Proximity Proteomics Has Potential for Extracellular Vesicle Identification.

Extracellular vesicles (EVs) are biomarkers and mediators of intercellular communication. In biological samples, EVs are secreted by various types of cells. The proteomic identification of proteins expressed in EVs has potential to contribute to research and clinical applications, particularly for cancer. In this study, the proximity-labeling method-based proteomic approach was used for EV identification, labeling membrane components proximal to a given molecule on the EV membrane surface. Due to the small labeling range, proteins on the surface of the same EVs are likely to be labeled by selecting a given EV surface antigen. The protein group of cancer cell-secreted EV (cEV), which abundantly expresses a close homologue of L1 (CHL1), was examined using a model mouse for lung cancer (LC). cEV-expressed proteins were identified by proteomic analysis of enzyme-mediated activation of radical sources by comparing serum EVs from wild-type and LC mice. SLC4A1 was found to be co-expressed in CHL1-expressing EVs, highlighting EVs expressing both CHL1 and SLC4A1 as candidates for cEVs. Serum EVs expressing both CHL1 and caspase 14 were significantly elevated in LC patients compared with healthy individuals. Thus, the combination of proximity labeling and proteomic analysis allows for effective EV identification.

Proximity proteomics identifies cancer cell membrane cis-molecular complex as a potential cancer target.

Cancer-specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane-resident "cis-bimolecular complex" as a possible cancer target (cis-bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme-mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co-expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

Tumor-dependent secretion of close homolog of L1 results in elevation of its circulating level in mouse model for human lung tumor.

Close homolog of L1 (CHL1) and its truncated form mainly play crucial roles in mouse brain development and neural functions. Herein, we newly identified that truncated form of CHL1 is produced and released from lung tumor tissue in a mouse model expressing human EML4-ALK fusion gene. Both western blot and direct ELISA analysis revealed that mouse CHL1 level in serum (including serum extracellular vesicles) was significantly elevated in EML4-ALK transgenic mice. The correlation between the tumor size and the amount of CHL1 secretion could be examined in this study, and showed a significant positive correlation in a tumor size-dependent manner. Considering these results, the measurement of circulating CHL1 level may contribute to assess a tumor progression in human lung tumor patients.

Analysis of lipid raft molecules in the living brain slices.

Neuronal plasma membrane has been thought to retain a lot of lipid raft components which play important roles in the neural function. Although the biochemical analyses of lipid raft using brain tissues have been extensively carried out in the past 20 years, many of their experimental conditions do not coincide with those of standard neuroscience researches such as neurophysiology and neuropharmacology. Hence, the physiological methods for lipid raft analysis that can be compatible with general neuroscience have been required. Herein, we developed a system to physiologically analyze ganglioside GM1-enriched lipid rafts in brain tissues using the "Enzyme-Mediated Activation of Radical Sources (EMARS)" method that we reported (Kotani N. et al. Proc. Natl. Acad. Sci. U S A 105, 7405-7409 (2008)). The EMARS method was applied to acute brain slices prepared from mouse brains in aCSF solution using the EMARS probe, HRP-conjugated cholera toxin subunit B, which recognizes ganglioside GM1. The membrane molecules present in the GM1-enriched lipid rafts were then labeled with fluorescein under the physiological condition. The fluorescein-tagged lipid raft molecules called "EMARS products" distributed differentially among various parts of the brain. On the other hand, appreciable differences were not detected among segments along the longitudinal axis of the hippocampus. We further developed a device to label the lipid raft molecules in acute hippocampal slices under two different physiological conditions to detect dynamics of the lipid raft molecules during neural excitation. Using this device, several cell membrane molecules including Thy1, known as a lipid raft resident molecule in neurons, were confirmed by the EMARS method in living hippocampal slices.