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 first 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, 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 immunofluorescent analyses confirmed 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-specific metastasis in clinical. 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. Given that a previous study has shown that ITGB4 acts as a cargo carried by exosome to exchange in tumor cells, 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-specific 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 flurocence protein dye, and then co-cultured with vascular endothelial cells. The results shown by fluorescence 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 benefit 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 significantly 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 confirmed 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 confirm 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 significantly (Fig. 5B). Moreover, the tubulogenic capacity was markedly increased when co-cultured with ITGA6/ ITGB4- overexpressing SW480 and Caco-2 cells (Fig. 5C). Taken together, our results showed that exosomal ITGA6 and ITGB4 could significantly 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. Meanwhile, a different type of metastatic cancer cells has a characteristic metastatic pattern, which defined as organ tropism. The lung is one of a common target organ for CRC metastasis. 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 significantly 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 significantly 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.