Introduction
Tumor metastasis to distant organs is the main cause of mortality in breast cancer (BC) patients [
1]. It is regulated by complicated processes that include the separation of tumor cells from the primary tumor, invasion into nearby blood and lymphatic vessels, extravasation from the vessel lumina into the parenchyma of distant organs, and subsequent growth from micrometastatic lesions into macroscopic tumors [
2]. Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and HER2/ERBB2 expression and known for being aggressive and having a poor prognosis [
3]. Because the therapeutic targets of TNBC are unknown, chemotherapy remains the mainstay of the treatment, even if some patients may not fully respond to chemotherapies [
4]. Therefore, identifying a possible target for TNBC therapy is an urgent need.
Tumor cells are heterogeneous, and not all subclones have the capacity to metastasize. This notion is supported by the successful cloning of a few highly metastatic cells from human and mouse tumors [
5]. Although the majority of subclones may not metastasize, they could contribute to the process by releasing tumor promoting factors that increase the capacity of highly metastatic cells and/or provide a favorable tumor microenvironment (TME). Several studies have shown that exosomes derived from TNBC cells promote the metastatic processes by transferring various information (i.e., RNA, DNA, and proteins) to recipient cells in original tumors or distant colonized organs [
6,
7]. Exosomes are a subtype of extracellular vesicles produced during the maturation of reticulocytes [
8], with a size of 30–150 nm and genesis in multivesicular bodies [
9].
The Wnt/β-catenin signaling pathway has been demonstrated to be involved in cancer cell proliferation [
10], metastasis [
11], stemness maintenance [
12], and cancer-associated systemic inflammation [
13].
Wnt1, originally called
Int1, is the first member of the Wnt family and was discovered as a potential cellular oncogene involved in mouse mammary tumor virus-induced mammary tumors [
14]. There have been 19 distinct Wnt ligands identified in the last four decades. Wnt proteins bind receptors, such as Frizzled and/or low-density-lipoprotein receptor-related protein 5/6, and activate either the β-catenin-dependent (canonical) or independent (non-canonical) signaling pathways, and the expression of Wnt receptors is elevated in BC, especially in TNBC [
15]. Exosomes were previously shown to transport Wnt ligands on their surface via the Evi exosomal protein and trigger signaling in target cells [
16,
17]. Even though an increasing number of studies are focused on the impact of aberrant Wnt signaling in BC, no Wnt inhibitors have been utilized in treatment to date.
The murine 4T1 cell line is a murine TNBC cell line derived from a mammary tumor that spontaneously developed in a BALB/c mouse foster-nursed by a C3H female mouse (BALB/cfC3H) [
18,
19]. In female BALB/c mice, 4T1 cells implanted at the orthotopic mammary fat pad spontaneously metastasize to multiple organs, including the bone, lung, and liver. 4T1 cells are also a mixture of subclones with different gene expression profiles and metastatic potentials [
20]. Several studies have been performed to evaluate the heterogeneity and to classify various subpopulations among 4T1 cells based on cell properties, such as cellular morphology [
21], cell proliferation, cancer stem cell-like [
22], and metastatic ability [
20]. Thus, 4T1 cells are an excellent model to explore the mechanisms of BC metastasis and study tumor cell heterogeneity. We previously observed that the levels of lung metastases by single-cell cloned 4T1 cells were significantly lower than those by parental 4T1 cells [
23], leading us to the hypothesis that interactions among 4T1 subclones, potentially via secretion of exosomes, are essential for the maximal lung metastasis by highly metastatic 4T1 subclones.
In this study, we tested the hypothesis by establishing two 4T1 subclones with low metastatic (LM) and high metastatic (HM) capacity and performing a series of experiments both in vitro and in vivo. Our study demonstrates, for the first time, that LM.4T1 cells have the capacity to enhance the metastatic ability of HM.4T1 cells via exosomal Wnt7a and Wnt7a is potentially a target for the treatment of TNBC patients. Our results also indicate that interclonal communications among cancer cell subclones play an import role in cancer progression.
Materials and methods
Cell lines
Murine breast cancer 4T1 cell line was obtained from American Type Culture Collection (ATCC, USA). The parental 4T1 and single-cell clone cells were grown in RPMI 1640 medium (Sigma-Aldrich, USA), supplemented 10% fetal bovine serum (FBS) (HyClone, USA), 100 μM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM sodium pyruvate (Wako, Japan) (complete medium).
