Ultrasensitive Detection of GRP78 in Exosomes and Observation of Migration and Proliferation of Cancer Cells by Application of GRP78-Containing Exosomes

Abstract

Cancer cells communicate with each other via exosomes in the tumor microenvironment. However, measuring trace amounts of proteins in exosomes is difficult, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated. In the present study, we attempted to quantify trace amounts of 78-kDa glucose-regulated protein (GRP78), which is involved in cancer progression, in exosomes released from cultured gastric cancer cells using an ultrasensitive ELISA combined with thio-NAD cycling. We also evaluated the cancer stemness-promoting effects by the application of high-GRP78-containing exosomes to cultured gastric cancer cells. The ultrasensitive ELISA enabled the detection of GRP78 at a limit of detection of 0.16 pg/mL. The stemness of cancer cultured cells incubated with high-GRP78-containing exosomes obtained from GRP78-overexpressed cells was increased on the basis of both an MTT assay and a wound healing assay. Our results demonstrated that the ultrasensitive ELISA has strong potential to measure trace amounts of proteins in exosomes. Further, exosomes with a high concentration of GRP78 promote the cancer stemness of surrounding cells. The technique for quantifying proteins in exosomes described here will advance our understanding of cancer stemness progression via exosomes.

Gastric Cancer Cell-Derived Exosomal GRP78 Enhances Angiogenesis upon Stimulation of Vascular Endothelial Cells

“…We confirmed that the commercially available, easy-to-use kit, which was mentioned in Section 2.2 ., was useful for exosome isolation [ 3 , 4 ]. Using this kit, the exosomes were collected from the culture medium of GRP78-OE, GRP78-KD, and GRP78-mock AGS cells.…”

“…Glucose-regulated protein 78 (GRP78) is involved in various aspects of cancer, such as invasion and metastasis [ 1 ], and GRP78 inhibitors are now considered potential cancer therapy targets [ 2 ]. In the tumor microenvironment, GRP78 contained in exosomes, transport vesicles of about 100-nm in size, is released from cancer cells [ 3 ], where it affects the surrounding cells and induces cancer malignancy [ 4 ]. Recent studies suggest that GRP78 also plays an important role in angiogenesis.…”

“…Our recent studies using an ultrasensitive ELISA with thionicotinamide-adenine dinucleotide (thio-NAD) cycling, however, enabled us to measure the concentration of GRP78 in exosomes derived from gastric cancer cells (AGS and MKN45) at the sub-attomolar level [ 3 , 4 ]. The migration and proliferation of cancer cells incubated with high-GRP78-containing exosomes were increased based on the results of both an MTT assay and a wound healing assay [ 4 ]. Furthermore, GRP78 was successfully quantified in both the lumen and membrane fractions of exosomes obtained from cancer cells [ 3 ].…”

Importance of Ultrasensitive ELISA in Cancer Research

“…Proteins in exosomes may play an important role in the communication between cancer cells (or between cancer cells and normal cells) in the tumor microenvironment [14]. Measuring trace amounts of proteins in exosomes is difficult, however, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated.…”

“…Measuring trace amounts of proteins in exosomes is difficult, however, and thus the cancer stemness-promoting mechanisms of exosomal proteins have not been elucidated. Our team attempted to quantify trace amounts of 78-kDa glucose-regulated protein (GRP78), which is involved in cancer progression, in exosomes released from cultured gastric cancer cells using the ultrasensitive ELISA [14]. We also evaluated the cancer stemness-promoting effects by applying high-GRP78-containing exosomes to cultured gastric cancer cells.…”