Presentation for Phi Sigma Fall 2015

TARGETING Breast Cancer:
What specific polymorphisms
in the immune pathways of
breast cancer increase risk of
breast carcinogenesis?
Caelie Kern
Lyon, France
UNH Mentor: Dr. Charles Walker
Foreign Mentor: Dr. David Cox
Centre Léon Bérard: Centre de Recherche en Cancérologie
Institut National de la Santé et de la Recherche Médicale

CK3 – mRNA levels in OIS cellules
L106
HMEC hTERT Rasv12 ± Doxycycline
L106: J2 J3 J4 J5
Éléments du RLR pathway
qPCR

Background – Axis 1
• Oncogene Induced Senescence (OIS)
• Anti-proliferative response
• Induced by:
• Activation of oncogene OR
• Inactivation of a tumor suppressor gene
• Ras vs. HRasG12V
http://edoc.hu-berlin.de/dissertationen/braig-melanie-2007-10-25/HTML/image002.jpg

• RLR – Rig-like receptors
• Respond to dsRNA
• Trigger antiviral, antitumor, and
anti-proliferative response
• Activated pathways plays
protective role against cancer
www.invivogen.com

• HTMM hTERT RasG12V
• Immortalized breast cancer cell line
• Telomerase reverse transcriptase
• Mutated Ras via Lentivirus vector
• Continuously proliferating
• Treatment with Doxycycline
• Induces cellular senescence
https://www.systembio.com/support/resources/faqs/lenti

Cinétique d’induction
Protocoles
Echantillons à tester (L106)
Cellules: HTMM Ras p34,5
Conditions: J2, J3, J4, J5 ± DOX
3 culture-dishes de 10Ø/conditions
Platées à 0,5%
J-1 J0 J1 J2 J3 J4 J5
+Medium
Medium ± dox (Iug/ml)
0,5%: 2,5.104
cel/cult dish 10Ø
Contrôles positifs et négatifs (L106)
Cellules: HTMM Ras p38
Conditions: ± IFNα pendant 8 heures
3 culture-dishes de 10Ø/conditions
Platées à 50%
J-1(soir) J0 J0+8heures
+Medium
Medium ± IFNα
(I000 U/ml)
HTMMRas à 50%: 2,5.106 cel/cult dish 10Ø

Experimental procedure
qPCR
RNA extrait: Macherey Nagel NucleoSpin RNA
Pour RT mRNA  cDNA: BioRad iScript cDNA Synthesis Kit, 1ug mRNA utilisé
Primers: FW/RV à 10mM - 10uL de Primer FW + 10uL de Primer RV + 80uL d’H2O = 100uL
Échantillons: cDNA 1 ug/mL dilué 1/20
Master Mix: pour les échantillons et l’H2O en duplicata (soit 4 échantillons + 2 H2O)*2)
Master Mix pour 12 each/gène Vol. Total: 15uL* Vol. Total :
SYBR Green 7,5 uL * 25 187,5 uL
Primers F/R (10uM) 0,4 uL * 25 10 uL
H2O 7,1 uL * 25 177,5 uL
Stage 1: 95 °C, 5min
Stage 2: 95 °C, 15s; 60 °C, 15s; 72 °C, 30s
Stage 3: 95 °C, 15s; 60° C, 20s; 95 °C, 15s
Sample volume: 20 uL
Data collection: Stage 2, Step 3
Check t°: 0,7
Appareils:
• Eppendorf Thermocycler
• BioSystems StepOnePlus
Real Time PCR System

RIG-I
L106 ± Dox
FW: 5’ – AGC TCA GCT TGA TGA GGG ACA – 3’
Rv: 5’ – GTC TGG CAT CTG GAA CAC CA – 3’
Primers from Ablasser JI 2009

RIG-I
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
J2 J3 J4 J5
FoldincreaseofRIGImRNAexpression(UA)
mRNA Expression of RIG-I on L106 ± Dox
no dox
avec dox
0
10
20
30
40
50
60
no IFNa IFNa
FoldincreaseofRIG-ImRNAexpression(UA)
mRNA Expression
of RIG-I on HTMM
Ras ± IFN-α

Results Axis 1
• Proteins up-regulated due to doxycycline
treatment:
• RIG-I, MDA-5, MAVS, IKKε, ISG20, CYLD, NLRP3,
ULBP2
• Conclusion:
• Cells treated with doxycycline exhibit:
• Change in morphology
• Changes in expression levels of RLR Pathway
Senescence!