Antibodies
Antibodies used in this study are listed in Additional file
1: Table S1.
Conventional RT-PCR and Real-time quantitative RT-PCR
For gene expression analysis, total RNA was extracted from cells and tumor tissues by using High Pure RNA Isolation Kit (Roche, Mannheim, Germany) or TRITMsure (Nippon Genetics, Tokyo, Japan) reagent. The complementary DNA (cDNA) was synthesized with the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA). KOD FX DNA polymerase (Toyobo, Osaka, Japan) was used in conventional RT-PCR for detection of Wnt7a expression in LM.4T1 and HM.4T1 cells. Annealing temperature was 57℃ for Wnt7a and β-actin. The primers sequences were as follows: Wnt7a: forward, 5′-GACAAATACAACGAGGCCGT-3′, reverse, 5′-GGCTGTCTTATTGCAGGCTC-3′. β-actin: forward, 5′-CAGCTGAGAGGGAAATCGTG-3′, reverse, 5′-CGTTGCCAATAGTGATGACC-3′. Real-time quantitative RT-PCR was performed using the Applied Biosystems Step One™ Real-Time PCR System (Life Technologies, Gaithersburg, MD). The expression of the Il6, Il1b, Tgfb1, Tnfa, Vegfa, Hgf, and Cola1a genes was analyzed by Taqman gene expression assay (Applied Biosystems, Foster City, CA). The total volume of each reaction was 10 µl containing 5.2 µl TaqManTM ProAmpTM Master Mix reagent, 0.4 µl primer and 2.8 µl UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, Carlsbad, CA). The primers of Wnt7a with probe for real-time PCR assay were purchased from Integrated DNA Technologies (Singapore, Republic of Singapore). The expression level of each gene was normalized to that of the Gapdh gene and presented as fold change over the expression of the control gene.
Western blotting
For total protein isolation, cells were lysed in a lysis buffer (Cell Signaling, Danvers, MA) containing protease inhibitor cocktail (Roche) and Halt™ Phosphatase inhibitor cocktail (ThermoFisher), incubated on ice for 30 min, and then centrifuged at 14,500×g for 10 min. Exosomes were resuspended in radioimmunoprecipitation assay (RIPA) buffer for western blot analysis. Tumor tissues were smashed in RIPA buffer containing phenylmethanesulfonyl fluoride. For assessment of β-catenin activation, nuclear and cytoplasmic extracts were prepared using a NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher) according to the manufacturer’s instructions. The protein concentrations were measured by BCA Protein Assay Kit (Takara, Tokyo, Japan). Equal amounts of protein samples were denatured at 100℃ for 10 min with 4 × NuPAGE LDS sample buffer and 10 × Sample Reducing agent (Invitrogen). Proteins (3 μg for exosome, 15–30 μg for cell lysates) were separated by 4–12% NuPAGE Bis–Tris precast gel (ThermoFisher) and transferred onto nitrocellulose blotting membranes (GE Healthcare Life science, Freiberg, Germany). The membranes were blocked with 5% milk in tris-buffer saline-Tween 20 for 1 h with at room temperature. After overnight incubation with a primary antibody at 4℃, the membrane was washed with TBS-T for 10 min three times, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. After secondary antibody incubation, the membranes were washed again for 10 min three times and the presence of the protein of interest was visualized and quantitated with C-DiGit Blot scanner (Scrum, Tokyo, Japan).
For spheroid generation, 1 or 2 × 103 cells were seeded in ultra-low attachment 96-well round-bottomed plates (Corning, Corning, NY) with sphere-forming medium DMEM/F12 (Invitrogen) supplemented with 1 × B27 serum substitute (Invitrogen), 20 ng/ml mouse recombinant epidermal growth factor (PeproTech, Rocky Hill, NJ) and 10 ng/ml basic fibroblast growth factor (PeproTech). HM.4T1 spheroid were stimulated with 100 ng/ml recombinant human Wnt7a protein (Abcam, UK) or 100 ng/ml recombinant mouse Wnt3a protein (BioLegend, San Diego, CA). To inhibit PI3K or mTOR, spheroids were pre-treated for 1 h with 10 μM LY294002 or 50 nM rapamycin, and then, 100 ng/ml Wnt7a was added. The size of spheroids was evaluated at 24 h, 48 h, and 72 h. Cell lysates were prepared at 6 h or 24 h after addition of Wnt7a. The radius of each spheroid was measured by using the Image J software, and the volume (μm3) was calculated by the following formula: V = 4/3πr3.
