Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET

 
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Breast Cancer Stem cells :
Stemming the tide of resistance

        Emmanuelle CHARAFE-JAUFFRET
                 Epithelial Stem Cell and Cancer team
                 Centre de Recherche en Cancérologie de Marseille
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Tumor cell heterogeneity: a complex ecosystem

                     Conventional Therapies

Genetic diversity   Functional diversity   CSC resistance

     Clone 1                                                              Tumor relapse
     Clone 2                                                          Metastatic progression
     Clone 3

                                              Adapted from Kreso & Dick et al., Cell Stem Cell, 2014
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Tumor cell heterogeneity: a complex CSC system

                             Identify sources
                             of heterogeneity
                               within cancer

Understanding CSC’s                             How essential is the
  biology to cure                               niche in regulating
      cancer                                      CSC metastatic
                                                    spreading?
                               Conventional
                               therapies

                 Anti-CSC
                 therapies
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Targeting CSC strategy

CSC Screening                                       Cancer stem cells                 Characterization

CSC Profiling                                         CSC        Cell lines
                                                CSC             Tumorspheres            Markers and targets
Existing
                                                                    PDXs
therapies
                                                         Primary tumors
Descriptive
genomic

                   Drug library
CSC drug        (FDA/Experimental)

screening                                                                             Drugs
                Targeted drugs/Chemo

                                                                                        Ab

                                                                 Xenograft assay
                                                                                      shRNA

                                       Clinical trials             Preclinical step
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Deciphering CSC biology to discover anti-CSC therapies
                                                                    Repertaxin
TGFb epigenetic                                                                        CXCR1/IL8 axis
                                                                                       Charafe-Jauffret et al., Cancer Research, 2009
network
                                                                                       Ginestier et al., JCI, 2010
El Helou et al., Stem cells, 2014
                                                  Fas-Ligand                 IL-8 IL-8
                                                          Fas            IL-8 IL-8

                      TGFbR
                                                          FADD                   CXCR1                 FRIDA Clinical Trial ,
       TGFb
              TGFb                                                                                    Phase II
                                                                       FAK
  TGFb                                                                         PI3-K
                                                          SCD1
          TGFb
                   SMADs                                                               PTEN

                                    SMADs
                                                         PORCN
                                                                        AKT                               Mir-600 molecular
                                                                                  FOXO3a
                                                        β-catenin
                                                                              GSK3β                       switcher of bCSC
                                      β-catenin
                                                                                                          El Helou et al., Cell rep, 2017
                                       TCF

                                                     Acetyl-CoA

                                                                    HMG-CoA           GGTI
                                            citrate

                                                                    Mevalonate

                                                   Farnesylation               Geranylation

                                                                    Cholesterol
                                                                                              Mevalonate Metabolism
                                                                                              Ginestier, (…), Charafe-jauffret, Stem cells,
                                                                                              2012
                                    Cancer Stem Cell
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
ALDEFLUOR+ population and tumorigenicity

                                        ALDEFLUOR-                   ALDEFLUOR+
                ALDEFLUOR assay

                     R1            R2

ALDEFLUOR-         ALDEFLUOR+                  ALDEFLUOR   –   ALDEFLUOR     +
 50,000 cells       50,000 cells                50,000 cells     500 cells
  5000 cells         5000 cells
  500 cells          500 cells
                                                      Ginestier et al. Cell stem cell, 2007
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
ALDEFLUOR+ population and tumorigenicity

                   ALDEFLUOR   -   ALDEFLUOR   +

ALDEFLUOR+ population regenerates
  heterogeneity of the initial tumor
                                                   Ginestier et al. Cell stem cell, 2007
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Targeting CSC strategy

CSC Screening                                       Cancer stem cells                 Characterization

CSC Profiling                                         CSC        Cell lines
                                                CSC             Tumorspheres            Markers and targets
Existing
                                                                    PDXs
therapies
                                                         Primary tumors
Descriptive
genomic

                   Drug library
CSC drug        (FDA/Experimental)

screening                                                                             Drugs
                Targeted drugs/Chemo

                                                                                        Ab

                                                                 Xenograft assay
                                                                                      shRNA

                                       Clinical trials             Preclinical step
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Patient Tumors‐derived xenografts(PDXs)

