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

Abstract

Improvements in cancer survival mean that long-term toxicities, which contribute to the morbidity of cancer survivorship, are being increasingly recognized. Late adverse effects (LAEs) in normal tissues after radiotherapy (RT) are characterized by vascular dysfunction and fibrosis causing volume loss and tissue contracture, for example, in the free flaps used for immediate breast reconstruction after mastectomy. We evaluated the efficacy of lentivirally delivered superoxide dismutase 2 (SOD2) overexpression and connective tissue growth factor (CTGF) knockdown by short hairpin RNA in reducing the severity of LAEs in an animal model of free flap LAEs. Vectors were delivered by intra-arterial injection, ex vivo, to target the vascular compartment. LVSOD2 and LVshCTGF monotherapy before irradiation resulted in preservation of flap volume or reduction in skin contracture, respectively. Flaps transduced with combination therapy experienced improvements in both volume loss and skin contracture. Both therapies reduced the fibrotic burden after irradiation. LAEs were associated with impaired vascular perfusion, loss of endothelial permeability, and stromal hypoxia, which were all reversed in the treatment model. Using a tumor recurrence model, we showed that SOD2 overexpression in normal tissues did not compromise the efficacy of RT against tumor cells but appeared to enhance it. LVSOD2 and LVshCTGF combination therapy by targeted, intravascular delivery reduced LAE severities in normal tissues without compromising the efficacy of RT and warrants translational evaluation as a free flap-targeted gene therapy.

Figure 1. Irradiation with 50 Gy/3 fractions generates a LAE phenotype characterized by SOD2 depletion and CTGF over-expression.

A) Representative photographs of bilateral SIEA flaps (edges tattooed in Indian ink) inset onto the hind limbs of Fischer (F344) male rats taken at 180 days after irradiation. The left limb flaps (ii and iv) were irradiated with 50 Gy/3 fractions on consecutive days whereas flaps on the contralateral limb did not undergo irradiation. Flap skin paddles were not significantly different prior to irradiation. Characteristic LAE features were observed in irradiated flaps including contracture, induration of the skin, telangiectasia and hair loss (un-irradiated flaps have been shaved to expose marked edges, whereas irradiated flaps have not) (supplementary figure 1). B) Changes in skin paddle surface area of irradiated and control SIEA flaps demonstrating significant losses in absolute (i) and relative (ii) skin paddle surface area (± 95% CI). Irradiated flaps were observed to lose up to 70% of their pre-irradiation skin paddle surface area (p<0.0001). C) Acute and late RTOG scores. i) Acute RTOG scores (mean ± SEM) demonstrating the duration and severity of acute toxicities observed in flaps irradiated with 50 Gy/3 fx. ii) Late RTOG scores (mean ± SEM) in flaps irradiated with 50 Gy/3 fx demonstrating progressive severity of LAEs in irradiated flaps. iii) Component scores for the RTOG late effects scores demonstrating that LAEs were first evident in the skin followed by the subcutaneous tissues and joint. iv) Passive ROM at the knee joint in irradiated and non-irradiated hind limbs demonstrating a significant reduction in knee joint excursion in irradiated hind limbs compared to controls. [*** p < 0.0001]. D) i) T₂-weighted MR image of bilateral SIEA flaps showing an irradiated flap (left) and un-irradiated control (right) at 180 days after irradiation (flaps outlined in green). Note the apparent shrinkage in flap cross-sectional area in the irradiated flap compared to control. ii) Mean MRI-derived absolute flap volume (± 95% CI) showing significant flap volume loss in irradiated flaps. iii) Relative MRI-derived flap volume changes (normalized to pre-irradiation volumes) (± 95% CI) demonstrating that irradiated flaps lose up to 50% of their pre-irradiation volume by 6 months. [* p < 0.05, *** p < 0.0001]. E) i) CTGF ELISA on flap tissues taken from both irradiated and control flaps (180 days post-RT) demonstrating an increase in CTGF protein concentration in irradiated flaps. ii) SOD2 activity measurement by biochemical assay demonstrating a significant reduction in SOD2 activity in irradiated flaps (52% decrease) compared to controls. [** p < 0.01]. F) Western blotting of matched control and irradiated SIEA flaps (at 180 days post-RT) showing reductions in SOD2 protein expression (upper panel) and activation of Wnt-signalling through phosphorylation of GSK-3β. G) Immunohistochemical analysis of irradiated flaps (180 days post-RT) with Masson’s trichrome (i-iii), CTGF (iv-vi) and fat necrosis (vii-ix) demonstrating significant increases in collagen deposition (i and ii; green staining), CTGF (iv and v) and fat necrosis (vii and viii; red dashed circle). Graphs represent mean ± SEM of counts per high powere field (hpf) or % section exhibiting fat necrosis. H) RT-QPCR for CTGF, Col1a2 and Col3a1 gene expression (mean fold increase in gene expression ± SEM) (flaps harvested at 180 days post-RT) demonstrating significant reductions in CTGF and Col3a1 expression but significant increases in Col1a2 gene expression [*p<0.05, **p<0.01, **** p < 0.0001].

