Fstl1 suppresses breast cancer lung metastasis by inhibiting M2-like tumor-associated macrophages recruitment towards the lung

doi:10.21203/rs.3.rs-1548160/v1

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Fstl1 suppresses breast cancer lung metastasis by inhibiting M2-like tumor-associated macrophages recruitment towards the lung

https://doi.org/10.21203/rs.3.rs-1548160/v1

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Immune cell infiltration in the tumor microenvironment is associated with cancer prognosis. Tumor-associated macrophages (TAMs) play an essential role in tumor initiation, progression, and metastasis. Follistatin-like protein 1 (Fstl1), a glycoprotein widely expressed in human and mouse tissues, is a tumor suppressor in various cancers and a regulator in macrophage polarization. However, how Fstl1 affects the crosstalk between breast cancer cells and macrophages remains unclear. Analyzing the public data, we found that Fstl1 expression significantly decreases in breast cancer tissues compared with normal breast tissues, and high expression of Fstl1 in patients means prolonged survival. Using flow cytometry, we found that total macrophages and M2-like macrophages increased dramatically in the metastatic lung tissues during breast cancer lung metastasis of Fstl1^+/- mice. Cellular experiments in vitro showed that Fstl1 inhibited macrophage migration towards 4T1 cells via decreasing colony stimulating factor 1 (CSF-1), transforming growth factor-β1 (TGFB1) and vascular endothelial growth factor-α (VEGF-α) expression of 4T1 cells. We demonstrated that Fstl1 inhibited M2-like tumor-associated macrophages recruitment towards the lung and suppressed TGFB1/Smad2/3 pathway and relevant cytokines expression of 4T1 cells. Thereby we find a potential therapeutic strategy contributing to breast cancer.

Breast cancer

Breast cancer lung metastasis

Follistatin-like protein 1 (Fstl1)

Tumor microenvironment

M2-like tumor-associated macrophages

There are about 4.2 million newly-diagnosed breast cancer patients in China every year, and its incidence is rising year by year, while the mortality rate has not been significantly reduced [1], One of the underlying causes of the increase in breast cancer is the changing risk factors for breast cancer, such as delayed and fewer births, which are most evident in countries undergoing social and economic transition. Breast cancer could be sorted into several molecular subtypes, including hormone-receptor-positive, human epidermal growth factor receptor-2 overexpressing (HER2⁺), and triple-negative breast cancer (TNBC) based on histological features. TNBC, which barely expresses ER or HER2, is the most malignant and intractable breast cancer type to treat. It is associated with poor prognosis due to its metastasis in the early stage [2–4]. Therefore, the suppression of TNBC metastasis is of significant importance.

Breast cancer development, metastatic ability, and therapy reactivity rely on both the cancer cells themselves and their interaction with the surrounding microenvironment [5]. The tumor microenvironment (TME) is comprised of various cellular components (cancer cells, immune cells, fibroblasts, endothelial cells, et al.), growth factors, proteases, and extracellular matrix (ECM) [6–8]. In TME, innate immune cells are highly represented, and the most abundant of these cells are tumor-associated macrophages (TAMs). TAMs, a subgroup of macrophages, could be conventionally subdivided into two groups owing to single cell sequencing, M1-like TAMs and M2-like TAMs. M1-like TAMs could exert anti-tumor effects when they are activated [9]. In contrast, M2-like TAMs undergo a promoting-tumor process, supporting tumorigenesis and metastasis [10]. M2-like TAMs, activated by IL-4, IL-13, IL-10, and M-CSF/CSF-1, could secrete cytokines like TGFB1, VEGF and EGF, which would further promote tumor pre-metastatic niche formation and angiogenesis around the niche [11].

Follistatin-like protein 1 (Fstl1) is a secreted glycoprotein widely expressed in human and mouse tissues, composed of non-functional extracellular calcium-binding domain [12] and the FK domain of murine Fstl1[13]. It plays an essential role in multiple processes, including cellular biology, organ development, carcinogenesis, and metastasis [14]. Fstl1, as a well-known tumor suppressor in different kinds of cancer types [15–17], had been recently reported to be a potential regulator in macrophage polarization [18]. We had elucidated that deficiency of Fstl1 could accelerate the growth of breast cancer lung metastatic tumor but not primary tumor growth in previous data, either the 4T1 cell proliferative ability [19], though our laboratory had reported the anti-proliferative effect of Fstl1 on the human TNBC cell line MDA-MB-231 via TGF-β/Smad2/3 mediated signaling pathway [20]. However, it is still unclear about the dynamic immune cell populations in the lung microenvironment.

