Interleukin 17A Promotes Cell Migration, Enhances anoikis resistance, and Favors Microenvironment for Triple Negative Breast Cancer Tumor Metastasis


 Background: Our previous study demonstrated that overexpression of multiple epidermal growth factor-like domains 11 was involved the recurrence mechanism of triple negative breast cancer (TNBC) via up-regulation of cytokines and chemokines, including IL-17A signaling pathway. However, information concerning the role of IL-17A in tumor behavior or cancer microenvironment remains lacking. The aim of this study was to investigate the role of IL-17A on TNBC recurrent mechanisms, including tumor behavior, circulating tumor cells (CTCs), and cancer microenvironment. Methods: Using human TNBC MDA-MB-231 and MDA-MB-468 lines, the role of IL17-A was elucidated by knocked down IL-17A (DIL-17A) or administration of different dose of IL-17A in the culture medium. Cell proliferation, migration assay, Western blot analysis and Real-time PCR for IL-17A related signaling were evaluated. Three groups of implanted 4T1 cells in BALB/c mice were designed, namely, wild type (WT), DIL-17A, and WT + neutralizing IL-17 antibody (WT+Ab). Tumor weight, necrosis area, and the number of CTCs were measured. Immunohistochemistry or Western blot for CD34, CD8, and TGF-b1 were evaluated. Anoikis resistance was analyzed by live/dead stain and flow cytometry. Finally, clinic-pathological correlation between IL-17A expression and patients’ outcome such as disease free survival (DFS) and overall survival (OS) was performed by Kaplan-Meier’s method.Results: Our results demonstrated that IL-17A stimulated migratory activity, but not growth rate, of MDA-MB-231/468 cells via increased Src, Rho, and COX2 expression. In vivo, there was an increased necrosis area, a decreased tumor CD34 expression and CTCs in DIL-17A group; while there was a decreased tumor CD34 expression, CD8(+) cells, and CTCs, but an increased TGF-b1 expression in WT+Ab group, compared to the WT group. Knocked down-IL-17A also decreased anoikis resistance in human TNBC and murine 4T1 cell lines. Kaplan-Meier’s analysis disclosed a negative correlation between tumor IL-17A expression and OS in TNBC patients. Conclusion: We conclude that IL-17A promotes migratory and angiogenic activity in the tumors, enhances anoikis resistance, and modulates the immune landscape of tumor microenvironment favoring subsequent cancer metastasis. The blockade of IL-17A might provide a co-treatment target to prevent tumor metastasis or recurrence in TNBC patients.


Introduction
Breast cancer is the most common diagnosed female cancer worldwide and ranks as the fourth most important cause of death in Taiwan [1]. Triple negative breast cancer (TNBC) represents 15% − 20% of all newly diagnosed breast cancer patients and is characterized as a large tumor size, frequently diagnosed among younger women, and a poor prognosis with high metastases potential [2][3][4]. Recent investigations have demonstrated that TNBC may be composed of a number of distinct subtypes [5][6][7].
However, early recurrent mechanisms as consequences of poor prognosis in TNBC patients remain further elucidation.
IL-17, a proin ammatory cytokine secreted by activated T cells, regulates the activities of MAPKs and NF-κB and increases the expression of IL6 and cyclooxygenase-2 (COX-2) [8]. Overexpression of IL-17A is known to be highly associated with in ammatory diseases such as multiple sclerosis, psoriasis and rheumatoid arthritis [9]. It is generally accepted that chronic in ammation may increase the risk of cancer [10], as the in ammatory response shares various molecules and corresponding signaling pathways with the carcinogenic process. The IL-17 related protumoral effects on cancer initiation are postulated through MAPK and NF-kB recruitment. By contrast, IL-17 cytokines also act as an antitumor role and the same duality makes IL-17 a "double-edged sword" [11,12]. However, the role of IL-17A on the survival of circulating tumor cells (CTCs) is unclear.
Evidence suggests that poor prognosis in breast cancer might relate to CTCs [13,14], and the survival of tumor cells in circulation requires three aspects: 1.resistance to anoikis, 2. escape immune surveillance, and 3. being stable under blood shearing force. The resistance to anoikis requires several signaling pathways related to anchorage-independent growth and epithelial-mesenchymal transition, including Akt, TrkB, Src, and so on [15]. Nonetheless, information concerning the role of IL-17A on the survival of CTCs remains to be elucidated.
Our previous results demonstrated that over-expression of MEGF11 in TNBC cells triggered the expression of many cytokines and chemokines, which effectively resulted in a cytokine cascade. Besides, a positive feedback between MEGF11 and IL-17A in TNBC cells was also demonstrated, which might explain the role of MEGF11 in TNBC recurrence [16]. Accordingly, the aim of this study was to investigate the role of IL-17A on TNBC recurrent mechanisms, including tumor behavior, cancer microenvironment, and the survival of CTCs.
Cell growth by trypan blue dye exclusion assay and cell cycle analysis MDA-MB-468/231 cells were cultured in low serum medium with a cell density (1 × 10 5 / well) in a 12-well plate, followed by treatment of different doses of IL17A (0-, 1-, 10 ng/mL). After 1, 2, and 3 days of treatment, cells were washed with phosphate-buffered saline (PBS), pH 7.4, and trypsinized with TE buffer (Gibco/Invitrogen, New York). Suspended cells were washed and cell numbers were counted with a hemocytometer. For cell cycle analysis, cells (2 × 10 5 / well) were cultured for 24 h in low serum medium (0.1% FBS), followed by another 24 h-culture, and then cells were harvested for cell cycle analysis by ow cytometry. Cell cycles were presented as percentages of cell cycle fraction, namely, sub G0/G1 phase, G0/G1 phase, S phase, and G2/M phase.

