Intraesophageal MnSOD-plasmid liposome enhances ... - Nature

Gene Therapy (2008) 15, 347–356 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

Intraesophageal MnSOD-plasmid liposome enhances engraftment and self-renewal of bone marrow derived progenitors of esophageal squamous epithelium Y Niu, MW Epperly, H Shen, T Smith, H Wang and JS Greenberger Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

We evaluated whether the improved esophageal radiation tolerance following Manganese Superoxide Dismutase (MnSOD)-Plasmid Liposomes was explained by improved engraftment of bone marrow-derived progenitors. C57BL/ 6NHsd female mice pretreated with intraesophageal MnSOD-PL were irradiated to 29 Gy to the esophagus and intravenously transplanted with marrow from male B6. 129S7Gt (ROSA) 26S OR/J ROSA (Lac-Z+, G418-resistant) mice. After 14 days, esophagi were removed and side population and non-side population cells evaluated for donor multilineage (endothelin/vimentin/F480) positive esophageal cells. Serial intravenous transplantability was tested in second generation 29 Gy esophagus-irradiated mice. Esophagi from recipients receiving swallowed MnSOD-PL 24 h prior to irradiation

demonstrated significantly increased esophageal repopulation with donor bone marrow-derived Lac-Z+, G418+, Y-probe+ multilineage cells (37.8±1.8450 cell Lac-Z+ foci per esophagus) compared to irradiated controls (19.8±1.8) Po0.0001. Serial transfer to second-generation irradiated C57BL/6NHsd mice of intravenously injected SP or NSP first generation recipient esophagus cells was also significantly enhanced by MnSOD-PL intraesophageal pretreatment (74.4±3.6 SPderived Lac-Z+ foci per esophagus, 48.6±5.4 NSP-derived) compared to irradiation controls (23.4±1.8 SP, 6.0±3.0 NSP), Po0.0001. Thus, intraesophageal MnSOD-PL administration enhances engraftment of marrow-derived progenitors. Gene Therapy (2008) 15, 347–356; doi:10.1038/sj.gt.3303089; published online 20 December 2007

Keywords: MnSOD-gene therapy; marrow progenitor engraftment

Introduction Ionizing irradiation-induced esophagitis constitutes a major dose-limiting toxicity in the chemoradiotherapy of lung and esophagus cancer.1–4 The mouse model of irradiation-induced esophagitis has established dose, fraction size and esophageal volume-dependent toxicity.5 MnSOD-PL intraesophageal administration 24 h prior to single fraction irradiation, or at serial time points during fractionated irradiation has been demonstrated to significantly improve radiation dose tolerance and reduce apoptosis, ulceration and associated dehydration and weight loss in the mouse model system.5–7 We have demonstrated that bone marrow-derived progenitors of esophageal squamous epithelium engraft to the irradiated esophagus via the circulation.8 Clearance of the esophageal progenitor cell niche is optimum about 5 days after single-fraction irradiation in an animal model system of GFP+ male donor bone marrow intravenous injection into esophageal-irradiated C57BL/6J female mice.9,10 Bone marrow origin progenitors of esophageal squamous epithelium demonstrate multilineage

Correspondence: Dr JS Greenberger, Department of Radiation Oncology, University of Pittsburgh Cancer Institute, 200 Lothrop Street, Pittsburgh, PA 15213, USA. E-mail: [email protected] Received 5 July 2007; revised 4 October 2007; accepted 4 October 2007; published online 20 December 2007

differentiation capacity in vitro forming endothelian+, vimentin+ and F480+ single cell derived colonies.11 In the present studies, we used the ROSA mouse male donor selection system (G418-resistant, Lac-Z+, Y-chromosome+, triply selectable) cells to allow us to quantitate the magnitude of enhancement by MnSOD-PL esophageal treatment on the relative engraftment capacity and the serial transplantability of bone marrow-derived esophageal progenitor cells. The results demonstrate a significant enhancement of cell engraftment by MnSOD-PL intraesophageal administration prior to irradiation of both first- and second-generation mouse recipients of serially transplanted esophageal stem cells.

Results ROSA male mouse bone marrow hematopoietic and stromal cells show G418 resistance in vitro and Lac-Z+ positivity We first evaluated the robustness of donor male C57BL/ 6J. ROSA.G418.Lac-Z+ bone marrow displaying G418 resistance in vitro. Bone marrow cells freshly removed from ROSA male control C57BL/6NHsd male mice were first cultured in 0.8% semi-solid Methylcellulosecontaining medium in the presence of G-CSF, IL-3, EPO and other cytokines that have stimulated its capacity for proliferation of hematopoietic progenitor cells and also non-adherent cells.10 We quantitated the

MnSOD-PL esophagus treatment increases engraftment Y Niu et al

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effect of G418 on colony formation by adherent bone marrow stromal cells (mesenchymal stem cells) and the bone marrow cells from each mouse were cultured in increasing concentrations of neomycin (to demonstrate G418 resistance). The results are shown in Table 1 and demonstrate a 50-fold resistance of ROSA male marrow stromal cells to G418 compared to control mouse marrow. Hematopoietic cells from ROSA mice forming nonadherent colonies in semi-solid medium showed a 100fold increased resistance to G418. Over 80% of both adherent and non-adherent (semi-solid medium) colonyforming cells were Lac-Z+. A mixture of 50% ROSA male and C57BL/6NHsd male bone marrow cells cultured in the optimal concentration of G418 gave a result of 50% colony formation compared to that of pure ROSA male hematopoietic or stromal cells. These results establish that the donor bone marrow cells showed selective capacity for growth in neomycin in vitro and that the cells continued to express Lac-Z+ in colony formation in the presence of neomycin. The results supported the high likelihood of detection of male bone marrow-derived cells in the esophagus of engrafted mice in vivo when explanted to in vitro culture conditions.