In our research, we analyzed a potential relationship between Fstl1 and lung microenvironment of breast cancer metastasis. The results showed that Fstl1 reduced the population of M2-like TAMs in the lung and might inhibited CSF-1, VEGF-α, and TGFB1 gene expression in 4T1 breast cancer cells via TGFB1/Smad2/3 signaling pathway. Immune cells infiltrated into the TME is closely associated with patient prognosis [21], and Fstl1 might be able to evolve as a cancer immunotherapy to treat malignant tumors and their metastasis.

2.1 Reagents and facilities

RPMI-1640 was bought from Thermo Fisher Scientific corporation (Waltham, USA). Reagent extracting RNA was bought from Sangon Biotech (Shanghai, China). Cytometry labelled antibodies: PE anti-mouse F4/80 Antibody (Catalog: #123110, Biolegend, USA), FITC anti-mouse CD206 (MMR) Antibody (Catalog: #141704, Biolegend, USA), Fixable Viability Stain 450 (FVS450) (Catalog: #562247, BD Biosciences, USA). Recombinant mouse interleukin IL-4 protein (Catalog: #214 − 14, PeproTech, USA), Recombinant mouse Fstl1 protein (Catalog: #1738-FN-050, R&D systems, USA). Recombinant human TGFB1 protein (Catalog: #240-B-010/CF, R&D systems, USA), Recombinant human BMP4 protein (Catalog: #314-BP-010, R&D systems, USA).

2.2 Experimental animals

129 background Fstl1^flox/+ mice were backcrossed to Balb/C background mice for more than 10 generations, and then our experimental animals were gained: Balb/C background Fstl1^+/− mice. Experimental procedures of generation of Fstl1^+/− mice were mentioned in previous report [20]. For mouse genotyping, Fstl1 primer sequences were used, Forward: 5’-CTCCCACCTTCGCCTCTAAC-3’; Reverse: 5’-CGGCTAGGAAAGACTTGGAA-3’. Mice were purchased from the Model Animal Research Center of Nanjing University and were bred in our animal institution with controlled conditions, a 12h/12h light-dark cycle. All animal experiments were performed in accordance with the Administration Regulations on Laboratory Animals of Beijing Municipality. All animals were raised in specific pathogen free (SPF) level conditions and given adequate irradiated food and water. All used animals were healthy female Balb/C mice (6–8 weeks).

2.3 Experimental cell lines

4T1 breast cancer cell line is a kind gift from professor Wen Ning of Nankai University (Tianjin, China). Cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin /streptomycin (P/S) and were cultured under 37°C and 5% CO₂. Macrophage RAW264.7 cell line is a gift from professor Ying Sun, and Ana-1 cell line is a gift from professor De-Shan Zhou of Capital Medical University (Beijing, China). These two macrophage cell lines were cultured in sterile RPMI 1640 medium containing 10% fetal bovine serum (FBS) and incubated at 37°C, 5% CO₂. Cells were cultured and generated for more than two generations and digested for following experiments.

2.4 Mouse genotyping

0.5-1cm mouse tail was cut and digested for genomic DNA solution. 50ul volume of DNA solution was removed from preceding solution, the double volume of 100% alcohol was added, and DNA was extracted visible to the naked eye. 500ml 75% alcohol was used to wash it, and finally, DNA was prepared to be diluted to a certain concentration.

2.5 Breast cancer model mice

6–8 week-old healthy female Balb/C background wild type mice (WT as control group) and Fstl1^+/− mice (as experimental group) were age-matched and weight-matched to construct the animal model of breast cancer. On the 0th day, the mice were anaesthetized with pentobarbital sodium 0.25 mg/kg and injected 1×10^6 4T1 cells into the second fat pad under the stereoscope. The mice were subsequently housed and fed in individual ventilation cages (IVC). On the 28th day, mice were euthanized. Tumors in situ were cut off subsequently, tumor weights were measured and lungs were removed from mice thoracic cavity preparing for the following total mRNA, protein assay, and other experiments.