                                                     Primary Tumor

            «Clearing»

                                                          Dissociation

                                                                             Tumoral growth monitoring
                                                         + Matrigel

                                      -oestradiol

Time from birth
(weeks)

                  3                     5                                7                               12
                         Human fibroblasts

           Humanization
Breast Cancer Stem cells : Stemming the tide of resistance - Emmanuelle CHARAFE-JAUFFRET
Patient Tumors‐derived xenografts (PDXs)

                                                                                                 50 PDXs obtained

           Proportion of tumor outgrowth
                                                          Normal-like
                                                                             Basal
                                           p=0.006e-2

                                                                             ERBB2-like
                                                                             Luminal B      1.     Conservation of histology
                                                                                                   and phenotype
                                                                             Luminal A

                                                Days after PT implantation

                                             Molecular subtypes

                                                                                          Charafe-Jauffret et al, Cancer Research 2013
CRCM 181 PT

              HES   ER   PR   ERBB2

CRCM 181X

              HES   ER   PR   ERBB2
CRCM 224 PT

              HES   ER   PR   ERBB2

CRCM 224X

              HES   ER   PR   ERBB2
Patient Tumors‐derived xenografts (PDXs)

200 Tm injected                                                                                              50 PDXs obtained

                   Proportion of tumor outgrowth
                                                                  Normal-like
                                                                                     Basal
                                                   p=0.006e-2

                                                                                     ERBB2-like   1.       Conservation of histology
                                                                                     Luminal B             and phenotype

                                                                                     Luminal A

                                                        Days after PT implantation

                                                     Molecular subtypes                           2.        Conservation of molecular
                                                                                                            phenotype

                                                                                                   3.       Maintain genomic profile

                                                                                                   4.        Maintain a hierarchical
                                                                                                             organization

                                                                                                       Charafe-Jauffret et al, Cancer Research 2013
25%
                                          engrafment rate
            High grade                                        Expression of CSC marker

                                                                                                      PT non-engrafter

                                                               Metastasis-free survival
 Variable    Hazard ratio (95% CI)   p-value
   ER          0.78 [0.27-2.31]        0,66
   PR          0.67 [0.23-1.92]        0,45
SBR Grade      2.94 [1.02-8.44]      4,60E-02                                                          PT engrafter
  KI67         1.32 [0.46-3.82]        0,61
 ALDH1         3.61 [1.12-11.65]     3,20E-02
                                                                                          p=0.00352

                                                            Charafe-Jauffret et al., Cancer Research 2013
Breast CSC transcriptional programs
      8 PDXs

                            Tumor dissociation
                                                                              Gene expression
                                                                                 profiling

                    dNTP metabolism                                HR machinery

Charafe-jauffret et al., Cancer Res, 2013
in vivo                                  in vitro

                     Cell tracker

    ALDH1/Ki67/DNA

                                                                     Cell tracking

                           R1       R2
                                                                     8 6 4 2 0 Days

                                                                     Cell tracking

                                                                     8 6 4 2 0 Days

                                                                                      non-CSC

                                          Cell proliferation
                                                                                      CSC

                                                               Tracking time (days)
bCSC transcriptional programs as a response to replicative stress?

Macharet & Halazonetis, 2015

                          dNTPs pool                                 DSB
                                                                                    Genomic
                                                                                    instability

                                                                      HR
                                                             Fork‐restart pathway
  DNA replication                         Stalled fork
     initiation

                                                                                    Completed
                                                                 Fork restart       replication
                                           HR
                               Replication fork « escort »
ɣH2AX

                                             ALDHlow
     ɣH2AX positive cell (%)                   count

G1
S
                                       DNA

G2
                               ɣH2AX
                                             ALDHbr
                                               count
     ɣH2AX positive cell (%)
G1
S
                                       DNA
                                                       Breast CSC present a limited replication stress

G2
in vivo

    ALDH1/γH2AX/DNA
Breast CSC present a limited replication stress

                                                   IDU

                                                          ALDHlow
                               20 min IdU

                           Replication cluster

                                                          ALDHbr
                                       *
Breast CSC present a limited replication stress

DNA combing
                                                                        *
     O

                     ALDHlow

                                                   Fork velocity w)
                     ALDHbr

                                                                      SUM159
RAD51 expression in bCSC

       RAD51 positive cell (%)

