Tumor Biology and Tumor Biology and Cell MetastasisCell Metastasis

By:By: Olympia Jarboe Olympia Jarboe

Mentor:Mentor: Mary Alpaugh, Mary Alpaugh, Cancer Biologist, Ph.D.Cancer Biologist, Ph.D.

City College Of New York, City College Of New York, CUNYCUNY

Introduction:Introduction:

• Tumor biologists study cancer cells.Tumor biologists study cancer cells.• The lab of Dr. Alpaugh is growing The lab of Dr. Alpaugh is growing

various cancer cells cultures: MCF-various cancer cells cultures: MCF-7s, 468s, 231s, C861s, and Mary X 7s, 468s, 231s, C861s, and Mary X (which are breast cancer cells); (which are breast cancer cells); Melanoma; MEF-GCP (stemcells). Melanoma; MEF-GCP (stemcells).

• Proteins in cells will be examined in Proteins in cells will be examined in Western Blot.Western Blot.

Introduction continued:Introduction continued:

• Terminology:Terminology:– Invitro: Working with cells in the lab.Invitro: Working with cells in the lab.– Invivo: Working with cells in animals.Invivo: Working with cells in animals.– Media: Food the cells (fetal bovine Media: Food the cells (fetal bovine

serum, salts, 1% antibiotics).serum, salts, 1% antibiotics).– Metastasis: Cancer cells have moved Metastasis: Cancer cells have moved

or spread in the body.or spread in the body.

Purpose of Research:Purpose of Research:

• To answer the question:To answer the question:

–What inhibitors and What inhibitors and activators are responsible in activators are responsible in aiding cancer cells to trick aiding cancer cells to trick and program blood cells and program blood cells into allowing cancer cells to into allowing cancer cells to enter the bloodstream?enter the bloodstream?

Reagents/Chemicals:Reagents/Chemicals:

•EthanolEthanol

•HH22O O

•Media Media •Cell Lysis Cell Lysis

BufferBuffer

•Distilled HDistilled H22OO

• Deionized HDeionized H22OO

• 1.5M Tris-HCI1.5M Tris-HCI• 10% (w/v) Sodium 10% (w/v) Sodium

Dodecyl SulfateDodecyl Sulfate• Acrylamide/Bis Acrylamide/Bis

(30% stock)(30% stock)• 10% Ammonium 10% Ammonium

PersulfatePersulfate• TEMEDTEMED

Materials/Apparatus:Materials/Apparatus:• PipettesPipettes• Pipette AidPipette Aid• BeakersBeakers• Tissue Culture Tissue Culture

Dishes Dishes • Micro-Micro-

centrifugecentrifuge• Steam ChamberSteam Chamber• Liquid Nitrogen Liquid Nitrogen

HolderHolder

• IncubatorIncubator• 5mL syringe 5mL syringe • Centrifuge Centrifuge

capsulescapsules• Western Blot Western Blot

ApparatusApparatus• HoodHood• Electron Electron

MicroscopeMicroscope

Procedure Overview:Procedure Overview:

•1) Remove Cells from 1) Remove Cells from Liquid NLiquid N22 monitor. monitor.

•2) Thaw cells in water.2) Thaw cells in water.•3) Place cells in media 3) Place cells in media within centrifuge capsules.within centrifuge capsules.

•

Continued…Continued…

•4) Centrifuge and remove 4) Centrifuge and remove supernatant.supernatant.

•5) Add more media.5) Add more media.•6) Grow tissue cultures of 6) Grow tissue cultures of

breast cancer cells, and stem breast cancer cells, and stem cells; place in incubator. cells; place in incubator.

Preparation of whole cell Preparation of whole cell lysis buffer solution:lysis buffer solution:

• 1) Add 2mL of H20 to capsule 1) Add 2mL of H20 to capsule with Protease Inhibitor Cocktail with Protease Inhibitor Cocktail Tablet (complete, EDTA-free), to Tablet (complete, EDTA-free), to make cocktail.make cocktail.

• 2) Dissolve tablet.2) Dissolve tablet.• 3) Add 4.8mL of cell lyis buffer 3) Add 4.8mL of cell lyis buffer

liquid to a 50mL capsule and liquid to a 50mL capsule and then add cocktail.then add cocktail.

Western Blot Overview:Western Blot Overview:

•1) Prepare separating and 1) Prepare separating and stacking gel and load in to stacking gel and load in to gel slabs.gel slabs.

•2) Pour samples into wells.2) Pour samples into wells.•3) Add dye to stain the 3) Add dye to stain the proteins.proteins.

Continued…Continued…

•4) Run gel electrophoresis to 4) Run gel electrophoresis to separate proteins according to separate proteins according to molecular weight.molecular weight.

•5) Wash gel slab and pre-5) Wash gel slab and pre-marked membrane.marked membrane.

•6) Develop film to detect 6) Develop film to detect protein bands.protein bands.

Expected Results:Expected Results:

• Expect to see specific protein band Expect to see specific protein band for the SDF-1 protein. for the SDF-1 protein.

• Hypothesis:Hypothesis:– The cancer cells may be using the The cancer cells may be using the

stemcells or are attracting the stemcells or are attracting the stem cells and are utilizing them to stem cells and are utilizing them to grow and metastasize to enter the grow and metastasize to enter the blood stream and lymphatic blood stream and lymphatic system. system.

References:References:

•Mini-Protean II Electrophoresis Mini-Protean II Electrophoresis Gel Instruction Manual (Bio-Gel Instruction Manual (Bio-Rad, Life Science Group USA)Rad, Life Science Group USA)

•BD Biosciences page 1 (BD Biosciences page 1 (www.bibiosciences.comwww.bibiosciences.com))

•Roche Applied Science (catalog Roche Applied Science (catalog number 118733580001)number 118733580001)

AcknowledgementAcknowledgement• Dr. AlpaughDr. Alpaugh• Yan XueYan Xue• KatharineKatharine• EthanEthan• AmberAmber• City College of New YorkCity College of New York• Dr. Sat BhattachryaDr. Sat Bhattachrya• Harlem Children SocietyHarlem Children Society• Memorial Sloan Kettering Cancer Memorial Sloan Kettering Cancer

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