Intraesophageal MnSOD-PL pretreatment enhances engraftment of ROSA male donor marrow progenitors of esophageal squamous epithelium to irradiated female C57BL/6NHsd mice Groups of 10 C57BL/6NHsd mice were treated with intraesophageal administration of MnSOD-PL (0.1 ml containing 100 mg plasmid) or received no intraesophageal pretreatment. Previous studies have documented the lack of effect of blank plasmid control liposomes in radiation protection of the esophagus.6,7 Mice were then irradiated to 29 Gy to the upper body. The abdomen and head were shielded according to the methods prescribed.2 Five days after esophageal irradiation (the time previously demonstrated to correlate to optimal apoptosis detection in the recipient esophagus),9 the mice received intravenous injection of 1 106 ROSA male whole bone marrow cells. Mice in the groups receiving no prior liposome administration showed 80% lethality by 20 days. These results confirm and extend the results from a previous publication.5 In contrast, mice receiving MnSOD-PL administration prior to irradiation and cells transplant showed 100% survival at 15 days.

Table 1

G418 resistance of ROSA male bone marrow hematopoietic and stromal cells in vitro

Marrow source

ROSA Male C57BL/6J Male

Colonies per 104 cells plated

Media

Control G418 Control G418

G418 cultures contained 250 mg ml1. *Po0.0001 compared to C57BL/6J. Gene Therapy

The esophagus from first-generation recipient mice was removed and 50% was prepared as a single cell suspension. The other 50% was fixed and stained in situ for Lac-Z+ positive foci, indicating the presence of donor marrow-derived cells. As shown in Figure 1a, positive control ROSA male mouse esophagus has shown uniform Lac-Z+ positivity. Figure 1b shows a lack of detectable Lac-Z+ in negative control C57BL/6J female esophagus. In Figure 1c, the esophagus of a C57BL/6NHsd female mouse engrafted with ROSA male donor bone marrow cells and examined at day 14 shows multiple foci of Lac-Z+ cells (first transfer). Figure 1d shows the esophagus of a mouse from the second transfer generation and a second recipient in the second generation is shown in Figure 1e. Y probe positive foci were increased in the MnSODPL-treated recipients of first- and second-generation cell transplants (Table 2). The results showed that the increased number of LacZ+ foci derived from donor bone marrow cells correlated with increased G418-resistant esophageal colonies in all mouse groups tested (Table 2). There was a significant increase in Lac-Z+ cell foci in the group that received MnSOD-PL prior to irradiation and cell transplant. These results confirm and extend a prior publication using GFP+ male marrow cells as the donor.8 Single cell suspensions of esophagus from all groups were sorted by Hoechst 33342 dye and propidium iodine by the sorting technique according to previous publications11 to separate the side population (SP) fraction known to contain multilineage stem cells and non-side population (NSP cells). SP and NSP cells from the MnSOD-PL pretreated mice showed an increase in both the total number and multilineage differentiation capacity in vitro of SP and NSP cells compared to similar cells from the controlirradiated mice. There was a significant increase in the in vitro colonies of adherent and non-adherent multilineage (endothelian/vimentin/F480) positive cells formed by the SP and NSP cells derived from the esophagus of the MnSOD-PL-treated mice compared to those harvested from the irradiated control mice (Figures 2 and 3). Y probe positive cells were detected in colonies grown in G418 (Figures 3c and d). Figure 4 shows Vimentin- and endothelin-positive cells detected by immunohistochemistry. Figure 5 shows the Lac-Z+ positivity of these cells. The relative percentages of Lac-Z+ and Y+ cells in colonies derived from SP and NSP sorted populations from explanted esophagus specimens from first

Adherent colonies at day 7

Nonadherent colonies at day 7

Adherent colonies at day 14

Nonadherent colonies at day 14

78.3±2.3 56.7±3.7* 81.3±3.2 o1

88.7±1.8 76.0±2.3* 94.7±2.6 o1

66.0±2.3 67.7±5.2* 59.0±3.5 o1

74.7±1.5 74.3±3.3* 85.3±4.3 o1


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Figure 1 Detection of Lac-Z+ expression in esophagus from first- and second-generation male ROSA marrow cell engrafted mice. The esophagus was excised from each irradiated female C57BL/6NHsd mouse (first generation) at day 14 after intravenous injection of bone marrow from ROSA male mice and from each second-generation recipient mouse at day 14 after intravenous injection of esophageal SP cells obtained from the esophagus of first-generation mice as described in the methods. Photographs of representative fields from: (a) ROSA male mouse esophagus, positive control, (b) C57BL/6NHsd female mouse, negative control, (c) MnSOD-PL-treated first-generation recipient of whole ROSA male mouse marrow and (d, e) MnSOD-PL-treated second-generation recipients of SP cells from first-generation recipient (two different mice). The esophagi were frozen in OCT, sectioned and stained for Lac-Z+ expression using an anti-Lac-Z+ antibody. Lac-Z+ expression is shown by the arrows in the ROSA male mouse (a), first generation recipient mouse (c) or second generation recipient mice (d, e) ( 100).

Table 2 Number of X50 cells containing foci of donor bone marrow origin male ROSA cells in recipient female esophagus 1st Generation Treated group (cell source)

2nd Generation

Number of Lac-Z+foci per esophagus (mean±s.e.m.)

Number of Y+foci per esophagus (mean±s.e.m.)

MnSOD-PL+29 Gy Whole marrow

37.8±1.8*

21.0±1.0*

29 Gy Whole marrow

19.8±1.8

11.1±1.2

Treated group (cell source)

MnSOD-PL+29 Gy (SP cells) (NSP cells) 29 Gy (SP cells) (NSP cells)

Number of Lac-Z+ foci per esophagus (mean±s.e.m.)

