PMC

Figure 7. In vitro wound/repair process upregulates NFκB-mediated pro-inflammatory cytokines. Levels of IL-6, IL-8, MCP-1 and GROa were determined from cell lysates obtained from control cultures (no wound) and from cells isolated from wound edge (WE) and wound back (WB) areas using xMAP technology.

Figure 6. Inhibition of TLR2 or MyD88 activity inhibits repair. CD44+/MyD88+ EOC stem cells were transfected with a dominant-negative form of TLR2 or a dominant-negative form MyD88 prior to wounding. The effect of blocking TLR2 or MyD88 function on repair is then determined. Note the significant decrease on wound repair in cells transfected with TLR2 or MyD88 dominant-negative plasmids. TLR2-dn, TLR2-dominant-negative; MyD88-dn, MyD88-dominant-negative. *p < 0.005.

Figure 8. Inhibition of NFκB inhibits repair and self-renewal. (A) CD44+/MyD88+ EOC stem cells were wounded and repair process determined in the presence of absence of the NFκB inhibitor, BAY 11-7082 (2.5 μM). (B) CD44, Sox2, Nanog expression levels were determined using qRT-PCR from wounded CD44+/MyD88+ EOC stem cells in the presence or absence of BAY 11-7082. *p < 0.05.

Figure 4. In vitro wound/repair process is associated with CD44+/MyD88+ EOC stem cell self-renewal. (A) Levels of CD44, Nanog, Sox2 and Oct4 were determined by qRT-PCR in control cultures (no wound) and in cells from wound edge (WE) 24h after wounding. (B) Levels of CD44, Oct-4, β-catenin and GAPDH were determined by western blot analysis from control cultures (no wound) and from cells isolated from WE and wound back areas. C, control no wound; WE, wound edge; WB, wound back.

Figure 2. Chemotherapy-induced tumor injury enriches for CD44+/MyD88+ EOC stem cells. (A) Paclitaxel treatment (20 mg/kg q3d) enhances growth of tumor implants. (B) Enrichment of CD44 positive cells in tumors from mice treated with Paclitaxel. Tumor implants treated as in (A) were dissociated at the end of the treatment and the number of CD44+ cells was determined by flow cytometry. Note the significant increase in CD44+ cells in the Paclitaxel-treated group.

Figure 3. CD44+/MyD88+ EOC stem cells repair in vitro wound. (A) CD44+/MyD88+ EOC stem cells (i) and CD44-/MyD88- EOC cells (ii), were grown to confluence and “wounded” as described in the “Materials and Methods” section. Note that only CD44+/MyD88+ EOC stem cells repair in vitro wound. (B) GFP+/CD44+/MyD88+ EOC stem cells and RFP+/ CD44-/MyD88- EOC cells were grown to confluence and wounded (i). Wound healing was determined by fluorescent microscope at 24 h (ii), 48 h (iii) and 72 h (iv). Note that only GFP+/CD44+/MyD88+ EOC stem cells can repair the wound. (C) Immunohistochemistry for Ki67 (green staining) was determined in (i) no wound control, (ii) wound edge 4 h after wounding, (iii) wound edge 24 h after wounding and (iv) in wound back 24h after wounding. w, wound area. Note: for wound edge and wound back definitions, please see text.

Figure 1. Tumor injury accelerates tumor growth in vivo. (A) Subcutaneous ovarian cancer xenografts were established on both sides of nude mice. Partial tumor debulking was performed on the right tumor, and skin was incised on the left side but the tumor was left untouched. (B) Size of tumor xenografts was measured ex vivo at day 24 post-surgery. Note that day 24 post-surgery, partially debulked or “wounded” tumor appears larger than the tumor that was not debulked. (C) Graph of tumor kinetics show accelerated growth in “wounded” tumors compared with non-debulked control. Day 0 indicates the measurement immediately preceding wounding. (D) Flow cytomety analysis for CD44 was performed on cancer cells dissociated from the excised tumor xenografts at day 24 post-surgery.

Figure 5. TLR2 signaling enhances repair and self-renewal. (A) qRT-PCR analyses for TLR2 expression was performed on cancer cells dissociated from the excised tumor fragments at day 24 post-surgery. *p < 0.05. (B) qRT-PCR analyses for TLR2 expression was performed on cancer cells dissociated from tumors of animals treated with Paclitaxel as described in . (C) Levels of TLR2, TLR3 and TLR4 were determined by qRT-PCR from control cultures (no wound) and from cells isolated from wound edge (WE) area 8 h post-wounding. Data represent the average from three independent experiments, measured in triplicate and normalized to GAPDH mRNA. (D) Wounded CD44+/MyD88+ EOC stem cultures were left untreated or treated with 2 µg/ml of PGN or 10 μg/ml of LPS and effect on wound repair was determined. (E) Levels of CD44 and Nanog were determined by qRT-PCR from wounded CD44+/MyD88+ EOC stem cells in the presence or absence of PGN.