                                                                        RAD51 positive cell (%)
                                          G1      S     G2/M                                                   G1      S   G2/M
                            DNA/RAD51

                                                                                                  DNA/RAD51
                                        ALDHlow                                                               ALDHbr

                         Restart of halted fork?
Protection of nascent forks by the formation of a stable Rad51 filament?
RAD51 inhibition induces replication stress in bCSC
            RAD51 inhibitor
         (Huang & Mazin, 2014)                DNA/RAD51                         ALDH1/γH2AX/DNA

                                       CTRL         BO2                  CTRL             BO2

                                               ***
RAD51 inhibition induces replication stress in bCSC

                                                                     ***
bCSC Targeting

                        Tumor regrowth

      Tumor debulking

B02                 Conventional therapy

                         Cisplatin

                                             Tumor
                                           eradication
BO2 sensitize bCSC to cisplatin

                                  *** < 0,001

Proportion of ALDHbr cells
 (normalized withcontrol)

                                 CTRL           Cisplatin
BO2 sensitize bCSC to cisplatin

                                                    *** < 0,001

Proportion of ALDHbr cells
 (normalized withcontrol)

                                 CTRL   Cisplatin     BO2         Cisplatin
                                                                     +
                                                                    BO2
Xenograft assay
D1                                   D33   D35   D37        D39    D41 D43   D45   D47

                            BO2

                            Cisplatin

                    300,0

                    225,0
     Volume (mm3)

                                                                                         BO2
                    150,0
                                                                                         Cisplatine
                                                                                         Combi
                                                                                         CTRL

                     75,0

                      0,0
                                33         36          40          44        48
                                                 Days post-graft
Take home message

CSC: limited Replicative Stress compare to mature cells
                                                           Are bCSC less
                                                          tolerant to RS ?
RS is limited by HR activation

HR inibition increase RS in bCSC

                                                          In vivo validation
Inhibition of HR sensitize bCSC to chemotherapies ?       in PDX is ongoing
Targeting CSC strategy

CSC Screening                                       Cancer stem cells                 Characterization

CSC Profiling                                         CSC        Cell lines
                                                CSC             Tumorspheres            Markers and targets
Existing
                                                                    PDXs
therapies
                                                         Primary tumors
Descriptive
genomic

                   Drug library
CSC drug        (FDA/Experimental)

screening                                                                             Drugs
                Targeted drugs/Chemo

                                                                                        Ab

                                                                 Xenograft assay
                                                                                      shRNA

                                       Clinical trials             Preclinical step
Personalized medicine: genome‐based approaches

                                   Rubio-Perez et al., Cancer Cell, 2015
44‐Drugs Panel
To improve Personalized medicine
                                               Dual Ex vivo
                                               drug screening
                                                     Bulk drug
                                                     screening

                             PDX

                                                 Short Term Culture
                                                  (STC) assay IC50

                                                CSC drug screening

                           Chemogram/          Mininiaturized Aldefluor
                                                        assay
                           Stemogram
                         Bulk drug screening

                         CSC drug screening
PDX genomic background

        MYC
       TP53
    PDGFRA
      ERBB2
     PIK3CA            Amplification
       PTEN            Deletion
FGFR1/ZNF703           mutation         18.7
       JAK2
       ROR2
      CCNE1
      HTERT
      ch1q21
       MYH9
      STK11
      PIK3R1
        RB1
       AKT1
      CCND1
     CDKN1B
     NOTCH2
       MYH2
        DMD
     ACVR1B
                                        4.9
       ANK3
      POLD2
        IRF4
      ICAM1
      PSMA6
PI3K/AKT pathway
                                                 PDXs ID
                               Doxorubicin                                                           Salinomycin
                               5-FU

          chemotherapies       Cisplatin
                               Docetaxel                        Temsirolimus (MTOR)           PRIMA1 (p53mut)
                               Gemcitabine
                               Eribulin
                               Crizotinib
                               LY2157299
                               Simvastatin
                               Lovastatin
     Mevalonate inhibitors     Pravastatin
                               GGTI-298
                               FTI-277
                               Tamoxifen
                               ATRA
                               vemurafenib
            RAF inhibitors     Sorafenib
                               Dabrafenib
                               Trastuzumab-DM1
                               Gefitinib
   EGFR-family inhibitors      Lapatinib
                               Cetuximab
                               Trastuzumab
                               BKM120                                        sensitivity     resistance
                               MK-2206
PI3K/AKT/mTOR inhibitors       Everolimus
                               Temsirolimus
                               SAHA                                               ERBB2
                               S78454
                  Epidrugs     Decitabine
                               Vidaza
                               Salinomycin
                                                               Gefitinib (EGFR)
                               Squalamine
                               Crenolanib