Number of Y+foci per esophagus (mean±s.e.m.)

74.4±3.6* 48.6±5.4*

41.2±2.4* 27.3±3.4*

23.4±1.8 6.0±3.0

13.0±2.0 3.0±1.2

Explants from first- and second-generation recipients. *Po0.05 compared to 29 Gy irradiation control mice. Gene Therapy


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Figure 2 Expression of vimentin, endothelin and F4/80 in G418 resistant (donor marrow derived) adherent colony-forming cells from first-generation recipient-esophageal SP and NSP cells from control- or MnSOD-PL-treated mice. C57BL/6NHsd female mice received intraesophageal MnSOD-PL (a) and were irradiated 24 h later along with control mice (b) to 29 Gy to the esophagus. Five days later, the mice were injected intravenously with bone marrow from male ROSA mice. The irradiated mice were killed 14 days later and the esophagus was removed, prepared into single cell suspensions, stained with Hoechst 33342 dye and the SP and NSP cells sorted by flow cytometry. The cells were plated in 96-well plates in DMEM containing 250 mg ml1 of G418. Fourteen days later, the colonies were fixed and immunohistochemistry was performed for expression of vimentin, endothelin or F4/80. The slides were scored under a fluorescent microscope and the percent of colonies positive determined. (Results are for three esophagus specimens per group, 100 wells per esophagus, 100 cells per well.) There were more colonies positive for vimentin, or endothelin, (*) in the esophagus from mice pretreated with MnSOD-PL (a) than in irradiated control mice (b). Control-irradiated mice had relatively more F4/80 positive colonies. More vimentin- and endothelinpositive colonies were detected in cultured SP cells compared to NSP cells in both groups (#) (Po0.05 for * and #).

generation (Figure 6) and second generation (Figure 7) recipients indicated that MnSOD-PL pretreatment enhanced engraftment of donor marrow-derived cells in both generations of recipients. There was a significant increase in overall Lac-Z+ cells and Lac-Z+ multilineage colony-forming cells derived from the esophagus of MnSOD-PL pretreated compared to control-irradiated mice. Thus, the total number of multilineage colony forming cells as well as the total SP and NSP cell population that was of donor bone marrow origin was most significantly increased by MnSOD-PL pretreatment of the esophagus (Figures 6 and 7). The sections of the esophagus that we studied contain approximately 1 106 cells of which SP cells make up 0.5–1% (5000 to 10 000 cells). The SP esophagus cells from the first-generation recipient MnSOD-PL treated mice had 18% or 900 to 1800 cells that were Lac-Z+. This number represents 0.09–0.18% of the donor ROSA bone marrow cells injected. In the control-irradiated firstgeneration mice, 450–900 SP cells were Lac-Z+ and came from donor cells (0.05–0.09% of the injected cells). In second-generation recipient mice, 4000 SP cells from Gene Therapy

Figure 3 Detection of vimentin, endothelin and F4/80 in G418resistant (donor marrow-derived) nonadherent colony-forming cells from first-generation recipient esophageal SP and NSP cells from control- or MnSOD-PL-treated mice. C57BL/6NHsd female mice were treated intraesophageally with MnSOD-PL and irradiated along with control mice. SP and NSP cells were isolated as described in the legend to Figure 2 and grown in 0.8% methylcellulose containing medium in G418 (250 mg ml1) for 14 days. Colonies were scored and cells isolated by the addition of water to the methylcelluose, centrifugation and cytospin. The cells were examined immunohistochemically for the expression of vimentin, endothelin or F4/80. The percent of cells expressing vimentin, endothelin or F4/80 was determined by examining the cells by fluorescent microscope. (Results are for three esophagus specimens per group, 100 wells, 100 cells per well.) There were more vimentinpositive SP cell-derived colonies in the MnSOD-PL-treated esophagus samples (a) than in the control-irradiated (b) SP cells (*). In the MnSOD-PL-treated mice, there were also more vimentin-positive SP-derived colonies compared to those derived from NSP cells (#) (Po0.05 for * and #).

first-generation recipient mice were injected. In MnSODPL treated second-generation recipient mice injected with esophageal SP cells from the first-generation MnSOD-PL-treated mice, 10% of the explanted esophageal SP cells were Lac-Z+, 500 to 1000 cells (which represent 12.5–25% of the injected first-generation SP cells). In contrast, second-generation control-irradiated mice had in their esophagus 4% Lac-Z+ SP cells (200–400 cells, 5–10%) from the irradiated control mouse firstgeneration esophageal SP cells. Therefore, pretreatment with MnSOD-PL allowed a higher percent of donor cells to engraft into the esophagus of first- and secondgeneration recipients.

MnSOD-PL pretreated second-generation esophagus-irradiated recipients of esophageal SP cells from MnSOD-PL-pretreated first-generation recipients show greatest increase in engraftment capacity The second generation recipients in the serial transfer experiment were prepared by dividing mice into two groups. One group received MnSOD-PL intraesophageally administered 24 h prior to radiation and the second group received irradiation alone. All groups were


Figure 4 Morphologic appearance of vimentin- or endothelinpositive first-generation recipient G418-resistant, Lac-Z+ esophageal cell-derived adherent colonies. SP and NSP adherent cells from first-generation recipient esophagus were isolated as described in the methods and in the legend to Figure 2 and stained for expression of vimentin (a) or endothelin (b). Results are for SP cells ( 400).

