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

SIMPLE SUMMARY: Cancer cells release exosomes to their surrounding cells, and it is believed that trace amounts of proteins included in exosomes promote cancer stemness. In the present study, we note 78-kDa glucose-regulated protein (GRP78), which is involved in cancer progression, and present the protocol for measurements of trace amounts of GRP78 in exosomes released from cultured gastric cancer cells using an ultrasensitive ELISA with thio-NAD cycling. We found that when high-GRP78-containing exosomes were incubated with cultured cancer cells, these cells increased their stemness, for example, an increase in indices of both an MTT assay and a wound healing assay. The technique for quantifying proteins in exosomes described here will advance our understanding of cancer stemness progression via 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.