TNBC-derived lipocalin-2 potentiates metastasis by inducing immunologic reprogramming of premetastatic and metastatic microenvironments

Triple negative breast cancer (TNBC) is distinguished by the absence of estrogen/progesterone receptors and HER2 overexpression. Generally, the median survival for metastatic TNBC is a dire 8-10 months, representing a sharp drop from the five-year survival for metastatic breast cancer of other subtypes (~40%) or non-metastatic TNBC (~75%). Metastatic TNBC is associated with anti-inflammatory tumor immune microenvironments (TIMEs) and little is known about how primary and secondary tumors communicate with one another during disease progression. The Lipocalin-2 gene (LCN2) encodes a cytosolic and secreted protein (Lcn2) that regulates receptor trafficking, inflammation, microbiome dynamics and iron homeostasis. Previous work from our group and others has shown that LCN2/Lcn2 promotes tumorigenesis; however, questions persist regarding the specific contexts in and mechanisms by which it functions to potentiate cancer progression. To define the molecular and cellular factors that govern LCN2-mediated tumor progression, we developed the Py230-C57Bl/6 syngeneic experimental metastasis model for interrogating mechanisms by which tumor cell-secreted factors condition premetastatic lung tissue to potentiate TNBC cell seeding/expansion. Using this model and TCGA data, we identify LCN2/Lcn2 as a molecular candidate that is upregulated in lung-tropic Py230 cells and other human TNBC cells, and that predicts poor TNBC patient outcomes. We demonstrate that TNBC cell-secreted Lcn2 potentiates premetastatic lung reprogramming to increase tumor cell seeding and expansion. Our analyses of global transcriptome and single-cell spatial proteome data in premetastatic lung tissue collected from mice systemically educated by mock media or breast cancer conditioned media from Py8119 (Lcn2-low), Py230-IgG2a (Lcn2-hi) or Py230-alphaLcn2 (Lcn2-blocked) cells reveal novel gene set enrichments affiliated with oxidative phosphorylation and proliferating B cells. Further single-cell spatial proteome analyses on Lcn2-potentiated lung metastases indicate an increase in proliferating regulatory T cells (FoxP3-pos) indicating that Lcn2-mediated reprogramming of the tumor-naïve lung microenvironment suppresses inflammation to support increased tumor burden. Finally, by using single-cell spatial transcriptomics on matched primary and metastatic TNBC samples, we determine that LCN2 expression localizes to a subpopulation of epithelial and hybrid EMT cells near T cells within primary tumor sites. Notably, within corresponding matched metastatic TNBC samples, we find immune cells that are enriched for genes affiliated with oxidative phosphorylation and anti-inflammatory signaling. Taken together, we report a novel immunologic reprogramming role for LCN2 in TNBC that may be targeted to block or reverse disease progression and improve patient outcomes.