irradiated to 29 Gy to the esophagus and 5 days later the subgroups were engrafted with either SP or NSP cells from first-generation recipient normal esophagus (1 105) I.V. 5 days after irradiation. At 14 days after irradiation, the esophagus from second-generation recipients was excised and prepared as described in the methods section in the same way as the preparation of cells from first-generation recipients. Second-generation recipient mouse esophagus-derived SP and NSP cells were removed and placed into in vitro assays for adherent and non-adherent G418resistant colonies. The highest percentage of Lac-Z+, G418 (neomycin)-resistant adherent and non-adherent colony-forming cells was found in the esophagus of MnSOD-PL pretreated mice (Figures 8 and 9). Y+ colonies were increased in G418-resistant colonies from MnSOD-PL-treated esophagus explants (Figures 7 and 10). The relative percentage of donor male ROSA bone marrow-derived esophageal colony-forming cells was found in the SP population of the MnSOD-PLpretreated second-generation mice who received SP cells from the MnSOD-PL pretreated first-generation recipient esophagus (Figure 7). This percentage was elevated above the level detected in first-generation recipient mice. While NSP cells removed from the secondgeneration recipient esophagus of MnSOD-PL pretreated mice showed an increase in the relative number of donor male ROSA+ derived esophageal colonies compared to all other groups, these numbers did not exceed the numbers detected using SP cells. In vitro colonies formed by G418-resistant SP and NSP cells from the MnSOD-PL pretreated second-generation mice also showed increased expression of vimentin, endothelin and F4/80 compared to the second-generation control mice (Figures 8 and 9). The esophagus from secondgeneration MnSOD-PL pretreated mice that had been injected with either esophageal SP or NSP first-generation MnSOD-PL pretreated mice also showed a significant increase in the expression of vimentin or endothelin in Lac-Z+, G418-resistant cells of donor marrow origin.

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Discussion

Figure 5 Morphologic appearance of Lac-Z+ SP cells in firstgeneration recipient esophagus. SP and NSP nonadherent cells were isolated as described in the legend to Figure 2 and were grown in G418 (250 mg ml1) in Methylcellulose-containing medium. Fourteen days later, the colonies and cells were cytospun and scored for Lac-Z+ expression. Lac-Z+ positive SP cells were detected at a frequency of 24% in MnSOD-PL-treated preirradiation esophageal SP cells (photo shown) compared to 3% in the irradiation alone SP cells (arrow shows Lac-Z+ positive cells) ( 400).

Ionizing irradiation induces cytotoxicity in organs through a complex interaction of acute and chronic responses. Radical oxygen species (ROS) induced by ionizing irradiation produced cellular DNA strand breaks,3 communication of stress response kinase signaling to the mitochondria,12,13 apoptotic events12 and then secondary waves of apoptosis induced by cytokines released from injured tissues.6 Such irradiation effects in the hours/days after exposure have been utilized to clear space in irradiated organs for cell engraftment.14,15 This technique has been the mainstay of bone marrow transplantation in both model systems and in the clinic. Irradiation also facilitates engraftment of intravenously delivered tissue-specific stem cell populations, embryonic stem cells or bone marrow stem cells.16,17 The notion that ionizing irradiation simultaneously damages the organ-specific microenvironment has recently become a subject of intense investigation.8,14,15,18 Production of ROS by cells of the organ-specific microenvironment has been demonstrated to persist for weeks to months after radiation exposure.6,9 Lipid peroxidation Gene Therapy


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Figure 6 Increased Lac-Z+, Y chromosome+ nonadherent cell-derived colonies in methylcellulose culture from SP and NSP cells isolated from the esophagus of MnSOD-PL intraesophageally pretreated compared to control-irradiated first-generation recipient mice. SP and NSP cells were isolated from first-generation recipient MnSOD-PL pretreated or control irradiated first generation mice as described in the methods. The cells were grown in methylcellulose in the presence (a, c) or absence (b, d) of G418 (250 mg ml1) for 14 days. The colonies were harvested from the methylcellulose, cytospun and scored for percent Lac-Z+ positivity (a, b) or Y+ positivity (c, d). Cells from irradiated MnSOD-pretreated esophagus grown in G418 (a) had a higher percentage of Lac-Z+ cells and Y+ cells (c) compared to cells grown without G418 (b, d) due to elimination of recipient SP and NSP cells. Colonies from MnSOD-PL-treated mice had a higher percent of Y chromosomepositive cells grown in G418 (c) compared to absence of G418 (d). Mice pretreated with MnSOD-PL had significantly increased esophageal SP and NSP cells positive for Lac-Z+ and Y+ (*) grown in G418 compared to the irradiated control mice (Po0.05 for *).

has been demonstrated to peak in activity days after irradiation to the esophagus.9 The question of whether radiation clearing of specific cell engraftment space by induction of early apoptotic events could be separated from delayed ROS-mediated toxicity has become a subject of recent interest, particularly as it has become clear that ROS-producing microenvironmental cells can induce toxicity in the engrafting nonirradiated cells. Toxicity expressed as DNA strand breaks, apoptosis, chromosome instability or induction of stress response genes in nonirradiated cells co-cultivated irradiated stromal cells in vitro has been demonstrated,19 and either humoral or cell contact transmission of toxicity has been described as the radiation bystander effect.20 The magnitude of bystander effect toxicity from engrafting stem cells in irradiated microenvironment has not been fully investigated. In the present studies, we determined whether ROS and cytokine production by the irradiated esophageal microenvironment, which is decreased by MnSOD-PL administration,6,7 would have a beneficial effect on either the magnitude or robustness of engrafting marrowderived progenitors of esophageal squamous epithelium. Two assay techniques were utilized to measure the effects of MnSOD-PL on stem cell engraftment: serial transplantability of donor-derived esophageal squamous cell foci producing cells in vivo and the number of donor bone marrow-derived multilineage progenitor cells in vitro. C57BL/6NHsd female mice were irradiated to the esophagus and injected intravenously with two sources of donor male bone marrow cells. In the first experiments, male ROSA marrow was utilized, carrying with it Gene Therapy

