Thrombosis for Anti-angiogenic Therapy of Breast Cancer

Min-Ying Su, Ph.D.
University of California, Irvine

Anti-angiogenic therapy is a promising alternative for treatment of cancer. It may also be used as a maintenance therapy to prevent the metastasis or recurrence. While 60% of the approximately 186,000 annual cases of breast cancer now present as node negative, 30% of these cases will recur after local therapy. Current approaches to target angiogenesis rely of inhibiting growth factors that stimulate vascular endothelial cells or blocking their receptors. Our interests are more directed towards the differences between the vessels on tumors and normal blood vessels in the body. At the level of the micro-circulation the tumor vasculature is ‘leaky’ and prone to becoming ‘plugged’ with blood clots. In fact, tumors appear to have an endogenous anti-coagulant system in place to maintain the blood flow essential for cell survival. Our approach is to explore the possibility of inhibiting this tumor-based anti-coagulant system as a novel means to attack metastatic breast cancer.

In this project we will test the efficacy of two pro-blood clotting (pro-thrombotic) drugs for their anti-angiogenic effects using breast cancer models. We will study both the formation of new tumor vessels and/or damage existing tumor vessels by causing selective clotting. One drug is aminocaproic acid, and the other drug is protamine sulfate. In addition to these two drugs alone, we will also study their combination effects. The two drugs to be investigated are already approved for human use (not for cancer therapy) and have well-defined toxicity profiles. We anticipate that by using protamine to promote blood clotting and also aminocaproic acid to prevent the body’s attempt to lysis the clot, the combined treatment approach will be the most effective.

Our experiments are performed in animals using rat or human breast cancer cell lines to form the tumors. We will employ ‘dynamic contrast enhanced’ MRI (magnetic resonance imaging) as a non-invasive means to measure the vascular characteristics in tumors. This will allow us to measure the effectiveness of our anti-angiogenic therapy. We are not only aiming for the determination of therapy outcome, but also investigating the mechanisms of how the therapy works or fails. The MRI-based methodology and our experimental results on tumor thrombosis modulation will provide information to improve the analysis of other anti-angiogenic drugs.

Progress Report, Year 1 (2001)

Introduction: Anti-angiogenic therapy is a promising alternative for treatment of cancer. It may also be used as a maintenance therapy to prevent the metastasis or recurrence. While 60% of the approximately 186,000 annual cases of breast cancer now present as node negative, 30% of these cases will recur after local therapy. Our approach is to explore the possibility of inhibiting this tumor-based anti-coagulant system as a novel means to attack metastatic breast cancer.

Progress towards specific aims: During the past year we tested the toxicity of the proposed treatment. The host animal tolerated the treatment without showing severe sick signs. We also studied the short-term effect of the treatment on R3230 AC adenocarcinoma. Protamine sulfate caused thrombosis of tumor vessels more than normal vessels. The enhancement kinetics measured after the protamine treatment was greatly reduced compared to the pre-treatment level, and the effect is much more pronounced in tumors compared to other normal tissues. We performed histological assessment to semi-quantitatively determine the degree of thrombosis. It was confirmed that there was more thrombosed vessels in tumors compared to normal tissues. The efficacy of long-term treatment monitored by MRI is currently in-progress. We have also developed CD31 vessel density staining technique to quantitatively count the vessel density for determination of the anti-angiogenic effect of this treatment.

Future direction: In the next year, we will apply the established study protocol to study the breast tumor model MCF7.

Progress Report, Year 2 (2002)

Note: This grant was extended 1-yr to complete the aims.

Introduction: Anti-angiogenic therapy is a promising alternative for treatment of cancer. It may also be used as a maintenance therapy to prevent the metastasis or recurrence. Our approach is to explore the possibility of inhibiting this tumor-based anti-coagulant system as a novel means for cancer treatment or prevention of metastasis.

Topic addressed: We studied the efficacy of two pro-thrombotic drugs for their anti-angiogenic effects. There are two current approaches to the design of anti-angiogenic therapy. The most common approach aims to prevent tumors from forming the new blood vessels necessary to nurture their growth. The second approach seeks to block blood vessels that have already been formed. We will study two agents that are intended to inhibit formation of neovasculature and/or damage existing vessels by causing selective thrombosis of tumor vessels, aminocaproic acid and protamine sulfate.

Progress towards specific aims: During the Year-02 of the study we started to test the effects of the thrombotic drugs in another cancer model MCF7. We have previously reported that in rats bearing R3230 AC tumors, the enhancement kinetics measured after the protamine treatment was greatly reduced compared to the pre-treatment level, and the effect is much more pronounced in tumors compared to other normal tissues. Protamine sulfate was injected into nude mice bearing MCF7 tumors, and the enhancement kinetics of two different contrast agents (Gd-DTPA and Gadomer-17) were measured before and after the treatment to assess the degree of thrombosis. The MCF7 tumor model did not respond to the treatment to the same degree as in the R3230 AC tumor model.

Future direction: In the following year, we will keep working on the long term treatment studies. We will try to use another rat model, MDA-MB-435. We previously proposed to measure euglobulin clot lysis time for assessment of in vivo fibrinolytic activity. Now we have access to a thromboelastography system which gives a much more accurate and quantitative picture of clotting and lysis. This will be used to replace the euglobulin clot lysis time measurements.

Impact on reducing the human/economic costs: The two drugs to be investigated here, protamine sulfate and aminocaproic acid, are already approved for human use and have well-defined toxicity profiles. The success of the tests may readily translate into human studiez in the near future. Our results should also improve the future design of other anti-angiogenic drugs. Another goal of this investigation is to find the MRI parameters that predict the final therapeutic response.

Publication:

1. Su MY, Samoszuk MK, Wang J, Nalcioglu O. Assessment of protamine-induced thrombosis of tumor vessels for cancer therapy using dynamic contrast-enhanced MRI.
NMR Biomed. 15(2):106-13. 2002