Differentiation Mechanisms of Myeloid-Lymphatic Progenitors that Promote Lymph Node Metastasis in Breast Cancer

Author

Nihit Bhattarai, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2020

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Ran, Sophia

Abstract

Metastasis to distant organs, a major cause of mortality from cancer, is often preceded by tumor spread to regional lymph nodes (LN). This process is supported exclusively by lymphatic vessels within or around the tumor. It is important to understand the mechanism by which tumors induce generation of new lymphatic vessels because interference with this early stage of tumor spread might prevent distant metastasis. Tumor lymphatic formation is thought to be induced by VEGF-C factor that activates its receptor VEGFR-3 expressed in lymphatic endothelial cells (LEC). However, this process also requires participation of tumor-recruited Myeloid-Lymphatic Endothelial Cell Progenitors (M-LECPs). We recently showed that M-LECPs are abundant in mouse and human breast tumors and that their density strongly correlates with both lymphatic formation and nodal metastasis. Here, we show that M-LECPs derived from bone marrow (BM) myeloid precursors can be differentiated in vitro by pretreatment with colony stimulating factor-1 (CSF-1) followed by activation of Toll-like Receptor-4 (TLR4) applied in this but not in a reverse order. We also identified CSF-1 as the best inducer of M-LECP differentiation out of 16 screened cytokines. Based on flow cytometry analysis of LEC- and myeloid-specific markers, CSF-1R-positive cells yielded significantly higher number of myeloid-lymphatic endothelial progenitors than BM cells lacking this receptor. Consistently, both anti-CSF-1R antibody and BLZ945 drug significantly reduced the number of myeloid-lymphatic progenitors compared with untreated controls. We also found that the requirement for CSF-1 to precede activation of TLR4 pathway is likely routed in CSF-1 dependent upregulation of essential components of TLR4 ligand recognition complex such as MD2, CD14, CD11b and CD18. In conclusion, we show that mouse BM cells can be differentiated into lymphatic endothelial progenitors by treatment with CSF-1 and TLR4 ligand, LPS. We also show that treatment with CSF-1 has to precede activation of TLR4 pathway as upregulation of several components of the TLR4 pathway is required for optimal M-LECP differentiation. To our knowledge, this is the first evidence identifying specific factors and sequence of their exposure that drive the formation of the lymphatic endothelial lineage from early myeloid precursors.