More Related Content
Similar to presentation symposium
Similar to presentation symposium (20)
presentation symposium
- 1. Analysis of eHsp90 Regulation of Secreted Exosomes from Cancer Cells Juan Felipe Toscano McCready Lab Department of Natural Science, Assumption College
- 3. • Under stress, cancer cells release a high amount of micro-vesicles named exosomes • Exosomes have a high amount of proteins that could potentially enhance cancer invasiveness and metastasis • Exosomes could be a way for cells to communicate and exchange genetic information outside the cell
- 4. • One of the most abundant proteins identified inside the exosomes was the Heat shock protein 90 (Hsp90) • Hsp90 is an important intracellular protein that activates many proteins • eHsp90 is secreted from tumor cells via exosomes • It activates pro-invasive proteins that contribute to cellular aggressiveness and motility McCready et al. 2010
- 5. Hypothesis We hypothesize that eHsp90 regulates exosome secretion from cancer cells
- 9. MDA-MB-231 100nM STA-12-7191Untreated DMSO 10nM STA-12-7191 Depleted media Determine protein concentration Western Blot Depleted media Depleted media Depleted media
- 10. 60 50 40 80 110 160 260 Control1Hsp90 Control2Hsp90 Hsp90controlBands untreated DMSO 10nM STA- 12- 7191 100nM STA-12- 7191 Hsp90 Depleted of exosomes Western blot from Drug treatment
- 11. Hsp90 concentration in media depleted of exosomes 0 10 20 30 40 50 Arbitraryunits Untreated DMSO 10nM STA-7191 100nM STA-7191
- 12. 60 50 80 110 160 260 Control1Hsp90 Control2Hsp90 Hsp90controlBands Untreated DMSO 10nM STA-12- 7191 100nM STA-12- 7191 Western blot from Drug treatment Hsp90 Exosomes
- 13. Hsp90 concentration in Exosomes isolated 0 10 20 30 40 50 Arbitraryunits Untreated DMSO 10nM STA-7191 100nM STA-7191
- 14. Inhibiting eHsp90 • eHsp90 levels remain high in exosome fraction • eHsp90 levels decrease in media depleted of exosomes
- 15. Conclusions • Data suggest that eHsp90 may be important for protein release from exosomes. Untreated Cells °° °° ° ° °° ° ° ° °°° ° ° °° ° ° ° ° ° ° Treated cells ° ° °° ° °° °°°
- 16. Acknowledgements • I want to thank Professor Jessica McCready for giving me the opportunity to work in her lab. • I would also like to thank the Jay Lab members at Tufts University for giving me the opportunity to continue this research that I started with them back in 2014
Editor's Notes
- A little bit of back ground Cancer is a genetic disease that happens after multiple mutations. This uncontrolled proliferation produce tumor cells that can communicate intercellularly, once the tumor reaches a specific size it can initiation a process called metastasis in which tumor cells invade the blood stream and migrate to other parts of the body. 90% of cancer deaths come from metastasis and not from the primary tumor. Current treatments do not target metastatic disease
- How do they communicate? contain microRNA (miRNA), which can lead to a mechanism of intercellular communication a mechanism of genetic exchange outside cancer cells that lead to distant cell interaction within the tumor microenvironment. Cancer cells are smart cells because tumor cells exposed to drugs are recognized to expel out drugs through exosomal mechanisms.
- McCready etal. did a wound healing assay to analyze how invasive and aggressive cancer cells could be depending on what they were exposed to These are pictures taken at 0h and 16 hours and length of the arrows represent how invasive the cells are. And It is seen how cancer cells when exposed exosomes became more invasive than cells exposed to recombinantHsp90 This means that Hsp90 secreted from exosomes make cancer cells more invasive Client proteins including kinases, steroid hormone receptors and transcription factors Pro-invasive proteins like MMP-2, HER-2 and plasmin are proteins that enhance aggressiveness and invasiveness of tumor cells
- Knowing how important Hsp90 can be to cancer cells, If the exosome release from cancer cells decreases by inhibiting Hsp90 with drugs, we will know that Hsp90 regulates the exosomal release process. Inhibition of Hsp90 could be a means to prevent cancer metastasis. We will test this hypothesis by monitoring exosome release from breast cancer cells in the presence of Hsp90 inhibitors
- Experimental design to show differences in Hsp90 concentration between exosomes and media depleted of exosomes
- Western blot Hsp90 concentration showing two bands because I did duplicates Based on the darkness of the band, HSP90 could be quantify N=3
- Exosomes had a 4 fold increase, meaning that Hsp90 in more abndant inside exosomes than floating around the extracellular space
- Drug treatment inhibiting Hsp90 only in the extracellular space How this affected exosome secretion from cancer cells Media ---- depleted of exosomes media and exosomes
- Hsp90 concentration in media depleted of exosomes Darkness of the bands shows the concentration of Hsp90 in the different samples you can even see a decrease based on the darkness of the bands when exposed to the drug
- Media that was depleted of exosomes shows an important decrease when it was exposed
- Each band represents the amount of Hsp90 found on each different sample And darkness of the bands let us quantify the amount of Hsp90
- Here we can see how cancer cells exposed to the drug showed an increase in Hsp90 concentration N=1
- schematic Shoing under normal conditions there are higher levels of Hsp90 inside the cell than floating in the extracellular space However, when the cells were exposed to the drug, Hsp90 levels was high in exosomes while Hsp90 levls was low in media depleted of exosomes Showing exosomes and Hsp90 and Hsp90 floating around