Irradiation-induced M1 Microglia Affect Brain Metastatic Colonization of A549 Cell Lines via miR- 9/CDH1 Axis

Yu Jin Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology https://orcid.org/0000-0002-3427-4937 Yalin Kang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Wan Qin Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Qianxia Li Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Qi Mei Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Xinyi Chen Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Guangyuan Hu Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Yang Tang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Xianglin Yuan (  yuanxianglin@hust.edu.cn ) Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology https://orcid.org/0000-0003-4653-5388

in inducing A549 cell lines into mesenchymal phenotype, and further decreased their localization capabilities in brain. Our ndings emphasized the modulating effect of irradiation on metastatic soil, and the cross-talk between tumor cells and the metastatic microenvironment. More importantly, our ndings provided new perspectives for effective anti-metastasis therapies, especially for NSCLC patients with high risk of brain metastasis.

Background
Lung cancer is among leading causes of premature mortality worldwide, accounting for over 142,670 cancer-related deaths in the United States in 2019 [1,2]. Metastasis is the main cause of death in lung cancer patients; with brain metastasis (BM) being common among both small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) patients [3]. Despite recent progress in diagnosis and treatment of BM, the associated overall median survival estimates remain at < 6 month [4]. Prophylactic cranial irradiation (PCI) was introduced in 1980 as a method to prevent brain metastasis [5]. Randomized clinical trials and a meta-analysis have shown that PCI was concerned with improved survival in patients with SCLC [6,7]. As for NSCLC patients, the RTOG 0214 trial demonstrated that the incidence of BM was lower in the PCI (7.7%) than in the non-PCI group (18%) but there is no overall survival bene t [8]. Thus, there is an urgent need to uncover the molecular mechanism of PCI in reducing brain metastasis and nd alternative therapies.
On the one hand, the irradiation dosage used in PCI (usually < 25 Gy in total) is insu cient to have tumoricidal effects. On the other, accounting for the persistent protective effect months after PCI treatment remains challenging. Some authors have suggested that irradiation has a modulating effect on the brain local immune system [9,10]. In fact, studies have demonstrated that a microenvironmental change in a distant organ can in uence the rate of colonization of metastatic tumor cells [11,12]. Based on these results, our group proposed that PCI might alter the "soil" in the brain protecting it against the colonization of a tumor "seed". Microglia, a resident macrophage, is a major constituent of the brain immune system, playing a vital role in the cerebral microenvironment [12]. Presence of different microglia phenotypes, including M1 and M2, has been previously suspected [13,14]. Moreover, it has been proposed that under LPS stimulation, microglia turn into M1 type and exhibit a pro-in ammatory phenotype, which leads to tumor growth inhibition [12,15,16]. However, activation of IL4 and IL10, microglia produce an M2 phenotype. M2 microglia produce anti-in ammatory and immune response-suppressive factors and create a microenvironment favorable to tumor proliferation [12,15,17].
Studies on metastases have demonstrated that lung cancer cells affect several vital pathways, including those associated with evading immune system surveillance [18] and developing chemotherapy resistance [19], both of which can facilitate metastatic behavior and disease progression. Notably, during the metastatic process, tumor cells undergo a morphological change that involves regulating multiple adhesion and cellular matrix molecules to acquire a mesenchymal phenotype. This phenomenon, called epithelial-to-mesenchymal transition (EMT), dramatically increases the rates of extravasation, blood/lymphatic vessel invasion, and distant organ reach of tumor cells [20,21]. When metastatic tumor cells travel to a distant organ, the reverse process occurs, mesenchymal-to-epithelial transition (MET), enabling these cells to up-regulate expression of various adhesion molecules, localizing then in proximity and forming metastatic loci [22]. Meanwhile, studies indicate that microRNA plays an important role in the regulation of the EMT/MET process [23]. To explore the detailed MET inhibitory mechanism of irradiated M1 microglia on A549 cells, we performed a meta-study on microRNA expression by pro ling a murine microglia cell model.
Therefore, we hypothesized that irradiated microglia might alter the phenotypical transition process of tumor cells and distant organ localization, which in turn lower the chances of brain metastasis. Here, we aimed to discover the polarization effect of irradiation on microglia, and to further explore the underlying mechanism of the inhibitory effects of microglia on tumor cell MET transition after ionized irradiation treatment.

