M2 macrophage-derived exosome facilitates metastasis in non-small-cell lung cancer via delivering integrin αvβ3

DOI: https://doi.org/10.21203/rs.3.rs-1594991/v1

Abstract

Background

The most prevalent cause of cancer death is metastasis. Immunological components of tumour microenvironment, especially tumour-associated macrophages, play a vital role in cancer metastasis. However, the underlying mechanisms of tumour-associated macrophages on non-small-cell lung cancer (NSCLC) metastasis remain largely unexplored.

Methods

The distribution of macrophages in tumor microenvironment, especially in lung cancer, was analyzed by online database and immunohistochemistry. The model of M2 macrophage was successfully established in vitro and M2 macrophage-derived exosomes were identified by transmission electron microscope imaging, nanoparticle tracking analysis and western blot. The role of M2 macrophage-derived exosomes (M2-exos) in promoting metastasis of lung cancer cells was identified by transwell assay, wound healing assay, immunofluorescence in vitro, and by tumor model in vivo. The mechanism that M2-exos facilitates metastasis via delivering integrin αvβ3 and activating the FAK signaling was investigated using western blot, transwell assays, immunofluorescence assays. Finally, the expression levels of integrin αvβ3 were assessed in clinical samples by immunohistochemistry.

Results

We demonstrated that M2-polarized phenotypic macrophages facilitate the migration and invasion of cancer cells in vitro and in vivo through intercellular delivering M2-exos. Importantly, we found that M2-exos had considerably higher levels of integrin αvβ3. The impact of M2 macrophage-mediated invasion and migration of NSCLC cells was clearly decreased when integrin αvβ3 was blocked. Mechanistically, exosomal integrin αvβ3 produced from M2 macrophages successfully triggered the FAK signaling pathway in recipient cells, boosting the migratory and invasive abilities of NSCLC cells. Clinically, we found that metastatic NSCLC patients had greater integrin αvβ3 expression, which was associated with worse prognosis.

Conclusions

This study reveals a novel mechanism that M2 macrophage-derived exosomal integrin αvβ3 significantly increased NSCLC cells migration and invasion. integrin αvβ3 can be used as a potential prognostic marker, and blocking integrin αvβ3 could be a viable treatment option for preventing tumour progression and metastasis.

1. Background

Lung cancer is the dominant forerunner of cancer-related fatalities globally, of which non-small-cell lung cancer (NSCLC) accounts for 80%-85% [1, 2]. Patients with NSCLC frequently develop metastases, the most common of which are to the brain and bone, with a 5-year survival rate of fewer than 15% [3]. Multiple gene mutations related with NSCLC metastasis have been identified, including EGFR, VEGF, KRAS, p53, and PTEN [4–6]. However, with the improvement of the understanding of various aspects of tumour, more and more evidences show that the tumor microenvironment (TME) has a crucial role in metastasis, whether it is primary site invasion or distant metastatic colonization [7–10].

The confrontation between tumor cells and immune cells determines the initiation and progression of tumour In TME [11]. Although adaptive immunity is widely regarded as the main force against the tumour, increasing evidence suggests innate immune cells, especially tumour-associated macrophages (TAM), also play an important role in this battle [12–14]. Tumour cells recruit and civilize macrophages in the tumour microenvironment to differentiate into M2-like macrophages by secreting various cytokines, such as CSF1 and CCL2 [15, 16]. TAM refers to M2-like macrophages in TME that have tumor-promoting and immunosuppressive activities [17]. TAM can release various cytokines such as TGF-β and EGF, which not only promote tumor proliferation and transformation but also facilitate the establishment of tumor tolerance microenvironment [18–20]. TAM can also secrete a series of inflammatory inhibitory molecules, including IL-10 and IL-13, which can directly inhibit CD8+T and CD4+T cell-mediated tumour killing [21, 22]. TAM infiltration in TME is linked to poor prognosis in breast, oral, ovarian and bladder cancers, and Hodgkin's lymphoma [23–25]. However, specific evidence linking TAM and NSCLC is still lacking, particularly in terms of NSCLC metastasis.

Exosomes are key mediators for intercellular cross-talk and are secreted by almost all cell types [26]. Exosomes are lipid bilayer membrane vesicles originated from endocytosis and have a diameter of about 30–150 nm [27]. Exosomes contain a variety of bio-active molecules, including proteins, lipids, RNAs and DNAs, which can be transferred to recipient cells and mediated their biological functions [28]. Accumulating research have shown that tumour-derived exosomes are associated with tumour growth, drug resistance, metastasis, and the remodeling of the tumour immune microenvironment [29, 30]. Exosomes from lung cancer, for instance, contribute to the polarization of macrophages toward an M2-like phenotype [31]. M2 macrophage-derived exosomes have been found to promote tumor progression and metastasis in colorectal cancer and liver cancer [32, 33]. However, there has been little investigation into the effect of M2 macrophage-derived exosomes (M2-exos) in metastatic NSCLC.

In this research, we demonstrated M2-exos were responsible for NSCLC progression and metastasis both in vitro and in vivo. Importantly, ITG αvβ3 was found to be highly enriched in M2-exos and was closely associated with NSCLC metastasis. The underlying mechanisms could be that M2-derived exosomes mediated ITG αvβ3 transmission to NSCLC cells, which triggered the FAK signaling of recipient cells, thus enhancing NSCLC cells migration and invasion. Our findings shed new light on the role of macrophages in tumor metastasis, suggesting that M2 macrophage-derived exosomes play an important role in tumor progression and may become a new target for tumor therapy.