We performed an invasion assay using the generated tumor spheroid. After 4 days of culture, the sphere-forming medium in each well was aspirated and switched to 100 μl Matrigel Matrix (Corning). Each spheroid was ensured to be in the center of the well. After 1-h incubation at 37 °C, each well was covered with 100 μl sphere-forming medium. The invasion area was monitored by IX2-SLP OLYMPUS microscope (Olympus, Tokyo, Japan), captured and measured by using the Image J software.
Immunofluorescence
One thousand cells were seeded into a Lab-Tek II chamber slide (Nalge Nunc, Rochester, NY). When cells became 60% confluent, they were stimulated with 100 ng/ml recombinant human Wnt7a protein or 100 ng/ml recombinant mouse Wnt3a protein. After 24-h incubation, medium was aspirated, and the cells were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were washed with phosphate-buffered saline (PBS) 5 min for three times, permeabilized with 0.5% Triton X-100 at room temperature for 20 min, washed with PBS 5 min three times, blocked with PBS containing 1% bovine serum albumin for 1 h at room temperature, and incubated with anti-β-catenin Ab at 4℃ overnight. After washing with PBS containing 0.1% TweenTM20 for 5 min three times, cells were incubated with Alexa Fluor 488-conjugated secondary antibody in the darkness for 1 h at room temperature. After washing for 5 min three times, slides were mounted using the ProLong™ Gold antifade mountant with DAPI (ThermoFisher). Images were acquired using fluorescence microscope BZ-X700 (Keyence, Tokyo, Japan).
Immunohistochemistry
Tumors were fixed overnight in 10% formalin and embedded in paraffin. Immunostaining was performed manually by a conventional method: Briefly, sections were deparaffinized in xylene and rehydrated in a sequence of descending concentrations of ethanol. Endogenous peroxidase reactivity was blocked with 3% H2O2 for 10 min. For antigen retrieval, sections were submerged in 10 mM citrate buffer (pH6.0) or 5 mM EDTA solution (pH8.0) and microwaved (700 W) continuously for 15 min in a pressure cooker and then incubated with a respective primary antibody for 1.5 h at room temperature. After washing, sections were incubated with an appropriate secondary antibody conjugated with horseradish peroxidase (Nichirei, Tokyo, Japan) and the signals were visualized using DAB (Dako, Santa Clara, CA) according to the manufacturer’s instructions. Finally, sections were counterstained with hematoxylin, dehydrated, and mounted. Images were acquired using an Olympus BX43 light microscope connected to a DP73 digital camera (Olympus).
Exosome isolation
Cells were grown to 80% confluent in complete medium and then in RPMI 1640 medium containing 2% exosome-depleted FBS for 48 h. The medium containing exosomes was collected and centrifuged for 10 min at 500×g, 20 min at 2000×g, and 30 min at 10,000×g. The exosomes pellet was harvested after 70 min of ultracentrifugation at 100,000×g (Beckman, Fullerton, CA) and then washed with PBS and ultracentrifugation again at 100,000×g for 70 min. Exosomes were then resuspended in PBS or RIPA for further studies. The concentration of exosomes suspension was measured by using Pierce™ BCA protein assay kit (Takara).
Transmission electron microscopy (TEM)
Exosomes were resuspended in PBS to be used for TEM analysis. Hydrophilic treatment was performed on a 400-mesh copper grid coated with formvar/carbon films. Exosome suspension (10 μl) was placed on Parafilm, and the grid was floating on top of the exosome suspension was left for 15 min. The sample was negatively stained with 2% uranyl acetate solution for 2 min. The grids were washed five times with PBS after each staining step and finally ten times in H2O before contrast staining with 2% uranyl acetate solution. Exosomes on the grid were imaged at 20,000 times magnifications using an H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) in the Central Research Laboratory, Okayama University Medical School.
Cell scratch assay
Cell scratch assay was used to evaluate cell migratory ability. Cells were seeded in a 6-well plate and cultured to 80% confluent. Cell monolayer was scratched with a sterile 1000 μl pipette tip and washed twice with PBS, and floated cells were aspirated. Finally, 2 ml of fresh medium was added to each well and cell monolayer was photographed with a microscope. Cell migratory ability was monitored for HM.4T1 cells at 24 h, 48 h, and 72 h. Using Image J software, wound recovery (%) = 100(A–B)/B was calculated, with A and B indicating the area of cell scratches before and after incubation, respectively.
