This is not such an easy question to answer. Firstly, what size are Your EVs? Secondly, are You sure they are EVs (if they are membranous) and not EPs?

While never having looked specifically at cancer cells in vitro, yes, I believe so. This is based on characterising Ex derived from 1° epithelial cells. Depending on donor patient and post-harvest conditions, we find that expression of CD81 and CD63 is variable; i.e not definitive for Ex. My interpretation is that Ex cargoes reflect subtle physiological changes cells. undergo during physiological trauma (aka harvest, isolation, 'in vitro' culture, etc). Do your controls confirm that your Abs are working in your assay?

Kamil Szeliski : Thank you for your reply. Our methodology allows us to detect vesicles effectively. With high precision and sensitivity, we can detect vesicles as small as 100 nm, as well as larger sizes specific to cancer-related vesicles. However, my question pertains to the suitability of tetraspanins as biomarkers for identifying cancer-related extracellular vesicles (EVs). Upon reviewing articles in the field of cancer biology, I discovered instances of tetraspanin downregulation in cancerous cells, which has prompted my inquiry.

David Ian Leavesley : Thank you for your response. I can affirm that our assay has been successfully validated using negative and positive controls with well-known tetraspanin antibodies. However, in my current research, I am utilizing antibodies that are recognized as cancer biomarkers. Although I am detecting vesicles that are enriched in these cancer biomarkers, I have observed a lower abundance of tetraspanins in these vesicles. On the other hand, when detecting vesicles from the same cancer sample using only tetraspanin antibodies, I have observed a higher enrichment of tetraspanins. Based on my observations, I have raised a question regarding whether cancer-related extracellular vesicles (EVs) may potentially lack expression of tetraspanins.

Ima. I suggest that you don't overlook the fact that antibodies are rather large molecules (180 kD), and encounter steric hindrance when interacting with target species in crowded environments. I wouldn't be surprised if some of your target 'cancer biomarkers' are coalesced with proximal tetraspanins (and/or other associated membrane-associated species) in membrane rafts. For example, even organised water (not part of these rafts) can interfere with ligand-receptor interactions (see work of Gerry Pollack).

Ima Ghaeli: Firstly, what technique are You using WB, NanoFlow, as it will also affect potential aswers? Higher abundance of tetraspanins on EVs surface is rather for small EVs like exosomes, (besides some exceptions like RBCs derived ones). Thus it will highly depend of the size distribution of Yours EVs whether You should expect high level of tetraspanins or not. Therfore as it is in MISEV recomendations, maybe You should also check some other classes of EVs markers, to be sure what are You working with? Nonetheless, did Your methodology of EVs tetraspanins detection worked on other (non-cancer) cells derived EVs ?

David Ian Leavesley : Thank you for providing your valuable feedback. I wholeheartedly agree with your point regarding steric hindrance of antibodies. However, I would like to mention that in our experiments, I performed separate stainings of the extracellular vesicles (EVs) using tetraspanin labelled antibodies and cancer biomarker labelled antibodies. Interestingly, the labeling observed in both cases was almost similar to the combination of cancerous and tetraspanin-labeled antibodies. Our assay has been specifically developed to address such challenges in capturing and labeling. Nonetheless, I acknowledge the importance of incorporating more controls and exploring various conditions to ensure the robustness and reliability of our findings. Your comments have further emphasized the need for careful consideration, and I appreciate your input.

Thank you once again for sharing your insights,

Kamil Szeliski : Thank you for your valuable comments. Indeed, our technique has demonstrated successful results with EVs derived from non-cancer cells. However, detecting the subpopulation of cancerous EVs presents a significant challenge, and we are currently in the development phase to address this issue. In my preliminary findings, I have observed a lower abundance of tetraspanins on cancer-derived EVs. This has prompted us to explore alternative cancer biomarkers that may be more effective in detecting these EVs. It appears that the use of tetraspanins alone may not capture the full spectrum of cancer-related EVs. I believe that exploring other relevant cancer biomarkers, in addition to tetraspanins, is crucial in order to successfully capture, identify, and characterize cancer-related EVs. In reference to your comment about the relationship between EV size and tetraspanin abundance, I would like to clarify. Are you suggesting that larger EVs typically exhibit lower levels of tetraspanin abundance, while small extracellular vesicles are known to have a high abundance of tetraspanins? Furthermore, considering our current method of identifying EVs, is there a possibility of bias towards the detection of EVs expressing tetraspanins? Could we potentially detect other subpopulations of sEVs by utilizing alternative biomarkers that are abundant in those specific populations but do not involve tetraspanins?