CK6 – In Vitro Generation of XCR1+ DC
from Cord Blood CD34+ Cells
Phéno de Différenciation et Activation par Ligands
STING

• Dendritic cells
• Antigen-presenting cells
• Interface between innate and adaptive immune systems
• BDCA3+  CD141+
• DC subset
• Superior capacity to present Ag
• Activated BDCA3+ = stronger response
• Excellent target for vaccine against cancer
http://www.blog.bryanmjones.com/2013/12/dendritic-cell-illustration.html

• STING  Stimulator of Interferon Genes
• Detect cytosolic DNA
• Target for cancer vaccine or immunotherapy
• Activation triggers natural anti-tumor response
https://blogs.shu.edu/cancer/2015/04/01/aduro-sting-pathway-in-cancer-immunology/

Echantillons à tester (CK2)
Cellules:
1. CBO26 – 500,000 cel/cult
2. CBO+55 – 400,000 cel/cult
4. C97.58 - ?#
5. C94_79 – 300 000 cellules
6. C96-180 – 350 000 cellules
7. C96-163 – 350 000 cellules
Purification HSC
J0 J7 J13 J17
Décongélation HSC
Mise en
différenciation
Ajoute cytokines de
différenciation
Prolifération
StemSpan +
SCF: 100ng/mL
Flt3-L: 100ng/mL
IL-3: 20ng/mL
IL-6: 20ng/mL
RMPIc: 20 uL/puit
GM-CSF:
20ng/mL
SCF: 20ng/mL
Flt3-L: 100ng/mL
IL-4: 100ng/mL
RMPIc +
GM-CSF:
20ng/mL
SCF: 20ng/mL
Flt3-L: 100ng/mL
IL-4: 100ng/mL
Phéno de
différenciation
Conditions: J0- J7 2x 25cm flasks
J7-J17 96 puits plaque (U-bottom)
Prolifération et Différenciation
Protocole

Donneur 1
Donneur 2
FSC/SSC Singulet FSC
Gate sur les cellules
vivantes =
Cellules DAPI- CD11b/BDCA3 CD11b/Clec9A BDCA3/Clec9A
FSC/SSC Singulet FSC
Gate sur les cellules
vivantes =
Cellules DAPI- CD11b/BDCA3 CD11b/Clec9A BDCA3/Clec9A
Petit Phenotype – FACS
Fluorescence-Activated Cell Sorting

Methods – Axis 2
• BDCA3+ Activation
• Increases expression of surface protein CLEC9A
• Controls:
• Lipofectamine
• Poly:IC
• Experimental Treatments: Cyclic Dinucleotides
• 2,3’ - cGAMP
• 2,3’ - cGAMP PS2

J18 J20
Activation par ligands
Phéno
d’activation
0,1 ug/ml 2-3' cGAMP
1 ug/ml 2-3' cGAMP
10 ug/ml 2-3' cGAMP
0,1 ug/ml 2-3' cGAMP PS2
1 ug/ml 2-3' cGAMP PS2
Mileau
PBS1x
Poly IC
Lipopolysaccharide
Lipofectamine
Donneur 1
0,1 ug/ml 2-3' cGAMP
1 ug/ml 2-3' cGAMP
10 ug/ml 2-3' cGAMP
50 ug/ml 2-3’ cGAMP
0,1 ug/ml 2-3' cGAMP PS2
50 ug/ml 2-3’ cGAMP PS2
Mileau
PBS 1x
Poly IC
Lipopolysaccharide
LipofectamineDonneur 2
Echantillons à tester (CK2)
Cellules:
1. CBO26 – 500,000 cel/cult
2. CBO+55 – 400,000 cel/cult
4. C97.58 - ?#
5. C94_79 – 300 000 cellules
6. C96-180 – 350 000 cellules
7. C96-163 – 350 000 cellules
Conditions: J0- J7 2x 25cm flasks
J7-J17 96 puits plaque (U-bottom)
Activation Protocole

