Abstract 12: Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature
Authors: Hall Kelly , Volk-Draper Lisa , Rajput Sandeep , DeNardo David , Ran Sophia 
Published: May 2015

Background: Lymph node (LN) metastasis, the strongest prognostic factor for breast cancer (BC) patient survival, directly correlates with increased density of lymphatic vessels. The formation of new lymphatic vessels (i.e. lymphangiogenesis) is strongly promoted by tumor-associated macrophages (TAMs) through mechanisms that are not fully understood. We recently discovered that inflammatory lymphangiogenesis is driven by bone marrow derived myeloid cells that transdifferentiate into lymphatic-like cells and structurally contribute to growing lymphatic vasculature. Clinical BC are characterized by an inflammatory environment and massive recruitment of macrophages. We, therefore, hypothesize that BC mobilized monocytic precursors may promote tumor lymphangiogenesis by induction of their transdifferentiation into Macrophage-derived Lymphatic Endothelial Cell Progenitors designated here as M-LECPs.

Methodology: To test this hypothesis, we analyzed 75 clinical specimens of invasive breast carcinoma and 5 normal breast tissues for the presence of M-LECPs and incidence of their integration into lymphatic vessels. M-LECPs were identified by double or triple staining using multiple specific markers for myeloid (CD68, CD14, and CD11b) and lymphatic lineages (LYVE-1, podoplanin and VEGFR-3). Integrated M-LECPs were identified by double staining of LYVE-1+ vascular structures with antibodies against myeloid-specific markers as well as confocal microscopy using anti-VE-cadherin staining.

Results: Out of 75 tumors, 58% had lymphatics, and 46% of these specimens showed integration of M-LECPs in the lymphatic vessels. In stark contrast, normal mammary tissues showed less than 5% of either double-stained macrophages or dual-identity vessels. The predominant M-LECP association with tumors was also evident in metastatic BC mouse models including MDA-MB-231 (human xenograft), R3L (transplantable syngeneic), and MMTV-PyMT (spontaneous syngeneic). All three models contained 40-50% of TAMs positive for lymphatic markers and up to 90% of lymphatic vessels positive for myeloid markers. In vitro studies showed that inflammatory triggers induce lymphatic proteins in myeloid cells but not myeloid markers in inflamed lymphatic endothelial cells.

Conclusions/Significance: These findings show, for the first time, that majority of TAMs in clinical BC exhibit the lymphatic phenotype and integrate into lymphatic vessels. Reproduction of this phenomenon in three BC mouse models and cultured myeloid cells treated with inflammatory mediators suggests a strong self-autonomous role of TAMs into outgrowth of tumor lymphatics. Further investigation of this novel mechanism of TAM-dependent lymphangiogenesis may identify new targets for inhibiting lymphatic metastasis leading to improved survival of breast cancer patients.

Citation Format: Kelly Hall, Lisa Volk-Draper, Sandeep Rajput, David DeNardo, Sophia Ran. Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 12. doi:10.1158/1538-7445.AM2014-12