2. Materials And Methods

Cell culture

The human NSCLC cell lines A549 and H1299 were cultured in DMEM medium (Gibco, USA), and the human acute monocytic leukemia cell line THP-1 was cultured in RIPA 1640 medium (Gibco, USA). Both medium contained with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). For macrophage polarization, THP-1 cells were treated with 100nM phorbol-12-myristate-13 acetate (PMA; Sigma-Aldrich) for 48 hours, after which the medium was discard and the cells were washed twice with pre-warmed phosphate buffered saline (PBS). The PMA-differentiated THP-1 macrophages were then cultured for another 24 h in the RPMI 1640 complete medium (without PMA) to obtain the resting state of macrophages (M0). For M1 or M2 macrophages polarization, M0 macrophages were cultured for 48 hours in the medium supplemented with 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL IFN-γ (PeproTech) or 20 ng/mL IL-4 and IL-13 (PeproTech), respectively. All cells were cultured at 37°C in a 5% CO2 atmosphere.

Exosome isolation

THP-1-differentiated M2 macrophages were cultivated for 24 hours in FBS-free RIPA 1640 medium, and which the medium was collected and exosomes were extracted by differential ultracentrifugation as described previously [34]. Briefly, to remove cells and debris, the conditioned media was centrifuged at 300 g for 5 minutes and 2,000 g for 15 minutes. Then, the supernatant was harvested and centrifuged at 15,000 g for 30 min at 4 ℃ to eliminate large extracellular vesicles. The exosomes were isolated by centrifugation (Beckman Coulter Avanti J30I) at 100,000 g for 90 minutes. Finally, the isolated exosomes were re-suspended in 200µL PBS and used immediately or stored at -80 degrees Celsius.

Exosome identification

TSG101, CD63, and Alix were utilized as positive controls in Western blot analysis, whereas as a negative control, calnexin, an endoplasmic reticulum protein, was used. The Nanosight NS300 system (Nanosight Technology, Malvern, UK) was employed to directly monitor the number and size distribution of exosomes.

Exosome uptake assays

The extracted exosomes were treated with PKH26 Fluorescent Cell Linker Kits (Sigma-Aldrich) according to the manufacturer's protocol to visualize exosome internalization. Next, the tagged exosomes were cultured with H1299 cells for 6 h. The cells were fixed in 4% paraformaldehyde for 30 minutes before being stained using Abcam's CytoPainter Phalloidin iFluor 488 Reagent for 30 minutes. The nuclei were then stained with Hoechst 33342 (Cell Signaling Technology, Danvers, MA) for 10 minutes. A confocal microscope was used to look at how H1299 cells took in exosomes.

Transwell assay

For cell migration experiments, 2×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the 24-well Transwell cell culturing chambers (8µm pore size, BD), and 650µl of medium containing 10% FBS was added into the lower chamber. For cell invasion assays, 4×104 NSCLC cells were resuspended in 200µL of FBS-free media and planted into the upper inserts with pre-coated Matrigel, and 650µl of media containing with 10% FBS were added to the lower chamber. For NSCLC cells and M2 macrophages indirect co-culture assays, 2×104 THP-1 cells were seeded into the lower chamber and they were induced to polarize towards M2 macrophages according to the above protocols. After then, NSCLC cells were harvested and suspended in 200µl of FBS-free DMEM before being transferred to the upper compartment. The cells in the upper chamber were wiped out after 24 hours, and the cells on the lower chamber were fixed with 4 percent paraformaldehyde and stained with 0.5 percent crystal violet. For identifying immune molecules in M2-exos that induce NSCLC cells migration and invasion, A549 and H1299 cells were cocultured with M2-exos, M2-exos + anti-IgG blocking antibody (M2-exos + IgG Ab), M2-exos + anti-ITG αvβ3 blocking antibody (M2-exos + ITG αvβ3 Ab, BioLegend, San Diego, CA) for 24 hours. Control group NSCLC cells were incubated with PBS. The results of NSCLC cells migration and invasion were photographed and counted. At least three random microscopic fields (magnification×100) were taken, and the cells were counted. All experiments were performed in triplicate.

Flow cytometry staining and analysis

Flow cytometric assays were used to evaluate the expression of CD206 and HLA-DR as previously described [35]. Briefly, 5×105 M1 and M2 macrophages were harvested and stained with PE-CD206 or FITC-HLA-DRα antibody (BioLegend, San Diego, CA) for 15–20 minutes, and subsequently analysis using flow cytometry. Flow cytometry data were analyzed by the Flowjo (Treestar, USA) software.

Western blot analysis

Briefly, Whole cell lysates were electrophoresed in an 8 percent SDS-PAGE gel and then transferred to 0.22 m PVDF membranes (Millipore, USA) after being lysed in RIPA buffer with protease inhibitors. The membranes were blocked for 1h at 37°C in TBST with 5% skimmed milk powder before being probed with the specific antibody (1:1000) overnight at 4°C. Then, the membranes were incubated with secondary antibody (1:5000) for 1h at 37°C. The protein bands were identified using an ECL detection system (Bio-Rad, USA).

Reverse transcription and quantitative real-time PCR

Total cellular RNA was extracted using TRIzol reagent (Invitrogen, USA), and 1µg total RNA was reverse transcribed into first-strand complementary DNA (cDNA) using cDNA Synthesis Kit (EZBioscience, USA) according to protocols. Afterwards, the cDNA was performed to measure the relative gene expression level using real-time PCR. The expression of target genes was normalized to GAPDH levels in the samples in triplicates. The 2−ΔΔCT method was used to calculate the relative variation in gene expression. Additional file: Table S1 contains a list of primers.

Animals

Male Balb/c nude mice aged 4 to 6 weeks were purchased from Guangdong Medical Laboratory Animal Center in China. For establishing human NSCLC lung metastasis model in nude mice, 3×106 A549luc cells were resuspended in 200µl FBS-free DMEM and injected intravenously into Balb/c nude mice. To study the blockade effects of ITG αvβ3, 10µg M2-exos, M2-exo + ITG αvβ3 Ab and M2-exos + IgG Ab were administered to Balb/c nude mice every four days, respectively. A similar volume of PBS was injected into the control group. Mice were sacrificed after 50 days, and the lungs were assessed for metastatic lesions by comparing biofluorescence signal intensities. Tissue morphology was identified by hematoxylin and eosin (H&E) staining.