  RTK (class III) Inhibitors   Imatinib
                               Sunitinib
                               Masitinib
                               RO-3306                            cx-4945 (CK2a)
                               BI2536                            MK-2206 (AKT)
                               cx-4945                           Doxorubicin (DNA)
                               smi4a
                               GSK1120212
                               MK-0752
                               TMI1
                               vismodegib
                               Olaparib
                               PRIMA-1
                               Nutlin-3
            SRC Inhibitors     Dasatinib
                               Trodusquemine
                                                                             sensitivity     resistance
                                                 R         S
PDXs ID         PDXs ID
                               Doxorubicin
                               5-FU

          chemotherapies       Cisplatin
                               Docetaxel
                               Gemcitabine
                               Eribulin
                               Crizotinib
                               LY2157299
                               Simvastatin
                               Lovastatin
     Mevalonate inhibitors     Pravastatin
                               GGTI-298
                               FTI-277
                               Tamoxifen
                               ATRA
                               vemurafenib
            RAF inhibitors     Sorafenib
                               Dabrafenib
                               Trastuzumab-DM1
                               Gefitinib
   EGFR-family inhibitors      Lapatinib
                               Cetuximab
                               Trastuzumab
                               BKM120
                               MK-2206
PI3K/AKT/mTOR inhibitors       Everolimus
                               Temsirolimus
                               SAHA
                               S78454
                  Epidrugs     Decitabine

                       No correlation between CSC score and genomic alterations
                               Vidaza
                               Salinomycin
                               Squalamine
                               Crenolanib

  RTK (class III) Inhibitors   Imatinib
                               Sunitinib
                               Masitinib
                               RO-3306
                               BI2536
                               cx-4945
                               smi4a
                               GSK1120212
                               MK-0752
                               TMI1
                               vismodegib
                               Olaparib
                               PRIMA-1
                               Nutlin-3
            SRC Inhibitors     Dasatinib
                               Trodusquemine

                                                 R         S   CSC
                                                                     = CSC
CSC and non-CSC present identical genomic background

        CNAs                                          Mutations

                                                  PANDORA6 (H2KD-)
                                                  PANDORA21

                                                  PANDORA18

                                                  PANDORA25

                                                  PANDORA11
                                                  T404 ALDH+
                                                  T404 ALDH-
                                                  PANDORA1

                                                  PANDORA7

                                                  PANDORA6

                                                  PANDORA3
                                                  T404

                                                  T330
                                                  T389

                                                  T433

                                                  T436

                                                  T272
                                                  T348
                                                  T168
                                                  T494

                                                  T434

                                                  T483
                                                  T392
                                                  T226
                                                  T442
                                   PANDORA1
                                         T404
                                  T404 ALDH+
                  T404            T404 ALDH-
                                         T330
                                         T389
                                 PANDORA21
                                         T433
                  T404 ALDH-     PANDORA18
                                   PANDORA7
                                         T436
                                   PANDORA6
                               PANDORA6 (H2KD-)
                                   PANDORA3
                  T404 ALDH+             T272
                                         T348
                                         T168
                                         T494
                                 PANDORA25
                                         T434
                  Merge          PANDORA11
                                         T483
                                         T392
                                         T226
                                         T442
pa           Pa          PA            pa           Pa           PA
                               T1 nd T4 T4 T4 nd T2 T3 T3 ND T3 T4    T1 nd T4 T4 T49 nd X2 T3 T34 ND T38 T43
                               68 ora 36 04 94 o2 26 92 48 OR 89 34   68 ora 36 04 4 o2 26 92 8 OR 9       4
                                   1            1          A6             1            1           A6

