Pegylated liposomal doxorubicin (PLD) is one of the most widely used nanotherapeutics for the treatment of advanced/metastatic breast cancer. PLD accumulates in tumors utilizing so-called the enhanced permeation and retention (EPR) effect. Nevertheless, therapeutic efficacy and long term survival remain variable due to the development of acquired resistance. Elucidating the mechanisms of acquired resistance to PLD shall help developing new strategies to improve therapeutic outcome. It has been largely overlooked that the transport of therapeutics across biological barriers can significantly affect the efficacy of cancer therapies. Previously, we showed that the transport of PLD to tumors depends both on the tumor type and organ site. This effect is controlled by the extent to which endothelial cells (ECs) are covered by the collagen type IV (Col4) in the basement membrane, which in turn is influenced by the levels of MMP-9. Here, we have developed 4T1 tumor model which develops acquired resistance to PLD and spontaneous lung metastases. Our objective is to elucidate the resistant mechanism by evaluating the changes in the transport of PLD to the sensitive and resistant/metastatic tumors.

BALB/c mice bearing 4T1 cancers were treated with PLD intravenously when tumor volumes reached a size of approximately 100-200 mm3 . Tumor volumes in all mice decreased after initial PLD injections (sensitive). However, tumors started to grow again after 20 days and didn't respond to the second/third injections (resistant). Furthermore, 13 out of 14 mice developed spontaneous lung metastases. To elucidate the mechanisms of the resistance, mice bearing sensitive or resistant tumors were sacrificed after 24 hours of PLD injection, respectively. PLD accumulation in tumors was evaluated by imaging fluorescence of doxorubicin. Immunofluorescence staining was performed to evaluate the expression of ECs, Col4, MMP-9, and efflux pump associated p-glycoprotein (P-gp) in the primary tumors, and the expression of ECs and Col4 were also evaluated in lung metastases.

PLD accumulation was significantly decreased in the resistant tumors compared to the sensitive tumors, although P-gp expression was not increased in the resistant tumors. The amount of ECs and Col4 increased in the resistant tumors. Interestingly, ECs were covered more tightly by Col4 in the resistant tumors as compared with the sensitive tumors, which could decrease the EPR effect in the tumors. MMP-9 expression decreased in the resistant tumors, suggesting less degradation of Col4 in the basement membrane. Coverage of ECs by Col4 was similar between the metastatic lung tumors and uninvolved lung tissue as well as the resistant primary tumors, indicating the EPR effect is not increased in the metastatic tumors.

In summary, ratio of ECs covered by Col4 is higher in the resistant/metastatic tumors as compared to that in the sensitive primary tumors. This structural change in the tumor microenvironment, impeding the sufficient PLD transport to the tumors after the initial PLD therapy, can be a cause of acquired resistance/development of lung metastasis. These changes should be taken into account to develop strategies for overcoming the acquired resistance and metastasis.

Citation Format: Kai M, Liu YT, Saito Y, Ferrari M, Yokoi K. Changes in the tumor microenvironment develop acquired resistance to pegylated liposomal doxorubicin in breast cancer mouse model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-07.