Figure 1. Irradiation with 50 Gy/3 fractions generates a LAE phenotype characterized by SOD2 depletion and CTGF over-expression.

A) Representative photographs of bilateral SIEA flaps (edges tattooed in Indian ink) inset onto the hind limbs of Fischer (F344) male rats taken at 180 days after irradiation. The left limb flaps (ii and iv) were irradiated with 50 Gy/3 fractions on consecutive days whereas flaps on the contralateral limb did not undergo irradiation. Flap skin paddles were not significantly different prior to irradiation. Characteristic LAE features were observed in irradiated flaps including contracture, induration of the skin, telangiectasia and hair loss (un-irradiated flaps have been shaved to expose marked edges, whereas irradiated flaps have not) (supplementary figure 1). B) Changes in skin paddle surface area of irradiated and control SIEA flaps demonstrating significant losses in absolute (i) and relative (ii) skin paddle surface area (± 95% CI). Irradiated flaps were observed to lose up to 70% of their pre-irradiation skin paddle surface area (p<0.0001). C) Acute and late RTOG scores. i) Acute RTOG scores (mean ± SEM) demonstrating the duration and severity of acute toxicities observed in flaps irradiated with 50 Gy/3 fx. ii) Late RTOG scores (mean ± SEM) in flaps irradiated with 50 Gy/3 fx demonstrating progressive severity of LAEs in irradiated flaps. iii) Component scores for the RTOG late effects scores demonstrating that LAEs were first evident in the skin followed by the subcutaneous tissues and joint. iv) Passive ROM at the knee joint in irradiated and non-irradiated hind limbs demonstrating a significant reduction in knee joint excursion in irradiated hind limbs compared to controls. [*** p < 0.0001]. D) i) T₂-weighted MR image of bilateral SIEA flaps showing an irradiated flap (left) and un-irradiated control (right) at 180 days after irradiation (flaps outlined in green). Note the apparent shrinkage in flap cross-sectional area in the irradiated flap compared to control. ii) Mean MRI-derived absolute flap volume (± 95% CI) showing significant flap volume loss in irradiated flaps. iii) Relative MRI-derived flap volume changes (normalized to pre-irradiation volumes) (± 95% CI) demonstrating that irradiated flaps lose up to 50% of their pre-irradiation volume by 6 months. [* p < 0.05, *** p < 0.0001]. E) i) CTGF ELISA on flap tissues taken from both irradiated and control flaps (180 days post-RT) demonstrating an increase in CTGF protein concentration in irradiated flaps. ii) SOD2 activity measurement by biochemical assay demonstrating a significant reduction in SOD2 activity in irradiated flaps (52% decrease) compared to controls. [** p < 0.01]. F) Western blotting of matched control and irradiated SIEA flaps (at 180 days post-RT) showing reductions in SOD2 protein expression (upper panel) and activation of Wnt-signalling through phosphorylation of GSK-3β. G) Immunohistochemical analysis of irradiated flaps (180 days post-RT) with Masson’s trichrome (i-iii), CTGF (iv-vi) and fat necrosis (vii-ix) demonstrating significant increases in collagen deposition (i and ii; green staining), CTGF (iv and v) and fat necrosis (vii and viii; red dashed circle). Graphs represent mean ± SEM of counts per high powere field (hpf) or % section exhibiting fat necrosis. H) RT-QPCR for CTGF, Col1a2 and Col3a1 gene expression (mean fold increase in gene expression ± SEM) (flaps harvested at 180 days post-RT) demonstrating significant reductions in CTGF and Col3a1 expression but significant increases in Col1a2 gene expression [*p<0.05, **p<0.01, **** p < 0.0001].

Figure 2. LAEs are characterized by vascular dysfunction, loss of endothelial perfusion and permeability and peri-vascular hypoxia.

