Cancer Exosome-derived Integrin α6 and Integrin β4 Promote Lung Metastasis of Colorectal Cancer

Background: Colorectal cancer (CRC) metastasis remains the major cause of the CRC mortality, while the underlying mechanisms remain to be fully understood. In this study we investigated the role of cancer exosomes in CRC lung metastasis in vivo and in vitro. Methods: Expressions of Integrinα6 and Integrin β4 were examined in CRC cells as well as released exosomes. Co-culture assay with vascular endothelial cells was also analyzed. Results: We found that Integrin α6 and Integrin β4 are overexpressed in highly tumorigenic and metastatic CRC cell lines HCT116 and SW620 and their secreted exosomes, compared to the low tumorigenic and non-metastatic CRC cell lines. Disruption of ITGA6 and ITGB4 expression in CRC decreased the proliferation and tubulogenic capacities of vascular endothelial cells signicantly, while ectopic expression of ITGA6 and ITGB4 gave rise to opposite effects. Further more, we demonstrated that exosomal ITGA6 and ITGB4 promoted the lung metastasis of CRCs in vivo. Conclusions: Our study provides new insight into the molecular mechanism of CRC metastasis by which CRC-derived exosomal ITGA6 and ITGB4 induce organotropism to the lung, leading to increased tubulogenic capacity and metastasis. It also reveals a biomarker-based prediction for CRC metastasis and a novel potential therapeutic targets for CRC. tumor-derived distinct roles niche Our demonstrate metastasis, thus providing a basis for the of novel therapeutic


Introduction
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide [1], and its mortality has been steadily increasing over the past decades [2]. Metastasis is one of the most lethal causes contributing to the poor outcome of colorectal cancers [3]. The ve-year survival rate of CRC patients diagnosed with localized CRC is 90.1%. However, it drops to 11.7% when tumors spread to adjacent organs or distant tissues [4]. The lung is one of the main target organs for CRC metastasis, which accounts for 10-20% of the diagnosed patient population and which contribute to a worse outcome [5]. However, the underlying mechanisms underlying CRC lung metastasis remains to be elusive.
Exosomes are small vesicles ranging in size between 30 and 150 nm, composed by a lipid bilayer containing membrane proteins that surrounds a lumen comprising proteins and nucleic acids, that vary according to cell type and mechanism of biogenesis [6]. Exosomes functions to transport the bioactive molecules between cells, such as mRNA and miRNA to induce the genetic or epigenetic change in the target cells [7,8]. Cancer-derived exosomes have been shown to promote tumor growth or metastasis by suppressing antitumor immune responses, increasing neoangiogenesis or migration to other cells [9].
Integrin functions as a cell surface receptor that mediates the transfer of extracellular mechanical and chemical signals into the cell interior, and then modulates the different signal transduction cascades [11,12]. Integrin also coordinates cell survival, apoptosis, proliferation and motility [13]. ITGA6 is also found to be critical for the migration of human thymic epithelial cells [14] and the progression of various types of malignant tumors [10].
Integrin β4 (ITGB4) also is the structural component of hemidesmosomes that maintains epithelial architecture and acts as a signaling adaptor driving tumor cell proliferation and movement, and metastasis [15][16][17]. Tang and his colleagues have indicated that ITGB4 taken part in regulating the Chromobox homolog 8 (CBX8)-mediated proliferation and metastasis of CRC [18]. ITGα6β4, a heterodimer of ITGα6 with ITGβ4 [19] showed increased expression in murine Lewis lung carcinoma variants with high metastatic potential [20]. In addition, tumor-derived exosomal integrins have a role in determining the organotropic metastasis [21]. However, these exosomal integrins have not been explored in colorectal cancer.
In this study, we have demonstrated a role of intracellular and extracellular ITGA6 and ITGB4 in CRC lung metastasis. We found that CRC-derived exosomal ITGA6 and ITGB4 is associated with CRC metastasis and is able to induce the proliferation and tubulogenic capacity of vascular endothelial cells. Furthermore, we analyzed the biodistribution and functions of high metastatic CRCs-secreted exosomes in vivo and demonstrated that exosomal ITGA6 and ITGB4 directed organ-speci c colonization which might help promote the metastasis of CRC to the lung. Our ndings could pave the way for the development of an effective method to predict organ-speci c metastasis and therapies to halt the metastatic spread.