Materials And Methods
Cell lines and irradiation treatment Human microglia cell lines CHME-5 and HMO6, human glioma cell line U87, human lung cancer cell line A549 and human renal epithelial 293T cells were purchased from Cellbank, Chinese Academy of Sciences, China. A549-F3 are brain metastatic cells derived from parental A549 cells through three rounds of in vivo selections with mice model. Cells were cultured in DMEM and RPMI 1640 medium (HyClone, Logan, UT), supplemented with 10% FBS (Gibco, Grand Island, NY), and cultured in a humidi ed incubator with 5% CO2 at 37 °C. The mesenchymal phenotype of A549 cells was induced with recombinant human TGF-β1 (PeproTech;100-21C). Cells were irradiated using a RS2000 X-ray Biological Research Irradiator (25 mA, 160 kV; Rad Source Technologies Inc., Suwanee, GA).

Western blot analysis
Western blot was conducted as described previously [24]. Proteintech) were used. The membranes were tested with an ECL detection system (Thermo Fisher Scienti c, Waltham, MA).

Animal studies
All animal work was done in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. For brain metastasis experiments, male BALB/c nude mice (6-to-8-week-old) were raised in pathogen-free conditions. The mice were randomly divided into two groups. In the irradiated group, mice were treated with 3 Gy*2f of irradiation to the whole brain and raised for 1 week.
Subsequently, 100 µl PBS containing 2×10 5 luciferase-labeled A549-F3 cells was injected into the left cardiac ventricle of each mouse. At the end of the experiment, mice were sacri ced and examined for brain metastases.

Intravital imaging and analysis
Mice received an intra-peritoneal injection of 150 ul of 1.5% D-luciferin and anaesthetized with 1.5% chloral hydrate. Imaging was completed with a Xenogen IVIS system (Caliper) coupled to Living Image Acquisition and Analysis software (Xenogen). For BLI plots, photon ux was calculated for each mouse by using a rectangular region of interest encompassing the mouse's thorax.

Statistical analyses
Statistical calculations were performed using GraphPad Prism 8.0.1 (GraphPad Software, CA, USA).
Statistical comparisons between experimental groups were performed with a two-tailed Student t-test. Data are shown as mean ± SD. P < 0.05 was considered indicative of signi cant ndings.

Irradiated microglia exhibited M1-type polarization and hampered A549 cells MET transition
To explore the impact of irradiation on microglial phenotype, CHME5 cells were treated with 0, 2, 3, 4 Gy of irradiation. Then the mRNA levels of iNOS and IL10 as the markers for M1 and M2 types, respectively, were measured. There was a noticeable up-regulation in iNOS expression and down-regulation of IL 10 expression in irradiated microglia, particularly in the group treated with 3 Gy (Fig. 1A). Western Blot analysis revealed up-regulation of iNOS expression and down-regulation of Arg1 expression after treatment with 3 Gy irradiation (Fig. 1B). Further analysis showed that the effects of irradiation were extremely obvious at 48 h using qRT-PCR and Western Blot (Fig. 1C and 1D). Immuno uorescence staining results were consistent with these observations (Fig. 1E).
Next, we plan to explore the role of irradiated M1 microglia on the phenotypical modulation of NSCLC cells. The expression of E-cadherin and Vimentin in A549 cells was analyzed by Western Blot, both of which vital markers of epithelial/mesenchymal phenotype. In the group with control culture media, the mesenchymal phenotype A549 cells quickly reversed into epithelial type with high E-cadherin and low Vimentin expression. In contrast, in the group treated with irradiated CHME5 supernatant, A549 cells retained their mesenchymal phenotype with low E-cadherin and high Vimentin expression. Introduction to these groups of CHME5 supernatant without irradiation or U87 supernatant did not affect the observed phenotype changes (Fig. 1F).