A) Parametric R₂* maps of control and irradiated flaps overlaid on T₂-weighted images acquired 6 months following irradiation. B) i) Absolute changes in R₂* (mean ± 1 SEM) demonstrating that, in irradiated flaps, basal R₂* was reduced significantly from 1 month onwards. Asterisks represent comparisons made with the pre-irradiation time point for each group separately. ii) Relative changes in R₂* (mean ± 1 SEM) demonstrating that irradiated flaps exhibited greater reductions in relative R₂* compared to control flaps at all post-irradiation time points. [ns = not significant, * p < 0.05, **** p < 0.0001]. C) Hoechst 33342 uptake, Evans blue leakage and pimonidazole adduct formation in control (i) and irradiated (ii) flaps (H&E and composite scan (x 10 magnification) of entire section inset with red box representing zoomed micrograph) demonstrating a significant reduction in Hoechst 33342 and Evans blue fluorescence and an increase in pimonidazole adduct immunofluorescence. D) Quantification of Hoechst 33342 uptake, Evans blue leakage and pimonidazole adduct formation. Bars represent mean percentage of section exhibiting staining (±SEM). Images are representative of the larger cohort (n = 3 animals with bilateral flaps). Scale bars (bottom right) represent 100 μm. [*** p < 0.001, **** p < 0.0001]. E, F and G) Post-radiation changes in microvascular function demonstrating reductions in Hoechst 33342 uptake (E), Evans blue fluorescence (F) and an increase in pimonidazole adduct immunofluorescence (G) (with H&E of section inset) in control and irradiated flaps with 50 Gy/3 fx. Note perivascular fibrosis around irradiated vessels (G.iii and vi; black arrow) and correlation with pimonidazole staining (G.ii and v; white arrow). Images are representative of the larger cohort. Scale bar (bottom right) is equal to 20 μm.

Figure 2. LAEs are characterized by vascular dysfunction, loss of endothelial perfusion and permeability and peri-vascular hypoxia.

A) Parametric R₂* maps of control and irradiated flaps overlaid on T₂-weighted images acquired 6 months following irradiation. B) i) Absolute changes in R₂* (mean ± 1 SEM) demonstrating that, in irradiated flaps, basal R₂* was reduced significantly from 1 month onwards. Asterisks represent comparisons made with the pre-irradiation time point for each group separately. ii) Relative changes in R₂* (mean ± 1 SEM) demonstrating that irradiated flaps exhibited greater reductions in relative R₂* compared to control flaps at all post-irradiation time points. [ns = not significant, * p < 0.05, **** p < 0.0001]. C) Hoechst 33342 uptake, Evans blue leakage and pimonidazole adduct formation in control (i) and irradiated (ii) flaps (H&E and composite scan (x 10 magnification) of entire section inset with red box representing zoomed micrograph) demonstrating a significant reduction in Hoechst 33342 and Evans blue fluorescence and an increase in pimonidazole adduct immunofluorescence. D) Quantification of Hoechst 33342 uptake, Evans blue leakage and pimonidazole adduct formation. Bars represent mean percentage of section exhibiting staining (±SEM). Images are representative of the larger cohort (n = 3 animals with bilateral flaps). Scale bars (bottom right) represent 100 μm. [*** p < 0.001, **** p < 0.0001]. E, F and G) Post-radiation changes in microvascular function demonstrating reductions in Hoechst 33342 uptake (E), Evans blue fluorescence (F) and an increase in pimonidazole adduct immunofluorescence (G) (with H&E of section inset) in control and irradiated flaps with 50 Gy/3 fx. Note perivascular fibrosis around irradiated vessels (G.iii and vi; black arrow) and correlation with pimonidazole staining (G.ii and v; white arrow). Images are representative of the larger cohort. Scale bar (bottom right) is equal to 20 μm.

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 3. Mitochondrial SOD2 over-expression in normal, but not tumour cells, mediates radioprotection across a range of fractionation schedules and durable SOD2 over-expression can be achieved in vivo.