three marker genes, Lac-Z+ positivity, G418 resistance and Y-chromosome. Recipient mice pretreated with MnSOD-PL prior to irradiation, but not irradiation alone, showed a significant increase in engraftment of donor marrow-derived cells. This was detected as donor marrow origin colonies in explanted esophagus in situ and also in G418-resistant colony formation by both adherent and non-adherent cells in semi-solid medium culture. Donor markers of Lac-Z+, neomycin and Yprobe positivity were consistently elevated in MnSODPL pretreated esophagus samples. Explanted esophagus samples were sorted into SP (stem cell-enriched) and NSP populations, and these were injected intravenously into a second generation of C57BL/6J female mice irradiated to the esophagus. This serial transfer experiment has been the mainstay of measuring self-renewal of bone marrow stem cells, and it is one of the established ‘gold standard’ assays for stem cell biology.21,22 Secondgeneration mice pretreated with MnSOD-PL to the esophagus prior to irradiation, but not those mice in groups receiving no pretreatment, showed increased engraftment of SP and NSP cells from first-generation esophagus explants. These assays reflected the original bone marrow Lac-Z+, G418-resistant, Y-probe+ cells from the original marrow donors. These assays in secondgeneration recipients measure the number of cells transplanting from the first-generation esophagus recipients as these were LAC-Z+ and G418-resistant. Utilizing the assays for donor bone marrow origin reflecting passage through the first generation recipients, those animals in the second generation that were pretreated with MnSOD-PL showed the greatest engraftment frequency.


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Figure 7 Increased Lac-Z+ and Y+ positive nonadherent cellderived colonies in methylcellulose culture from SP and NSP cells isolated from MnSOD-PL intraesophageally pretreated compared to control-irradiated second-generation recipient mice. SP and NSP cells were isolated from the esophagus of second-generation recipient mice of SP or NSP cells from MnSOD-PL pretreated or control-irradiated first-generation recipient mice, as described in the methods, and were grown in methylcellulose for 14 days in the presence of G418 (250 mg ml1). The cells were then harvested, cytospun and scored for detection of Lac-Z+ or Y+ expression. (SP SP refers to the SP cells from second-generation recipient esophagus of mice that received first-generation esophagus SP cells; SP NSP refers to SP cells from the second-generation recipient esophagus of some that received first generation esophagus NSP cells.) Secondgeneration recipient mice pretreated with intraesophageal MnSODPL had increased Lac-Z+ cells (a) and Y+ (b) in SP and NSP populations (*) compared to cells from the esophagus of controlirradiated mice that received the same first-generation recipient esophagus-derived cells. (five mice per point) (Po0.05).

Injection of bone marrow into mice receiving esophageal irradiation does have a functional role in protecting the esophagus from irradiation. Previously, it was demonstrated that C57BL/6NHsd mice irradiated to 30 Gy and then injected intravenously with bone marrow (5 104 to 1 106 cells) showed increased survival.8 Mice receiving 1 106 bone marrow cells in that study just as mice in our present experiments had increased survival compared to control-irradiated mice (P ¼ 0.0214).8 The present data further demonstrate that the engraftment of bone marrow cells into the esophagus has a role in decreasing irradiation-induced esophagitis. These results establish that intraesophageal MnSODPL administration increases the engraftment capacity of donor bone marrow-derived progenitors of esophageal squamous epithelium. They also confirm and extend a previous publication that the marrow is a source of reconstitution of the irradiation damaged esophagus and that it provides a source of multilineage epithelial cells.8 In a prior report, the more limited definition of ‘progenitors of esophageal squamous epithelium’ was used. In the present report, these cells have now been

Figure 8 Detection of vimentin, endothelin and F4/80 in adherent SP and NSP cell-derived colonies from the esophagus of secondgeneration MnSOD-PL intraesophageal pretreated or control-irradiated mice. Second-generation recipient mice that received MnSOD-PL 24 h before irradiation (a) or second-generationirradiated control mice (b) were killed 14 days after intravenous injection of either SP or NSP cells from first-generation MnSOD-PL pretreated or control-irradiated esophageal cell suspensions. The SP and NSP cells from second-generation esophagus were isolated and grown in liquid culture in DMEM media plus 250 mg ml1 G418. The colonies at day 14 were stained immunohistochemically for expression of vimentin, endothelin or F4/80. (In the legend, SP-SP refers to SP cells isolated from second-generation recipient mice that had been injected with first-generation esophagus harvested SP cells; NSP-SP refers to SP cells isolated from second-generation recipient mice that had been injected with harvested NSP cells from first-generation mouse esophagus.) There was significantly increased expression of vimentin and endothelin (*) in SP and NSP cell-derived colonies isolated from second-generation recipients that received intraesophageal MnSOD-PL (a) compared to the control-irradiated mice receiving the same cells (b). Controlirradiated second-generation mouse-derived NSP cells from recipients of first-generation NSP and (NSP-NSP) had increased relative expression of endothelin (five mice per point) (Po0.05).

shown to demonstrate serial transfer capacity, or serial self-renewal8,23 suggesting that they may be esophageal organ-specific stem cells, which were of marrow origin. We do not know if all of the donor ROSA cells isolated in the SP and NSP fractions have retained the ability to proliferate. Some of the isolated SP and NSP cells maintain their ability to grow in tissue culture and differentiate into vimentin- or endothelin-positive colonies. In the esophagus, primitive cells lining the mucosal layer are thought to divide, differentiate and migrate through the squamous layer and are lost into the esophageal lumen over time. These primitive dividing cells are thought to be esophageal stem cells. Stem cells are located in the SP fraction, but some stem cell activity is detected in NSP cells as well. This result is supported by our data, which show that cells from both the firstgeneration SP and NSPs fraction can engraft into the Gene Therapy


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Figure 10 Detection of Y+ probe progenitor cells in G418-resistant colony from second-generation recipient esophagus. The esophagus was removed from a second-generation MnSOD-PL-treated mouse, prepared into a single cell suspension, sorted for SP and NSP cells and the cells were grown in tissue culture medium supplemented with 250 mg ml1 G418. Colonies were fixed and probed for the Y chromosome. Shown is a Y chromosome (arrows) positive SP-cellderived colony stained with DAPI ( 400).