Construction and transfection of ITG αvβ3 shRNA and overexpression plasmids

As previously described, reliable knockdown and overexpression cell lines were established[36]. lentiviral vectors were used to create ITG αvβ3 shRNA and overexpressed plasmid. The generated plasmid was co-transfected into 293T cells for 48 hours with the viral packaging plasmids psPAX2 and pMD.2G. Lentiviral supernatants were harvested and filtered through 0.45µm filter before being cultivated for 24 hours with H1299 cells. Puromycin selection (2 g/ml) was applied to the cells. Additional file: Table S2 shows the targeting sequences for specific genes. Table S3 shows the primers of overexpressed genes.

Immunohistochemistry

Patients’ clinical tumour specimens were gathered at the Sun Yat-sen University Cancer Center in Guangzhou, China, who had been diagnosed with NSCLC. For patient specimens, all patients gave their agreement and enrolled in an IRB approved protocols at Sun Yat-sen University Cancer Center, which allowed the collecting and analysis of clinical data, archival, and paraffin specimens in compliance with ethical principles (Ethics Document No. SL-B2022-139-01). Tumour specimens were formalin-fixed and paraffin-embedded, as is standard laboratory pathology technique, and stored at the Sun Yat-sen University Cancer Center’s pathology department. The paraffin slices from patients' tissues were treated with primary anti-human antibodies at various dilutions (ITG αv, 1:400, ITG β3, 1:200) overnight at 4°C. They were then treated for 60 minutes at room temperature with the second antibody. The staining was identified by using DAB Kit (Zisbio) as directed by the manufacturer. Hematoxylin staining was measured using at least 5 randomly selected 200 or 400 fields of view after slides were stained for 6 minutes. Two pathologists independently evaluated the protein expression.

Statistical analysis

Unless otherwise specified, the results were presented as means SEM and analyzed using one-way ANOVA or Student's t-test analysis. The statistical significance level was set at p < 0.05. SPSS 22.0 or GraphPad Prism 7 were used for all statistical analyses.

3. Results

3.1 M2 macrophage infiltration is associated with poor clinical outcome

To look into the relationship between macrophages and lung cancer. The Cancer Immunome Atlas (https://tcia.at/) is an online database used to assess the macrophage distribution in TME. According to an analysis of macrophage distribution in various cancer, macrophages were highly enriched in lung adenocarcinoma (LUAD) samples (Fig. 1A). In LUAD, M2 macrophages made up the largest fraction of immune cells (Fig. 1B). TAMs are distinguished by specific surface molecules, such as the Mannose Receptor CD206, which related to angiogenesis and cancer metastasis [37]. Lung adenocarcinoma specimens were collected to better understand the distribution of M2 macrophages in the LUAD. M2 macrophages were found to be more prevalent in metastatic lung adenocarcinoma specimens (n = 59) than in non-metastatic lung adenocarcinoma specimens (n = 67) (Fig. 1C, D). Furthermore, high M2 macrophages infiltration was linked to a poor prognosis (Fig. 1E). Overall, macrophages are the most common immune subgroup in lung adenocarcinoma, and lung adenocarcinoma with high M2 macrophages infiltration is more prone to metastasis.

3.2 M2-polarized macrophages enhance NSCLC cells migration and invasion

It is reported that tumour-associated macrophages extensively regulate tumour progression and metastasis of a variety tumours [38]. Typically, tumour-associated macrophages exhibit an M2-like phenotype. To explore the impact of M2 macrophages on lung adenocarcinoma, we first successfully constructed an M1/M2 polarized macrophage model in vitro using THP-1 monocytes (Fig. 2A). In contrast to M1-polarized macrophages, M2-polarized macrophages showed increased expression of CD206, CD163, Arg-1 (M2 macrophages-associated marker), along with diminished level of HLA-DRα, TNF-α, iNOS (M1 macrophages-associated marker), which was confirmed by flow cytometry and qPCR assays (Fig. 2B, C). The induced M2 macrophages were then co-cultured with NSCLC cells for 24h, or NSCLC was pretreated with M2 conditioned medium. We found that both treatments markedly enhanced the migration and invasion abilities of H1299 and A549 cells (Fig. 2D, E). Therefore, we demonstrated in vitro that M2-type macrophages assist the progression and metastasis of NSCLC.

3.3 M2 macrophage-derived exosomes enhance NSCLC cells migration and invasion

After conditioned medium pretreatment with M2 macrophages, we found that A549 and H1299 cells migrated and invaded substantially more. Therefore, we wondered if substances derived from M2 macrophages were responsible for the remarkable effect. Accumulated evidences suggested that exosomes derived from tumour cells or tumour-associated stomal cells involve in tumour metastasis [39]. To investigate whether M2-exos are related to cancer metastasis, we extracted exosomes from M2 macrophages culture medium and subsequently treated NSCLC cells in vitro with these exosomes. Exosomes were validated by Western blot analysis using exosome-specific markers, TSG101, CD63, Alix as well as the negative marker calnexin (Fig. 3A). In addition, nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to quantify particle size and morphology (Fig. 3B, C). To exposed whether M2-exos can be internalized by NSCLC cells, we pre-treated H1299 cells with PKH26-labeled exosomes for 12h. Confocal fluorescence imaging revealed that M2-exos tagged with PKH26 were highly absorbed by H1299 cells (Fig. 3D).

We hypothesize that M2-exos promote NSCLC cells migratory and invasive capacities. Therefore, H1299 cells and A549 cells were cocultured with M2-macrophage-derived medium (M2-CM), M2-exos, and M2-CM depleted exosomes (M2-CM dexo), respectively. Transwell assays were employed to assess cancer cells' migration and invasion ability. As expected, M2-exos notably increased the migration and invasion of NSCLC cells, but had little impact on cell proliferation (Fig. 3E-H). Collectively, our results revealed that M2 macrophages enhanced the mobility and aggressiveness of NSCLC cells, which were predominantly dependent on exosomes.