A) Graph of SOD2 activity (mean ± SEM) in rat fibroblasts (RF) at 2, 6 and 24 hours after irradiation with 0, 8 or 16 Gy of radiation. The graph shows a dose-dependent reduction in SOD2 activity that occurs earlier with higher radiotherapy doses. B) Post-irradiation changes in SOD2 activity in RF cells over-expressing SOD2 (RF-LVSOD2) compared to vector (RF-LVGFP) and naïve (RF) controls demonstrating significant preservation of SOD2 activity in the RF-LVSOD2 cell line compared to controls. C) MTT assays performed using endothelial cells (YPEN1) and endothelial cells over-expressing SOD2 (YPEN1 SOD2) at 120 hours post-irradiation demonstrating significantly greater cell survival in the presence of SOD2 over-expression across a variety of biologically equivalent fractionation schedules. D) Three-dimensional spheroid assays using YPEN1 and YPEN1 SOD2 following irradiation across a range of biologically-equivalent fractionation schedules demonstrating significantly greater spheroid volume preservation after RT in cells over-expressing SOD2. E) MTT assay (120 hours) investigating the effect of silencing transiently SOD2 expression using siRNA in cells over-expressing SOD2. Control (YPEN1) endothelial cells (ECs) and ECs over-expressing SOD2 were irradiated and demonstrated a survival benefit in cells over-expressing SOD2, which was mitigated by the addition of SOD2 siRNA but not the addition of a scrambled siRNA control. F) Confirmation of SOD2 knock-down using siRNA by RT-QPCR demonstrating that SOD2 gene expression was significantly reduced (to almost 10% of basal levels). G) i) Quantification of clonogenic assays (mean SF ± SEM) of HeLa and HeLa-LVSOD2 demonstrating a significant increase in SF in HeLa-LVSOD2 at 2 Gy. The trend at 4 and 6 Gy (ii) is suggestive of improved survival in HeLa-LVSOD2. H) i) Quantification of clonogenic assays comparing FaDu and FaDu-LVSOD2 (mean SF ± SEM). This graph shows a trend suggestive of greater survival in FaDu-LVSOD2 at all radiation doses (ii). I) i and ii) Confocal immunofluorescent microscopy of control and SOD2 over-expressing fibroblasts (RF) using an anti-cytochrome C oxidase antibody (MTCO1) and anti-SOD2 primary antibody demonstrating visible over-expression of SOD2 in the RFSOD2 cells (ii) and co-localization of SOD2 with MTCO1. Cell lysates were collected and split into mitochondrial and cytosolic lysates. Biochemical SOD2 activity was found to be increased significantly in whole cell and mitochondrial lysates of RFSOD2 cells compared to RF controls but this difference did not reach statistical significance for the cytosolic compartment. J) Immunohistochemical staining for SOD2 protein expression in superficial inferior epigastric arteries of flaps infected with LVSOD2 (10⁸ TUs) (i) compared with sham (PBS) (ii) controls demonstrating greater protein expression within the vascular compartment. These increases were associated with increased SOD2 activity (iii) and equated to a 50% increase in basal SOD2 activity in flap tissues. SIEA flaps infected with LVSOD2 did not have significantly lower levels of CTGF protein expression (iv) but those infected with both LVSOD2 (10⁸ TUs) and LVshCTGF (10⁸ TUs) exhibited a significant reduction in CTGF concentration (relative 40% decrease). K and L) The flap pedicle, containing only artery and vein, was dissected from the flaps (sham and 10⁸ TUs of LVSOD2) and RT-QPCR was performed using pedicle RNA (K) to demonstrate significant over-expression of the SOD2 gene in vascular tissues of flaps infected with LVSOD2. This was also associated with significantly increased SOD2 biochemical activity (L) in flaps infected with LVSOD2. M) Immunofluorescent staining for GFP demonstrating vascular transgene expression at 6 months post-infection with LVeGFP in the SIEA (x 40 (i) and x100 (ii)), SIEV (iii) alongside a negative experimental control (PBS sham infection) (iv). GFP expression was also observed in the microvasculature as demonstrated by co-localization of GFP (green) (v and vi; x100). Nuclei are counter-stained with DAPI (blue). Extra-vascular GFP expression was also observed in the stromal compartment (adipocytes) of the flap as demonstrated by co-localization with fatty acid binding protein 4 (FABP4) (vii (x40) and viii (x100)) at 6 months post-infection (red square represents the area of higher magnification in vii). N) Q-PCR for the quantification of viral copy number in SIEA flaps at 6 months post-infection showing that flap infection with 10⁸ TUs of LVeGFP results in an approximate 10,000-fold increase in viral copy number compared to un-infected flap tissues. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 4. LVSOD2 and LVshCTGF therapy reduce volume loss and skin contracture following RT.

A) Phenotypic appearance of irradiated (50 Gy/3 fractions) SIEA flaps at 180 days following irradiation. Significant improvements in the LAE phenotype were observed in flaps infected with LVSOD2 and LVshCTGF either alone, or, in combination (i-v). Note the appearance of LAEs such as telangiectasia in adjacent tissues into which neither LVSOD2 nor LVshCTGF were delivered (iii red arrows). Quantification of the skin paddle changes (mean skin paddle surface area ± 95% CI) demonstrated significant differences between therapy groups (vi and vii) at 180 days post-irradiation. Specifically, flaps infected with LVSOD2 or LVshCTGF as single agents exhibited significantly less skin paddle contracture than sham (PBS) or vector control (LVscram) infected flaps. LVshCTGF monotherapy showed a trend towards greater skin paddle preservation compared to LVSOD2 alone however this did not reach statistical significance. Combination therapy with LVSOD2 plus LVshCTGF achieved the greatest improvements in skin paddle contracture with irradiated flaps losing approximately 15% of their pre-irradiation surface area, compared to controls that lost approximately 70%. B) RTOG scores for acute toxicities following irradiation demonstrating that flaps infected with LVSOD2 (either alone or in combination) experienced a shorter duration of acute toxicities that resolved sooner (i and iii), however, the maximum severity of acute toxicities was no different between groups. C) RTOG severity scoring for LAEs in irradiated flaps (I, iii, v and vii) and RTOG score component breakdown (ii, iv, vi, viii) showing that flaps receiving LVSOD2 monotherapy attained significantly lower RTOG scores for subcutaneous tissue effects (ii). Flaps infected with LVshCTGF achieved lower RTOG scores for cutaneous effects (iv) in the early and intermediate term but these did not persist to the end of the experiment. Flaps receiving dual therapy achieved sustained, lower severity scores for both cutaneous and subcutaneous effects (vi). Of note, no therapeutic group experienced a reduction in severity scores for joint-related effects suggesting that radioprotective effects do not leech out of the flap to its bed. D) T₂-weighted in vivo MRI of irradiated flaps (red arrows) at 180 days following the cessation of radiotherapy showing that flaps infected with LVSOD2 retained significantly more subcutaneous volume than other therapeutic groups. MRI-derived volumetric analysis (vi) revealed that flaps infected with LVSOD2, either alone or in combination, retained up to 80% of their pre-irradiation volumes, and were also seen to experience significant improvements in relative basal R₂* and were not statistically different from un-irradiated flaps (vii). [*p<0.05; **p<0.01; ***p<0.001].

