Real-time nanoscale imaging shows that how much of a protein is present does not necessarily reveal how accessible it is to antibodies
, /PRNewswire/ — Researchers at the Nano Life Science Institute (WPI-NanoLSI) at Kanazawa University have developed a way to observe how antibodies interact with a cancer-related protein carried on tiny membrane-bound vesicles released by cells. Using high-speed atomic force microscopy, they found that measuring how much of the protein is present does not necessarily show how accessible it is to antibodies. The findings reported in the journal Small Methods provide a new way to investigate differences between individual cancer-derived vesicles although further research is needed before the approach can be used clinically.
Seeing whether a cancer protein can actually be reached
Cancer cells can avoid attack by the immune system in several ways. One involves a protein called programmed death-ligand 1 or PD-L1. When PD-L1 interacts with immune cells it can act as a brake that reduces their ability to attack a tumor. Medicines that block this interaction have transformed the treatment of some cancers but patients respond very differently.
Tests often measure how much PD-L1 is present. However, quantity is only part of the picture. A protein may be present but partly hidden by nearby molecules or positioned in a way that makes it difficult for an antibody to reach. This physical availability is known as accessibility.
The Kanazawa University team investigated PD-L1 on small extracellular vesicles. These are tiny membrane-bound packages released by cells that carry proteins and other biological material. They can travel between cells and help cells communicate. Vesicles released by tumors can also influence immune responses. Their surfaces vary from one vesicle to another so measurements made across large groups can conceal important differences.
Watching antibodies bind to individual vesicles
Reon Imakawa and colleagues led by Richard W. Wong used high-speed atomic force microscopy to examine individual vesicles in liquid under conditions close to those found in the body. The technique uses an extremely fine probe to trace a surface and build rapid nanoscale images. It requires neither fluorescent labels nor chemical fixation so the researchers could follow antibody interactions in real time.
The researchers compared vesicles from two breast cancer cell lines. MCF7 cells have relatively low levels of PD-L1 while MDA-MB-231 cells are a model of triple-negative breast cancer with much higher PD-L1 expression. Triple-negative breast cancer is an aggressive form of the disease that lacks three biological markers commonly used to guide treatment.
Antibodies targeting PD-L1 behaved very differently on the two groups of vesicles. On vesicles with high PD-L1 levels the antibodies frequently docked at the surface and remained there for longer periods. On vesicles with low PD-L1 levels the interactions were mostly brief. The antibodies moved along the surface and soon disappeared from view.
More than 80 percent of the high-PD-L1 vesicles showed persistent antibody association. By contrast more than 90 percent of the low-PD-L1 vesicles showed no detectable stable interaction. A control antibody rarely remained attached to either type. These results show that the method can distinguish stable binding from fleeting contact on individual vesicles.
Differences hidden by average measurements
The team assessed both how often an antibody docked and how long it remained in place. Together these measurements provide a functional picture of how accessible PD-L1 is on each vesicle. Even vesicles released by the same cell type varied substantially. This variation would be difficult to see using conventional tests that report an average across a large population.
“We usually ask how much of a protein is present, but our results show that this is only part of the picture,” said Richard W. Wong, who led the study at Kanazawa University’s Nano Life Science Institute (WPI-NanoLSI). “What also matters is whether the protein is exposed in a way that allows other molecules to reach and interact with it.”
The researchers call their descriptive approach the Checkpoint Ligand Interface Phenotype or CLIP framework. It organizes vesicles according to observable features such as the frequency and duration of antibody binding. At this stage CLIP is an experimental framework rather than a diagnostic classification system.
A second antibody targeting CD63 which is commonly found on extracellular vesicles produced broadly similar binding patterns across the different samples. This indicates that the differences observed for PD-L1 were linked to the particular target rather than a general tendency of some vesicles to bind more antibodies.
A first test with patient-derived material
The researchers also examined vesicles released by breast cancer cells grown from a tumor sample donated by one patient with triple-negative breast cancer. Detectable antibody interactions occurred on 93.5 percent of these vesicles. However, the duration of binding fell between the two laboratory cell-line models. Individual patient-derived vesicles also differed in the number of antibody docking sites that could be detected.
These findings demonstrate that the imaging method can be applied to patient-derived material. They do not show that it can predict whether a patient will respond to immunotherapy. The patient-derived cells came from a single person and were grown in the laboratory. The vesicles were not taken directly from blood.
Potential significance
The study introduces protein accessibility as a measurable property that complements information about how much of a protein is present. The approach could help researchers understand why apparently similar vesicles behave differently and could be extended to other proteins carried on extracellular vesicles and other nanoscale biological structures.
Study limitations
This is an early laboratory study. It used two breast cancer cell lines and cells derived from one patient. Antibody accessibility under the imaging conditions should not be treated as a direct measure of immune activity in the body or as evidence of treatment response. The vesicles were held on a surface for imaging and the scanning probe may influence very weak interactions. The method is also lower-throughput than existing population-based tests. Clinical relevance will require larger patient groups direct analysis of vesicles from body fluids and comparison with treatment outcomes.
Figure
https://nanolsi.kanazawa-u.ac.jp/wp/wp-content/uploads/Small-Meth-E-eye-cat.jpg
High-speed AFM reveals heterogeneous antibody accessibility on single small extracellular vesicles (sEVs).
Real-time HS-AFM imaging directly visualizes long-lasting stable and transient antibody binding to individual sEVs. MDA-MB-231-derived and patient-derived TNBC sEVs showed markedly higher antibody co-localization and longer dwell times than MCF7-derived sEVs, revealing substantial differences in the accessibility of surface PD-L1 at the single-vesicle level.
Reference
Reon Imakawa, Keesiang Lim, Takeshi Yoshida, Tomoyoshi Yamano, Tsunaki Hongu, Uryo Onishi, Masaharu Hazawa, Toshio Ando, Noriko Gotoh, Rikinari Hanayama and Richard W. Wong. “Single-Vesicle Functional Nanophenotyping Reveals Heterogeneous PD-L1 Accessibility in Triple-Negative Breast Cancer.” Small Methods (2026); e71041.
DOI: 10.1002/smtd.71041.
https://doi.org/10.1002/smtd.71041
Funding
This research was supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan; MEXT/JSPS KAKENHI grant JP24K18449 and grants JP24H01276, JP25H02360, JP26K01637 and JP26K22904; JST CREST grant JPMJCR22E3; and grants from the Hokuriku Bank, the Astellas Foundation for Research on Metabolic Disorders, the Takeda Science Foundation and the Shimadzu Science Foundation.
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About the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University
The Nano Life Science Institute at Kanazawa University develops advanced nanoprobe technologies to directly image, analyze and manipulate biomolecules in living systems. By exploring previously inaccessible nanoscale phenomena, the institute seeks to uncover fundamental principles of life and disease.
https://nanolsi.kanazawa-u.ac.jp/en/
About the World Premier International Research Center Initiative (WPI)
The WPI program was launched in 2007 by Japan’s Ministry of Education, Culture, Sports, Science and Technology (MEXT) to establish globally visible research centers with outstanding research environments and a high degree of autonomy. The program is administered by the Japan Society for the Promotion of Science (JSPS).
https://www.jsps.go.jp/english/e-toplevel/
About Kanazawa University
Founded in 1862 in Ishikawa Prefecture, Kanazawa University is one of Japan’s leading comprehensive national universities. The university promotes interdisciplinary research and international collaboration across science, medicine, engineering, the humanities and social sciences.
https://www.kanazawa-u.ac.jp/en/
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