3.4 Exosomal ITG αvβ3 derived from M2 macrophages is a key player in mediating NSCLC metastasis

According to the evidence presented above, exosomes released by M2 macrophages deliver certain components to NSCLC cells, enhancing cancer cells motility and invasion. Previous research demonstrated that NSCLC cell-derived exosomes played a key role in mediating tumour metastasis through targeting integrin signaling pathways. Integrins, a heterodimeric transmembrane receptor family capable of regulation intercellular interactions with the extracellular matrix (ECM), have a key impact in the regulation in a range of tumor cell behaviors such as proliferation, adhesion, migration, invasion and survival. Thus, we assumed that integrin might played a key role in M2-exos in mediating NSCLC metastasis. We discovered that M2-exo treatment significantly increased the protein expression levels of ITG αv and β3 in H1299 and A549 cells in a concentration-dependent manner. However, there was no significant change in the mRNA levels of ITG αvβ3 (Fig. 4A, B). Therefore, the increased protein expression of ITG αv and β3 in A549 and H1299 cells is not endogenous. We investigated the expression of ITG av and β3 in M2-exos to investigate whether exosomes mediate direct intercellular transmission of ITG αvβ3. The Western blot experiment revealed that ITG αv and β3 were considerably more abundant in M2-exos than in M2 macrophage cell lysate (Fig. 4C). Furthermore, colocalization of ITG αvβ3 and exosomes was seen in A549 and H1299 cells cocultured with M2-exos, indicating that ITG αvβ3 was transported from M2 macrophages to NSCLC cells via exosomes (Fig. 4D). In conclusion, these results revealed that M2-exos enriched ITG αvβ3 meaningfully and could be directly transferred to NSCLC cells.

To ascertain whether M2-exos-generated ITG αvβ3 on mediating NSCLC cells metastasis, M2-exos were pre-incubated with or without anti-ITG αvβ3 blocking antibody (ITG αvβ3 Ab), and subsequently cocultured with NSCLC cells to detect their migration and invasion abilities. In this investigation, an IgG blocking Ab (IgG Ab) was used to evaluate the specificity of ITG αvβ3 Ab. As shown, when contrasted to the M2-exos + IgG Ab group, the M2-exos + ITG αvβ3 Ab group effectively prevented the invasion and migration of H1299 and A549 cells (Fig. 4E, F). To further confirm the critical role of exosomal ITG αvβ3 derived from M2-macrophages, ITG αv and β3 expression were suppressed in H1299 cells employing two distinct shRNAs, and western blot assays were used to confirm the knock-efficacy (Fig. 4G). Moreover, the down-regulation of ITG αv and β3 protein expression in H1299 cells significantly repressed their capacities of migration and invasion, and M2-exos treatment can salvage this inhibitory effect (Fig. 4H, I).

3.5 M2 macrophage-derived exosomal ITG αvβ3 promotes NSCLC metastasis in vivo

To investigate whether M2-exos and its component ITG αvβ3 prime NSCLC lung metastasis in vivo, we constructed A549 cells stably expressing luciferase gene (A549luc), followed by treated with M2-exos, M2-exos + IgG Ab, M2-exos + ITG αvβ3 Ab and PBS. Then, A549luc cells with different treatment were injected into caudal veins of male nude mice, and various treatment interventions were performed as illustrated in scheme (Fig. 5A). There was no notable variation in body mass between the groups throughout the experiment (Fig. 5B). When the M2-exos and M2-exos + IgG antibody groups were compared to the control group, we discovered a substantial increase in lung metastases. When compared to the M2-exos and M2-exos + IgG group lung metastasis was considerably reduced in the M2-exos + ITG αvβ3 Ab group (Fig. 5C, D). These results suggested that ITG αvβ3 was indeed the main effector molecule mediating M2-exos to promote tumor metastasis. Histologic investigation revealed that M2-exos dramatically enhanced the metastatic nodules in lung, but blocking exosomal ITG αvβ3 would inhibit this effect (Fig. 5E). These results implied that M2 macrophage-derived exosomal ITG αvβ3 could be transmitted to cancer cells and increased cancer migration and invasion in vivo.

3.6 ITG αvβ3 improve tumour metastasis via activating the FAK signaling pathway.

Exosomes have been proven in numerous studies to have a vital function in signal transduction [40–42]. However, the involvement of transportable ITG αvβ3 from M2-exos in the NSCLC migratory and invasive signaling pathway remains unknown. To further explore the relevant molecular mechanisms, we constructed A549 and H1299 cells overexpressing ITG αvβ3 (Fig. 6A, B). We found that A549-ITG αvβ3 and H1299-ITG αvβ3 had dramatically improved migration and invasion abilities (Fig. 6C). We also carried out wound-healing assay. The horizontal mobility of A549-ITGαvβ3 and H1299-ITG αvβ3 was higher than that of the control group, as expected (Fig. 6D). These results further suggested that ITG αvβ3 could be a key effector molecule promoting tumor metastasis. Focal adhesion kinase (FAK) is a non-receptor kinase that is primarily responsible for adhesion signaling and cell migration, but it can also promote cell survival in the absence of stress [43]. Many studies have shown that integrins primarily trigger the FAK signaling pathway, regulating various biological function [44, 45]. Therefore, we further investigated whether ITG αvβ3 delivered by M2-exos activates the downstream FAK/p-FAK signaling pathway of NSCLC cells. We discovered that p-FAK protein expression was considerably increased in A549 cells after M2-exos treatment compared to the control group, while p-FAK protein expression was down-regulated after pre-incubation with M2-exo and ITG αvβ3 blocking antibody (Fig. 6E). More importantly, FAK inhibitor treatment significantly offset the increased migration and motility of tumor cells induced by M2-exos. These results further demonstrated that ITG αvβ3 promoted tumor metastasis by activating FAK signaling pathway (Fig. 6F). Taken together, these results indicated that exosomal ITG αvβ3 derived from M2 macrophages is essential for migration and invasion of NSCLC cells. Mechanically, intercellularly transferred exosomal ITG αvβ3 primarily activated the FAK signaling pathway to execute biological functions.