Cell proliferation assay
In a 96-well microplate, cells were seeded at a density of 2 × 103 cells per well. After 4 days of culture, 10 μl of MTT labeling reagent (Roche) was added to each well. After a 4-h incubation in a humidified environment, 100 μl of the solubilization solution (Roche) was added to each well. Plate was allowed to stand overnight in an incubator in a humidified atmosphere (+ 37 ℃, 5–6.5% CO2). After complete solubilization of the purple formazan crystals, the spectrophotometrical absorbance of the samples was measured using a microplate reader. The wavelength to measure the absorbance of the formazan product was between 570 and 690 nm.
Generation of green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing cells
LM.4T1 or HM.4T1 cells were transfected with GFP or RFP expression vector [
24] using Neon™ Transfection system (ThermoFisher) and then incubated in complete medium containing 20 μg/ml puromycin for GFP-expressing cells or 5 μg/ml blasticidin for RFP-expressing cells (InvivoGen, San Diego, CA).
Generation of Wnt7a- and Rab27a-deficient cell lines by CRISPR/Cas9 system
To stably delete the expression of Wnt7a or Rab27a in LM.4T1 and HM.4T1 cells, cells were transfected with each targeting vector using Neon™ Transfection system. We designed three sgRNA oligonucleotides for each gene and tested their effects on gene silencing. All sgRNA oligonucleotides were against the two genes, and one of each oligonucleotide was chosen for the following experiments. The sgRNA sequence for Wnt7 gene was 5′-TCCGGAGGTAGACTATGCCC-3′. The sgRNA sequence for Rab27a gene was 5′-CCACCTGCAGTTATGGGACA-3′. Seven days after transfection, medium was changed to antibiotic-containing selection medium. When single-cell clone cells grew, several clones were picked, and protein was extracted for western blot analysis. The clones that lost the expression of targeted gene were also verified by DNA sequencing. Wnt7a-deficient and Rab27a-deficient cells were grown in complete medium containing 20 μg/ml puromycin.
Tumor transplantation model
Female BALB/c mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). MD. One million tumor cells were seeded in a T-75 tissue culture flask and grown to 50–80% confluence. Cells were detached with 0.2% trypsin–EDTA, washed once with complete medium and three times with PBS, and resuspended in PBS at 1 × 106 cells/ml. One hundred and thousand cells in 100 μl PBS were injected into the third left mammary pad. Tumor tissues were excised and fixed in 10% formalin. Lungs were perfused with Bouin’s solution (Wako, Osaka, Japan) and fixed in the same solution, and then, the number of tumor nodules was counted by eye. After fixation in Bouin’s solution, subpleural lung surface metastases were easily identified by their light, white appearance. Tumor length and width were measured using a caliper, and tumor volume was calculated using the following formula: Volume = (width)2 × length/2.
LM.4T1-RFP and HM.4T1-GFP cells were implanted into nude (BALB/c-nu/nu) mice (Japan SLC, Inc.). Four weeks later, lungs were removed and metastases foci of GFP and RFP expression cells were immediately visualized using a fluorescence microscope (Olympus stereoscopic microscope, SZX12). Lungs were then fixed in Bouin's solution to detect tumor nodules. LM-Wnt7a KO, HM-Wnt7a KO, and LM-Rab27a KO cells were also implanted into the mammary pad of BALB/c-nu/nu mice.
Analysis of database
The ID of the RNA sequencing data of 23 4T1 subclones from the Gene Expression Omnibus (GEO) database is GSE63180 [
20], which we then re-analyzed using a web-application GREIN (GEO RNA-seq Experiments Interactive Navigator), and GREIN is accessible at:
https://shiny.ilincs.org/grein [
25]. We utilized the Kaplan–Meier Plotter database for survival analysis, which is available at
https://kmplot.com/analysis/ [
26].
Statistical analysis
Results were analyzed by the GraphPad Prism 9.0 software (San Diego, CA) and presented as the mean ± standard error of mean (SEM). Each experiment was carried out in technical and biological triplicate. The Student’s t test was used to compare two groups. One-way ANOVA and two-way ANOVA were used to determine multiple group comparisons. A value of p < 0.05 was deemed statistically significant.
