Effect of Irradiation Dose on Breast Cancer Cell Proliferation Erin Rieke Mentor: Dr. Christine Kelly.

Effect of Effect of Irradiation Dose Irradiation Dose on Breast Cancer on Breast Cancer Cell ProliferationCell Proliferation

Erin RiekeErin RiekeMentor: Dr. Christine KellyMentor: Dr. Christine Kelly

Breast CancerBreast Cancer

Most prevalent cancer in female Most prevalent cancer in female population, except skin cancerpopulation, except skin cancer

1 in 7 (13.4%) chance of developing 1 in 7 (13.4%) chance of developing invasive breast cancerinvasive breast cancer

Currently, 2 million women living Currently, 2 million women living with breast cancerwith breast cancer

Chance of dying is 1 in 33 (3%)Chance of dying is 1 in 33 (3%)

BackgroundBackground

Current treatment of breast cancerCurrent treatment of breast cancer Removal of tumor followed by external beam Removal of tumor followed by external beam

radiotherapy to the whole breastradiotherapy to the whole breast

Recent TherapyRecent Therapy BrachytherapyBrachytherapy

Radiation is delivered through catheters inserted Radiation is delivered through catheters inserted through the target area through the target area

Radioactive solution flows through the catheters Radioactive solution flows through the catheters for a short period of time and irradiates the tumor for a short period of time and irradiates the tumor cavitycavity

Background cont.Background cont. Problem with catheter Problem with catheter

based brachytherapybased brachytherapy Requires the catheters to Requires the catheters to

be inserted and remain in be inserted and remain in the breast for the length of the breast for the length of treatmenttreatment

Requires one to two day Requires one to two day hospital stayhospital stay

Problem SolutionProblem Solution Radioactive pellet instead Radioactive pellet instead

of radiation fluidof radiation fluid However, has only been However, has only been

tested with prostate cancer tested with prostate cancer – needs to be tested with – needs to be tested with breast cancerbreast cancer

ObjectivesObjectives

Culture breast cancer cellsCulture breast cancer cells Observe and analyze the effect of Observe and analyze the effect of

irradiation on breast cancer cell irradiation on breast cancer cell proliferationproliferation

Characteristics to be examinedCharacteristics to be examined Time After ExposureTime After Exposure Exposure StrengthExposure Strength

Cell CulturingCell Culturing Cells cultured in T Cells cultured in T

flasks flasks Medium changed Medium changed

every 2-3 daysevery 2-3 days At confluency, cells At confluency, cells

passaged and split passaged and split into more flasksinto more flasks

When adequate cell When adequate cell number reached, number reached, cells frozen and cells frozen and stored in liquid stored in liquid nitrogennitrogen

Optimizing Culture Optimizing Culture EnvironmentEnvironment

Adherent cells – form a discrete net Adherent cells – form a discrete net on the bottom of the culture flaskon the bottom of the culture flask

However, sometimes they don’t like to However, sometimes they don’t like to stick – reduced number of retained stick – reduced number of retained cellscells

Attachment factors used to increase Attachment factors used to increase number of cells that lay downnumber of cells that lay down

Fibronectin used to coat flasks before Fibronectin used to coat flasks before seeded with cells seeded with cells

FibronectinFibronectin

A multi-domain glycoprotein found in A multi-domain glycoprotein found in connective tissue, on cell surfaces, connective tissue, on cell surfaces, and in plasma and other body fluidsand in plasma and other body fluids

Interacts with a variety of Interacts with a variety of macromolecules including macromolecules including components of the cytoskeleton and components of the cytoskeleton and the extracellular matrix the extracellular matrix

Binds cell surfaces and various Binds cell surfaces and various compounds including collagen, fibrin, compounds including collagen, fibrin, heparin, DNA, and actin heparin, DNA, and actin

Fibronectin and Cell Fibronectin and Cell AdhesionAdhesion

Fibronectin required for cell adhesionFibronectin required for cell adhesion Most cells do not produce enoughMost cells do not produce enough Surface coated with 1-5 ug/cmSurface coated with 1-5 ug/cm22

fibronectinfibronectin Test run to examine usefulness of Test run to examine usefulness of

fibronectin coating with breast cancer fibronectin coating with breast cancer cellscells

Two T-25 flasks seeded with same number Two T-25 flasks seeded with same number of cells – one coated and one notof cells – one coated and one not

After 4 days, cell count performed to After 4 days, cell count performed to determine number of adherent cellsdetermine number of adherent cells