3.7 Integrin αvβ3 related to NSCLC metastasis and poor prognosis

We revealed that M2 macrophage-derived exosomes mediate ITG αvβ3 transmission to increase NSCLC migration and invasion in vitro and in vivo models. In order to verify the reliability of this conclusion in real data. We collected 126 lung adenocarcinoma specimens, including 59 metastatic cases and 67 non-metastatic cases (Table 1). Immunohistochemistry was used to determine the levels of ITG αv and β3 expression in lung cancer specimens. We discovered that lung adenocarcinomas with metastasis had greater levels of ITG αv and β3 expression than those without metastasis (Fig. 7A, B). Then the specimens were divided into high or low ITG αv group and ITG β3 group, based on immunohistochemical scores. We found that the rate of metastasis was higher in the high ITG αv and high ITG β3 groups than in the correspondingly low score groups, respectively (Fig. 7C). This suggested that high expression of ITG αv and ITG β3 in tumour cells could indicate a poor prognosis. Therefore, we analyzed metastasis-free survival in each group. When compared to the low expression group, the metastasis-free survival rate of the high expression group was lower than expected (Fig. 7D, E). Overall, these clinical evidences suggested that high expression of ITG αv and ITG β3 was associated with a poor prognosis.

Table 1

correlation between metastasis and clinical pathology characteristics in Lung cancer

Variable

NO.

metastasis

X2

P Valve

non-meta

meta

Age

          

< 60

59

32(54.2%)

27(45.8%)

0.924

0.3

> 60

67

42(62.7%)

25(37.3%)

Gender

          

Female

57

35(61.4%)

22(23.5%)

0.307

0.6

Male

69

39(56.5%)

30(43.5%)

Smoking

          

non-smoking

79

46(58.2%)

33(41.8%)

0.022

0.9

smoking

47

28(59.6%)

19(40.4%)

ITG β3

          

low expression

62

40(64.5%)

22(35.5%)

6.306

0.012

high expression

64

27(42.2%)

37(57.8%)

ITG αv

          

low expression

66

41(62.1%)

25(37.9%)

4.455

0.035

high expression

60

26(43.3%)

34(56.7%)

4. Discussion

Currently, the vast majority of cancer-related deaths (about 90%) are caused by metastatic disease [46]. Tumour metastasis is a complex biological process involving multiple cascade steps, and there are still many unexplained mechanisms [47]. A mass of studies have shown that TME enrich a variety of immunosuppressive cells, which have a vital role in mediating the invasiveness of primary tumours and the ability to metastasize to distant sites through direct contact with cancer cells or paracrine pathways [48, 49]. Macrophages are the main tumour-infiltrating leukocytes in almost all cancers. They can be "domesticated" by tumour cells to polarize toward M2-like macrophages phenotype, therefore supporting tumour progression [49]. Many studies have reported that the invasion and metastasis of NSCLC is closely related to its microenvironment [50]. However, the link between the metastasis of NSCLC and M2 macrophages and the underlying molecular mechanism have not yet been clarified.

It is reported that TAMs in the TME can be differentiated from bone marrow-derived macrophages, or tissue colonized macrophages, according on where the tumor is located in the body [49]. In NSCLC, the tumour-promoting TAMs are mainly derived from bone marrow macrophages [51]. Therefore, in this study, we simulated the physiological effects of TAMs in vivo by inducing the polarization of acute leukemia monocytes THP-1 to M2 macrophages in vitro. M2 macrophages have been shown to highly express genes such as CD163, CD206, Arg-1, and IL-10, while low expression of M1 macrophage markers, such as HLA-DRα, iNOS and TNF-α. In this study, we successfully induced M2 macrophages in vitro using THP-1 cell line. More importantly, CD206+ M2 macrophages have the characteristics of significantly promoting the migration and invasion phenotype of NSCLC. A study performed by Lee et al. has shown that TAMs in metastatic tumours are mainly M2 macrophage phenotypes [52]. Many studies have reported that M2 macrophages released a wide range of chemokines, cytokines to enhance tumour invasion and metastasis [53]. Nevertheless, the molecular mechanism by which they interact with tumor cells is unknown.

More and more studies have shown that exosomes produced from tumour-associated stromal cells have a significant role in mediating intercellular communication [32]. For example, exosomes derived from tumour-associated fibroblasts promoted the metastasis of colorectal cancer cells and chemotherapy resistance [39]. Similarly, M2-exos were confirmed to increase the metastasis of colorectal cancer cells [32]. In addition, Wu et al. found that M2 macrophages-derived exosomes delivered integrin αMβ2 to hepatocellular carcinoma cells and activated the expression of MMP9, thereby promoting the invasion and metastasis of cancer cells [54]. Consistently, our research has shown that exosomes secreted by M2 macrophages possess the ability to enhance the metastasis of NSCLC cells in vivo and in vitro.

Exosomes are made up of a range of biologically active components, including proteins, RNA, DNA and lipids [27, 55]. Importantly, exosomal contents derived from different cell types are unique [56]. Our study found that M2-exo showed a significant enrichment of ITG αvβ3. ITG αvβ3 is a marker of tumour angiogenesis, and its expression on tumour cells is related to cancer progression, drug resistance and EMT [57]. In addition, Wettersten et al. showed that ITG αvβ3 has a significant positive correlation with TAMs markers in various cancers [58]. Additionally, the enrichment of ITG αvβ3 was found in prostate cancer cell-derived exosomes, which could promote the migration phenotype of non-tumourigenic cells through intercellular deliver of exosome [59]. In this study, we demonstrated that M2-exos-mediated invasion and metastasis of NSCLC cells are dependent on ITG αvβ3. Exogenous blocking of ITG αvβ3 derived from M2-exos prevented the migration and invasion of NSCLC induced by M2-exos.