Irradiation-induced M1 microglia increased both intra and extra-cellular miR-9 level
Several recent studies indicated microRNAs played crucial roles in regulation of EMT/MET process [26,27]. Thus, we analyzed microRNA expression in LPS-treated group and control groups. miR-9 levels increased dramatically in LPS-induced M1 microglia ( Fig. 2A). We con rmed that the level of miR-9 was signi cantly elevated in irradiated CHME5 and HMO6 cells (3 Gy and 3 Gy*2f), when compared with nonirradiated microglia, both intracellularly ( Fig. 2B and 2D) and extracellularly ( Fig. 2C and 2E). These results showed that miR-9 expression was up-regulated in irradiated microglia and it was secreted into the extracellular space, suggesting that miR-9 secreted by irradiated microglia played an important role in inducing a mesenchymal phenotype in metastatic A549 cells.
Irradiated M1-type microglia elevated intracellular level of miR-9 in A549 cell lines To con rm the effects of miR-9 produced by irradiated M1 microglia on the MET process, we modulated the expression of miR-9 in A549 or brain metastatic A549-F3 cells, using an miR-9 mimic and inhibition of plasmid transduction (Fig. 3A and 3B). Levels of intracellular miR-9 were detected via qRT-PCR in each of three groups: negative control, over-expression, and inhibition group. Signi cantly elevated/decreased miR-9 levels were detected in the overexpression/inhibition group respectively, con rming the success of in vitro miR-9 expression modulation in A549 and A549-F3 cells ( Fig. 3C and 3D).
We added irradiated or non-irradiated microglia culturing supernatant into A549 and A549-F3 cells transfected with control/miR-9 mimic/miR-9 inhibition plasmid, respectively, observing the highest level of miR-9 in miR-9 mimic group treated with irradiated conditioned medium. In contrast, the lowest miR-9 level were detected in the miR-9 inhibition group, where the non-irradiated microglia supernatant was added ( Fig. 3E and 3F). When the miR-9 inhibition A549/A549-F3 group was treated with irradiated microglia supernatant, their intracellular levels of miR-9 were signi cantly higher than those in the miR-9 inhibition group treated with non-irradiated microglia supernatant. This result suggests that irradiated M1 microglia increased their production and secretion of miR-9; and so, the uptake of miR-9 by A549 and A549-F3 was increased. This absorbed miR-9 might play a role in reducing NSCLC brain metastasis.
Irradiated M1 type microglia inhibited the MET via miR-9/CDH1 To explore the in uence of miR-9 levels on phenotypic conversions of A549 and A549-F3 cells we conducted Western blot analysis. In Fig. 4A, groups with controlled culture media and negative plasmid the mesenchymal phenotype A549 cells quickly reversed into epithelial-type with high E-cadherin and low Vimentin expression. In contrast, groups with up-regulated miR-9 retained their mesenchymal phenotype (low E-cadherin and high Vimentin expression). Among groups with up-regulated miR-9, those treated with irradiated microglia supernatant retained the most typical mesenchymal phenotype. Meanwhile, miR-9 downregulation could promote E-cadherin expression. Furthermore, A549 cells with down-regulated miR-9 treated with non-irradiated microglia supernatant transformed into the most typical epithelial phenotype (the highest E-cadherin and the lowest Vimentin expression). Consistent with the above, A549-F3 cells transfected with down-regulated miR-9 plasmid were able to develop an epithelial phenotype, while irradiated microglia supernatant could inhibit the MET process to keep the A549-F3 cells in a mesenchymal state. Meanwhile, A549-F3 cells with up-regulated miR-9 treated with irradiated CHME5 supernatant maintained the most typical mesenchymal phenotype (Fig. 4B).
Low dose irradiation reduced brain metastases of A549-F3 cells in brain mice model To con rm the effects of irradiation on NSCLC-BM in vivo, we selected 7 days after irradiation to inject tumor cells for the higher level of miR-9 (Fig. S1). We use bioluminescence imaging (BLI) to assess the incidence rate of BM in a mouse model (Fig. 5A and 5B). BM incidence was reduced in irradiated mice (40%, n = 10), when compared to the control group (70%, n = 10) (Fig. 5C). There are signi cant differences between the two groups by analysis of the photon ux (Fig. 5D). Furthermore, irradiation increased miR-9 levels in mice brain microenvironment (Fig. 5E). These ndings suggest that, in a mouse model, low dose irradiation reduces A549-F3 cells-mediated brain metastases, plausibly by elevating miR-9 expression levels, which inhibit tumor cells MET in brain microenvironment.

