Tumor Biology and Cell Metastasis By: Olympia Jarboe Mentor: Mary Alpaugh, Cancer Biologist, Ph.D. City College Of New York, CUNY

Introduction: Tumor biologists study cancer cells. The lab of Dr. Alpaugh is growing various cancer cells cultures: MCF-7s, 468s, 231s, C861s, and Mary X (which are breast cancer cells); Melanoma; MEF-GCP (stemcells). Proteins in cells will be examined in Western Blot.

Introduction continued: Terminology: Invitro: Working with cells in the lab. Invivo: Working with cells in animals. Media: Food the cells (fetal bovine serum, salts, 1% antibiotics). Metastasis: Cancer cells have moved or spread in the body.

Purpose of Research: To answer the question: What inhibitors and activators are responsible in aiding cancer cells to trick and program blood cells into allowing cancer cells to enter the bloodstream?

Reagents/Chemicals: Ethanol H2O Media Cell Lysis Buffer Distilled H2O Deionized H2O 1.5M Tris-HCI 10% (w/v) Sodium Dodecyl Sulfate Acrylamide/Bis (30% stock) 10% Ammonium Persulfate TEMED

Materials/Apparatus: Pipettes Pipette Aid Beakers Tissue Culture Dishes Micro-centrifuge Steam Chamber Liquid Nitrogen Holder Incubator 5mL syringe Centrifuge capsules Western Blot Apparatus Hood Electron Microscope

1) Remove Cells from Liquid N2 monitor. 2) Thaw cells in water. Procedure Overview: 1) Remove Cells from Liquid N2 monitor. 2) Thaw cells in water. 3) Place cells in media within centrifuge capsules.

Continued… 4) Centrifuge and remove supernatant. 5) Add more media. 6) Grow tissue cultures of breast cancer cells, and stem cells; place in incubator.

Preparation of whole cell lysis buffer solution: 1) Add 2mL of H20 to capsule with Protease Inhibitor Cocktail Tablet (complete, EDTA-free), to make cocktail. 2) Dissolve tablet. 3) Add 4.8mL of cell lyis buffer liquid to a 50mL capsule and then add cocktail.

Western Blot Overview: 1) Prepare separating and stacking gel and load in to gel slabs. 2) Pour samples into wells. 3) Add dye to stain the proteins.

Continued… 4) Run gel electrophoresis to separate proteins according to molecular weight. 5) Wash gel slab and pre-marked membrane. 6) Develop film to detect protein bands.

Expected Results: Expect to see specific protein band for the SDF-1 protein. Hypothesis: The cancer cells may be using the stemcells or are attracting the stem cells and are utilizing them to grow and metastasize to enter the blood stream and lymphatic system.

References: Mini-Protean II Electrophoresis Gel Instruction Manual (Bio-Rad, Life Science Group USA) BD Biosciences page 1 (www.bibiosciences.com) Roche Applied Science (catalog number 118733580001)

Acknowledgement Dr. Alpaugh Yan Xue Katharine Ethan Amber City College of New York Dr. Sat Bhattachrya Harlem Children Society Memorial Sloan Kettering Cancer Center