Quantification of Transforming Growth Factor- β in Radiation-Induced

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Quantification of Transforming Growth Factor- β in Radiation-Induced Muscle Fibrosis: A Pilot Study Evander Britt, Daniel Bracey, MD, Jeffrey Willy, PhD, Ann Tallant , PhD, Patricia Gallagher, PhD, Thomas Smith, PhD, Cynthia Emory, MD - PowerPoint PPT Presentation

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Quantification of Transforming Growth Factor- in Radiation-InducedMuscle Fibrosis: A Pilot Study

Evander Britt, Daniel Bracey, MD, Jeffrey Willy, PhD, Ann Tallant, PhD, Patricia Gallagher, PhD, Thomas Smith, PhD, Cynthia Emory, MD

Department of Orthopaedic Surgery, Wake Forest School of Medicine

MethodsIntroductionResultsConclusionOver 1,000,000 patients are diagnosed with cancer annually, with 50% receiving radiotherapy (RT). Radiation-induced fibrosis (RIF) is a debilitating effect of RT causing muscle weakness and functional limitations. The cytokine Transforming Growth Factor- (TGF-) is believed to play a part in the fibrotic response by activating fibroblast cells that secrete collagen. The current investigation sought to identify when the TGF- response occurs after receiving radiotherapy. Modeled Sarcoma Radiotherapy

Figure 2. Timeline of irradiations and sacrifice points for four groups of CD-1 miceundergoing modeled sarcoma radiotherapy

10 day course of fractionated radiation (7.3 Gy/fraction; 4 fractions; 29.2 Gy total)

Quantification of TGF-ELISA assays were conducted on tissue homogenates and blood plasma. Immunohistochemistry assays were conduced on tissue samples.Quantification of Collagen DepositionPicro-Sirius Red Stains were conducted on tissue samples.ELISAs, Immunohistochemistry, and Pic-Red staining, did not show a significant rise in TGF- at any time point.

The TGF- response to irradiation likely occurs at a later time point than originally hypothesized.

Figure 3. Average TGF- concentrations in blood plasmaFigure 4. AverageTGF-concentrations ingastrocnemius-soleus complex

Figure 1. TGF- signaling pathway.Taken from Olman, M. (2009) Beyond TGF-: a prostaglandin promotes fibrosis. Nature Medicine 15: 1360-1361.Figure 5. Immunohistochemistry showing TGF- in muscle cross sections. Color indicates TGF-.A- sacrifice day 4 controlB- sacrifice day 4 irradiatedC- sacrifice day 7 controlD- sacrifice day 7 irradiated

Figure 6. Picro-Sirius Red stain for collagen deposition. Picture A is sacrifice day 0 control, and Picture B is sacrifice day 0 irradiated. More collagen deposition (as shown in B by diagonal red shading) could suggest that fibrosis is developing.

Future StepsOnce the time point at which the TGF- response occurs is identified, the molecular mechanism of RIF therapeutics may be further investigated.ABCD

AcknowledgementsThanks to the Wake Forest URECA Center for generousfunding and to Mark Landrum for laboratory training.