Patients and plasma samples
The study was approved by the Guangxi Medical University Cancer Hospital Ethics Committees, and all patients were enrolled by written informed consent. This study was conducted under the principles of the Helsinki Declaration. Five patients with histologically con rmed CRC with or without lung metastasis only at Department of colorectal and anal surgery, Guangxi Medical University Cancer Hospital at 2016 were recruited for this study. Their diagnoses were independently re-reviewed by two pathologists and classi ed by WHO criteria. Plasms were collected from these patients.

Cell culture
Human CRC cell lines HCT116, SW620, SW480, and Caco2 from American Type Culture Collection (ATCC) were authenticated by short tandem repeat pro ling and were passaged for less than six months. The vascular endothelial cells were obtained from the Laboratory of Tumor Immunopathology (Nanjing, China). Cells were maintained as a monolayer culture in Dulbecco's modi ed Eagle medium (DMEM) (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS) and were incubated at 37 °C in a humidi ed incubator with 5% CO2.

ELISA assay
The ITGA6 and ITGB4 concentration in the supernatant of cultured CRC and serum of patients were measured by human ITGA6 and ITGB4 ELISA kit (Boster, Shanghai, China) according to the manufacturer's instructions. Brie y, CRC cells cultured in DMEM medium without for 24 h. The cells and supernatants or the serum from patients were collected for cell counting and ELISA assay, respectively.

Exosomes Isolation and characterization
Isolation of exosomes from the cell culture medium using ExoQuick-TC Exosome Precipitation kit (EXOTC50A-1, SBI, Palo Alto, CA). The conditioned medium was harvested from cells cultured in advanced DMEM medium (Life Technologies, Carlsbad, CA, USA). Brie y, 1 × 10 6 cells were seeded on 100-mm dish, and the conditioned medium was collected two days later. The supernatant was centrifuged at 3,000 g for 15 min to remove debris. Added 3.3 ml ExoQuick-TC volume in 10 ml culture media, and then, resuspended the pellets with the mixture. Incubation of the mixture at 4℃for 12 hours. Next, the supernatant was centrifuged at 1500 g for 30 min to remove supernatant, then the pelleted resuspended in PBS and stored at -80˚C for using. Characterization was obtained via transmission electron microscopy (TEM) according to the protocol described by Zaharie et al [23].

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed as previously described [24]. The sequences of the speci c primer sets for ITGA6, ITGB4 and ACTN were listed in Supplementary Table S1. The level of ACTN was used as internal control.
Tumor cell proliferation assay Cell proliferation was examined using Cell Counting Kit-8 (Beyotime, Shanghai, China), for ve days. Brie y, cells (1000 cells) were seeded into 96-well plates. At each interval, added ten µl CCK-8 solution to each well and incubated for 2 hours at 37 °C. The absorbance value was measured at 450 nm using a microtiter plate reader. The experiment was conducted for in triplicate.

Lentiviral infection
The human ITGA6 and ITGB4-speci c short hairpin RNA (shRNA) vectors, the non-targeting shRNA

Statistical analysis
All statistical analyses were performed using SPSS 18.0 software. The unpaired Student's t-test was used to determine the statistical difference between two groups. Data were presented as the mean ± SD. p < 0.05, p < 0.01 and p < 0.001 was considered with statistical signi cance.

Results
Upregulation of ITGA6 and ITGB4 expression in highly metastatic CRC cells ITGA6 and ITGB4, members of integrin, have been reported to be involved in tumor metastasis [25,26]. To explore a possible role of ITGA6 and ITGB4 expression in CRC metastasis, we rst evaluated their expression in metastatic vs. non-metastatic CRC cell lines. As shown by Western blot in Fig. 1A, ITGA6 and ITGB4 were highly expressed in high-metastatic-potential HCT116 and SW620 cell lines [27], compared to low tumorigenic and metastatic SW480 and Caco2 cell lines [28,29]. A similar result was also obtained in RT-PCR analysis (Fig. 1B). Further immuno uorescent analyses con rmed that ITGA6 and ITGB4 were located in the cytoplasm, and their expressions were higher in HCT116 and SW620 cells compared to SW480 and Caco2 cells (Fig. 1C-D). These results revealed a possible association of ITGA6 and ITGB4 with CRC metastasis.
Upregulation of extracellular ITGA6 and ITGB4 protein in metastatic CRC patients and cell lines Previous research found that exosomal integrins could be used to predict organ-speci c metastasis in clinical [30]. We next used the ELISA assay to examine the abundance of ITGA6 and ITGB4 protein in the blood plasma CRC patients. The result showed that ITGA6 and ITGB4 protein levels were expressed in higher levels in patients with distant metastasis compared to those without distant metastasis ( Fig. 2A).
Furthermore, ITGA6 and ITGB4 proteins levels were also found in higher levels in supernatants of HCT116 and SW620 cell culture compared to that of SW480 and Caco2 cells (Fig. 2B). Thus, based on the data obtained from both patient's blood samples and cancer cell culture supernatant, we conclude that ITGA6 and ITGB4 proteins are highly abundant in metastatic CRC compared to low-metastatic ones, which suggest a possible role of CRC-secreted ITGA6 and ITGB4 in metastasis.