Fig. 4. LVSOD2 and LVshCTGF therapy reduce volume loss and skin contracture following RT.

A) Phenotypic appearance of irradiated (50 Gy/3 fractions) SIEA flaps at 180 days following irradiation. Significant improvements in the LAE phenotype were observed in flaps infected with LVSOD2 and LVshCTGF either alone, or, in combination (i-v). Note the appearance of LAEs such as telangiectasia in adjacent tissues into which neither LVSOD2 nor LVshCTGF were delivered (iii red arrows). Quantification of the skin paddle changes (mean skin paddle surface area ± 95% CI) demonstrated significant differences between therapy groups (vi and vii) at 180 days post-irradiation. Specifically, flaps infected with LVSOD2 or LVshCTGF as single agents exhibited significantly less skin paddle contracture than sham (PBS) or vector control (LVscram) infected flaps. LVshCTGF monotherapy showed a trend towards greater skin paddle preservation compared to LVSOD2 alone however this did not reach statistical significance. Combination therapy with LVSOD2 plus LVshCTGF achieved the greatest improvements in skin paddle contracture with irradiated flaps losing approximately 15% of their pre-irradiation surface area, compared to controls that lost approximately 70%. B) RTOG scores for acute toxicities following irradiation demonstrating that flaps infected with LVSOD2 (either alone or in combination) experienced a shorter duration of acute toxicities that resolved sooner (i and iii), however, the maximum severity of acute toxicities was no different between groups. C) RTOG severity scoring for LAEs in irradiated flaps (I, iii, v and vii) and RTOG score component breakdown (ii, iv, vi, viii) showing that flaps receiving LVSOD2 monotherapy attained significantly lower RTOG scores for subcutaneous tissue effects (ii). Flaps infected with LVshCTGF achieved lower RTOG scores for cutaneous effects (iv) in the early and intermediate term but these did not persist to the end of the experiment. Flaps receiving dual therapy achieved sustained, lower severity scores for both cutaneous and subcutaneous effects (vi). Of note, no therapeutic group experienced a reduction in severity scores for joint-related effects suggesting that radioprotective effects do not leech out of the flap to its bed. D) T₂-weighted in vivo MRI of irradiated flaps (red arrows) at 180 days following the cessation of radiotherapy showing that flaps infected with LVSOD2 retained significantly more subcutaneous volume than other therapeutic groups. MRI-derived volumetric analysis (vi) revealed that flaps infected with LVSOD2, either alone or in combination, retained up to 80% of their pre-irradiation volumes, and were also seen to experience significant improvements in relative basal R₂* and were not statistically different from un-irradiated flaps (vii). [*p<0.05; **p<0.01; ***p<0.001].

Fig. 4. LVSOD2 and LVshCTGF therapy reduce volume loss and skin contracture following RT.

