–glycogen synthase kinase 3β (GSK3β) (Ser9), β-catenin, and LEF1, with β-actin used for loading control. (H to J) Immunohistochemical analysis and quantification of collagen deposition [(H) Masson’s trichrome (collagen is green)], CTGF expression (I), and hematoxylin and eosin (H&E) staining for the assessment of fat necrosis [(J), red circle)]. Graphs represent means ± SEM of counts of collagen fibrils (H) or cells with membranous CTGF expression (I) per high-powered field (hpf) or % of section exhibiting fat necrosis (J) (n = 6 per group; scale bar, 50 μm). (K) Real-time quantitative polymerase chain reaction (RT-qPCR) for Ctgf, Col1a2, and Col3a1 gene expression (mean fold increase in gene expression ± SEM) in irradiated flap tissues (n = 6 per group). All tissue-based analyses were performed on flaps harvested at 180 days after RT. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 2 LAEs are characterized by vascular dysfunction, loss of endothelial perfusion and permeability, and perivascular hypoxia.

    (A) Parametric R2* maps of control and irradiated flaps overlaid on T2-weighted images acquired 6 months after RT (image representative of n = 6 animals per group). (B) Absolute transverse relaxation rates (R2*) in irradiated versus control flaps at 6 months after RT and relative changes R2* (ΔR2*) after RT (n = 6 animals per group) (mean ± SEM). ns, not significant. (C) Vascular staining for perfused endothelium [Hoechst 33342 (H33342)], vascular permeability [Evans blue (EB)], and immunofluorescent (IF) staining for stromal hypoxia (pimonidazole adduct formation) in irradiated and control flaps at 180 days after RT (scale bars, 100 μm; images representative of n = 3 animals per group). Images depict whole-section H&E (inset left) and whole-section tile scan (inset right), and main image represents higher-magnification view of area depicted by red box. (D) Quantification of H33342 uptake, EB leakage, and pimonidazole adduct formation (mean ± SEM). (E) Fluorescence microscopy for H33342 staining in irradiated and control flaps at day 180 after RT. (F) Fluorescence microscopy for EB staining. (G) Immunofluorescent staining for pimonidazole adduct formation in irradiated and control flaps at 180 days after RT, with H&E staining of images depicting perivascular fibrosis (black arrow; all images representative of n = 3 animals per group). (E to G) Scale bars, 20 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 SOD2 overexpression preserves ROS scavenging capacity after RT.

    (A) SOD2 activity in rat fibroblasts (RF) at 2, 6, and 24 hours after irradiation with 0, 8, or 16 Gray (Gy) of radiation. (B) After irradiation changes in SOD2 activity in RF cells overexpressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls. (C) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in YPEN1 and YPEN1_SOD2 cells at 120 hours after RT across a variety of biologically equivalent fractionation schedules. (D) Three-dimensional spheroid assays using YPEN1 and YPEN1_SOD2 after RT across a range of biologically equivalent fractionation schedules. Graph shows mean relative spheroid area (± SEM) and representative spheroid images (n = 3 repeats). (E) MTT assay (120 hours) investigating the effect of transiently silencing SOD2 expression with small interfering RNA (siRNA) in cells overexpressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs overexpressing SOD2 were irradiated and evaluated for survival. (F) Confirmation of SOD2 knockdown using siRNA by RT-qPCR. (G) Clonogenic assays (bottom) and quantification (top, shown as mean surviving fraction ± SEM) for HeLa and HeLa-LVSOD2. (H) Clonogenic assays (bottom) and quantification (top, shown as mean surviving fraction ± SEM) for FaDu and FaDu-LVSOD2 at different radiation doses. Images are representative of n = 3 repeats. (I) Confocal IF microscopy of RF and RFSOD2 (MTCO1, anti-cytochrome C oxidase antibody; SOD2, anti-SOD2 antibody; scale bars, 10 μm; images representative of n = 3 repeats). (J) Biochemical SOD2 activity (mean ± SEM) in mitochondrial and cytosolic compartments of YPEN1 and YPEN1_SOD2 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 4 Lentiviral transgene expression penetrates tissue and provides durable effects in vivo.

    (A) Immunohistochemical staining for SOD2 protein expression in SIEA of flaps infected with LVSOD2 [108 transducing units (TUs)] compared with sham [phosphate-buffered saline (PBS)] controls at 180 days after RT. Representative images of n = 3 animals per group are shown. (B) Biochemical assessment of SOD2 activity in flaps infected with LVSOD2 and controls (mean ± SEM) (n = 3 per group). (C) ELISA for CTGF expression in flaps infected with LVSOD2 or LVSOD2 + LVshCTGF (n = 3 per group) compared to controls (mean ± SEM). (D) RT-qPCR for Sod2 gene expression and biochemical SOD2 activity (mean ± SEM) in flap pedicles (artery and vein only) infected with LVSOD2 and controls (n = 3 per group). (E) Immunofluorescent (IF) staining for green fluorescent protein (GFP) expression at 6 months in flaps infected with LVeGFP (108 TUs) and PBS-sham controls (PBS), depicting macrovascular (SIEA and superficial inferior epigastric artery expression; L, lumen; scale bars, 50 μm) and microvascular expression (CD31 colocalization with GFP; scale bars, 25 μm; images representative of three animals per group). Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI). (F) IF staining for extravascular GFP expression (FABP4 colocalization with GFP; scale bar, 50 μm) in flaps infected with LVeGFP (108 TUs; images representative of three animals per group). Nuclei counterstained with DAPI. (G) qPCR for viral gag gene expression in flap tissues infected with LVeGFP at 6 months after infection (n = 3 animals per group). *P < 0.05, **P < 0.01.