No stimulation
cGAMP ps2 1ug/ml
cGAMP ps2 10ug/ml
cGAMP ps2 50ug/ml
CLEC9A CLEC9ACD11b/BDCA3
Ungated
Donor 2 Activation_CD11bFITC BDCA3APC CLEC9APE.fcs
Event Count: 50000
0102
103
104
105
SSC-A
0
102
103
104
10
5
FSC-A
99.2
Morphology
Event Count: 49606
0102
103
104
105
FSC-A
101
10
2
103
10
4
FSC-H
89.2
Singulets FSC
Event Count: 44252
0102
103
104
105
SSC-A
101
10
2
103
10
4
SSC-H
99.7
Singulets SSC
Event Count: 44141
0 103
104
105
<APC-A>
0
102
103
104
105
<FITC-A>
0.324
1.31
BDCA3+ DC
Event Count: 143
0 102
103
104
105
<PE-A>
0
1
2
3
#Cells
94.45.59
CD11b+ DC
Event Count: 580
0 102
103
104
105
<PE-A>
0
2
4
6
8
10
#Cells
74.325.7
Ungated
Donor 2 cGAMP_1ug cGAMP ps2 CD11bFITC BDCA3APC CLEC9APE.fcs
Event Count: 50000
0102
103
104
105
SSC-A
0
102
103
104
10
5
FSC-A
99.5
Morphology
Event Count: 49743
0102
103
104
105
FSC-A
101
10
2
103
10
4
FSC-H
91.9
Singulets FSC
Event Count: 45720
0102
103
104
105
SSC-A
101
10
2
103
10
4
SSC-H
99.1
Singulets SSC
Event Count: 45297
0 103
104
105
<APC-A>
0
102
103
104
105
<FITC-A>
0.892
1.07
BDCA3+ DC
Event Count: 404
0 102
103
104
105
<PE-A>
0
2
4
6
8
10
#Cells
963.96
CD11b+ DC
Event Count: 483
0 102
103
104
105
<PE-A>
0
2
4
6
#Cells
61.938.1
Ungated
Event Count: 50000
0102
103
104
105
SSC-A
0
102
103
104
10
5
FSC-A
98.3
Morphology
Event Count: 49131
0102
103
104
105
FSC-A
101
10
2
103
10
4
FSC-H
70.4
Singulets FSC
Event Count: 34600
0102
103
104
105
SSC-A
101
10
2
103
10
4
SSC-H
99.1
Singulets SSC
Event Count: 34294
0 103
104
105
<APC-A>
0
102
103
104
105
<FITC-A>
0.691
0.869
BDCA3+ DC
Event Count: 237
0 102
103
104
105
<PE-A>
0
2
4
6
#Cells
972.95
CD11b+ DC
Event Count: 298
0 102
103
104
105
<PE-A>
0
1
2
3
#Cells
55.444.6
Ungated
Event Count: 50000
0102
103
104
105
SSC-A
0
102
103
104
10
5
FSC-A
99.2
Morphology
Event Count: 49591
0102
103
104
105
FSC-A
101
10
2
103
10
4
FSC-H
83.2
Singulets FSC
Event Count: 41265
0102
103
104
105
SSC-A
101
10
2
103
10
4
SSC-H
99.1
Singulets SSC
Event Count: 40899
0 103
104
105
<APC-A>
0
102
103
104
105
<FITC-A>
0.628
0.929
BDCA3+ DC
Event Count: 257
0 102
103
104
105
<PE-A>
0
2
4
6
#Cells
94.25.84
CD11b+ DC
Event Count: 380
0 102
103
104
105
<PE-A>
0
3
6
9
12
#Cells 95.54.47

Results Axis 2
• Success of cord blood differentiation:
• Donor 1: 3% BDCA3+
• Donor 2: 12% BDCA3+
• cGAMP PS > cGAMP
• Increased expression of CLEC9A
• Problem:
• Fixation technique reduced viability of cells

References
http://www.jbc.org/content/282/21/15315
http://jem.rupress.org/content/207/6/1247
http://www.cs.tau.ac.il/~spike/maps/spike00028.html
http://www.ncbi.nlm.nih.gov/pubmed/19330805
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3354961/
http://www.ncbi.nlm.nih.gov/pubmed/20032638
http://www.abcam.com/protocols/flow-cytometry-immunophenotyping
http://www.nature.com/nrc/journal/v6/n6/fig_tab/nrc1884_F1.html
http://edoc.hu-berlin.de/dissertationen/braig-melanie-2007-10-25/HTML/chapter1.html
https://blogs.shu.edu/cancer/2015/04/01/aduro-sting-pathway-in-cancer-immunology/
http://www.invivogen.com/review-rlr
https://books.google.com/books?id=Vwu7BAAAQBAJ&pg=PA115&lpg=PA115&dq=RLR+and+cancer&source=bl&ots=I75ndUvA_H&sig=Nq8bVnXDs9voVHtKeOpz-
QVtb1s&hl=en&sa=X&ved=0CEgQ6AEwCGoVChMIlpaP8-yjyAIVgTI-Ch15jAku#v=onepage&q=RLR%20and%20cancer&f=false

Presentation for Phi Sigma Fall 2015

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (20)

En vedette

En vedette (16)

Similaire à Presentation for Phi Sigma Fall 2015

Similaire à Presentation for Phi Sigma Fall 2015 (20)

Presentation for Phi Sigma Fall 2015

Notes de l'éditeur