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. 2022 Apr 5;24(1):25.
doi: 10.1186/s13058-022-01514-2.

Breast cancer metastasis to brain results in recruitment and activation of microglia through annexin-A1/formyl peptide receptor signaling

Sok Lin Foo  1   2   3   4 , Karishma Sachaphibulkij  1   2   3 , Corinne L Y Lee  1   2   3 , Gracemary L R Yap  1   2   3   4 , Jianzhou Cui  1   2   3 , Thiruma Arumugam  1   5 , Lina H K Lim  6   7   8   9
Affiliations

Breast cancer metastasis to brain results in recruitment and activation of microglia through annexin-A1/formyl peptide receptor signaling

Sok Lin Foo et al. Breast Cancer Res. .

Abstract

Background: Despite advancements in therapies, brain metastasis in patients with triple negative subtype of breast cancer remains a therapeutic challenge. Activated microglia are often observed in close proximity to, or within, malignant tumor masses, suggesting a critical role that microglia play in brain tumor progression. Annexin-A1 (ANXA1), a glucocorticoid-regulated protein with immune-regulatory properties, has been implicated in the growth and metastasis of many cancers. Its role in breast cancer-microglia signaling crosstalk is not known.

Methods: The importance of microglia proliferation and activation in breast cancer to brain metastasis was evaluated in MMTV-Wnt1 spontaneous mammary tumor mice and BALBc mice injected with 4T1 murine breast cancer cells into the carotid artery using flow cytometry. 4T1 induced-proliferation and migration of primary microglia and BV2 microglia cells were evaluated using 2D and coculture transwell assays. The requirement of ANXA1 in these functions was examined using a Crispr/Cas9 deletion mutant of ANXA1 in 4T1 breast cancer cells as well as BV2 microglia. Small molecule inhibition of the ANXA1 receptor FPR1 and FPR2 were also examined. The signaling pathways involved in these interactions were assessed using western blotting. The association between lymph node positive recurrence-free patient survival and distant metastasis-free patient survival and ANXA1 and FPR1 and FPR2 expression was examined using TCGA datasets.

Results: Microglia activation is observed prior to brain metastasis in MMTV-Wnt1 mice with primary and secondary metastasis in the periphery. Metastatic 4T1 mammary cancer cells secrete ANXA1 to promote microglial migration, which in turn, enhances tumor cell migration. Silencing of ANXA1 in 4T1 cells by Crispr/Cas9 deletion, or using inhibitors of FPR1 or FPR2 inhibits microglia migration and leads to reduced activation of STAT3. Finally, elevated ANXA1, FPR1 and FPR2 is significantly associated with poor outcome in lymph node positive patients, particularly, for distant metastasis free patient survival.

Conclusions: The present study uncovered a network encompassing autocrine/paracrine ANXA1 signaling between metastatic mammary cancer cells and microglia that drives microglial recruitment and activation. Inhibition of ANXA1 and/or its receptor may be therapeutically rewarding in the treatment of breast cancer and secondary metastasis to the brain.

Keywords: Annexin-A1; Brain metastasis; Breast cancer; FPR2; Microglia; STAT3.

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Conflict of interest statement

The authors declare no other competing interests or conflicts of interest.