A) Phenotypic appearance of irradiated (50 Gy/3 fractions) SIEA flaps at 180 days following irradiation. Significant improvements in the LAE phenotype were observed in flaps infected with LVSOD2 and LVshCTGF either alone, or, in combination (i-v). Note the appearance of LAEs such as telangiectasia in adjacent tissues into which neither LVSOD2 nor LVshCTGF were delivered (iii red arrows). Quantification of the skin paddle changes (mean skin paddle surface area ± 95% CI) demonstrated significant differences between therapy groups (vi and vii) at 180 days post-irradiation. Specifically, flaps infected with LVSOD2 or LVshCTGF as single agents exhibited significantly less skin paddle contracture than sham (PBS) or vector control (LVscram) infected flaps. LVshCTGF monotherapy showed a trend towards greater skin paddle preservation compared to LVSOD2 alone however this did not reach statistical significance. Combination therapy with LVSOD2 plus LVshCTGF achieved the greatest improvements in skin paddle contracture with irradiated flaps losing approximately 15% of their pre-irradiation surface area, compared to controls that lost approximately 70%. B) RTOG scores for acute toxicities following irradiation demonstrating that flaps infected with LVSOD2 (either alone or in combination) experienced a shorter duration of acute toxicities that resolved sooner (i and iii), however, the maximum severity of acute toxicities was no different between groups. C) RTOG severity scoring for LAEs in irradiated flaps (I, iii, v and vii) and RTOG score component breakdown (ii, iv, vi, viii) showing that flaps receiving LVSOD2 monotherapy attained significantly lower RTOG scores for subcutaneous tissue effects (ii). Flaps infected with LVshCTGF achieved lower RTOG scores for cutaneous effects (iv) in the early and intermediate term but these did not persist to the end of the experiment. Flaps receiving dual therapy achieved sustained, lower severity scores for both cutaneous and subcutaneous effects (vi). Of note, no therapeutic group experienced a reduction in severity scores for joint-related effects suggesting that radioprotective effects do not leech out of the flap to its bed. D) T₂-weighted in vivo MRI of irradiated flaps (red arrows) at 180 days following the cessation of radiotherapy showing that flaps infected with LVSOD2 retained significantly more subcutaneous volume than other therapeutic groups. MRI-derived volumetric analysis (vi) revealed that flaps infected with LVSOD2, either alone or in combination, retained up to 80% of their pre-irradiation volumes, and were also seen to experience significant improvements in relative basal R₂* and were not statistically different from un-irradiated flaps (vii). [*p<0.05; **p<0.01; ***p<0.001].

Fig. 5. Combination therapy with LVSOD2 and LVshCTGF reduces fibrosis post-RT but the preservation of microvascular function is attributable to LVSOD2 only.

A) Western blotting for SOD2 in flap tissues taken at 180 days after irradiation with 50 Gy/3 fractions. These data show that the observed reductions in SOD2 expression after RT seen in control groups (PBS and LVscram) are mitigated by the delivery of LVSOD2. Of note, LVshCTGF therapy alone does not appear to impact post-irradiation changes in SOD2 expression. B) Masson’s trichrome staining for the quantification of fibrosis in flap tissues taken at 180 days after irradiation from each therapeutic group (i-vi). Photos are representative of the larger cohort and whole sections are presented inset (top left). Reductions in collagen deposition (green) were observed in both flaps infected with LVSOD2 and LVshCTGF as monotherapies (iii and iv). Combination therapy with LVSOD2 plus LVshCTGF yielded further reductions in collagen deposition compared to controls (i and vi). C) i) Quantification of Masson’s trichrome staining showing significant differences in collagen deposition between groups. Specifically, flaps infected with LVSOD2 and LVshCTGF as monotherapies exhibited significantly less fibrosis than controls (p<0.01) whereas flaps infected with both vectors exhibited further significant reductions in collagen deposition (p<0.05). Correlative RT-QPCR for Col1a2 gene expression in flap tissues at 180 days post-irradiation demonstrated muting of expression in flaps infected with LVSOD2 (either alone or in combination). LVshCTGF monotherapy resulted in greater Col1a2 expression compared to sham (PBS) and vector (LVscram) controls (p<0.05) but this did not result in greater collagen deposition (figure 6.b.iv). D) Immunofluorescent imaging of functional vasculature (Hoechst 33342 (H33342) and Evans blue (EB)) and hypoxia (pimonidazole (P)) in flap tissues taken at 180 days following the end of radiotherapy (i-vi). Images are presented as merged composites (whole section upper panel and x40 middle panel) and split channels (lower panel). These data show differences in H33342, EB and P across therapeutic groups. Specifically, flaps infected with LVSOD2 (either alone or in combination) have preservation of H33342 (viable endothelium) and EB (vascular permeability) and a commensurate reduction in P (hypoxia) staining (iii and v). E) Thresholded imaging analysis of immunofluorescent staining for H33342, EB and P demonstrating statistically significant changes. Flaps infected with LVSOD2 (either alone or in combination) showed significant improvements in perfused vasculature (H33342) (i), vascular permeability (EB) (ii) and a reduction in hypoxia (P) (iii). Flaps infected with LVshCTGF showed reductions in perfused vasculature (H33342) (i) and greater hypoxia (P) compared to flaps receiving LVSOD2. However, LVshCTGF flaps showed modest, but significant, improvements in vascular permeability (EB) (ii) compared to sham (PBS) and vector (LVscram) controls. However, these improvements remained significantly less than those seen with LVSOD2 therapy. F) Multiplexed immunofluorescent staining for GFP and RFP expression at 180 days post-irradiation in flaps that have been infected with LVSOD2-RFP and LVshCTGF-GFP (10⁸ TUs each) and irradiated with 50 Gy/3 fractions. Images are shown alongside negative controls at x20 (i) and x40 (iii) magnification Images show co-localization of GFP and RFP expression (ii and iv), particularly within vessel walls (L = lumen) but also in the extra-vascular compartment. Cells demonstrating GFP expression alone (white arrows) or RFP expression alone (red arrows) are also observed. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 5. Combination therapy with LVSOD2 and LVshCTGF reduces fibrosis post-RT but the preservation of microvascular function is attributable to LVSOD2 only.