Studies so far have shown that exosomes are important mediators of intercellular signal transduction. However, the mechanism of ITG αvβ3 transferred by M2-exo in driving the migration and invasion of NSCLC is still unclear. FAK is a major regulator of growth factor receptor and integrin-mediated signals, which controls the basic processes of normal cells and cancer cells through kinase activity and scaffold function [60]. FAK activity and expression are upregulated in many cancers, and are usually relevant to poor clinical outcomes, implying that FAK could be used to predict tumour progression [60]. In this study, we found that M2-exos ITG αvβ3 mainly facilitated the invasion and metastasis of NSCLC through activating the FAK signal transduction.

5. Conclusions

Our research showed that M2-exos were rich in ITG αvβ3, which could be directly transferred to NSCLC cells, resulting in accelerated migration and invasion in NSCLC. ITG αvβ3 facilitated NSCLC invasion and metastasis by increasing the phosphorylation of FAK. Blocking exosomal ITG αvβ3 weakened the potential of M2-exos to increase NSCLC cells migration and invasion. Therefore, our study supported that ITG αvβ3 as a biomarker of TAMs activation in NSCLC. In addition, blocking the ITG αvβ3-FAK signal transduction pathway may be a promising treatment to control the metastasis of NSCLC. However, more research is still needed to further clarify its potential mechanism. In addition, our future research will need to isolate TAMs from human specimens for further verification

Abbreviations

NSCLC: Non-small-cell lung cancer 

M2-exos: M2 macrophage-derived exosomes

ITG αvβ3: Integrin αvβ3

TME: Tumor microenvironment

TAM: Tumour-associated macrophages

EGFR: Epidermal growth factor receptor

VEGF: Vascular endothelial Growth Factor

CSF1: Colony-stimulating factor 1

CCL2: The C-C motif chemokine ligand 2

TGF-β: Transforming growth factor-beta

EGF: Epidermal growth factor

IL-10: Interleukin-10

IL-13: Interleukin-13

FAK: Focal adhesion kinase

LUAD: lung adenocarcinoma

TNF-α: Tumor necrosis factor-alpha

HLA-DRα: Human leukocyte antigen-DRα

iNOS: Inducible nitric oxide synthase 

TSG101: Tumor susceptibility gene 101

NTA: Nanoparticle tracking analysis

TEM: Transmission electron microscopy

M2-CM: M2-macrophage-derived medium

ECM: Extracellular matrix

EMT: Epithelial-Mesenchymal Transition.

Declarations

Ethics approval and consent to participate This study involves human subjects and was approved by the institutional research ethics committee of Sun Yat-sen University Cancer Center (SL-B2022-139-01).  Subjects gave informed consent to participate in the study before taking part.

Consent for publication Not applicable

Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests The authors have no relevant or potential conflicts of interest to declare. 

Funding This work was supported by National Key R&D Program of China (2021YFE0202000), National Natural Science Foundation of China (81773888).

Authors' contributions LF and FW conceived of the study.  LF and JZ designed it. LH, JZ, CS, SW, CY and ML carried out the experiments.  LH and JZ analyzed and interpreted the data.  LH and JZ drafted the manuscript with comments from all authors.  FW and LF reviewed the manuscript.  All authors read and approved the final manuscript.

