ExoHCR: a sensitive assay to profile PD-L1 level on tumor exosomes for immunotherapeutic prognosis

Abstract

Cancer immunotherapy has made recent breakthrough, including immune checkpoint blockade (ICB) that inhibits immunosuppressive checkpoints such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). However, most cancer patients do not durably respond to ICB. To predict ICB responses for patient stratification, conventional immunostaining has been used to analyze the PD-L1 expression level on biopsied tumor tissues but has limitations of invasiveness and tumor heterogeneity. Recently, PD-L1 levels on tumor cell exosomes showed the potential to predict ICB response. Here, we developed a non-invasive, sensitive, and fast assay, termed as exosome-hybridization chain reaction (ExoHCR), to analyze tumor cell exosomal PD-L1 levels. First, using αCD63-conjugated magnetic beads, we isolated exosomes from B16F10 melanoma and CT26 colorectal cancer cells that were immunostimulated to generate PD-L1-positive exosomes. Exosomes were then incubated with a conjugate of PD-L1 antibody with an HCR trigger DNA (T), in which one αPD-L1-T conjugate carried multiple copies of T. Next, a pair of metastable fluorophore-labeled hairpin DNA (H1 and H2) were added, allowing T on αPD-L1-T to initiate HCR in situ on bead-conjugated exosome surfaces. By flow cytometric analysis of the resulting beads, relative to αPD-L1-fluorophore conjugates, ExoHCR amplified the fluorescence signal intensities for exosome detection by 3–7 times in B16F10 cells and CT26 cells. Moreover, we validated the biostability of ExoHCR in culture medium supplemented with 50% FBS. These results suggest the potential of ExoHCR for non-invasive, sensitive, and fast PD-L1 exosomal profiling in patient stratification of cancer immunotherapy.

Exosomal PD-L1 in cancer and other fields: recent advances and perspectives

“…A pair of metastable fluorophore-labeled hairpin DNA (H1 and H2) was added subsequently, initiating HCR in situ on bead-conjugated exosome surfaces. Finally, ExoHCR amplified the fluorescence signal intensities by 3-7 times and facilitated detection of exoPD-L1 greatly ( 97 ).…”

Molecular Docking and Intracellular Translocation of Extracellular Vesicles for Efficient Drug Delivery

“…[ 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 ]. PD-L1 is present in exosomes released from melanoma cells [ 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 ]; however, exosomes derived from metastasised melanoma cells have a higher PD-L1 content than those derived from primary focal melanoma cells. Moreover, an electron microscope analysis revealed the PD-L1 protein was carried on the surface of the protein [ 187 ].…”

“…Interestingly, based on PD-L1 levels in exosomes, patients who are most likely to respond to checkpoint inhibitors can be identified and the response to these drugs can be evaluated [ 162 , 194 , 196 , 197 , 198 , 199 ]. For example, compared to patients with high exosomal PD-L1 levels before treatment, those with lower levels responded remarkably better to treatments with the checkpoint inhibitor pembrolizumab (Keytruda), which can block PD-1, the immune cell binding partner of PD-L1 [ 185 , 196 , 198 , 200 , 201 , 202 , 203 , 204 , 205 ]. In contrast, after the start of treatment, higher exosomal PD-L1 levels reflected a reduction in tumour size [ 206 , 207 , 208 ], indicating that two different mechanisms occur.…”

Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle

“…The traditional ELISA method for detection of EVs PD-L1 was always suffered by the large sample consumption and insufficient sensitivity. , Some new emerging techniques were expected to take over ELISA, among which, sandwich structure-based method was most commonly used and combined with signal amplification technologies, such as rolling circle amplification, , hybrid chain reaction (HCR), CRISPR/Cas system, and terminal deoxynucleotidyl transferase-mediated amplification to achieve high sensitivity. The recognition molecules can be divided into two categories: (1) antibodies/aptamer that recognize transmembrane proteins, such as the universal protein CD63 , and PD-L1; (2) hydrophobic molecules that identify EVs phospholipid membranes, such as distearyl phosphate ethanolamine, cholesterol, titanium dioxide (TiO 2 ), etc.…”