Abstract B045: Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells
Authors: Mohd Nauman, Yookyung Jung, Burcu Karadal-Ferrena, Camille Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John Condeelis, David Entenberg, Maja H. Oktay
Journal: Cancer Research (2024)

Abstract Background: Breast cancer is the most frequently diagnosed cancer, and the second leading cause of cancer-related death in women. Most breast cancer related deaths are due to metastasis to distant organs. The primary tumor and secondary site microenvironments provide cues that activate cancer cell plasticity programs such as those associated with invasion and stemness, which are needed for dissemination and the establishment of metastatic foci. We demonstrated that in the primary tumor &amp;gt;50% of cancer stem cells (CSCs) co-express the invasive phenotype marker, MenaINV. Given that ∼80% of newly arriving circulating tumor cells to the lung are CSCs, we questioned if extravasation of CSCs to the lung is mediated solely by MenaINV or if other compensatory pathways are at play. We describe here our discovery that CSCs require MenaINV for extravasation to the lung parenchyma. Methods: To visualize breast cancer stem cell extravasation into the lung parenchyma, we expressed the stemness biosensor, SORE6, in MDA-MB-231 breast cancer cells. SORE6 is composed of 6 tandem repeats of the Sox2 and Oct4 response elements placed upstream of destabilized Green Fluorescent Protein (GFP). To distinguish CSCs (green) from non-CSCs, all cells were also labeled with red tracker dye. Breast cancer cells transduced with lenti-SORE6 were FACS sorted into low SORE6-GFP cells (non- CSCs) and high SORE6-GFP (CSCs). Activation of the stem program in FACS sorted cells was validated by qRT-PCR analysis of the widely used stem cell markers CD44, CD133, P63, ALDH1, Sox9, Sox2, and Oct4. The extravasation efficiency of non-CSCs vs. CSCs was assessed in vitro by the extravasation directed transendothelial migration (eTEM) assay, which mimics extravasation by allowing migration of cancer cells through endothelial cells cultured on a porous membrane, as well as in vivo, by intravital imaging. The impact of MenaINV on CSC extravasation was evaluated through CRISPR/Cas9 mediated knockout of MenaINV. In in vivo experiments, a 50/50 mixture of non-CSCs and CSCs expressing either wild type or knocked-out MenaINV were injected into the vasculature of Rag2-/- MacBlue mice. in vivo visualization of cancer cell extravasation was accomplished by implantation of an indwelling lung imaging window prior to the injection of cancer cells, followed by high-resolution intravital imaging. Results: CSCs exhibit significantly higher expression of all stem cell markers and express more MenaINV compared to non-CSCs. Moreover, CSCs show significantly better transendothelial migration in eTME assay (p=0.03) and lung extravasation (p&amp;lt;0.0001) in intravital imaging, compared to non-CSCs. Finally, knocking out MenaINV reduced lung extravasation to the levels comparable to non-CSCs. Conclusions: This study highlights the crucial role of MenaINV in facilitating lung metastasis by CSCs. Thus, targeting MenaINV may represent a promising therapeutic strategy to reduce metastatic burden in breast cancer patients. Citation Format: Mohd Nauman, Yookyung Jung, Burcu Karadal-Ferrena, Camille Duran, Madeline Friedman-DeLuca, Nicole Barth, Prachiben Patel, John Condeelis, David Entenberg, Maja H. Oktay. Requirement of MenaINV for metastatic colonization of the lungs by breast cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B045.