Discussion
In this study, we showed a signi cant shift towards M1 microglia phenotype after irradiation. Moreover, M1 microglia inhibited the MET process of A549 cell lines, a crucial step in their capacity for adhesion to and colonization of distant organs, in particular, the brain. MiRNA array analysis revealed up-regulated expression of miR-9 in M1 microglia. In addition, increased miR-9 secreted by irradiated M1 microglia played an important role in inhibition of the MET process. Finally, miR-9 exerted these effects by targeting CDH1, a gene vital to this process. Evidence from a mouse model supported the effects of irradiationinduced miR-9 elevation on lowering the incidence of brain metastasis in A549-F3 cells.
It has been well established that microglia localizing in brain parenchymal are main cellular constituent that are involved in brain innate immunity [29]. They detect "danger signals," including presence of infectious agent, toxins and cell damage among others, using danger-associated-molecular-pattern receptors (DAMP) and trigger in ammatory responses [9,13]. Here, our ndings are consistent with previous studies that demonstrated that irradiation promoted M1 microglia activation and anti-metastatic effects [9,10]. Concurrently, numerous studies have suggested that the pro-in ammatory status of M1 microglia showed anti-tumoral capacity via improved antigen presenting capabilities [30] and direct suppression of tumor growth [31]. This study revealed a novel mechanism of inhibitory effects on NSCLC cells localization, presumably by promoting miR-9 production and secretion from irradiated M1 microglia.
Recent studies have indicated that miR-9 exhibits anti-tumor effects by inhibiting tumor cell motility. Speci cally, Ben-Hamo et al. demonstrated that overexpression of miR-9 in glioblastoma hampered tumor cell mobility, possibly via inhibition of the MAPK pathway, which subsequently disrupted cellular actin cytoskeleton organization [32]. In addition, Xu et al. showed that ectopic expression of miR-9 in melanoma cells suppressed the tumor capacity of migration and invasion. These authors identi ed the downstream target gene NRP1, negatively regulated by miR-9, as responsible for the change in motility of tumor cells [33]. Meanwhile, growing evidences have indicated that miR-9 plays a vital role in regulating the MET process and tumor cell metastasis [27,34,35]. In our research, through high-throughput micro-RNA pro ling analysis, we observed signi cant miR-9 up-regulation in irradiated microglia. This increased miR-9 expression within A549 cell lines, in turn, promoted their mesenchymal phenotype. Eventually, decreased expression of adhesive molecules in metastatic tumor cells hampered their localizing capacity in the brain.
However, we must point out that our research has some limitations. For one hand, we only got preliminary results and have not veri ed the possibility of the target as an intervention treatment. For another, the present ndings are based on cellular and animal models, while elucidating the molecular mechanism of irradiated effects on brain metastasis in NSCLC patients requires further studies involving human subjects.

Conclusions
In short, we found signi cant M1 polarization shifting of microglia after irradiation treatment. We demonstrated irradiation induced M1-type microglia signi cantly modulate metastatic A549 cell lines into mesenchymal phenotype through up-regulating and secreting of miR-9, which eventually shifted tumor cell into mesenchymal phenotype and further decreased localization capabilities in brain. Our study rstly demonstrated that irradiated microglia modulated tumor cell MET process via miR-9/CDH1, providing new insights into PCI's mechanism. It might inform further studies on irradiation effects to tumor microenvironment. Clinically, miR-9 expression might act as predictive biomarker for brain metastasis in NSCLC patients. Moreover, treatments targeting the miR-9 pathway combined with radiotherapy might help reduce the risk of brain metastasis, providing novel perspectives for effective anti-metastatic therapies. Abbreviations

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Figure 3
Irradiated M1-type microglia elevated intracellular level of miR-9 in A549 cell lines (A) and (C) Transfection of miR-9 mimic and inhibition plasmid into A549 cells and A549-F3 cells. Magni cation was Low dose irradiation reduced brain metastases of A549-F3 cells in brain mice model (A) Schematic diagram illustrates the treatment schedule of irradiation. (B)Brain metastases were determined by bioluminescence imaging in control and irradiated group. (C) Histogram showed incidence of brain metastasis in control and irradiated group. (D) Photon ux detected from brain metastatic focus in control and irradiated group. (E) qRT-PCR results of miR-9 from brain tissue of mouse treated with or without irradiation. Data are mean ± SD. *P<0.05, **P<0.01, ***P<0.001.

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