Fibronectin Test ResultsFibronectin Test Results

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

7.0E+05

8.0E+05

9.0E+05

Coated Non-Coated

Number of Adherent Cells

General Experimental General Experimental MethodsMethods

Culture breast cancer cells Culture breast cancer cells in in

laboratorylaboratory Cell line ZR-75-1Cell line ZR-75-1 Plate in 48-well and 96-well Plate in 48-well and 96-well

plate for testingplate for testing Expose cells to radioactive Expose cells to radioactive

source at various strengthssource at various strengths Perform proliferation and Perform proliferation and

cytotoxicity assays at cytotoxicity assays at various time points after the various time points after the irradiationirradiation

AnalysisAnalysis Efficacy of irradiation to be tested with Efficacy of irradiation to be tested with

various assaysvarious assays

The multi-well plates will be useful in The multi-well plates will be useful in following the effects over a period of timefollowing the effects over a period of time

Assays to be performed:Assays to be performed: LDH Cytotoxicity AssayLDH Cytotoxicity Assay MTT Cell Proliferation AssayMTT Cell Proliferation Assay Live/Dead Staining with Trypan BlueLive/Dead Staining with Trypan Blue Caspase 3/CPP32 Colorimetric AssayCaspase 3/CPP32 Colorimetric Assay

LDH Cytotoxicity AssayLDH Cytotoxicity Assay

Tests levels of lactate dehydrogenase, Tests levels of lactate dehydrogenase, enzyme present in all cells in the mediumenzyme present in all cells in the medium

Facilitates conversion of lactate into Facilitates conversion of lactate into pyruvate, creating NADH in the process pyruvate, creating NADH in the process

LDH Cytotoxicity AssayLDH Cytotoxicity Assay LDH usually impermeable to cell membraneLDH usually impermeable to cell membrane When cell membrane damaged, released into When cell membrane damaged, released into

surrounding mediumsurrounding medium NADH used to convert tetrazolium salt INT NADH used to convert tetrazolium salt INT

into a formazan product (purple color).into a formazan product (purple color).

LDH ProtocolLDH Protocol

Samples of cell-free medium taken at Samples of cell-free medium taken at 0,12,48,72, and 96 hours after exposure 0,12,48,72, and 96 hours after exposure and frozenand frozen

Samples thawed and placed into 96-well Samples thawed and placed into 96-well plateplate

Added LDH dye solution (Biovision K311-Added LDH dye solution (Biovision K311-400) and allowed 30 min for color 400) and allowed 30 min for color developmentdevelopment

Analyzed with a microplate reader at 490 Analyzed with a microplate reader at 490 nmnm

Percent CytotoxicityPercent Cytotoxicity

Percent cytotoxicity calculated:Percent cytotoxicity calculated:

Low control = normal cells (normal Low control = normal cells (normal LDH levels)LDH levels)

High Control = cells treated with 1% High Control = cells treated with 1% Triton X-100 (full LDH release)Triton X-100 (full LDH release)

LDH ResultsLDH ResultsLDH Levels After Irradiaiton Dose of 20 Gy

-10

0

10

20

30

40

50

60

70

0 12 24 36 48 60 72 84 96 108

Time After Exposure (hrs)

Cytotoxicity (%)

MTT Cell Proliferation MTT Cell Proliferation AssayAssay

Tests for metabolic Tests for metabolic activity of viable cellsactivity of viable cells

Yellow tetrazolium Yellow tetrazolium salt MTT cleaved into salt MTT cleaved into purple formazan by purple formazan by mitochondrial mitochondrial dehyrogenases found dehyrogenases found in active cellsin active cells

Similar idea to LDH Similar idea to LDH Cytotoxicity AssayCytotoxicity Assay

MTT ProtocolMTT Protocol

10 uL of 5 mg/ml MTT solution 10 uL of 5 mg/ml MTT solution (Chemicon CT02) added to test and (Chemicon CT02) added to test and control wellscontrol wells

Allowed to react for 4 hours – black Allowed to react for 4 hours – black crystals form on bottom of flaskcrystals form on bottom of flask

Isopropanol and HCl solution added to Isopropanol and HCl solution added to dissolve crystals and negate neutralize dissolve crystals and negate neutralize medium colormedium color

Color development analyzed by Color development analyzed by spectrophotometer at 570 nmspectrophotometer at 570 nm

MTT ResultsMTT ResultsCell Proliferation Response to 20 Gy

00.5

11.5

22.5

33.5

44.5

0 24 48 72 96 120Time After Exposure (hrs)

Cell Number (OD570nm)

Control Experimental

Live/Dead StainingLive/Dead Staining

Trypan Blue used to stain dead cells.Trypan Blue used to stain dead cells.