Cell migration assay
In vitro cell migration of MDA-MB-468/231 cells was performed using a trans-well system (ThinCertTM cell culture inserts, 24 well, 8 µm, Greiner bio-one, Switzerland) [18]. In brief, MDA-MB-468/231 cells were cultured in the upper chamber and different doses of IL-17A (0-, 1-, 10 ng/mL) were administrated in the lower chamber. After 4 h (MDA-MB-231) or 8 h (MDA-MB-468) of culture, the cells on the reverse side of upper chamber membrane were xed and stained with 2% crystal violet for 10 min, then washed and photographed. The migrating cells were examined under a light microscope and quanti ed. Measurement of IL-17A level IL-17A level in the culture medium was quanti ed with ELISA method under the guidance of commercially available protocol (Quantikine ELISA Human IL-17A). In brief, MDA-MB-468/231 (4 × 10 5 / well) cells were cultured for 1 d, followed by administration of different doses (0-, 1-, 10 ng/mL) of IL-17A in low serum medium for 24 h. Then, the culture medium were discarded and cells were cultured for another 48 h, followed by harvesting the cells and the supernatants were collected, centrifuged (300 x g) for 5 min, and the nal supernatants were ready for IL-17A measurement.
RNA extraction and reverse transcription-PCR Total RNA were isolated by using a modi ed single-step guanidinium thiocyanate method [20]. (TRI REAGENT, T-9424, Sigma Chem. Co., St. Louis, MO) Complementary DNA (cDNA) was prepared from the total RNA complied with the First Strand cDNA Synthesis Kit (Invitrogen, CA). The de novo gene synthesis changed by each treatment group was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Primers pairs were purchased commercially [16]. The possible contamination of any PCR component was excluded by performing a PCR reaction in the absence of RT product in each set of experiment. Quanti cation of RNA transcripts was analyzed according to the method described previously [16]. For statistical comparison, the relative expression of speci c genes mRNA was normalized to the amount of GAPD in the same RNA extracts. All samples were analyzed in triplication.

In vivo tumor xenograft
In this study, any protocols that involved experimental mice followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Institutional Animal Committee of Taipei Veterans General Hospital (No. 2018-029). Immunode cient NU-Foxn1nu 8-wk-old mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan, ROC). They were given ad libitum access to water and food and maintained in a speci c pathogen-free environment with a 12 h dark-light cycle at 22-24 °C with 50% humidity. Wild type (WT) and knocked down IL-17A (∆IL-17A) MDA-MB-231 cells (1 × 10 7 in 0.1 mL PBS) containing a luciferase gene were injected into back of immunode cient NU-Foxn1nu mice, which gave rise to a solid tumour that was noticeable around the injection site. At the 2nd, 3rd, and 5th week, the progression of the tumor was visualized using an in vivo imaging system (IVIS).

