Abstract 1318: Potential mechanism of re-dissemination from lung metastases to tertiary sites in breast carcinoma: Tumor microenvironment of metastasis doorways and related pro-metastatic tumor phenotypes
Authors: Karadal-Ferrena Burcu , Duran Camille L. , Friedman-DeLuca Madeline , Barth Nicole , Patel Prachiben , Condeelis John S. , Entenberg David , Oktay Maja H. 
Published: April 2023

Abstract
Background: The five-year relative survival rate of female breast cancer patients with distant metastases is only 29%, compared to 99% for localized disease. Among all subtypes, triple negative breast cancer (TNBC) has the poorest outcome due to a high risk of early relapse with visceral organ metastases, and a lack of targeted therapies. Targeting metastatic burden could improve patient survival. Recent mouse and human genomic profiling studies show that metastasis is a complex and multidirectional process. Cancer cells can re-disseminate from one metastasis to tertiary sites. But the mechanism of re-dissemination is unknown. Breast cancer cells disseminate from primary tumors via Tumor MicroEnvironment of Metastasis (TMEM) doorways, composed of three cells: a tumor cell, a perivascular macrophage, and an endothelial cell. The direct and stable interaction of these cells cause transient and local vascular opening through which metastasis-competent tumor cells co-expressing invasive, stem, and dormant phenotypes that preferentially reside around TMEM doorways disseminate to distant sites. We hypothesize that TMEM doorways also form in lung metastases, enabling cancer cell re-dissemination to tertiary sites, which increases overall cancer burden and risk of death.
Methods: We used MDA-MB-231 and MDA-MB-468 human TNBC cells to generate tumors in the 4th mammary fat pad of 6-8 weeks old NOD/SCID mice. Primary tumors were resected when 5-6 mm and fixed with 10% formalin. When lung metastases were visible via micro-Computed Tomography (microCT) fluorescent dextran was injected intravascularly, and lungs collected and fixed with 10% formalin. Triple immunohistochemistry was used to visualize TMEM doorways. To test TMEM doorway-mediated vascular opening, sequential slides were stained for extravascular dextran. Cells co-expressing stem (SOX9), invasive (MenaINV), and dormant (NR2F1) markers were visualized by immunofluorescent staining. The density of TMEM doorways, CSCs, invasive, and dormant cells was determined by automated image analysis.
Results: We demonstrated the presence and activity of TMEM doorways in lung metastases. Within lung metastases, we also documented the presence of cancer cells with a pro-metastatic phenotype indicated by their co-expression of invasive (MenaINV), stemness (SOX9), and dormancy (NR2F1) markers. We will report how the expression of pro-metastatic markers relates to TMEM doorway niches.
Conclusion: We documented functional TMEM doorways in lung metastases of TNBC, which could explain the occurrence of an early overall metastatic burden in patients with TNBC. Thus, targeting TMEM doorways after the removal of the primary tumor in patients with TNBC could block cancer cell re-dissemination, decrease metastases, and improve survival.
Citation Format: Burcu Karadal-Ferrena, Camille L. Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John S. Condeelis, David Entenberg, Maja H. Oktay. Potential mechanism of re-dissemination from lung metastases to tertiary sites in breast carcinoma: Tumor microenvironment of metastasis doorways and related pro-metastatic tumor phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1318.