Upregulation of exosomal ITGA6 and ITGB4 proteins in metastatic CRC cells
A mount of studies has demonstrated that exosomes released from CRCs can be taken by various types of cells [31]. Given that a previous study has shown that ITGB4 acts as a cargo carried by exosome to exchange in tumor cells [32], we next explored whether ITGA6 and ITGB4 can be secreted via exosomes. We incubated the CRC cell lines in an exosome-free medium made from exosome-free FBS and then isolated the CRC-secreted exosomes using ExoQuick-TC Exosome Precipitation kit. Transmission electron microscope (TEM) was used to characterize the morphology and relative purity of isolated exosomes. As shown in Fig. 2E, the exosomes secreted in the CRC cell culture supernatant were a double-layer membrane with a uniformly cup-shaped morphology; ranged from 40 nm to 100 nm. To further explore the characteristics of those exosomes and to quantify them in the cell culture supernatants, we examined CD63 and CD9, exosome-speci c markers [33,34]. The western blot analysis showed that exosomal ITGA6 and ITGB4 expression were higher in the supernatant of HCT116 and SW620 cells culture compared to SW480 and Caco2 cells (Fig. 2F). Together, these results demonstrate that the vesicles isolated from the conditional media were the exosomes and exosomal ITGA6, ITGB4 were highly expressed in metastatic CRC cells.
The exosomal ITGA6 and ITGB4 prompt the proliferation and tube formation capacity of endothelial cells.
Vascular endothelial cells, which line tumor blood vessel, are well known to be required for a tumor to metastasize. To evaluate the functional role of exosomal ITGA6 and ITGB4 in promoting metastasis, we labeled the exosome with PKH67, a green urocence protein dye, and then co-cultured with vascular endothelial cells. The results shown by uorescence microscope indicated that vascular endothelial cells could effectively acquire the exosome from CRC cells (Fig. 3A). Meanwhile, we investigated the effect of exosomal ITGA6 and ITGB4 on the proliferation in vascular endothelial cells in vitro. The results showed that the exosomes derived from HCT116 and SW620 cells could dramatically promote the proliferation of vascular endothelial cells compared to the exosome from Caco2 and SW480 (Fig. 3B). The tubulogenic capacity is a critical feature of vascular endothelial cells, which is bene t to tumor growth. We further assessed the effect of ITGA6 and ITGB4-containing exosomes on the Tubulogenesis of vascular endothelial cells. The result showed that HCT116 and SW620-derived exosomes, when co-cultured with vascular endothelial cells signi cantly increased the tubulogenic capacity as compared to that Caco2 and SW480-derived exosomes (Fig. 3C).
To further demonstrate the role of exosomal ITGA6 and ITGB4, we silenced the expressions of ITGA6 and ITGB4 in HCT116 and SW620 cells using different, non-overlapping small hairpin RNA (shRNA) as con rmed by qRT-PCR (Supplementary Fig. S1A) and Western blot (Supplementary Fig. S1B). Knockdown of ITGA6 and ITGB4 in CRC cells also resulted in downregulation of ITGA6 and ITGB4 in exosome collected from SW620 and HCT116 cells, respectively (Fig. 4A). Accordingly, exosomes from SW620-shITGA6/ITGB4 and HCT116-shITGA6/ITGB4 showed markedly reduced ability to promote the proliferation of vascular endothelial cells (Fig. 4B). Furthermore, when co-cultured with vascular endothelial cells, the tubulogenic capacity was markedly decreased upon ITGA6/ITGB4 knockdown (Fig. 4C).
The above results suggested that exosomal ITGA6 and ITGB4 directly regulate the proliferation and tubulogenesis of vascular endothelial cells. To further con rm this conclusion, we over-expressed the ITGA6 and ITGB4 in SW480 and Caco2 cells ( Supplementary Fig. S2A-B), which leads to increased expression of ITGA6 and ITGB4 in exosomes (Fig. 5A). Consistently, the ITGA6/ITGA4-enriched exomes from SW480 and Caco2 promoted the proliferation of vascular endothelial cells signi cantly (Fig. 5B).
Moreover, the tubulogenic capacity was markedly increased when co-cultured with ITGA6/ ITGB4overexpressing SW480 and Caco-2 cells (Fig. 5C). Taken together, our results showed that exosomal ITGA6 and ITGB4 could signi cantly promote the proliferation and the tubulogenesis of vascular endothelial cells.
The exosomal ITGA6 and ITGB4 promote the lung metastasis of CRC.
Tumor spread to the distant organ is an obstacle for cancer treatment [35]. Meanwhile, a different type of metastatic cancer cells has a characteristic metastatic pattern, which de ned as organ tropism. The lung is one of a common target organ for CRC metastasis [36]. To further explore the biological effects of exosomal ITGA6 and ITGB4 on CRC metastasis in vivo, we used a xenograft mouse model. The CRC cells were injected thought the caudal vein to establish an animal model for colon cancer metastases to the lung. The exosomes derived from HCT116 and SW620 cells were also injected through the caudal vein (25 µg/ml/100 µl; 3 injections per week). As shown in Fig. 6A, there is no obvious lung metastasis in the mice injected with SW480 cells alone. However, the exosomes derived from highly metastatic counterpart SW620 could signi cantly increase the lung metastasis of SW480 cells (Fig. 6A-B). The SW620-xenograft gave rise to obvious metastasis in lung; but treatment with GW4869, an inhibitor of exosome biogenesis/release [37] signi cantly inhibited the CRC cells metastasize to the lung (Fig. 6C-D). Moreover, exosome derived from SW620-shITGA6/ITGB4 has reduced the ability to promote lung metastasis (Fig. 6E-F). Taken together, those data has validated a role of exosomal ITGA6 and B4 in underlying the lung metastasis of CRC.