In vivo metastasis study
Three groups of animal studies were designed, namely, WT, knocked down IL-17A (∆IL-17A), and anti-IL-17A neutralizing antibody (250 µg/100µL, twice a week) (WT + anti-IL-17A antibody). Mouse mammary 4T1 cells (5 × 10 6 in 0.1 mL PBS) were orthotopically injected into the fat pads (left upper and/or right lower mammary glands) of 8-wk-old female BALB/c mice. The mice were sacri ced at 8 weeks thereafter or when the tumour sizes being more than 2% of the body weight. The tumor sizes and weights were measured, and the circulating tumor cells were isolated as mentioned below.

Subjects
Under the approval of the Institutional Review Board of Taipei Veterans General Hospital (2013-10-020BC), human tumour tissues from a bio-bank were obtained. From Jan. 2001 to Dec. 2010, eighty three patients diagnosed as female breast cancer in department of pathology, Taipei Veterans General Hospital were enrolled. Records such as oestrogen receptor (ER) status, progestin receptor (PR) status, HER2 status and clinical outcome, including overall survival (OS) and disease-free survival (DFS), were retrospectively reviewed. All data had been collected during clinical care and did not involve direct contact with the patients. Besides, written consent by the study subjects was waived by the Institutional Review Board. Every subject participated in this study (n = 83) was followed up for ≥ 5 years. Patients' outcome was subjected to a Kaplan-Meier survival analysis. ER or PR values of ≥ 1% were de ned as positive, while ER or PR values of < 1% were de ned as negative.

Statistic analysis
Results were expressed as the mean ± SEM. Differences between two groups were identi ed by Mann-Whitney U test or Student t test. Differences between groups on each time point or each dose were identi ed by one-way ANOVA, followed by Dunnet's post hoc test. Statistical comparison between two independent variables was determined by Two-way ANOVA with Bonnforonni post-hoc to correct multiple comparisons. A p-value < 0.05 is considered statistically signi cant compared to vehicle or no treatment group.
DFS was de ned as the time between initial breast cancer diagnosis and the date of recurrence as con rmed by pathology or an imaging study. OS was calculated from the time of initial breast cancer diagnosis to the date of death or last visit in outpatient clinic. The Kaplan-Meier method was used to estimate the cumulative incidence of RFS and OS, and log-rank tests were then used for the various comparisons (GraphPad Prism 5).

Role of IL-17A in tumor behavior
Knocked-down IL-17A did not affect cell growth in human TNBC cell lines.
After IL-17A gene was silenced in TNBC MDA-MB-231 and MDA-MB-468 lines, there was no signi cant change of growth rate in ΔIL-17A group compared to the WT group, both in cell lines studies (Fig. 1a) and in tumor-bearing nude mice studies (Fig. 1b).
IL-17A up-regulated gene expression of pro-in ammatory cytokines and chemokines When IL-17A was administrated in the culture medium of TNBC cells, there was an increased mRNA transcripts of pro-in ammatory cytokines (TNF-α, IL-1β) and chemokines (CXCL2, CCL20) and COX2 in MDA-MB-231 and MDA-MB-468 (Fig. 3c). Of note, IL-17A up-regulated IL-17A gene expression both at transcript level and protein level (Fig. 3d). These data suggest that IL-17A has autoregulation activity and enhances in ammatory cytokines and chemokines cascades.

In vivo metastasis study
Role of IL-17A on the tumor behaviour Using murine mammary 4T1 cell line as in vivo metastatic model, three groups of animal studies were designed, namely, WT, knocked down IL-17A (∆IL-17A), and WT + anti-IL-17A neutralizing antibody (WT + Ab). The roles of IL-17A in tumors (∆IL-17A group) and around tumor microenvironment (WT + Ab group) were compared to the WT group. The results showed that there was a signi cant decreased tumor volume and an increased tumor necrosis area (Fig. 4a) in ∆IL-17A group compared to the WT group. To investigate the role of IL-17A on angiogenesis in vivo, CD34 expression by immunohistochemistry and Western blot was performed. The results demonstrated that there was a signi cant decreased CD34 expression (Fig. 4b) in ∆IL-17A and WT + Ab groups compared to the WT group.

