Development of a novel in vitro human tissue-based angiogenesis assay to evaluate the effect of antiangiogenic drugs

Abstract

Objective: To describe a novel in vitro human tissue-based angiogenic model that can predict an individual tumor's response to antiangiogenic drugs.

Summary background data: A number of in vitro and in vivo angiogenesis assays exist, but they do not provide potentially useful information for the treatment of an individual patient. Clonogenic assays have been used to evaluate the response of an individual's tumor to antineoplastic agents, but these tumor fragments are cultured in an environment that does not lead to neovessel growth. The authors have previously demonstrated that human vein disks or human tumor xenograft fragments incorporated into a 0.3% fibrin-thrombin clot will develop angiogenic vessel growth from the cut edge of the vessel disk or xenograft fragment.

Methods: Fresh human tumor or normal tissue disks (2 x 1 mm) from fresh surgical specimens were incorporated into fibrin-thrombin clots overlain with nutrient medium containing either 20% fetal bovine serum alone or in combination with Epothilone B, a tubulin inhibitor with antiangiogenic properties. Tissue disks were visually assessed over time to determine the percentage of wells that developed an angiogenic response. Neovessel growth, density, and length were graded at intervals using a semiquantitative visual neovessel growth-rating scheme (angiogenic index, 0-16 scale) devised in the authors' laboratory.

Results: Epothilone B treatment at doses of 10-6 mol/L and 10-8 mol/L decreased the number of wells that developed an invasive angiogenic response and limited the development of vessels that invaded the matrix. At these doses, Epothilone B also caused regression of vessels in wells that had been allowed to develop an angiogenic response. Treatment of tumors or normal tissues with Epothilone B at doses less than 10-8 mol/L was ineffective.

Conclusions: Epothilone B may be an effective antiangiogenic agent in a variety of tumor types. The authors speculate that this in vitro model might provide useful information to the clinician on the effect of specific antiangiogenic agents on individual tumors. This may be particularly useful in patients with tumors that, as a group, are unresponsive to treatment with antineoplastic agents.

Figure 1. Photomicrograph of human parathyroid tissue following culture. (A) Hematoxylin and eosin stain (100×). (B) Immunohistochemical localization of factor VIII on the endothelial tubes invading the fibrin-thrombin clot matrix (100×).

Figure 2. Effect of Epothilone B treatment at different doses on the percentage of wells that develop evidence of invasion (% I) (A) and the development of the angiogenic response (AI) (B). % I, % of wells demonstrating invasion of neovessels into the fibrin-thrombin clot ([# of wells with invasion/# of wells plated] × 100). AI ± SEM, mean angiogenic index ± SEM of wells. Data from wells that demonstrated invasion into the clot and those that did not invade the clot are included. Wells that did not demonstrate invasion are represented by a score of zero.

Figure 3. Time course for the development of an angiogenic response in a carcinoid liver metastasis in vitro. In this experiment, wells were exposed to Epothilone B with 10⁻⁸, 10⁻¹⁰, or 10⁻¹² mol/L from day 1 in culture. Epothilone B treatment was effective at the 10⁻⁸ mol/L concentration but was ineffective at higher doses. % I, % of wells demonstrating invasion of neovessels into the fibrin-thrombin clot ([# of wells with invasion/# of wells plated] × 100). AI ± SEM, mean angiogenic index ± SEM of wells. Data from wells that demonstrated invasion into the clot and those that did not invade the clot are included. Wells that did not demonstrate invasion are represented by a score of zero.

Figure 4. Regression of established neovessels following therapy with Epothilone B at 10⁻⁶ and 10⁻⁸ mol/L. Wells were allowed to grow until day 18, when therapy was initiated. Epothilone B treatment at these doses inhibited further neovessel development and caused a decrease in the percentage of wells that exhibited evidence of neovessel invasion (A) and decreased the mean AI in treated wells (B). % I, % of wells demonstrating invasion of neovessels into the fibrin-thrombin clot ([# of wells with invasion/# of wells plated] × 100). AI ± SEM, mean angiogenic index ± SEM of wells. Data from wells that demonstrated invasion into the clot and those that did not invade the clot are included. Wells that did not demonstrate invasion are represented by a score of zero.