Discussion
Exosomes are small extracellular membrane vesicles secreted by most cultured cells. They are involved in regulating intercellular communication, providing an opportunity for the exchange of DNA, mRNAs, microRNAs (miRNAs), proteins, and other molecules between donor cells and recipient cells and acting to regulate their function [38][39][40]. The previous study showed that exosomes take part in mediating the tumor invasion and metastasis [41]. In this study, we demonstrated a close correlation of intracellular and extracellular ITGA4 and ITGB6 with CRC metastasis through both clinical sample and CRC cell line analysis. We further provided functional validation of exosomal ITGA4 and ITGB6 in vitro and in vivo. Our work demonstrates that CRCs secret ITGA4 and ITGB6-carrying exosomes, to promote lung metastasis of CRC.
Our nding is consistent with the previous reporting indicating a role of ITGA6 or ITGB4 in cancer invasion, metastasis and poor prognosis in human gallbladder carcinoma [26]. Kwon et al. have shown that ITGA6 was highly expressed in esophageal squamous cell carcinoma and plays a vital role in the progression of cancer cells by regulating the proliferation and invasiveness of these cells [42]. ITGB4 has been reported they participate in regulating the invasive and metastatic behavior in cancer cells [43]. Chen et al. showed that ITGB4 is involved in regulating migration and invasion of ovarian cancer cells [44].
Angiogenesis plays a vital role in survival, growth, and development of solid tumors, and it has become clear that the establishment of new microvessels may provide nutrition for cancer cells proliferation [45][46][47]. It has been shown that cancer-associated exosome take parts in the establishment of a premetastatic microenvironment which contributes to metastasis formation [48]. In this study, we demonstrated that exosomal ITGA6/ITGB4 could promote the proliferation and tubulogenic capacities of endothelia cells, which may organize a pre-metastatic microenvironment and contribute to CRC metastasize to the lung.

Conclusions
In conclusion, our ndings might imply that circulating tumor-derived exosomes perform distinct roles in completing pre-metastatic niche evolution. Our ndings demonstrate an important role for exosomes in dictating distant organ metastasis, thus providing a basis for deciphering the mystery of organotropism and developing the novel therapeutic strategy for CRCs.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Informed consent forms have been signed by all patients before this study. The research protocol has been approved by the Ethics Committee of the Guangxi Medical University Cancer Hospital and is based on the ethical principles of medical research involving human subjects in the Helsinki Declaration.

Consent for publication
Not applicable.