2.6 Quantitative real-time polymerase chain reaction (q-PCR) and reverse transcription polymerase chain reaction (RT-PCR)

β-actin primers, Forward: 5’-CATCCGTAAAGACCTCTATGCCAAC-3´; Reverse: 5’-ATGGAGCCACCGATCCACA-3´; IL-4 primers, Forward: 5´-GGTCTCAACCCCCAGCTAGT-3´; Reverse: 5´-GCCGATGATCTCTCTCAAGTGAT-3´; IL-13 primers, Forward: 5´-CCTGGCTCTTGCTTGCCTT-3´; Reverse: 5´-GGTCTTGTGTGATGTTGCTCA-3´; Arg-1 primers, Forward: 5´-CAAGGTGATGGAAGAGACCTT-3´; Reverse: 5´-TAAGGTAGTCAGTCCCTGGCTT-3´; IL-10 primers, Forward: 5´-ATGCTGCCTGCTCTTACTGACTG-3´; Reverse: 5´-CCCAAGTAACCCTTAAAGTCCTGC-3´; CCL2 primers, Forward: 5´- TTAAAAACCTGGATCGGAACCAA-3´; Reverse: 5´- GCATTAGCTTCAGATTTACGGGT-3´; CCL5 primers, Forward: 5´-GCTGCTTTGCCTACCTCTCC-3´; Reverse: 5´-TCGAGTGACAAACACGACTGC-3´;

CSF-1 primers, Forward: 5´-GTGTCAGAACACTGTAGCCAC-3´; Reverse: 5´-TCAAAGGCAATCTGGCATGAAG-3´; VEGF-α primers, Forward: 5´- GCACATAGAGAGAATGAGCTTCC-3´; Reverse: 5´-CTCCGCTCTGAACAAGGCT-3´; TGF-β primers, Forward: 5´- CTCCCGTGGCTTCTAGTGC-3´; Reverse: 5´-GCCTTAGTTTGGACAGGATCTG-3´. PCR analyses was performed as described previously [23].

2.7 Western blot analyses

The lung tissues and breast tissues were lysed in RIPA buffer (Applygen, Beijing, China) containing a proportional mixture of protease inhibitor (1:50) and protein phosphatase inhibitor (1:100). Detection of mouse Fstl1 antibody (Catalog: AF1738, R&D Systems, dilution 1:1000), β-actin (Catalog: 4970, Cell Signaling Technology, dilution 1:2000), Arg-1 (Catalog: sc-271430, Santa Cruz Biotechnology, dilution 1:1000), TGF-β (Catalog: ab66043, Abcam, dilution 1:1000), Smad2/3 (Catalog: 8685, CST, dilution 1:1000), P-Smad2/3 (Catalog: 8828S, CST, dilution 1:1000), Smad1/5/8 (Catalog: sc-6031-R, Santa Cruz, dilution 1:1000), P-Smad1/5/8 (Catalog: sc-12353, Santa Cru, dilution 1:1000), Smad1/5 (Catalog: sc-6031-R, Santa Cruz, dilution 1:1000); P-Smad1/5 (Catalog: 9516T, CST, dilution 1:1000), MMP-9 (Catalog: ab58803, Abcam, dilution 1:1000) was performed. Western blot analyses were performed using standard protocols as described previously [23].

2.8 Flow cytometry

Cytometric analyzed single-cell suspensions were prepared from lung tissues. The lung tissues were cut into 2–4 mm³ and placed in a sieve for mechanical digestion. To identify immune cell populations, cells were first incubated with Fixable Viability Stain 450 (FVS450) for 15 minutes, and then they were incubated with a monoclonal antibody CD16/32 for 10 minutes to block cellular Fc receptor. Ultimately cells were stained with PE anti-mouse F4/80 antibody and FITC anti-mouse CD206(MMR) antibody for 60 minutes. CD206 is a transmembrane protein and needed both cellular surface staining and intracellular staining. If necessary, the samples were sustained overnight using 2% paraformaldehyde (PFA). Data were integrated by Tree Star Flowjo software.