A) Western blotting for SOD2 in flap tissues taken at 180 days after irradiation with 50 Gy/3 fractions. These data show that the observed reductions in SOD2 expression after RT seen in control groups (PBS and LVscram) are mitigated by the delivery of LVSOD2. Of note, LVshCTGF therapy alone does not appear to impact post-irradiation changes in SOD2 expression. B) Masson’s trichrome staining for the quantification of fibrosis in flap tissues taken at 180 days after irradiation from each therapeutic group (i-vi). Photos are representative of the larger cohort and whole sections are presented inset (top left). Reductions in collagen deposition (green) were observed in both flaps infected with LVSOD2 and LVshCTGF as monotherapies (iii and iv). Combination therapy with LVSOD2 plus LVshCTGF yielded further reductions in collagen deposition compared to controls (i and vi). C) i) Quantification of Masson’s trichrome staining showing significant differences in collagen deposition between groups. Specifically, flaps infected with LVSOD2 and LVshCTGF as monotherapies exhibited significantly less fibrosis than controls (p<0.01) whereas flaps infected with both vectors exhibited further significant reductions in collagen deposition (p<0.05). Correlative RT-QPCR for Col1a2 gene expression in flap tissues at 180 days post-irradiation demonstrated muting of expression in flaps infected with LVSOD2 (either alone or in combination). LVshCTGF monotherapy resulted in greater Col1a2 expression compared to sham (PBS) and vector (LVscram) controls (p<0.05) but this did not result in greater collagen deposition (figure 6.b.iv). D) Immunofluorescent imaging of functional vasculature (Hoechst 33342 (H33342) and Evans blue (EB)) and hypoxia (pimonidazole (P)) in flap tissues taken at 180 days following the end of radiotherapy (i-vi). Images are presented as merged composites (whole section upper panel and x40 middle panel) and split channels (lower panel). These data show differences in H33342, EB and P across therapeutic groups. Specifically, flaps infected with LVSOD2 (either alone or in combination) have preservation of H33342 (viable endothelium) and EB (vascular permeability) and a commensurate reduction in P (hypoxia) staining (iii and v). E) Thresholded imaging analysis of immunofluorescent staining for H33342, EB and P demonstrating statistically significant changes. Flaps infected with LVSOD2 (either alone or in combination) showed significant improvements in perfused vasculature (H33342) (i), vascular permeability (EB) (ii) and a reduction in hypoxia (P) (iii). Flaps infected with LVshCTGF showed reductions in perfused vasculature (H33342) (i) and greater hypoxia (P) compared to flaps receiving LVSOD2. However, LVshCTGF flaps showed modest, but significant, improvements in vascular permeability (EB) (ii) compared to sham (PBS) and vector (LVscram) controls. However, these improvements remained significantly less than those seen with LVSOD2 therapy. F) Multiplexed immunofluorescent staining for GFP and RFP expression at 180 days post-irradiation in flaps that have been infected with LVSOD2-RFP and LVshCTGF-GFP (10⁸ TUs each) and irradiated with 50 Gy/3 fractions. Images are shown alongside negative controls at x20 (i) and x40 (iii) magnification Images show co-localization of GFP and RFP expression (ii and iv), particularly within vessel walls (L = lumen) but also in the extra-vascular compartment. Cells demonstrating GFP expression alone (white arrows) or RFP expression alone (red arrows) are also observed. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 5. Combination therapy with LVSOD2 and LVshCTGF reduces fibrosis post-RT but the preservation of microvascular function is attributable to LVSOD2 only.