Discussion
Intratumor heterogeneity appears in all kinds of cancer and the risk of mortality increased when more than two clones coexisted in the same tumor sample [
48]. Many prevalent mutations have been discovered in BC, resulting in genetic and phenotypic diversity. Aneuploid rearrangements occur early in tumor evolution and remain highly stable as tumor masses expand [
49,
50]. This implies that each subclone derived from BC may represent a stable biological state, while some subclones may retain the fitness advantage over others by preserving the fitness of the entire tumor or subpopulations. By using the murine TNBC cell line 4T1, we established two distinct subclones with low (LM.4T1) or high (HM.4T1) metastatic potential. In contrast to previous studies showing the ability of highly metastatic cells to assist low metastatic cells in obtaining a metastatic ability [
51‐
53], we discovered that low metastatic cells could assist lung metastasis of highly metastatic cells using at least two mechanisms: by secreting exosomal Wnt7a that increases the capacity of highly metastatic cells via activation of the PI3K/Akt/mTOR signaling pathway and by increasing angiogenesis in the tumor microenvironment for a better escape of highly metastatic cells.
The process of tumor metastasis begins with local invasion of cancer cells in the primary tumor, and epithelial–mesenchymal transition (EMT) is a critical mechanism in cancer cells invasion [
54]. Compared to LM.4T1 cells, HM.4T1 cells exhibited a mesenchymal cell morphology with a higher level of Snail expression and metastatic potential, suggesting that HM.4T1 cells are at a more advanced stage of EMT. To further investigate the components that differentiate these two types of cells, we analyzed previously reported RNA sequencing data of 23 individual 4T1 subclones in the GEO database and divided them into two subgroups, epithelial cell-like and mesenchymal cell-like, based on the gene expression correlation matrix. Wnt7a was subsequently discovered to be one of the genes differentially expressed between the groups and expressed significantly higher in epithelial cell-like than the mesenchymal cell-like subgroup. We detected a higher level of Wnt7a in LM.4T1 cells than HM.4T1 cells, leading to a question whether there is a link between Wnt7a expression and EMT. In a previous study, there was a clear correlation between the expression of the EMT suppressor grainyhead transcription factor Grhl2 and the epithelial marker E-cadherin: Grhl2 was significantly down-regulated in disseminated cancer cells that had undergone EMT [
55]. In an in vitro human mammary epithelial cell culture model, WNT7A was found to be up-regulated in cell populations expressing epithelial markers and down-regulated in cell populations expressing mesenchymal markers [
56]. Similarly, we found that a highly metastatic HM.4T1 cells with mesenchymal cell-like phenotypes expressed a low level of Wnt7a, whereas low metastatic LM.4T1 cells with epithelial cell-like phenotypes expressed a high level of Wnt7a, suggesting a link between Wnt7a expression and EMT. Interestingly, HM.4T1 cells were able to receive a significant amount of Wnt7a from LM.4T1 cells. In a
MMTV-Wnt1 transgenic mouse model, Wnt1 secreted by luminal subtype of breast cancer cells promoted the proliferation of basal-like recipient cells [
57]. Given that tumors consisted of diverse subclones, the various kinds of subclones likely interact in a paracrine way using multiple molecules and contribute to the tumor progression.
Extracellular vesicles (EVs) secreted from cancer cells function in cancer cell dissemination by working as modulators of tumor microenvironments. Different kinds of EVs are classified based on their sub-cellular origin [
58]. Exosomes, enriched in late endosome components, were defined as a small EV subtype containing the tetraspanins CD63 and CD9/CD81 proteins [
59]. So far, tumor cell-derived exosomes have been shown to be involved in nearly all steps of the invasion–metastasis cascade by activating the EMT process in neoplastic epithelial cells, fostering a premetastatic niche in distant organ, and modulating host immunity [
60]. Wnt proteins can be transferred by a paracrine mechanism, but it remains unclear how they travel in the extracellular space. In Drosophila, wingless (Wg) is transferred via exosomes by binding to the exosomal protein Evi, implying that Wnt proteins can travel with exosomes [
16,
17]. Here, we showed that exosomes from LM.4T1 cells increased the lung metastasis of HM.4T1 cells. When the release of exosomes from LM.4T1 cells was blocked or the
Wnt7a gene was mutated in LM.4T1 cells, the number of metastases in the lungs of mice injected with a mixture of LM.4T1 and HM.4T1 cells decreased, strongly suggesting that Wnt7a on exosomes was responsible.