Figures

Fig. 1
Fig. 1
Activation of CD11b+CD45lo brain resident microglial cells in primary and secondary mammary tumors-bearing MMTV-Wnt1 mice. a Representative FACS showing microglia from the brains of mice bearing primary tumor or MMTV-Wnt1 mice bearing secondary tumors. b Flow cytometry analysis of number of microglial cells in the brains of indicated mice. c CD115, d CD86 pro-inflammatory marker, and e CD206 anti-inflammatory marker expression on microglial cells from tumor mice. MFI, median fluorescence intensity. Data represents mean ± standard error of mean (SEM) of n = 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 2
Fig. 2
Secretome from 4T1 metastatic mammary cancer cells promotes microglial proliferation, directional migration, and activation. a Representative brightfield images of primary adult mouse microglia. b Growth curves of BV-2 microglial cells upon exposure to SFM, 4T07 CM and 4T1 CM analyzed using MTT assay. c Microglial cells were plated in the upper compartment of a boyden chamber chemotaxis assay, with either SFM, 4T07 CM or 4T1 CM the as chemoattractant for 24 h. Representative brightfield images of migrated BV-2 microglial cells captured at 100× magnification and quantification of migrated cells after 24 h. d BV-2 microglia were treated with either SFM, 4T07 CM or 4T1 CM and gene expression of inflammatory markers was determined by qRT-PCR. Data represent mean ± SEM; n = 3 independent experiments. Brightfield images captured at 100× magnification, representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Fig. 3
Fig. 3
ANXA1 in 4T1 metastatic mammary cancer cells secretome promotes BV-2 microglial directional migration. a–d BV-2 microglial cells were treated with indicated concentrations of a SFM, 4T1 CM or 4T1 ΔANXA1 CM, b recombinant ANXA1 protein, c 4T1 CM supplemented with Boc-MLF FPR1 antagonist or d WRW4 FPR2 antagonist for 24 h in transwell migration assay. Data represent mean ± SEM; n = 3 independent experiments. Images are representative of three independent experiments. ****P < 0.0001
Fig. 4
Fig. 4
ANXA1 expressed in 4T1 cells injected into the carotid artery may enhance initial tumor growth. a Diagram depicting model and cells injected into BALB/c mice. b Mice were injected with 4T1-luminescent cells into the intracarotid artery. Representative bioluminescence images and c violin plot quantification of brain metastases at Day-5 and Day-14 after intracarotid injection of 4T1-12b cells. d, e Representative bioluminescence images of the brains and violin plot quantification of brain metastases 14 days after intracarotid injection of 4T1-12B cells. f Weight loss and g Survival rates after injection of parental 4T1 or the ΔANXA1-4T1 tumor cells. h The ratio of CD45lo/CD11b+ Microglia in the brain at Day 14 analyzed by flow cytometry. i Expression of CD11b, CX3CR1, CD40, CD86 and CD206 in microglia. *P < 0.05, **P < 0.01, versus Sham, #P < 0.05 versus 4T1
Fig. 5
Fig. 5
Extracellular and intracellular ANXA1 elicited different effects on migratory profiles in BV-2 microglial cells. a Primary adult microglia treated with either SFM, 4T07 CM, or 4T1 CM for 24 h and stained with ANXA1 (green), microglial marker Iba1 (red), and DNA-binding dye DAPI (blue). Graph (rightmost) showing the immunoreactivity against ANXA1 on primary adult microglial cells. b and c BV-2 or BV-2 ΔANXA1 microglia were treated with SFM, 4T1 CM or 4T1 ΔANXA1 CM for 24 h in the transwell migration assay. Representative brightfield images and quantification of migrated BV-2 or BV-2 ΔANXA1 microglia. c BV-2 or BV-2 ΔANXA1 microglia were treated with SFM, 4T1 CM or 4T1 ΔANXA1 CM for 24 h and gene expression of inflammatory markers were analyzed using qRT-PCR. Data represent mean ± SEM; n = 3 independent experiments. *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
ANXA1 regulates STAT3 signaling through FPRs in microglia. a, c and d BV-2 microglia were treated with SFM, 4T1 CM, 4T1 ΔANXA1 CM or 4T1CM with the indicted inhibitors for 24 h. b BV-2 microglia were treated with SFM or 4T1 CM for 24 h. c BV-2 or BV-2 ΔANXA1 microglial cells were treated with SFM, 4T1 CM or 4T1 ΔANXA1 CM for 24 h. Indicated proteins were analyzed using western-blot analysis. f BV-2 microglia were treated with SFM or 4T1 CM for 24 h. The interaction between ANXA1 and phosphorylated STAT3 was assessed using immunoprecipitation (IP). Images shown are representative of at least 3 independent biological replicates. Loading control: β-actin
Fig. 7
Fig. 7
Expression and metastatic prognosis of ANXA1, FPR1 and FPR2 in clinical breast cancer samples. a The gene expression pattern analysis of ANXA1 in the various subtypes of breast cancers b Kaplan–Meier survival curve analysis for recurrence-free survival (RFS) for lymph node positive breast cancer patients c Kaplan–Meier survival curve analysis for distant metastasis free survival (DMFS) for lymph node positive breast cancer patients
Fig. 8
Fig. 8
ANXA1-FPR-STAT3 and ERK1/2-STAT1 signaling in breast cancer to brain metastasis. ANXA1 is involved in an autocrine/paracrine signaling network between metastatic mammary cancer cells and microglial cells. Exogenous ANXA1 from tumors (red) or microglia (green) promotes microglial migration via regulation of the expression of several pro-inflammatory and anti-inflammatory genes, and this in turn, benefits cancer migration through its immune-modulatory effects on microglial cells via formyl-peptide receptors (FPRs) 1 and 2. Meanwhile, the endogenous ANXA1 expression (yellow) was triggered simultaneously in microglia, leading to enhanced activation of STAT3 induced by activated FPRs, and thus antagonizes the anti-tumorigenic ERK1/2-STAT1 pathway
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