2.9 Macrophage migration experiment

To mimic the lung tumor microenvironment, the transwell (pore size: 8.0µm) was used to study the co-culture of 4T1 breast cancer cells and RAW264.7/Ana-1 macrophages. RAW264.7/Ana-1 cells (3×10^4 cells/well, 150ul 1640) were placed in the upper well while 4T1 cells (1.2×10^5 cells/well, 500ul complete medium containing 10%FBS) were placed in the lower well. RAW264.7/Ana-1 macrophages:4T1 cells = 1:4. Fstl1 and IL4 were administered into the lower solution separately or both. After 12h co-culture, the 8.0µm transwell was washed and stained by 4% paraformaldehyde (PFA) containing 0.1% crystal violet for 30 mins. 5 fields per slide were observed with 100× magnification and migrated macrophages were calculated using Photoshop software compared with blank control.

2.10 Cell proliferation assays

4T1 breast cancer cells were grown and digested by 0.25% trypsin for cell proliferation analysis. 4T1 cells (1.2×10^5 cells/well) were inoculated in 96-well plates and were cultured at 37°C, 5% CO₂ for three time points (12h, 24h, 48h), treated with different concentrations of recombinant mouse Fstl1 protein (0ng/ml, 250ng/ml, 500ng/ml and 1000ng/ml). Ultimately, the absorption data were assayed by enzyme-labelled instrument for every single time point under the particular OD value (450nm).

2.11 Hematoxylin-Eosin staining

Paraffin embedded lung tissue was cut into sections. The lung tissue sections were put into distilled water and were dyed in hematoxylin solution for several minutes. Then they were put into the acid water and ammonia water for color separation, a few seconds each. Followed rinsing with water for 1 hour and then with distilled water for a while. Sections were dehydrated in 70% and 90% alcohol for 10 minutes each and stained by alcohol eosin staining solution for 2–3 minutes. The stained sections were dehydrated by pure alcohol and were transparented by xylene. Finally, dropping gum on the sections and putting the cover glass to seal it.

2.12 Wound healing experiment

4T1 cell migration was evaluated by the wound healing experiment, also known as the “scratch” assay. The experiment was divided into 3 groups including 4T1 group, 4T1 negative control (4T1NC) group and 4T1 overexpressed (4T1OE) group. When the cells reached 90% confluence, a scratch was made through each well using a 100ul sterile pipette tip. Cells were monitored with 100× magnification at 0h, 12h and 24h after wounding. Images of cells were captured at the same marked position to document the repair process. The experiment had been repeated for 3 times.

2.13 Immunohistochemistry (IHC) assay

Firstly, lung samples were pretreated in 4% formaldehyde fixed for 24h and were immersed in 70% alcohol saved for 24h. Paraffin-embedded lung sections were deparaffinized in xylene, dehydrated through gradient ethanol and then heated in citrate buffer (pH ≈ 6.0) for antigen retrieval. After blocked with 3% H₂O₂ for 15 mins, the tissue sections were incubated at 4°C overnight with Arg-1 antibody (Catalog: sc-271430, Santa Cruz Biotechnology, dilution 1:500), followed by incubation with HRP-conjugated secondary antibody (ZSGB-bio, Beijing, China) at 37°C for 30 mins. After washing three times with PBS, color was developed using DAB Chromogen (ZSGB-bio, Beijing, China). Slides were rinsed in tap water and counterstained with hematoxylin. Five random fields per section were viewed under the microscope.

2.14 Database

UALCAN is used for digging TCGA (The Cancer Genome Atlas) data and CPTAC (Clinical Proteomic Tumor Analysis Consortium) data. Website address: http://ualcan.path.uab.edu. Kaplan-Meier survival analysis is capable to access the survival rate of 21 cancer types including breast cancer. Website address: https://kmplot.com/analysis/.

2.15 Statistical analyses

Statistical analysis was performed using Microsoft excel 2019 (Microsoft Corp.), GraphPad Prism 6.0 (GraphPad Software Inc.) and Image J. All results were calculated as mean ± standard deviation and all analysis were considered p < 0.05 statistically significant. All graphs show mean ± SD.