  • Fig. 5 LVSOD2 and LVshCTGF therapy reduces volume loss and skin contracture after RT.

    (A) Phenotypic appearance of irradiated (50 Gy/3 fractions) SIEA flaps (dashed white line, skin paddle outline) at 180 days after RT (scale bars, 10 mm). Note the appearance of LAEs such as telangiectasia in adjacent tissues into which neither LVSOD2 nor LVshCTGF were delivered (red arrows; images representative of n = 6 animals per group). (B) Quantification of the relative skin paddle surface area changes (mean ± 95% CI). (C) Comparison of relative skin paddle surface area means at day 180 after RT (mean ± SEM). (D) RTOG scores for acute toxicities after RT in flaps infected with LVSOD2 and/or LVshCTGF compared to PBS sham and LVsh-scram controls (mean ± SEM). (E) RTOG severity scoring for LAEs in irradiated flaps (left) and RTOG score component breakdown (right) for flaps infected with LVSOD2 and/or LVshCTGF compared to PBS sham and LVsh-scram controls (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Combination therapy with LVSOD2 and LVshCTGF preserves flap volume and reduces fibrosis after RT.

    (A) T2-weighted in vivo MRI of irradiated flaps (red arrows) at 180 days after RT (images representative of n = 3 animals per group). (B) MRI-derived volumetric analysis (mean ± SEM) comparing volume changes between groups (n = 3 animals per group). (C) Relative transverse relaxation rate (R2*) differences between groups at 180 days after RT (mean ± SEM) (n = 3 animals per group). (D) Western blot for SOD2 in irradiated and control flap tissues taken from different animals at 180 days after RT with 50 Gy/3 fractions (β-actin loading control). (E) Masson’s trichrome staining for collagen deposition in flap tissues taken at 180 days after RT from each therapeutic group. Images are representative of the larger cohort (n = 6), and whole sections are presented inset (top left). Scale bars, 100 μm. (F) Quantification of Masson’s trichrome staining shown in (E) expressed as total percentage of section exhibiting fibrotic change (mean ± SEM) and correlative RT-qPCR for Col1a2 gene expression in flap tissues 180 days after RT. (G) Multiplexed IF staining for GFP and red fluorescent protein (RFP) expression 180 days after RT in flaps infected with LVSOD2-RFP and LVshCTGF-GFP (both at 108 TUs) or negative controls (PBS) (scale bars, 100 μm). GFP and RFP colocalization is observed within vessel walls (L, lumen). Nuclei are counterstained with DAPI. Cells demonstrating either GFP expression alone (white arrows) or RFP expression alone (red arrows) are also highlighted (images representative of n = 6 animals per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 7 LVSOD2 acts through the preservation of microvascular function.

    (A) Immunofluorescent imaging of perfused vasculature (H33342 and EB) and hypoxia [pimonidazole (P)] in flap tissues on day 180 after RT. Images are presented as merged composites (whole section in the upper panel and hpf of area depicted by white box shown in the middle panel) and split channels (lower panel) (scale bars, 100 μm; images representative of n = 6 animals per group). (B to D) Thresholded imaging analysis of IF staining for H33342 (B), EB (C), and P (D) showing percentage of section stained (mean ± SEM) (n = 6 animals per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 8 SOD2 overexpression in normal tissues does not compromise the cytotoxic efficacy of RT.

    (A) Conditioned medium experiment to assess transmissibility of SOD2 overexpression after exposure of target cells (HeLa) to supernatant (HeLa-SOD2 supernatant) taken from producer cells (HeLa_SOD2) (blot representative of n = 3 biological repeats). (B) Schematic of the in vivo tumor recurrence model showing tumor (red arrow) growing in an SIEA flap (paddle outline dashed white). Animals with tumors greater than 2 cm in diameter were deemed to have exceeded the experimental severity limit and euthanized. (C) Tumor volume growth (mean ± SEM) for Mat B III tumors after RT in flaps infected with LVSOD2, LVeGP, or sham (PBS) (n = 5 animals per group). (D) Individual growth curves for tumors growing in sham (PBS)–infected and unirradiated, sham (PBS)–infected and irradiated (20 Gy/5 fractions), LVSOD-infected and irradiated (20 Gy/5 fractions), or LVeGFP-infected and irradiated (20 Gy/5 fractions) flaps (n = 5 animals per group). (E) Kaplan-Meier plot of survival to severity limit (2- cm tumor diameter) for animals with Mat B III tumors grown in sham (PBS)–, LVSOD2-, or LVeGFP-infected flaps. Median survival is shown at the bottom right. n/a, not applicable.

Supplementary Materials

  • Supplementary Material for:

    Genetically modified lentiviruses that preserve microvascular function protect against late radiation damage in normal tissues

    Aadil A. Khan, James T. Paget, Martin McLaughlin, Joan N. Kyula, Michelle J. Wilkinson, Timothy Pencavel, David Mansfield, Victoria Roulstone, Rohit Seth, Martin Halle, Navita Somaiah, Jessica K. R. Boult, Simon P. Robinson, Hardev S. Pandha, Richard G. Vile, Alan A. Melcher, Paul A. Harris, Kevin J. Harrington*

    *Corresponding author. Email: kevin.harrington{at}icr.ac.uk

    Published 24 January 2018, Sci. Transl. Med. 10, eaar2041 (2018)
    DOI: 10.1126/scitranslmed.aar2041

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Development and validation of a model of free flap LAEs.
    • Fig. S2. Validation of SOD2 overexpression using LVSOD2 and CTGF knockdown using LVshCTGF.
    • Table S1. Antibodies used, dilutions, and suppliers.
    • References (6971)

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