Wnt7a can activate both canonical and non-canonical Wnt signaling pathways and play diverse functions in various malignancies. In bladder and oral squamous carcinomas, it promoted cancer cell invasion and migration via a canonical β-catenin-dependent signaling pathway [
31,
32], whereas Wnt7a overexpressed in non-small cell lung cancer enhanced tumor radiosensitivity via a non-canonical Wnt/JNK pathway [
34]. We observed that when recombinant Wnt7a was added to HM.4T1 cells in 3D cell culture, the volume of tumor spheroids increased significantly. However, we did not detect the accumulation of β-catenin in the nucleus, revealing that Wnt7a did not activate the canonical Wnt signaling pathway. Wnt7a/Fzd7 interaction was shown to maintain muscle stem/progenitor cell expansion via a non-canonical Wnt pathway, called planar cell polarity pathway, driven by VangL2 and induce muscle cell hypertrophy via the PI3K/Akt/mTOR pathway [
33,
36]. We detected the activation of the PI3K/Akt/mTOR signaling pathway in Wnt7a-stumulated HM.4T1 cells. The PI3K/Akt/mTOR signaling pathway is a prominent intracellular signaling pathway that plays an important role in tumor cell growth and proliferation [
61]. mTOR is a protein kinase that is inactivated by a bacterial toxin, rapamycin. There are two different multiprotein mTOR complexes: mTORC1 and mTORC2. p70S6 kinase is a downstream target of mTORC1, and its target substrate is S6 ribosomal protein. The phosphorylation of p70S6 kinase induces protein synthesis in ribosome [
62]. In HM.4T1 cells, a low concentration of rapamycin (50 nM) significantly inhibited sphere formation and constitutive and Wnt7a-induced p70S6K phosphorylation. In BC, p70S6K was associated with angiogenesis and lung metastasis [
63,
64], and elevated p70S6K level was found to correlate with a poor prognosis and survival in BC patients [
65]. We also discovered rapamycin reduced the phosphorylation of Akt, which can also be activated via mTORC2. A previous study revealed that higher concentrations of rapamycin (0.2–20 μM) could target mTORC2, while lower concentrations (0.5–100 nM) could target mTORC1 [
66]. Aside from the effect by different concentrations, chronic rapamycin administration can also impact mTORC2 assembly, which in turn suppresses Akt activation [
67]. Since we used a low concentration of rapamycin, we concluded that its effects were on mTORC1. However, additional experiments are needed to clarify the role of mTORC2 in the process.
Angiogenesis is a critical component of cancer metastasis. We detected higher numbers of ERG- or CD31-positive cells with higher
Vegfa mRNA expression in LM.4T1 tumors than in HM.4T1 tumors, suggesting that LM.4T1 cells have a higher capacity to induce angiogenesis than HM.4T1 cells. This could be another way LM.4T1 cells assist HM.4T1 cell metastasis. We investigated whether Wnt7a was responsible for the higher level of angiogenesis in LM.4T1 tumors and found that tumors of Wnt7a (−) LM.4T1 cells had lower numbers of ERG- or CD31-positive cells with a lower level of
Vegfa mRNA expression than those of Wnt7a (+) LM.4T1 cells. Although the evidence linking Wnt7a to tumor angiogenesis is rather limited, astrocyte-derived Wnt7a was shown to stimulate angiogenesis [
68], suggesting that Wnt7a may play a role in tumor angiogenesis which also contributes to cancer cell metastasis.
To explore the possibility that a similar mechanism is also present in human TNBC, we evaluated the expression of
WNT7A mRNA in human TNBC cell lines using the preexisting data in the GEO database [
69]. Interestingly, a high level of
WNT7A mRNA expression was often detected in the basal-like subtype of TNBC but not in the mesenchymal-like subtype (Additional file
1: Fig. S7A). We also analyzed the association between the level of
WNT7A mRNA expression and patients’ prognosis using the existing microarray database and found that higher
WNT7A mRNA expression was associated with poor overall survival (OS) of all patients with BC (HR 1.24) and patients with basal-like subtype (HR 1.54) (Additional file
1: Fig. S7B) that is often diagnosed as TNBC and comprises about 50 to 75% of the TN subtype [
70]. Although it remains unclear whether interclonal communication by WNT7A plays a role in the progression of human TNBC, it is interesting to see the association between
WNT7A mRNA expression and patients’ survival in human BC cases.
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