3.1 Fstl1 barely affected proliferation of TNBC in situ but enhanced its metastasis to the lung

To investigate the functional mechanism of Fstl1 in breast cancer, we established a breast cancer lung metastasis model in mice as described above. WT mice served as a control group, and Fstl1^+/− hemizygous mice as an experimental group (Fstl1^−/− homozygous mice were unable to breathe after birth and subsequently died [22]). Moreover, primary tumor growth in situ of WT and Fstl1^+/− mice had no difference in primary breast cancer tissue weights (Fig. 1A) compared with WT mice. However, lung tissues removed from Fstl1^+/− mice had been confirmed to present more metastatic nodules than WT group (Fig. 1B). Furthermore, pulmonary metastasis on the HE-staining section of WT and Fstl1^+/− mice groups apparently proved the former argument (Fig. 1C). Given that 4T1 expressed no Fstl1 (Supplementary Fig. 1A) and Fstl1 expression decreased in metastatic lungs of Fstl1^+/− mice (Supplementary Fig. 1B), we treated 4T1 cells with recombinant mouse Fstl1 protein to test its biological behaviors, and the result showed that Fstl1 had no effect on the proliferative ability of 4T1 cells (Fig. 1D). We analyzed several Epithelial-Mesenchymal Transition (EMT) markers including N-Cadherin and E-Cadherin expression in 4T1 cells treated with different Fstl1 concentrations, which also showed no difference between control and Fstl1 treated group (Fig. 1 1E). Fstl1 overexpressed 4T1 cells also showed no significant difference of cell proliferative and migrative ability compared with 4T1 cells and 4T1 negative control (Supplementary Fig. 2A, Supplementary Fig. 2B, Supplementary Fig. 2C). Those evidences indicated that Fstl1 might accelerated lung metastasis by shifting the microenvironment at lung metastatic sites.

3.2 M2-like macrophages increased in the lung tissues during breast cancer lung metastasis in Fstl1^+/− mice compared with WT mice

As hypothesized that Fstl1 played a vital role in the tumor microenvironment, and macrophages are described as a large population of immune cells and a kind of important components in tumor microenvironment [25], therefore we next investigated macrophage ratios in the lung on the 0th day and the 28th day. As expected, the result displayed that total macrophage and had no difference between the two groups on the 0th day (Fig. 2A, Supplementary Fig. 3A), either the M2-like macrophages ratios (F4/80 + CD80-CD206+) by flow (Fig. 2C, Supplementary Fig. 3C). However, both total and M2-like macrophages significantly increased on the 28th day lung tissues of Fstl1^+/− mice (Fig. 2B, Fig. 2D, Supplementary Fig. 3B, Supplementary Fig. 3D). Furthermore, M2 polarization stimulators (IL4 and IL13 gene expression) increased in the 28th lung tissues of Fstl1^+/− mice compared with tumor bearing WT mice and normal lung tissues (Fig. 2E, Fig. 2F). M2-like macrophages related markers (Arg-1 and IL10 gene expression) also remarkably increased on the 28th day (Fig. 2G, Fig. 2H). M2-like macrophages secreted protein markers Arg-1 gene expression were also significantly upregulated in lung tissues of Fstl1^+/− mice to support fore-mentioned experimental results (Fig. 2I, Fig. 2J). Above analyzed data showed that Fstl1 deficiency promoted the accumulation of F4/80 + CD80-CD206+ (M2-like) lung macrophages in the tumor microenvironment.

3.3 Fstl1 exhibited no effects on CCL2 and CCL5 but inhibited CSF-1, TGFB1 and VEGF-α expression of 4T1 cells

We detected monocyte populations within the blood to distinguish the source of increasing lung macrophages. The blood routine test displayed no significant difference between WT and Fstl1^+/− mice on the 0th and 28th day. Nevertheless, we discovered that on the 14th day, blood monocytes of Fstl1^+/− mice had an elevating tendency compared with WT mice. To investigate how Fstl1 modulated the interaction relationship between blood monocytes, M2-like TAMs and breast cancer cells during breast cancer lung metastasis, we treated 4T1 cells with Fstl1/IL4 and detected some associated chemokine markers of monocytes and M2-like TAMs by q-PCR (Fig. 3D, Fig. 3G). Fstl1 could not exert any suppressive effects on monocytes related chemokines during this process, though CCL2 (Fig. 3E, Fig. 3H) and CCL5 (Fig. 3F, Fig. 3I) mRNA expression significantly increased with IL4 treatment. On the contrary, we found that Fstl1 could suppress CSF-1 (Fig. 5A), TGFB1 (Fig. 5B), and VEGF-α (Fig. 5C) mRNA expression induced by IL4 protein to a distinct degree. Thus, we demonstrated that Fstl1 could inhibit CSF-1, TGFB1 and VEGF-α gene expression produced by 4T1 breast cancer cells to slow down the intercellular crosstalk process.