A) Western blotting for SOD2 in flap tissues taken at 180 days after irradiation with 50 Gy/3 fractions. These data show that the observed reductions in SOD2 expression after RT seen in control groups (PBS and LVscram) are mitigated by the delivery of LVSOD2. Of note, LVshCTGF therapy alone does not appear to impact post-irradiation changes in SOD2 expression. B) Masson’s trichrome staining for the quantification of fibrosis in flap tissues taken at 180 days after irradiation from each therapeutic group (i-vi). Photos are representative of the larger cohort and whole sections are presented inset (top left). Reductions in collagen deposition (green) were observed in both flaps infected with LVSOD2 and LVshCTGF as monotherapies (iii and iv). Combination therapy with LVSOD2 plus LVshCTGF yielded further reductions in collagen deposition compared to controls (i and vi). C) i) Quantification of Masson’s trichrome staining showing significant differences in collagen deposition between groups. Specifically, flaps infected with LVSOD2 and LVshCTGF as monotherapies exhibited significantly less fibrosis than controls (p<0.01) whereas flaps infected with both vectors exhibited further significant reductions in collagen deposition (p<0.05). Correlative RT-QPCR for Col1a2 gene expression in flap tissues at 180 days post-irradiation demonstrated muting of expression in flaps infected with LVSOD2 (either alone or in combination). LVshCTGF monotherapy resulted in greater Col1a2 expression compared to sham (PBS) and vector (LVscram) controls (p<0.05) but this did not result in greater collagen deposition (figure 6.b.iv). D) Immunofluorescent imaging of functional vasculature (Hoechst 33342 (H33342) and Evans blue (EB)) and hypoxia (pimonidazole (P)) in flap tissues taken at 180 days following the end of radiotherapy (i-vi). Images are presented as merged composites (whole section upper panel and x40 middle panel) and split channels (lower panel). These data show differences in H33342, EB and P across therapeutic groups. Specifically, flaps infected with LVSOD2 (either alone or in combination) have preservation of H33342 (viable endothelium) and EB (vascular permeability) and a commensurate reduction in P (hypoxia) staining (iii and v). E) Thresholded imaging analysis of immunofluorescent staining for H33342, EB and P demonstrating statistically significant changes. Flaps infected with LVSOD2 (either alone or in combination) showed significant improvements in perfused vasculature (H33342) (i), vascular permeability (EB) (ii) and a reduction in hypoxia (P) (iii). Flaps infected with LVshCTGF showed reductions in perfused vasculature (H33342) (i) and greater hypoxia (P) compared to flaps receiving LVSOD2. However, LVshCTGF flaps showed modest, but significant, improvements in vascular permeability (EB) (ii) compared to sham (PBS) and vector (LVscram) controls. However, these improvements remained significantly less than those seen with LVSOD2 therapy. F) Multiplexed immunofluorescent staining for GFP and RFP expression at 180 days post-irradiation in flaps that have been infected with LVSOD2-RFP and LVshCTGF-GFP (10⁸ TUs each) and irradiated with 50 Gy/3 fractions. Images are shown alongside negative controls at x20 (i) and x40 (iii) magnification Images show co-localization of GFP and RFP expression (ii and iv), particularly within vessel walls (L = lumen) but also in the extra-vascular compartment. Cells demonstrating GFP expression alone (white arrows) or RFP expression alone (red arrows) are also observed. [* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001].

Fig. 6. SOD2 over-expression in normal tissues does not compromise the cytotoxic efficacy of RT.

A) Western blotting for SOD2 to demonstrate that despite SOD2 over-expression (double band) in stable, producer (LVSOD2-HeLa) cells SOD2 over-expression is not seen in naïve target cells following inoculation with media taken from producer cells. B) Schematic of the in vivo tumour recurrence model using MatBIII cells (rodent breast adenocarcinoma). Animals underwent SIEA flap surgery as described previously, with the delivery of LVSOD2, LVeGFP or sham (PBS). One month later MatBIII cells were engrafted into the flap and established tumours (photo bottom right of panel showing established MatBIII tumour (red arrow) in an SIEA flap (paddle outline dashed white)) were irradiated with 20 Gy/5 fractions (n= 5 animals per group). C) Tumour volume growth for MatBIII tumours in flaps infected with LVSOD2, LVeGP or sham (PBS) demonstrating significant differences in tumour volume growth. Following irradiation with 20 Gy/5 fractions tumours growing in LVSOD2-infected flaps exhibited significantly slower tumour growth compared to those growing in flaps infected with control vectors. D) Individual growth curves for tumours growing in: sham (PBS) infected and un-irradiated (i), sham (PBS) infected and irradiated (20Gy/5 fractions) (ii), LVSOD-infected and irradiated (20 Gy/5 fractions) (iii) and LVeGFP-infected and irradiated (20 Gy/5 fractions) (iv). These data show that 4 out of 5 tumours irradiated in LVSOD2-infected flaps achieved remission whereas only 1 out of 5 tumours in each control group (sham (PBS) and vector (LVeGFP)) achieved remission. E) Kaplan-Meier plot of survival to humane end-point for animals with MatBIII tumours grown in sham (PBS), LVSOD2 or LVeGFP flaps showing significant differences in survival. Animals with LVSOD2-infected flaps did not achieve median survival whereas animals with control (sham (PBS) or vector (LVeGFP)) flaps had significantly shorter median survival (PBS: 27 days, LVeGFP: 25 days).