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The Role of Fibronectin on Spatial Organization in 3D Culture: A Model for Wound Healing After Breast Reconstruction and Post-Mastectomy Radiation Therapy
Henry C. Hsia, M.D., Sibo Tian, B.A., Sharad Goyal, M.D., Bruce G. Haffty, M.D., Ting Chen, Ph.D., Ramsey A. Foty, Ph.D..
Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, USA.
Breast cancer patients undergoing reconstruction after mastectomy and radiation therapy are at risk for significant morbidity. Though much variation exists among patients in their ability to heal after irradiation, the underlying mechanism is poorly understood; and no consistent method exists for modeling wound healing after radiotherapy. This study's purpose was to determine how changes to the wound microenvironment affect cell behavior in the presence of ionizing radiation, and whether spatial organization of cell aggregates in 3-D culture can be used to model wound healing after radiation therapy.
Skin and soft tissue samples from benign breast tissue specimens were prepared into 3-D cultures using a hanging drop technique. Cultures were supplemented with different concentrations of fibronectin (FN), and with or without FUD, a fibronectin assembly inhibitor. Spheroid cell aggregates in culture received 2Gy, 4Gy or 8Gy, given in 2 equal fractions 24 hours apart. The strength of cell cohesion, as approximated by compaction, was derived from the aggregate area, relative cell density, and isoperimetric quotient of each spheroid. Radioresistance was defined as the relative change in spheroid compaction per 1Gy increase in absorbed dose. Fibronectin matrix assembly was assayed using deoxycholate (DOC) solubility.
Using patient-derived dermal fibroblasts, fibronectin matrix assembly was inhibited by increasing doses of ionizing radiation, with a significant difference between lower doses (0Gy, 2Gy) and higher doses (4Gy, 8Gy). Both primary fibroblasts and endothelial cells in 3-D culture exhibited dose-dependent behavior towards ionizing radiation; higher absorbed doses inhibited cohesion when assayed via spheroid compaction. In the absence of radiation, optimal cell cohesion occurred in aggregates cultured in 6.25 ng/μL of fibronectin. Significant interpatient variability exists in the response to exogenous fibronectin, such that the conditions of optimal cell aggregation varied from patient to patient, occurring at different concentrations of exogenous fibronectin. The fibronectin assembly inhibitor, FUD, effectively abolished aggregation regardless of absorbed dose. An optimal cellular environment, (6.25 ng/μL fibronectin), was able to promote increased cell cohesion with increasing absorbed dose. The relative radioresistance of different conditions were: -1.04 (FUD 0.3ng/μL), 1.55 (FN depleted), 4.75 (FN 6.25 ng/μL), -4.0 (FN 9.4 ng/μL), 1.9 (FN 25 ng/μL). Patients varied in their ability to produce matrix-assembled and unassembled fibronectin. Fibronectin concentration at optimal aggregation was inversely related to DOC-insoluble fibronectin concentration (r = -0.85) and DOC-soluble fibronectin (Figure). Optimal exogenous fibronectin was more closely related to the amount of assembled fibronectin (r2 = 0.72). Fibronectin matrix assembly was inhibited by increasing doses of ionizing radiation, with a significant difference between lower doses (0Gy and 2Gy) and higher doses (4Gy, 8Gy).
Ionizing radiation, in a dose-dependent manner, inhibits both fibronectin matrix assembly and cell cohesion. A specific level of fibronectin in 3-D culture was most effective in abrogating the anti-aggregating effects of ionizing radiation. The 3-D culture system used here is a valuable in vitro model for characterizing cell-cell interactions after radiation therapy.
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