A heterotopic model for tumor-associated (lymph)angiogenesis in the murine cornea - feasibility of intrastromal tumor cell injections

Abstract

Purpose: The avascular cornea is predestined to study neovascular responses. First experiences with this model date back to 1972, when Gimbrone et al heterotopically implanted tumor fragments in rabbit corneal pockets. Different assays in rabbit, rat, and mouse corneas have since been published including placement of (anti-)angiogenic growth factor releasing pellets or proangiogenic corneal sutures. Using murine corneas is surgically more intricate but advantageous due to the well-defined genetic background and availability of genetically modified animals. In mice, so far tumor-associated angiogenesis has been studied by inserting pre-grown tumor fragments into corneal pockets. Here we describe an alternative approach, where a suspension of cultured tumor cells is directly injected into the corneal stroma.

Methods: One µl of B16F10 melanoma cells suspended in PBS (100.000 cells/µl) and pre-stained with FITC+ CellTracker Green was injected into the paracentral corneal stroma of C57Bl/6 mice (n=10, OD) using a Hamilton 33G microsyringe. After 7 days, eyes were enucleated and fixated either in aceton for wholemount preparation, or in formalin for paraffin-embedded sections. LYVE1+ stained wholemounts were evaluated for the relative corneal area covered by FITC+ tumor cells (RAT, in relation to the entire corneal area) and the smallest distance between the tumor cell cluster and the LYVE1+ lymphatic limbus (DTL). Paraffin-embedded sections were stained for HMB45 or Ki67. The rate of Ki67+ tumor cells was calculated.

Results: Macroscopic evaluation in 9 eyes revealed a smooth corneal surface with localized pigmented stromal areas. One mouse was euthanized on post-op day 2 due to corneal perforation/endophthalmitis. All wholemounts (n=4) showed a paracentral area of FITC+ tumor cells. RAT was 5,05% (±2.27, mean ±SD). DTL was 1.32mm (±0.34). In paraffin-embedded sections (n=5) pigmented HMB45+ tumor cells were observed within the corneal stroma. No tumor cells were found in the adjacent anterior chamber. Thirty-seven percent of intrastromal tumor cells were Ki67+.

Conclusions: Intrastromal tumor cell injection into the murine cornea appears feasible, allowing for malignant cell survival and ongoing proliferation. Further studies are needed to assess tumor growth and the potential manifestation of associated (lymph)angiogenesis over a longer time period.

Keywords: 554 immunohistochemistry • 744 tumors • 748 vascular endothelial growth factor