Abstract 1577: Thrombus formation inside liver metastasis of breast cancer by pegylated liposomal doxorubicin
Authors: Kai Megumi , Liu Yan Ting , Ferrari Mauro , Yokoi Kenji 
Published: July 2016

Abstract
A relationship between cancer and thrombosis has been long recognized. In the patients with advanced breast cancer, chemotherapy increases risk of deep thrombosis from 2-10% to around 18%. In 1856, Virchow proposed three pathophysiological risk factors for cancer patients who will likely develop deep thrombosis, such as stasis of circulation, blood components and vessel damages. Virchow's triad explains risk only for deep thrombosis, but whether the risk for thrombus formation inside tumor can also be increased is not determined yet. In preclinical studies, it has been reported that doxorubicin can induce endothelial cell damage and induce thrombus formation. Based on these evidences, we hypothesized that thromboembolic events inside tumor can be increased by doxorubicin, therefore these events may affect transport of therapeutics, oxygen and nutrients to tumor cells to show anti-tumor effect.
In current study, we evaluated thrombus formation by IF staining of 4T1 murine breast tumors metastasized to mouse liver using antibody to fibrinogen before or after therapy with pegylated liposomal doxorubicin (PLD). There was a significant increase in the amount of fibrinogen accumulated inside tumor after PLD therapy. We also quantified the amount of blood vessels using antibody to endothelial cells. There was a significant reduction in the amount of vessels only inside, but not outside metastatic liver tumors after PLD. Next, we imaged transport of systemically injected fluorescently labeled albumin inside and outside liver metastases using intravital microscopy. The amount of circulation inside metastatic tumor was much limited as compared to those in outside tumors. We also imaged diffusion of labeled albumin into tumor. After PLD therapy, the amount of albumin inside tumor was reduced as compared to that in untreated liver metastases. Then, we evaluated relative level of hypoxia inside tumor between untreated and PLD treated tumors using antibody to CA9. There was a significant increase in the level of hypoxia inside liver metastases after the therapy.
Previously we reported accumulation of PLD in 4T1 primary breast tumors by imaging fluorescence of doxorubicin. PLD accumulated and extravasated predominantly inside tumor, suggesting direct anti-tumor effect by PLD in the primary tumors. In contrast, we imaged PLD accumulated in macrophages located in surrounding area of metastatic tumors in liver. Doxorubicin may be released, diffused close distance and affect adjacent endothelial cells, therefore inducing thrombus in vessels surrounding tumor. These changes may reduce circulation/diffusion, induce hypoxia inside tumor and kill tumor associated endothelial cells. Thus, PLD may show indirect anti-tumor cell effect in liver metastases. To determine whether thrombus formation inside tumors is key mechanism for (indirect) antitumor effect in liver metastases, combination therapy using PLD with anticoagulant will be performed.
Citation Format: Megumi Kai, Yan Ting Liu, Mauro Ferrari, Kenji Yokoi. Thrombus formation inside liver metastasis of breast cancer by pegylated liposomal doxorubicin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1577.