Figure 9 Expression of vimentin, endothelin and F4/80 in nonadherent SP and NSP cell-derived colonies from secondgeneration MnSOD-PL intraesophageal pretreated, irradiated control mice. Cell populations from the same second-generation recipients as described in the legend to Figure 8 were grown in 0.8% semi-solid methylcellulose medium containing G418 (250 mg ml1) and colonies scored on day 14. SP and NSP cells were isolated from second-generation MnSOD-PL-treated (a) or control-irradiated (b) second generation recipient mice 14 days after intravenous injection of SP or NSP cells from esophagus of firstgeneration recipient mice. (In the legend, SP-SP refers to SP cells isolated from second-generation recipient mice receiving firstgeneration recipient-derived esophageal SP cells. NSP-SP refers to SP cells from second-generation recipient mice receiving firstgeneration recipient esophagus NSP cells.) Colonies were recovered from methylcellulose at day 14, cytospun onto slides and stained immunohistochemically for vimentin, endothelin or F4/80 expression. Cells from the esophagus of MnSOD-PL pretreated secondgeneration mice receiving cells from MnSOD-PL pretreated, irradiated first-generation mice had significantly higher relative expression of vimentin compared to cells from second-generation irradiated control mice receiving the same first-generation SP cells (*) (panels a and b compare the respective groups). SP cells from the second-generation esophagus of mice that received first-generation recipient-derived MnSOD-PL-pretreated esophageal SP cells (panel a, SP-SP, white bars) had increased expression of vimentin compared to SP cells from second-generation MnSOD-PL pretreated mice receiving first-generation MnSOD-PL-treated esophagus-derived NSP cells (#) (five mice per point). The difference was significant (*) if P-value was less than 0.05.

irradiated esophagus of second-generation recipient mice. More cells engraft from injection of SP cells than the NSP fraction data, which supports the idea that stem cells are primarily located in the SP fraction. The present results may have wide application for bone marrow-derived stem cell engraftment to other organs, including lung,24 oral cavity,25,26 liver, pancreas and bone marrow. Antioxidant gene therapy treatment of the irradiated or cytotoxic drug-treated microenvironment may prove to be a valuable technique by which to ‘stretch’ donor bone marrow stem cell numbers to provide therapeutic recovery of a greater number of Gene Therapy

recipients. Such technology would have potential application in the treatment of thoracic cancer patients in whom the esophagus receives a high dose of irradiation or in the therapy of total body-irradiated survivors of a nuclear accident or deliberate nuclear terrorism event.

Materials and methods Mice Adult female C57BL/6NHsd mice (Harlan Sprague– Dawley, Indianapolis, IN, USA) and male B6. 129S7-Gt (ROSA) 26S OR/J mice referred to as (ROSA Lac-Z+ transgenic mice)27 (Jackson Laboratories, Bar Harbor, ME, USA) were housed five per cage according to institutional IACUC protocols. MnSOD-PL treatment and esophageal irradiation Female C57BL/6NHsd mice (groups of 10) received intraesophageal administration of 100 ml of water followed by 100 ml of MnSOD-PL (100 mg plasmid DNA) by placing a feeding tube attached to a 1 cc syringe at the top of the esophagus and placing 100 ml into the top of the esophagus allowing the mice to swallow.5 Twentyfour hours later, the MnSOD-PL-treated mice and control mice were irradiated to a dose of 29 Gy to the esophagus using a linear accelerator as published.5 Only the pulmonary cavity was irradiated, with the remainder of the body shielded from the irradiation as described previously.8 Five days later, bone marrow was isolated from the femurs of ROSA male mice and 1 106 single-cell suspended bone marrow cells were injected intravenously into the irradiated mice. These first-generation recipient mice were killed 14 days later, the esophagus was removed and analyzed by histologic evaluation5 or the cells in single-cell suspension were sorted for SP and NSP cells as described.11 The SP and NSP populations were injected i.v. into second-generation recipient 29 Gy upper body-irradiated MnSOD-PL intraesophageal treated or control mice 5 days after irradiation as described above for firstgeneration recipients. The second-generation recipient mice were killed 14 days later, the esophagus was


removed and analyzed for the percent of Lac-Z+ cells, or multilineage colony-forming cells as described.8,10

