Objective: To utilize Multiphoton Microscopy (MPM) as a novel imaging technique to characterize and quantify collagen at the Renal Cell Carcinoma Pseudocapsule, to assess for both intra-tumoral and inter-tumoral variation of collagen characteristics. MPM combines Second Harmonic Generation and Two Photon Excitation Fluorescence to image extracellular matrix architecture.
Methods: Twenty partial nephrectomy specimen tissues were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The pseudocapsules (PCs) were imaged with 2X and 20X objective at selected Regions of Interest. Corresponding clinical information was retrieved. PC thickness was determined. Collagen parameters measured included quantification by the Collagen Area Ratio, and qualitative measurements by the Collagen Fiber Density and Collagen Reticulation Index.
Results: The boundaries between tumor, PC and normal renal parenchyma were distinguished by MPM without need for staining. In the thickest areas of the PC, collagen content and density were quantitatively higher compared to the thinnest areas. Median Collagen Area Ratio was higher in the thickest compared to the thinnest areas of the PC (P = .01). Clear Cell RCC specimens had a consistently higher Collagen Fiber Density in both the thickest and thinnest areas compared to non-Clear Cell RCC specimens (P = .02).
Conclusions: We demonstrated the ability of MPM to quantify collagen characteristics of PCs without fluorescent labeling. Tumor enucleation for Renal Cell Carcinoma along its PC remains debatable with regards to oncological safety. Even with a complete and intact PC, the PC is not a homogenous structure, and varies in its thickness and its collagen characteristics within, and between tumors.
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