Abstract
Abstract
Background: Breast cancer cells use tumor microenvironment of metastasis (TMEM) doorways as portals for hematogenous dissemination to distant sites. Each TMEM doorway is composed of one perivascular macrophage in direct contact with one endothelial cell and one Mena expressing cancer cell. TMEM density is prognostic of metastatic outcome in breast cancer patients. Since the development of metastases requires tumor initiating properties in cancer cells, and we recently demonstrated that cancer cells expressing cancer stem cell biosensors and other stem cell markers accumulate around TMEM (Sharma et al 2020 AACR abstract), we wanted to assess the relationship between stem cell marker expression and TMEM in mouse and human breast cancer.
Methods: We evaluated the distribution of cancer stem cells relative to TMEM doorways in MDA-MB-231breast cancer cell xenografts that express a reporter (SORE6) and in transgenic PyMT mammary carcinomas using Sox9 as marker of stemness. We also evaluated the correlation of the percentage of cancer cells expressing stem cell markers (CD44high/CD24low, CD133, ALDH, and Sox9) with TMEM and the distribution of cancer cells expressing stem cell markers relative to TMEM in 49 breast cancers collected from patients. The stem cell markers were assessed by flow cytometry, fluorescence in situ hybridization (FISH), and qRT-PCR in Fine Needle Aspirates (FNA), as well as in fixed tissues by immunofluorescence assay. TMEM density was assessed in corresponding formalin-fixed and paraffin-embedded (FFPE) tumors by immunohistochemistry. The correlation of TMEM with stemness and the distance analysis of stem cells relative to TMEM was evaluated by Spearman's and Pearson's rank correlation respectively.
Results: We observed a 7- and 3.4-fold enrichment of cancer stem cells (CSCs) close to TMEM (<200µm) compared to away from TMEM in MDA-MB-231 xenografts and PyMT tumors respectively. This CSC enrichment was specific to TMEM doorways, as we did not see any enrichment of CSCs around blood vessels lacking TMEMs. In breast cancer patients, we also observed strong positive correlation between percentage of CD44high/CD24low cells and TMEM scores (r=0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r=0.88 and 0.86 respectively). FISH results were validated using qRT-PCR and showed very strong correlation with TMEM scores (r=0.76 and 0.73 for CD133 and ALDH1 respectively). Moreover, we found strong positive correlation between TMEM and Sox9 and CD133 (r=0.84 and 0.47 respectively) in FFPE tissues.
Conclusion: The density of TMEM doorways positively correlates with the number of cancer stem cells in human breast cancer. Moreover, stem cells accumulate around TMEM consistent with the findings that TMEM doorways represent an educational niche for stemness in breast cancer.
Citation Format: Gina Kim, Ved P. Sharma, Gargi Bandyopadhyaya, Eli Grunblatt, Sweta Roy, Nathan Agi, Binwu Tang, Esther Adler, Joan Jones, George S. Karagiannis, Xianjun Ye, David Entenberg, Sumanta Goswami, Lalage Wakefield, John S. Condeelis, Maja H. Oktay. The relationship of TMEM (tumor microenvironment of metastasis) doorways with breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6022.