Acknowledgements Not applicable

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F: Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021.
  2. Herbst RS, Morgensztern D, Boshoff C: The biology and management of non-small cell lung cancer. Nature 2018, 553(7689):446–454.
  3. Lim Z-F, Ma PC: Emerging insights of tumor heterogeneity and drug resistance mechanisms in lung cancer targeted therapy. J Hematol Oncol 2019, 12(1):134.
  4. Wu YL, Tsuboi M, He J, John T, Grohe C, Majem M, Goldman JW, Laktionov K, Kim SW, Kato T et al: Osimertinib in Resected EGFR-Mutated Non-Small-Cell Lung Cancer. The New England journal of medicine 2020, 383(18):1711–1723.
  5. Kim J, Lee HM, Cai F, Ko B, Yang C, Lieu EL, Muhammad N, Rhyne S, Li K, Haloul M et al: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1 mutant lung cancer. Nature metabolism 2020, 2(12):1401–1412.
  6. Planchard D, Smit EF, Groen HJM, Mazieres J, Besse B, Helland Å, Giannone V, D'Amelio AM, Jr., Zhang P, Mookerjee B et al: Dabrafenib plus trametinib in patients with previously untreated BRAF(V600E)-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial. The Lancet Oncology 2017, 18(10):1307–1316.
  7. Wu Y, Yang S, Ma J, Chen Z, Song G, Rao D, Cheng Y, Huang S, Liu Y, Jiang S et al: Spatiotemporal Immune Landscape of Colorectal Cancer Liver Metastasis at Single-Cell Level. Cancer discovery 2022, 12(1):134–153.
  8. Dai J, Cimino PJ, Gouin KH, 3rd, Grzelak CA, Barrett A, Lim AR, Long A, Weaver S, Saldin LT, Uzamere A et al: Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain. Nat Cancer 2022, 3(1):25–42.
  9. Kfoury Y, Baryawno N, Severe N, Mei S, Gustafsson K, Hirz T, Brouse T, Scadden EW, Igolkina AA, Kokkaliaris K et al: Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment. Cancer Cell 2021, 39(11):1464–1478.e1468.
  10. Lim B, Woodward WA, Wang X, Reuben JM, Ueno NT: Inflammatory breast cancer biology: the tumour microenvironment is key. Nat Rev Cancer 2018, 18(8):485–499.
  11. Bayik D, Lathia JD: Cancer stem cell-immune cell crosstalk in tumour progression. Nat Rev Cancer 2021, 21(8):526–536.
  12. DeNardo DG, Ruffell B: Macrophages as regulators of tumour immunity and immunotherapy. Nat Rev Immunol 2019, 19(6):369–382.
  13. Güç E, Pollard JW: Redefining macrophage and neutrophil biology in the metastatic cascade. Immunity 2021, 54(5):885–902.
  14. Wellenstein MD, Coffelt SB, Duits DEM, van Miltenburg MH, Slagter M, de Rink I, Henneman L, Kas SM, Prekovic S, Hau CS et al: Loss of p53 triggers WNT-dependent systemic inflammation to drive breast cancer metastasis. Nature 2019, 572(7770):538–542.
  15. Martinez FO, Helming L, Gordon S: Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol 2009, 27:451–483.
  16. Morrissey SM, Zhang F, Ding C, Montoya-Durango DE, Hu X, Yang C, Wang Z, Yuan F, Fox M, Zhang HG et al: Tumor-derived exosomes drive immunosuppressive macrophages in a pre-metastatic niche through glycolytic dominant metabolic reprogramming. Cell metabolism 2021, 33(10):2040–2058.e2010.
  17. Cassetta L, Pollard JW: Targeting macrophages: therapeutic approaches in cancer. Nat Rev Drug Discov 2018, 17(12):887–904.
  18. Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004, 4(1):71–78.
  19. Mantovani A, Marchesi F, Malesci A, Laghi L, Allavena P: Tumour-associated macrophages as treatment targets in oncology. Nat Rev Clin Oncol 2017, 14(7):399–416.
  20. Salmaninejad A, Valilou SF, Soltani A, Ahmadi S, Abarghan YJ, Rosengren RJ, Sahebkar A: Tumor-associated macrophages: role in cancer development and therapeutic implications. Cell Oncol (Dordr) 2019, 42(5):591–608.
  21. Noy R, Pollard JW: Tumor-associated macrophages: from mechanisms to therapy. Immunity 2014, 41(1):49–61.
  22. Jackaman C, Tomay F, Duong L, Abdol Razak NB, Pixley FJ, Metharom P, Nelson DJ: Aging and cancer: The role of macrophages and neutrophils. Ageing Res Rev 2017, 36:105–116.
  23. Zhang Q-w, Liu L, Gong C-y, Shi H-s, Zeng Y-h, Wang X-z, Zhao Y-w, Wei Y-q: Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature. PLoS One 2012, 7(12):e50946.
  24. Zhao X, Qu J, Sun Y, Wang J, Liu X, Wang F, Zhang H, Wang W, Ma X, Gao X et al: Prognostic significance of tumor-associated macrophages in breast cancer: a meta-analysis of the literature. Oncotarget 2017, 8(18):30576–30586.
  25. Guo B, Cen H, Tan X, Ke Q: Meta-analysis of the prognostic and clinical value of tumor-associated macrophages in adult classical Hodgkin lymphoma. BMC Med 2016, 14(1):159.
  26. O'Brien K, Breyne K, Ughetto S, Laurent LC, Breakefield XO: RNA delivery by extracellular vesicles in mammalian cells and its applications. Nature reviews Molecular cell biology 2020, 21(10):585–606.
  27. Pegtel DM, Gould SJ: Exosomes. Annu Rev Biochem 2019, 88:487–514.
  28. Kalluri R, LeBleu VS: The biology function and biomedical applications of exosomes. Science 2020, 367(6478).
  29. Lin Y, Xu J, Lan H: Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. J Hematol Oncol 2019, 12(1):76.
  30. Huang W, Yan Y, Liu Y, Lin M, Ma J, Zhang W, Dai J, Li J, Guo Q, Chen H et al: Exosomes with low miR-34c-3p expression promote invasion and migration of non-small cell lung cancer by upregulating integrin α2β1. Signal Transduct Target Ther 2020, 5(1):39.
  31. Pritchard A, Tousif S, Wang Y, Hough K, Khan S, Strenkowski J, Chacko BK, Darley-Usmar VM, Deshane JS: Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization. Cells 2020, 9(5).
  32. Lan J, Sun L, Xu F, Liu L, Hu F, Song D, Hou Z, Wu W, Luo X, Wang J et al: M2 Macrophage-Derived Exosomes Promote Cell Migration and Invasion in Colon Cancer. Cancer Res 2019, 79(1):146–158.