Compromised cell membranes allow Compromised cell membranes allow uptake of blue dyeuptake of blue dye

Cell counted using a hemocytometer Cell counted using a hemocytometer and % viability foundand % viability found

Live/Dead Staining Live/Dead Staining ResultsResultsPerecnt Viability from Live/Dead Staining with

Trypan Blue - 20 Gy

0

20

40

60

80

100

120

0 24 48 72 96 120

Time After Exposure (hrs)

Percent Viability

Control Experimental

Caspase 3/CPP32 Colorimetric Caspase 3/CPP32 Colorimetric AssayAssay

Caspase 3 know Caspase 3 know mediator of apoptosis mediator of apoptosis (programmed cell death)(programmed cell death)

Member of family of Member of family of asparate-specific asparate-specific cysteinyl proteases cysteinyl proteases

Can cleave artificial Can cleave artificial substrates consisting of substrates consisting of an appropriate sequence an appropriate sequence of four amino acidsof four amino acids

Resulting compounds Resulting compounds can be analyzed can be analyzed fluorometrically or fluorometrically or colorimetricallycolorimetrically

Caspase 3/CPP32 Caspase 3/CPP32 Colorimetric Assay ProtocolColorimetric Assay Protocol Cells collected, pelletted, and lysedCells collected, pelletted, and lysed Cytosolic extract allowed to react Cytosolic extract allowed to react

with DEVD-pNA (N-acetyl-Asp-Glu-with DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-pNA)Val-Asp-pNA)

Active caspase 3 cleaves at Asp Active caspase 3 cleaves at Asp residue and leaves free pNA (p-residue and leaves free pNA (p-nitroanilide) – chromogenicnitroanilide) – chromogenic

pNA levels analyzed with pNA levels analyzed with spectrophotometer at 400 nmspectrophotometer at 400 nm

Caspase 3/CPP32 Caspase 3/CPP32 Colorimetric Assay ResultsColorimetric Assay Results

Caspase 3 Response to 30 Gy

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

24 hours 72 hours

Caspase Activity (OD 400 nm)

Experimental Control

SummarySummary LDH – Cell stress/death increased 48 LDH – Cell stress/death increased 48

hours and beyond with 20 Gyhours and beyond with 20 Gy MTT – No significant change in cell MTT – No significant change in cell

proliferation within 96 hours of proliferation within 96 hours of exposure to 20 Gyexposure to 20 Gy

Live/Dead Staining – Cell viability Live/Dead Staining – Cell viability declined starting at 48 hours with 20 declined starting at 48 hours with 20 Gy, compared to the controlGy, compared to the control

Caspase3/CPP32 Assay – No Caspase3/CPP32 Assay – No significant increase in caspase significant increase in caspase activity seen with in 72 hours of activity seen with in 72 hours of exposure to 30 Gy exposure to 30 Gy

Further ResearchFurther Research

Examine multiple exposure Examine multiple exposure strengthsstrengths

Determine optimum culture Determine optimum culture conditions for irradiation conditions for irradiation experimentsexperiments

Perform experiments with Matrigel Perform experiments with Matrigel basement membrane matrix – basement membrane matrix – resembles the mammalian cellular resembles the mammalian cellular basement membrane basement membrane

Thank YouThank You Dr. Christine Kelly – OSU Chemical Dr. Christine Kelly – OSU Chemical

Engineering DepartmentEngineering Department Dr. Frank Chaplen – OSU Biological Dr. Frank Chaplen – OSU Biological

Engineering DepartmentEngineering Department HHMI ProgramHHMI Program Dr. Chris Mathews – OSU Biochemistry and Dr. Chris Mathews – OSU Biochemistry and

BiophysicsBiophysics Dr. Kevin Ahern – OSU Biochemistry and Dr. Kevin Ahern – OSU Biochemistry and

BiophysicsBiophysics Dr. Alena Paulenova – OSU Nuclear Dr. Alena Paulenova – OSU Nuclear

Engineering DepartmentEngineering Department

ReferencesReferences Ingham, Kenneth. Fibronectin – Molecular Ingham, Kenneth. Fibronectin – Molecular

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