Role of IL-17A on tumor microenvironment
When dissecting the tumors away from the mice chest wall, it was surprising to notice that the tumors of the WT + Ab group, which were no smaller in size than those of the WT group, were easily freed from adjacent chest wall without any adhesion nor increased vascularity (Fig. 4a). To investigate the role of IL-17A on immune landscape in vivo, CD8 and TGF-β1 expressions by immunohistochemistry and TGF-β1 expression by Western blot were performed. The results demonstrated that there was a signi cant decreased CD8 (+) T cells (Fig. 5a), and also decreased COX2 but increased TGF-β1 expression in ∆IL-17A and WT + Ab groups compared to the WT group (Fig. 5b).

Role of IL-17A on anoikis resistance in circulating tumor cells (CTCs)
The CTCs obtained from the blood of sacri ced mice were isolated, subcultured, and selected by colony assay. The results disclosed that there were signi cant decreased CTCs colonies in both ∆IL-17A group and WT + Ab group compared to the WT group (Fig. 6a). Using anoikis plate as in vitro model, the results showed that there was a decreased anoikis resistance via caspase 3 activation (Fig. 6b) in ∆IL-17A 4T1 group compared to the WT 4T1 group. Similarly, there was a decreased anoikis resistance (Fig. 6c) in ∆IL-17A MDA-MB-231/468 group compared to the WT MDA-MB-231/468 group. These data suggest that IL-17A plays important roles in maintaining CTCs survival against anoikis. Clinically, the expression of IL-17A correlated negatively with patients' OS (Fig. 7a), but a trend with patients' DFS (Fig. 7b).

Discussion:
Using paired (non-recurrent and recurrent) resected tumor tissues obtained from breast cancer patients, we have identi ed a novel gene, MEGF11, is highly associated with tumor recurrence in TNBC patients.
Besides, Overexpression of MEGF11 gene up-regulates IL-17A gene expression in TNBC MDA-MB-231 and MDA-MB-468 cells. Although IL-17A is well known to be highly associated with in ammatory diseases such as rheumatoid arthritis and psoriasis [9], the role of IL-17A in TNBC recurrence remains further elucidation. In this study, we have demonstrated that IL-17A promotes migration-promoting activity in the tumors and changes the immune landscape favoring tumor microenvironment for cancer metastasis.
While knocked down IL-17A group was designed to investigate the role of IL-17A in tumor behavior, the WT + anti-IL17A antibody group was designed to elucidate that in tumor microenvironment. Tumor necrosis, a form of cell death, is commonly associated with hypoxia, rapidly growing, and aggressive forms of breast cancer [22]. There is evidence that angiogenesis is essential for tumor growth and metastasis [23]. and IL-17A promotes tumor angiogenesis via stimulation of endothelial fatty acid βoxidation [24]. Using CD34 as a marker for hematopoietic stem cells [25], our results showing a signi cantly increased necrosis area and a decreased CD34 expression in ΔIL-17A group compared to the WT group suggest that IL-17A enhances angiogenesis and hence against ischemic necrosis in rapid progress tumor tissue.
By contrast, when dissecting the tumors away from the mice chest wall, tumors of the WT + Ab group were easily detached from adjacent chest wall without any adhesion nor increased vascularity (Fig. 5A).
A decreased CD34 expression level supports the speculation of decreased angiogenesis in this group. Recent investigations suggest that the immune system plays a dual role in tumor progression, namely, inhibiting or promoting tumor expansion. For example, TGF-β1 produced during chronic in ammation actively promotes growth and metastasis [10,26]. Besides, the TGF-β signaling pathway plays key roles in the early development of Treg cells in the thymus [27], in maintaining the homeostasis of peripheral CD4 + T cells via by increasing IL-7Rα expression [28], and in controlling CD8 + T-cell homeostasis [29]. Of note, several lines of evidence demonstrate the opposing effects of TGF-β on Th17 cells, namely, the immunoregulatory or pathogenic role of the Th17 cells in clinical settings [30,31].
Our results that there was a decreased CD8(+) cells and an increased TGF-β expression in WT + Ab (anti-IL-17A) group lead us to speculate that TGF-β plays an immunosuppressive role leading to immune escape of the implanted 4T1 tumors, which is in agreement with previous investigation [32].
The presence of circulating tumor cells (CTCs) is well known as a mechanism of tumor metastasis or cancer recurrence [33]. Several hypotheses about CTCs evading immune surveillance have been proposed, such as adhered to platelet and myeloid-derived suppressor cells [34,35], or mediated by PD-L1 [36]. Our results that there was signi cantly decreased CTCs colony number in ΔIL-17A and WT + anti-IL17A Ab groups compared to the WT group demonstrated a negative correlation of IL-17A expression and CTCs in the blood, which is consistent with previous ndings observed in colon cancer [37].
There is consensus that persistent chronic in ammation increases the risk of cancer [10] and necrosis has proinlfammatory and tumor promoting potential [38]. The use of nonsteroidal anti-in ammatory drugs (NSAIDs) is well demonstrated to decrease incidence and mortality of many cancers [39]. IL-17E/IL-17E receptor axis may underlie TNBC resistance to EGFR inhibitors and suggest that inhibiting IL-17E or its receptor in combination with EGFR inhibitor administration may improve TNBC management [40]. Recent studies suggest that IL-17A inhibitor might play important role in immune therapies for psoriasis and ankylosing spondylitis patients [41].