  33. Zhang Y, Meng W, Yue P, Li X: M2 macrophage-derived extracellular vesicles promote gastric cancer progression via a microRNA-130b-3p/MLL3/GRHL2 signaling cascade. J Exp Clin Cancer Res 2020, 39(1):134.
  34. Ying W, Gao H, Dos Reis FCG, Bandyopadhyay G, Ofrecio JM, Luo Z, Ji Y, Jin Z, Ly C, Olefsky JM: MiR-690, an exosomal-derived miRNA from M2-polarized macrophages, improves insulin sensitivity in obese mice. Cell metabolism 2021, 33(4):781–790.e785.
  35. Tan-Garcia A, Wai LE, Zheng D, Ceccarello E, Jo J, Banu N, Khakpoor A, Chia A, Tham CYL, Tan AT et al: Intrahepatic CD206(+) macrophages contribute to inflammation in advanced viral-related liver disease. Journal of hepatology 2017, 67(3):490–500.
  36. Tai HC, Wang SW, Swain S, Lin LW, Tsai HC, Liu SC, Wu HC, Guo JH, Liu CL, Lai YW et al: Melatonin suppresses the metastatic potential of osteoblastic prostate cancers by inhibiting integrin α(2) β(1) expression. Journal of pineal research 2022.
  37. Sun L, Kees T, Almeida AS, Liu B, He XY, Ng D, Han X, Spector DL, McNeish IA, Gimotty P et al: Activating a collaborative innate-adaptive immune response to control metastasis. Cancer Cell 2021, 39(10):1361–1374.e1369.
  38. Fu L-Q, Du W-L, Cai M-H, Yao J-Y, Zhao Y-Y, Mou X-Z: The roles of tumor-associated macrophages in tumor angiogenesis and metastasis. Cell Immunol 2020, 353:104119.
  39. Hu JL, Wang W, Lan XL, Zeng ZC, Liang YS, Yan YR, Song FY, Wang FF, Zhu XH, Liao WJ et al: CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer. Mol Cancer 2019, 18(1):91.
  40. Xu R, Rai A, Chen M, Suwakulsiri W, Greening DW, Simpson RJ: Extracellular vesicles in cancer - implications for future improvements in cancer care. Nat Rev Clin Oncol 2018, 15(10):617–638.
  41. Nam GH, Choi Y, Kim GB, Kim S, Kim SA, Kim IS: Emerging Prospects of Exosomes for Cancer Treatment: From Conventional Therapy to Immunotherapy. Advanced materials (Deerfield Beach, Fla) 2020, 32(51):e2002440.
  42. Hoshino A, Kim HS, Bojmar L, Gyan KE, Cioffi M, Hernandez J, Zambirinis CP, Rodrigues G, Molina H, Heissel S et al: Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers. Cell 2020, 182(4):1044–1061.e1018.
  43. Dawson JC, Serrels A, Stupack DG, Schlaepfer DD, Frame MC: Targeting FAK in anticancer combination therapies. Nat Rev Cancer 2021, 21(5):313–324.
  44. Oguri Y, Shinoda K, Kim H, Alba DL, Bolus WR, Wang Q, Brown Z, Pradhan RN, Tajima K, Yoneshiro T et al: CD81 Controls Beige Fat Progenitor Cell Growth and Energy Balance via FAK Signaling. Cell 2020, 182(3):563–577.e520.
  45. Cooper J, Giancotti FG: Integrin Signaling in Cancer: Mechanotransduction, Stemness, Epithelial Plasticity, and Therapeutic Resistance. Cancer Cell 2019, 35(3):347–367.
  46. Altorki NK, Markowitz GJ, Gao D, Port JL, Saxena A, Stiles B, McGraw T, Mittal V: The lung microenvironment: an important regulator of tumour growth and metastasis. Nat Rev Cancer 2019, 19(1).
  47. Dasgupta A, Lim AR, Ghajar CM: Circulating and disseminated tumor cells: harbingers or initiators of metastasis? Mol Oncol 2017, 11(1):40–61.
  48. Hinshaw DC, Shevde LA: The Tumor Microenvironment Innately Modulates Cancer Progression. Cancer research 2019, 79(18):4557–4566.
  49. Davies LC, Jenkins SJ, Allen JE, Taylor PR: Tissue-resident macrophages. Nat Immunol 2013, 14(10):986–995.
  50. Liang Z-W, Ge X-X, Xu M-D, Qin H, Wu M-Y, Shen M, Zhang Y, Liu X-M, Chen K, Li W et al: Tumor associated macrophages (TAMs) promote the metastasis and growth of non-small cell lung cancer (NSCLC) cells through NF-κB/PP2Ac positive feedback loop. Cancer Sci 2021.
  51. Cortez-Retamozo V, Etzrodt M, Newton A, Rauch PJ, Chudnovskiy A, Berger C, Ryan RJH, Iwamoto Y, Marinelli B, Gorbatov R et al: Origins of tumor-associated macrophages and neutrophils. Proceedings of the National Academy of Sciences of the United States of America 2012, 109(7):2491–2496.
  52. Lee C-C, Lin J-C, Hwang W-L, Kuo Y-J, Chen H-K, Tai S-K, Lin C-C, Yang M-H: Macrophage-secreted interleukin-35 regulates cancer cell plasticity to facilitate metastatic colonization. Nat Commun 2018, 9(1):3763.
  53. Zeng X-Y, Xie H, Yuan J, Jiang X-Y, Yong J-H, Zeng D, Dou Y-Y, Xiao S-S: M2-like tumor-associated macrophages-secreted EGF promotes epithelial ovarian cancer metastasis via activating EGFR-ERK signaling and suppressing lncRNA LIMT expression. Cancer Biol Ther 2019, 20(7):956–966.
  54. Wu J, Gao W, Tang Q, Yu Y, You W, Wu Z, Fan Y, Zhang L, Wu C, Han G et al: M2 Macrophage-Derived Exosomes Facilitate HCC Metastasis by Transferring α β Integrin to Tumor Cells. Hepatology 2021, 73(4):1365–1380.
  55. Wu S, Luo M, To KKW, Zhang J, Su C, Zhang H, An S, Wang F, Chen D, Fu L: Intercellular transfer of exosomal wild type EGFR triggers osimertinib resistance in non-small cell lung cancer. Mol Cancer 2021, 20(1):17.
  56. Su C, Zhang J, Yarden Y, Fu L: The key roles of cancer stem cell-derived extracellular vesicles. Signal Transduct Target Ther 2021, 6(1):109.
  57. Demircioglu F, Hodivala-Dilke K: αvβ3 Integrin and tumour blood vessels-learning from the past to shape the future. Curr Opin Cell Biol 2016, 42:121–127.
  58. Wettersten HI, Weis SM, Pathria P, Von Schalscha T, Minami T, Varner JA, Cheresh DA: Arming Tumor-Associated Macrophages to Reverse Epithelial Cancer Progression. Cancer research 2019, 79(19):5048–5059.
  59. Singh A, Fedele C, Lu H, Nevalainen MT, Keen JH, Languino LR: Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype. Mol Cancer Res 2016, 14(11):1136–1146.
  60. Lee BY, Timpson P, Horvath LG, Daly RJ: FAK signaling in human cancer as a target for therapeutics. Pharmacol Ther 2015, 146:132–149.