3.4 Fstl1 attenuated M2-like tumor-associated macrophages migrated towards 4T1 breast cancer cells

To mimic macrophage recruitment and the crosstalk between immune cells and cancer cells in vivo, we designed co-culture transwell experiments utilizing the macrophage cell lines in the upper well and the 4T1 cells in the lower well (Fig. 4B, Fig. 5E). The experiments revealed that the migration ability of macrophages showed no difference after 1ug/ml Fstl1 treatment with or without 20ng/ml IL4 combined reaction (Fig. 4A, Fig. 4C, Fig. 5D, Fig. 5F). It's worth noting that the migrated macrophages significantly increased in the 4T1 group, but those migrated cells reduced dramatically after treated with Fstl1 protein for 12h co-culture (Fig. 4A, Fig. 4C, Fig. 5D, Fig. 5F). Those results indicated that 4T1 cells are one of chemotactic factors to macrophage migration, and Fstl1 could slow down the intercellular crosstalk process. Consistently, the migration cells increased after treating 4T1 cells with IL4 protein while reduced in the 4T1 + IL4 + Fstl1 groups (Fig. 4A, Fig. 4C, Fig. 5D, Fig. 5F), without regard to the proliferative effect of Fstl1 and IL4 cytokine on 4T1 cells or RAW264.7 macrophages (Fig. 4D, Fig. 4E). Exactly as IL4 is a M2-like TAM stimulator, those results furtherly demonstrated that Fstl1 might be a potential suppressor of M2-like TAM migration.

3.5 Fstl1 diminished arginase-1 expression in RAW264.7 macrophages in vitro and elevated arginase-1 in lungs of Fstl1^+/− mice in vivo

We treated RAW264.7 macrophages with IL4 protein and detected arginase-1 expression, one of characteristic markers of M2-like TAMs by q-PCR. Arginase-1 mRNA expression was markedly increased after IL4 stimulation (Fig. 6A). Meanwhile, low concentration of Fstl1 (250ng/ml) could regulate arginase-1 protein expression, negatively and effectively (Fig. 6B, Fig. 6C). Additionally, we observed that arginase-1 expression was elevated in lung metastatic tissues of Fstl1^+/− mice and distributed surrounding around tumor metastatic site. Interestingly, comparing with Fstl1^+/− mice, it was obvious that arginase-1 marked cells were accumulating towards lung metastatic tumor at a much slower speed in WT mice (Fig. 6D). On the 28th day, more MMP-9 expression in Fstl1^+/− mice was produced for degrading and remodeling extracellular matrix contributing to lung metastatic progression, which was mainly derived from recruited macrophages at advanced stage of breast cancer (Fig. 6E, Fig. 6F).

3.6 Fstl1 affected relevant 4T1 characteristics via TGFB1/Smad2/3 but not BMP4 signaling pathway

As previously demonstrated that Fstl1 had no effect on 4T1 biological behaviors such as its proliferative and migrative abilities, we further discovered that several genes expression of 4T1 cells could be functionally reduced by recombinant Fstl1 treatment. To explore the potential underlying mechanism of the phenomenon, two classical pathways of the transforming growth factor (TGF) superfamily were investigated in the following experiments. As the functional ligand, TGF-β homodimer in lung tissues of Fstl1^+/− mice was much higher than in WT mice compared with TGF-β monomer (Fig. 7A, Fig. 7B). The downstream Smad2/3 protein appeared the same changing tendency in lung tissues of Fstl1^+/− mice (Fig. 7C, Fig. 7D). Cellular experiments showed that 4T1 cell itself did not express phosphorylated-Smad2/3 and did not response to Fstl1 treatment, whereas Fstl1 could inhibit the P-Smad2/3 expression induced by TGFB1 protein (Fig. 7G). However, Fstl1 did not affect the BMP4/Smad1/5 pathway, either in lung tissues or in 4T1 cells (Fig. 7E, Fig. 7F, Fig. 7H).