Explant of esophagus for histology and sorting of SP population cells Fourteen days after injection of bone marrow, the first- or second-generation recipient mice were euthanized and the esophagus excised and removed. The esophagi were either prepared into single-cell suspensions as described11 and sorted for SP and NSP cells, or the esophagus was frozen in OCT, sectioned and examined for Lac-Z+ expression in epithelial cells in situ using an anti-Lac-Z+ antibody (Sigma B-9271, Sigma Chemical Company, St Louis, MO, USA) and Mouse Rapid Staining Kit (Quik1, Sigma Chemical Company, St Louis, MO, USA). For histology, the sections were allowed to air dry for 30 min, fixed in 10% formalin for 20 min at room temperature, washed in PBS three times and non-specific binding blocked by incubation of the sections in 10% sheep serum (Sigma Chemical Company, St Louis, MO, USA) for 1 h at room temperature. The sections were then incubated in a 1:200 dilution of the biotinylated antiLac-Z+ antibody for 2 h in a humidity box at room temperature. The slides were then washed in PBS and two drops of biotinylated labeled anti-goat secondary antibody was added to each section for 5 min, washed in PBS followed by the addition of two drops of ExtrAvidinPeroxidase reagent for 5 min, washed in PBS and two drops of 3-amino-9-ethylcarbazole and incubated at 37 1C until the peroxidase activity was detected. To sort the SP and NSP cell populations, the esophagi were individually digested in a solution of 0.1% trypsin, 0.2% type II collagenase and 240 U of grade II dispase (Sigma Chemical Company, St Louis, MO, USA) for 1 h at 37 1C. The cells were cytocentrifuged and resuspended in DMEM media, drawn through proportionately smaller gauge needles to a 27 gauge needle and filtered through a 100 mM filter and then a 45 mM filter to remove cell clumps. The cells were resuspended at 1 106 cells ml1 in prewarmed DMEM+ (DMEM, 2% FBS and 10 mM Hepes buffer) and stained with Hoechst 33342 dye at a final concentration of 6 mg ml1 for 90 min at 37 1C. The cells were washed and resuspended in 150 ml of cold HBSS+ containing 2% FCS and 10 mM Hepes buffer containing anti-CD45 and TER119 for 30 min at 37 1C. Propidium iodide (2 mg ml1) was added to the cells for discrimination of dead cells. The SP and NSP cell populations were isolated by flow cytometer.11 Assay for single and multilineage esophageal progenitor cell colony-forming cells in vitro SP and NSP cells were cultured in 96-well plates in DMEM media alone or media containing G418 (250 mg ml1), 10% FBS, 1% glutamine and 1% penicillin/strep or in 4% methylcellulose containing 10% FBS, 250 mg ml1 G418, 10% sodium bicarbonate and 1% penicillin/strep as published.10 Seven days later, the cells growing in DMEM media were fixed with methanol and stained with antibodies for Lac-Z+, vimentin, endothelin and F4/80 as described below. Fourteen days after plating, the non-adherent cells grown in methylcellulose were isolated by adding water to the methylcellulose and spinning down the cells. These cells were

cytospun onto slides and fixed with methanol. The cells were stained for Lac-Z+ using the methods described above.11 Following fixation of the cells, they were analyzed for expression of vimentin, endothelin or F4/ 80, as published,11 by washing the slides twice in PBS and incubating the cells in a 1:200 dilution of goat antivimentin (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-endothelin (Ab-2, PC266 Calbiochem, San Diego, CA, USA) or PE-conjugated F4/80 (L11209, Caltag Laboratories, Burlingame, CA, USA) for 2 h at 37 1C. The cells were washed with PBS and stained with antibodies to vimentin or endothelin incubated with FITC anti-mouse or FITC anti-goat IgG secondary antibodies for 1 h at room temperature. The slides were washed twice with PBS and covered with anti-fade and coverslipped. The sections were examined under a fluorescent microscope and the percent of cells positive for each marker protein determined by scoring 1000 cells in each of 10 samples per esophagus for at least 5 samples per point.

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In vitro or in situ hybridization for detection of the Y chromosome To demonstrate the presence of the Y chromosome in cells or colonies grown in vitro or in situ in the esophagus of female C57BL/6NHsd mice following irradiation, colonies on coverslips or esophagus sections were stained using a murine Y chromosome hybridization kit from CamBio (Cambridge, UK). Briefly, colonies or esophagus sections were fixed with a 3:1 mixture of methanol/acetic acid for 30 min, incubated in proteinase K (10 mg ml1) for 45 min at 37 1C and dehydrated by serial ethanol washes for 2 min each in 70% (vol/vol) ethanol, 90%, and 5 min in 100% ethanol. The sections were air dried overnight and denatured by incubating the sections in prewarmed denaturation solution (70% deionized formamide and 30% 2 saline sodium citrate (SSC) (8.76 g NaCl, 4.41 g Na citrate, 500 ml doubledistilled water, pH 7.4)) at 86 1C for 2 min. The denaturation was quenched by placing the slides in icecold 70% (vol/vol) ethanol for 4 min and dehydration by serial washing in ethanol as described above. Twentyfour hours later, the sections were hybridized overnight in the dark at 37 1C with the Y probe. The sections were washed in 2 SSC buffer for 5 min, twice in stringency wash solution (50 ml deionized formamide plus 50 ml 1 SSC) for 5 min at 45 1C, twice in 1 SSC at 45 1C and incubated for 4 min in detergent wash solution (500 ml of 4 SSC and 250 ml Tween 20) at 45 1C. The slides were drained and mounted with 50 ml of working reagent B (15 ml working reagent A (DAPI stain) plus 500 ml of mountant (antifade)). The slides were covered with glass coverslips, sealed with nail varnish and stored in the dark at 4 1C. The possibility of cell fusion, detected by scoring number of X chromosome in each Y probe positive cell could not be excluded.28,29 Statistics Different groups were compared for statistical significance using a Student’s t-test.6

