AOS-HCI Comparing the effects of alligator and horseshoe crab blood on cancer cell viability

1. AOS-HCIComparing the effects of alligator and horseshoe crab blood on cancer cell viability Estelle Gong AnuVenkatachalam Ryan Wong Joshua Foo

2. Background AOS HCI Robust innate immune system of the Singapore horseshoe crab (Carcinoscorpiusrotundicauda) Infection studies on them have demonstrated that a large amount of bacteria was rapidly suppressed. Proteins found in the blood of horseshoe crabs potentially provides a more effective way of treating cancer • American alligators (A. missippiensis) have remarkable immune systems • Blood serum components • Muscle tissue peptides • Cannot develop tumors; cannot develop cancers

3. Purpose AOS HCI Elucidate specific proteins in Horseshoe crab blood that recognize and bind surface antigens/ PAMPs of cancer cells To propose potential peptide-based drugs for cancer detection & treatment • To find what components of alligator serum, if any, have anticancer properties • Testing serum components against cancer cell membrane proteins • Isolating specific

4. Hypothesis & IV/DV AOS HCI Proteins present in horseshoe crab blood recognize and bind PAMPs of cancer cells. IV: Horseshoe crab blood hemolymph DV: Proteins that bind PAMPs of cancer cells • If alligator serum has anticancer properties, then proteins in alligator serum will bind to cancer cell membrane proteins. • IV: Alligator blood serum • DV: Proteins that bind to cancer cell membrane proteins

5. Experimental Design • AOS: • Test alligator blood serum on whole colorectal cancer cell cultures to confirm its effectiveness, if any, against cell viability. • Both AOS and HCI: • Separate serum/hemolymph from whole alligator and horseshoe crab blood, respectively • AOS – culture and lyse colorectal cells; HCI buys colorectal lysates • Isolate the proteins in the cell membrane using the QIAGEN protein kit. • Load affinity columns with membrane proteins and run serum/hemolymph through so proteins present in serum bind to proteins in the membrane. • Use a chaotropic agent such as urea to elute the proteins in the column for extraction. • Use gel electrophoresis to analyze the extracted proteins. • Eliminate the proteins of the plasma membrane from the extracted proteins to get the active components (proteins) of the whole serum/hemolymph. • Analyse the isolated active components by MALDI-TOF or other methods.

6. Experimental Design – Control setups • Run alligator serum/horseshoe crab hemolymph through a hydrophobic column not preloaded with any plasma membrane proteins. • Helps identify proteins that bind to the matrix of the column and not to the preloaded plasma membrane proteins. • Run alligator serum/horseshoe crab hemolymph through a hydrophobic column preloaded with plasma membrane proteins of normal (healthy) epithelial cells. • Helps identify proteins that already bind to non-cancer cells. • With these, we will be able to distinguish the proteins that actually help fight off cancer from the proteins that do not.

7. Experimental Design – Control setups • Analyze cancer cell plasma membrane proteins via SDS-PAGE for step 6 of ED.

8. Timeline • Present – February – AOS works, HCI break • AOS tests alligator blood serum on whole colorectal cancer cells • February – May –AOS & HCI works • Both test blood serum/hemolymph against colorectal cell membrane proteins • Isolate most effective components of • May – August – AOS break, HCI works

9. References