3.7 Fstl1 mRNA expression decreases in human breast cancer and its various subtypes

Our laboratory had previously reported that Fstl1 deficiency could accelerate the growth of breast cancer cells in lung metastatic sites. To confirm the suppressor role of Fstl1 in human breast cancer, we collected invasive breast cancer samples from TCGA samples and sorted them into different types. The results showed that Fstl1 significantly decreased in all the invasive breast cancer compared with normal breast tissues (Fig. 8A). All individual cancer stages of invasive breast cancers were remarkably lacking Fstl1 expression compared with normal breast tissues (Fig. 8B). The similar result occurred in different nodal metastasis status (Fig. 8C). Furthermore, invasive breast cancer tissues sorted by luminal, HER2, and triple-negative showed a lower Fstl1 expression compared with normal breast tissues (Fig. 8D). Considering that Fstl1 is deficient in all types of human breast cancer, we regard Fstl1 as a possible human breast cancer suppressor.

3.8 Breast cancer patients with higher Fstl1 expression show relative prolonged survival rate

To further predict the role of Fstl1 in breast cancer, we collected mRNA expression data of invasive breast cancer tissues from TCGA samples and grouped them into different types. As shown in Fig. 8E, though breast cancer patients from the TCGA database with high Fstl1 expression had the same survival rate before 4000 days, it seemed to have a better survival rate in a more extended period. Further, there were fewer breast cancer patients with high-level Fstl1 and better survival (Fig. 8F). Another survival curve dawn by the Kaplan Meier plotter showed that overall survival rate (OS) decreased dramatically in lower Fstl1expression breast cancer patients compared with the higher groups (Fig. 8G). The same trend appeared in breast cancer patients with positive metastasis (Fig. 8H). Therefore, those pieces of evidence indicated that Fstl1 could be potentially benefiting for breast cancer prognosis, especially for TNBC patients.

This research was a sequential extension of our previous research. Our previous data had elucidated that deficiency of Fstl1 could accelerate the growth of breast cancer lung metastatic tumor but not primary tumor growth [19]. However, the underlying mechanism was still unclear. In our following research, 4T1 breast cancer cells expressed no Fstl1 and its proliferation and EMT markers had no change after recombinant Fstl1 treatment, thus we speculated that different cell types expressed Fstl1 and different cancer microenvironment might be the key cause of animal breast cancer primary tumor and metastatic tumor respective growth status. In the present research, we firstly analyzed the potential function of Fstl1 in breast cancer patients based on the public database. The results indicated the potential benefit of Fstl1 for breast cancer clinical patients, especially for poorly prognostic TNBC patients. Next, we confirmed that Fstl1 did not affect the proliferation of 4T1 cells in vivo and in vitro again. However, metastatic cancer in the lung increased in the Fstl1 deficiency mice. Therefore, we hypothesized that Fstl1 might exert an immune suppressive effect on the lung microenvironment to affect breast cancer progression.

The tumor microenvironment (TME) remains poorly understood due to its complex components, especially the immunity status. However, tumor-associated macrophages (TAMs) are one of predominant components [24–26]. TAMs are essential initiator of tumor progression, metastasis, and resistance to therapy. There are anti-tumor M1-like and pro-tumor M2-like TAMs that coexisted in TME [27]. Hence, the percentage of M2-like TAMs in TME directly impacts the metastasis of cancer. Our result displayed M2-like TAMs significantly increased in the lung of Fstl1 heterozygous mice during breast cancer lung metastasis (BCLM). Furthermore, IL4 and IL13, the stimuli of M2-like TAMs [28–29], also increased in the metastatic lung tissues. Accordingly, Arg-1, IL10, MMP-9 and TGF-β, the secretor of M2-like TAMs [11, 30–31], upregulated dramatically in the metastatic lung tissues. Those results demonstrated that Fstl1 was a suppressor of M2-like TAMs in the lung TME. To simulate in vivo lung microenvironment, we designed a co-culture transwell experiment in vitro. IL4 served as a macrophage and 4T1 cell stimulator, while 4T1 cells performed as an attractive factor. The cellular results showed that Fstl1 could reduce macrophages attracted by 4T1 cells and IL4-treated 4T1 cells.