Acknowledgements Supported by NIH Grant No CA083876. Gene Therapy


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References 1 Zhang Z, Liao Z, Jin J, Ajani J, Chang JY, Jeter M et al. Doseresponse relationship in locoregional control for patients with stage II–III esophageal cancer treated with concurrent chemotherapy and radiotherapy. IJROBP 2005; 61: 656–664. 2 Ahn SJ, Kahn D, Zhou S, Yu X, Hollis D, Shafman TD et al. Dosimetric and clinical predictors for radiation-induced esophageal injury. IJROBP 2005; 61: 335–347. 3 Socinski MA, Rosenman JG, Schell MJ, Halle J, Russo S, Rivera MP et al. Dose-escalating conformal thoracic radiation therapy with induction and concurrent carboplatin/paclitaxel in unresectable stage IIIA/B nonsmall cell lung carcinoma. A modified phase I/II trial. Cancer 2001; 92: 1213–1223. 4 Rosenman JG, Halle JS, Socinski MA, Deschesne K, Moore DT, Johnson H et al. High-dose conformal radiotherapy for treatment of stage IIIA/IIIB non-small cell lung cancer: technical issues and results of a phase I/II trial. Int J Radiation Oncology Biol Phys 2002; 54: 348–356. 5 Stickle RL, Epperly MW, Klein E, Bray JA, Greenberger JS. Prevention of irradiation-induced esophagitis by plasmid/ liposome delivery of the human manganese superoxide dismutase transgene. Radiat Oncol Investig 1999; 7: 204–217. 6 Epperly MW, Gretton JA, DeFilippi SJ, Sikora CA, Liggitt D, Koe G et al. Modulation of radiation-induced cytokine elevation associated with esophagitis and esophageal stricture by manganese superoxide dismutase-plasmid/liposome (SOD-PL) gene therapy. Radiat Res 2001; 155: 2–14. 7 Epperly MW, Kagan VE, Sikora CA, Gretton JE, Defilippi SJ, BarSagi D et al. Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) administration protects mice from esophagitis associated with fractionated irradiation. Int J Cancer (Radiat Oncol Invest) 2001; 96: 221–231. 8 Epperly MW, Guo HL, Shen H, Niu YY, Zhang X, Jefferson M et al. Bone marrow origin of cells with capacity for homing and differentiation to esophageal squamous epithelium. Radiat Res 2004; 162: 233–240. 9 Epperly MW, Zhang X, Nie S, Cao S, Kagan V, Tyurin V et al. MnSOD-plasmid liposome gene therapy effects on ionizing irradiation induced lipid peroxidation of the esophagus. In vivo 2005; 19: 997–1004. 10 Epperly MW, Shen H, Zhang X, Nie S, Cao S, Greenberger JS. Protection of esophageal stem cells from ionizing irradiation by MnSOD-plasmid liposome gene therapy. In vivo 2005; 19: 965–974. 11 Epperly MW, Shen H, Jefferson M, Greenberger JS. In vitro differentiation capacity of esophageal progenitor cells with capacity for homing and repopulation of the ionizing irradiation damaged esophagus. In vivo 2004; 18: 675–685. 12 Epperly MW, Sikora C, Defilippi S, Gretton JA, Zhan Q, Kufe DW et al. Manganese superoxide dismutase (SOD2) inhibits radiation-induced apoptosis by stabilization of the mitochondrial membrane. Radiation Res 2002; 157: 568–577. 13 Epperly MW, Gretton JE, Bernarding M, Nie S, Rasul B, Greenberger JS. Mitochondrial localization of copper/zinc

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15

16

17

18

19

20 21

22

23 24

25

26

27

28 29

superoxide dismutase (Cu/ZnSOD) confers radioprotective functions in vitro and in vivo. Radiation Res 2003; 160: 568–578. Prigozhina TB, Gurevitch O, Slavin S. Nonmyeloablative conditioning to induce bilateral tolerance after allogeneic bone marrow transplantation in mice. Exp Hematol 1999; 27: 1503–1510. Blomberg M, Rao S, Reilly J, Tiarks C, Peters S, Kittler E et al. Repetitive bone marrow transplantation in nonmyeloablated recipients. Exp Hematol 1998; 26: 320–324. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001; 107: 1395– 1402. Krause D, Theise N, Collector M, Henegariu O, Hwang S, Gardner R et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001; 105: 369–377. Terry NH, Travis EL. The influence of bone marrow depletion on intestinal radiation damage. Int J Radiat Oncol Biol Phys 1989; 17: 569–573. Gorbunov NV, Pogue-Geile KL, Epperly MW, Bigbee WL, Draviam R, Day BW et al. Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation. Radiat Res 2000; 154: 73–86. Mothersill C, Seymour CB. Radiation-induced bystander effects—implications for cancer. Nat Rev Cancer 2004; 4: 158–164. Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation: effects of donor age, cell number, and transplantation procedure. J Exp Med 1982; 156: 1767–1779. Harrison DE, Astle CM, Delaittre JA. Loss of proliferative capacity immunohematopoietic stem cells caused by serial transplantation rather than aging. J Exp Med 1978; 147: 1526–1531. Zipori D. The nature of stem cells: state rather than entity. Nat Rev 2004; 5: 873–883. Carpenter M, Epperly MW, Agarwal A, Nie S, Hricisak L, Niu Y et al. Inhalation delivery of manganese superoxide dismutaseplasmid/liposomes protects the murine lung from irradiation damage. Gene Therapy 2005; 12: 685–693. Guo HL, Seixas-Silva JA, Epperly MW, Gretton JE, Shin DM, Greenberger JS. Prevention of irradiation-induced oral cavity mucositis by plasmid/liposome delivery of the human manganese superoxide dismutase (MnSOD) transgene. Radiat Res 2003; 159: 361–370. Epperly MW, Carpenter M, Agarwal A, Mitra P, Nie S, Greenberger JS. Intra-oral manganese superoxide dismutase plasmid liposome radioprotective gene therapy decreases ionizing irradiation-induced murine mucosal cell cycling and apoptosis. In vivo 2004; 18: 401–410. Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 1991; 5: 1513–1523. Menon SG, Goswami PC. A redox cycle within the cell cycle: ring in the old with the new. Oncogene 2007; 26: 1101–1109. Ogle BM, Cascalho M, Platt JL. Biological implications of cell fusion. Mol Cell Biol 2005; 6: 567.