Conclusion
Our results demonstrate that IL-17A stimulates migratory activity of TNBC cells and modulates the immune landscape of tumor microenvironment favoring subsequent cancer metastasis. The blockade of IL-17A might provide a co-treatment target to prevent tumor metastasis or recurrence in TNBC patients.

Declarations
Ethics approval and consent to participate: Study protocols involving experimental mice followed ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Institutional Animal Committee of and Taipei Veterans General Hospital (No. 2018-029).
The human study for tumour tissue utilization from the bio-bank was approved by the Institutional Review Board of Taipei Veterans General Hospital (# 2013-10-020BC).

Consent of publication: Not applicable
Data availability: All data generated or analysed during this study are included in this published article (and its Supplementary Information les) Competing interest: We declare that we have no con icts of interest, including nancial and non-nancial interests such as the following items.   2nd, 3rd, and 5th week, the progression of the tumors was visualized using an in vivo imaging system (IVIS) and tumour volume was quanti ed (b). Two-way ANOVA and Mann-Whitney U test were used for statistical analysis for cell growth in vitro and in vivo, respectively.  2nd, 3rd, and 5th week, the progression of the tumors was visualized using an in vivo imaging system (IVIS) and tumour volume was quanti ed (b). Two-way ANOVA and Mann-Whitney U test were used for statistical analysis for cell growth in vitro and in vivo, respectively.  2nd, 3rd, and 5th week, the progression of the tumors was visualized using an in vivo imaging system (IVIS) and tumour volume was quanti ed (b). Two-way ANOVA and Mann-Whitney U test were used for statistical analysis for cell growth in vitro and in vivo, respectively.  cell proliferation rate was evaluated by trypan blue assay (a). For cell cycle analysis, cells (2 x 105 / well) were cultured for 24 h in low serum medium, followed by another 24 h-culture, and then cells were harvested for cell cycle analysis (b). Cell cycles were presented as percentages of cell cycle fraction, namely, sub G0/G1 phase, G0/G1 phase, S phase, and G2/M phase. Growth related signalling proteins expression, such as Src, AKT, CDK4, and cyclinD1, were analyzed by Western blot (c) and quanti ed (d).
Asterisk indicates a p value < 0.05 (one-way ANOVA). followed by treatment of different doses of IL17A (0-, 1-, 10 ng/mL). After 1, 2, and 3 days of treatment, cell proliferation rate was evaluated by trypan blue assay (a). For cell cycle analysis, cells (2 x 105 / well) were cultured for 24 h in low serum medium, followed by another 24 h-culture, and then cells were harvested for cell cycle analysis (b). Cell cycles were presented as percentages of cell cycle fraction, namely, sub G0/G1 phase, G0/G1 phase, S phase, and G2/M phase. Growth related signalling proteins expression, such as Src, AKT, CDK4, and cyclinD1, were analyzed by Western blot (c) and quanti ed (d).
Asterisk indicates a p value < 0.05 (one-way ANOVA).      Immunohistochemistry and Western blot for CD34 expression was quanti ed (b). Bar indicated 50 μm.