In historically typical course of TAMs polarization, CCL2 and CCL5 chemokines recruited monocytes from the circulation system to the targeted organ [32]. The monocytes infiltrated in the targeted organ were polarized to M2 subtype stimulated by several cytokines (such as IL4, IL10, CSF-1, TGFB1 and VEGF-α) [33–36]. Our research found that the 4T1 cells expressed CCL2/CCL5, CSF-1, TGFB1 and VEGF-α, and those factors significantly increased after IL4 treatment. Furthermore, Fstl1 could inhibit CSF-1 and TGFB1 gene expression induced by IL4. Unfortunately, CCL2 and CCL5 chemokines had no change in this study. Thereafter, our research had drawn a relatively orchestrated pattern to elucidate how Fstl1 inhibited the interaction between breast cancer cells and M2-like TAMs. Collectively, we illustrated the increased lung metastasis of breast cancer in Fstl1 heterozygous mice. 4T1 cancer cells could secrete CCL2/CCL5, CSF-1, TGFB1 and VEGF-α, which are stimuli of M2-like TAMs. CCL2 and CCL5 recruited monocytes from the circulation system to the lung tissues. Then CSF-1, TGFB1 and VEGF-α promoted monocyte/macrophage recruitment and monocytes differentiation towards M2-like TAMs, which expressed Arg-1 and IL10 to promote breast cancer progression through immunosuppressive microenvironment. Afterwards, more CCL2/CCL5, CSF-1, TGFB1 and VEGF-α cytokines were released by 4T1 cancer cells. As a consequence, a circle was formed between breast cancer cells and M2-like TAMs. Fstl1 restrained CSF-1 and TGFB1 mRNA expression to blockade the circle, modulate their downstream molecules and finally reduce breast cancer lung metastases.

Triple-negative breast cancer (TNBC) has the characteristics of aggressive biological behaviors and highest risk of distant recurrence [37–39]. Immune checkpoint inhibitors like anti-programmed cell death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) agents are in the course of TNBC investigation. And tumor-associated macrophages (TAMs) have been reported to modulate PD-1/PD-L1 expression in cancer microenvironment [40]. Our laboratory had been previously reported that Fstl1 deficiency could impair T cell development in thymuses and decrease T cell ratios in lungs [41], and also had been next discovered to decrease M2-like TAM ratios in lung metastatic sites. As reported that M2-TAM subsets could be redistributed by lactic acid level to upregulate PD-L1 level and assist tumor immune escape [42], it’s worthwhile looking forward to Fstl1 immune checkpoint value in breast cancer immunotherapy as TAM agonist or antagonist cellularly or molecularly.

Ethical Approval and Consent to participate

The experimental animal department had passed the ethical review.

Consent for publication

When research is accepted for publication, agreed to publish using either immediate gold open access or the traditional publishing route.

Availability of supporting data

Materials in the manuscript are the original and available work of the authors.

Competing interests

The authors declare no conflicts of interest, financial or otherwise.

Funding

This study was supported by the Natural Science Foundation of Beijing Municipality (5202004)

Authors' contributions

GY conceived the experiments. YY and TL performed the experiments and analyzed the experimental data and generated the figures. YY and JXW wrote the manuscript. All of the authors performed literatures review and approved the final manuscript.

Acknowledgements

We thank Prof. Ying Sun and Prof. De-Shan Zhou for the gift of macrophage RAW264.7 cell line and Ana-1 cell line, respectively.

Authors' information

ying yang <[email protected]>, Tao Lu <[email protected]>, Xiao-wei Jia <[email protected]>, Yan Gao (corresponding author) <[email protected]>.

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Fstl1 suppresses breast cancer lung metastasis by inhibiting M2-like tumor-associated macrophages recruitment towards the lung

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Reagents and facilities

2.2 Experimental animals

2.3 Experimental cell lines

2.4 Mouse genotyping

2.5 Breast cancer model mice

2.6 Quantitative real-time polymerase chain reaction (q-PCR) and reverse transcription polymerase chain reaction (RT-PCR)

2.7 Western blot analyses

2.8 Flow cytometry

2.9 Macrophage migration experiment

2.10 Cell proliferation assays

3 Results

3.1 Fstl1 barely affected proliferation of TNBC in situ but enhanced its metastasis to the lung

3.4 Fstl1 attenuated M2-like tumor-associated macrophages migrated towards 4T1 breast cancer cells

3.6 Fstl1 affected relevant 4T1 characteristics via TGFB1/Smad2/3 but not BMP4 signaling pathway

3.7 Fstl1 mRNA expression decreases in human breast cancer and its various subtypes

3.8 Breast cancer patients with higher Fstl1 expression show relative prolonged survival rate

4 Discussion

Declarations

References

Additional Declarations

Supplementary Files

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