               Doxorubicin
                        5‐FU
                   Cisplatin
                  Docetaxel
              Gemcitabine
                    Eribulin
                  Crizotinib
                 LY2157299
               Simvastatin
                 Lovastatin
                Pravastatin
                  GGTI‐298
                     FTI‐277
                 Tamoxifen
                       ATRA
              vemurafenib
                  Sorafenib
                Dabrafenib
        Trastuzumab‐DM1
                   Gefitinib
                  Lapatinib
                 Cetuximab
              Trastuzumab
                   BKM120
                   MK‐2206
                Everolimus
             Temsirolimus
                       SAHA
                     S78454
                 Decitabine
                      Vidaza
               Salinomycin
               Squalamine
                Crenolanib
                   Imatinib
                   Sunitinib
                  Masitinib
                   RO‐3306
                     BI2536
                    cx‐4945
                       smi4a
              GSK1120212
                   MK‐0752
                        TMI1
                vismodegib
                   Olaparib
                   PRIMA‐1

R   S               Nutlin‐3
                  Dasatinib
                                                                                                                CSC
                                                                                                                      = CSC
           Trodusquemine
PDXs ID             PDXs ID
                               Doxorubicin
                               5-FU

          chemotherapies       Cisplatin
                               Docetaxel
                               Gemcitabine
                               Eribulin
                               Crizotinib
                               LY2157299
                               Simvastatin
                               Lovastatin
     Mevalonate inhibitors     Pravastatin
                               GGTI-298
                               FTI-277
                               Tamoxifen
                               ATRA
                               vemurafenib
            RAF inhibitors     Sorafenib
                               Dabrafenib
                               Trastuzumab-DM1
                               Gefitinib
   EGFR-family inhibitors      Lapatinib
                               Cetuximab
                               Trastuzumab
                               BKM120
                               MK-2206
PI3K/AKT/mTOR inhibitors       Everolimus
                               Temsirolimus
                               SAHA
                               S78454
                  Epidrugs     Decitabine
                               Vidaza
                               Salinomycin
                               Squalamine
                               Crenolanib

  RTK (class III) Inhibitors   Imatinib
                               Sunitinib
                               Masitinib
                               RO-3306
                               BI2536
                               cx-4945
                               smi4a
                               GSK1120212
                               MK-0752
                               TMI1
                               vismodegib
                               Olaparib
                               PRIMA-1
                               Nutlin-3
            SRC Inhibitors     Dasatinib
                               Trodusquemine

                                                 R         S   CSC
                                                                        = CSC
PA                   pa                            PA
                                           pan              Pa                                                  Pa
                                       T16     T43 T40 T49     T22 T39 T34 ND T38 T43       T16 nd T43 T40 T49     X22 T3 T34 ND T38 T43
                                           dor             ndo                                                  nd
                                        8       6 4 4           6 2 8 OR 9 4                 8 ora 6 4 4            6 92 8 OR 9       4
                                           a1               21                                                 o21
                                                                           A6                    1                            A6

        Doxorubicin
        5‐FU
        Cisplatin
        Docetaxel
        Gemcitabine
        Eribulin
        Crizotinib
        LY2157299

                                    T404 (ALDH+)

                                                                        T434 (ALDH+)
                     T404 (ALDH-)

                                                         T434 (ALDH-)
        Simvastatin
        Lovastatin
        Pravastatin
        GGTI‐298
        FTI‐277
        Tamoxifen
        ATRA
        vemurafenib
        Sorafenib
        Dabrafenib
        Trastuzumab‐DM1
                                                                                        - P-src (Y416)
        Gefitinib
        Lapatinib                                                                       - GAPDH
        Cetuximab
        Trastuzumab
        BKM120
        MK‐2206
        Everolimus
        Temsirolimus
        SAHA
        S78454
        Decitabine
        Vidaza
        Salinomycin
        Squalamine
        Crenolanib
        Imatinib
        Sunitinib
        Masitinib
        RO‐3306
        BI2536
        cx‐4945
        smi4a
        GSK1120212
        MK‐0752
        TMI1
        vismodegib
        Olaparib
R   S   PRIMA‐1
        Nutlin‐3
                                                                                                                                           CSC
                                                                                                                                                 =   CSC

        Dasatinib
        Trodusquemine                                                                                                                                      Src inhibitor
In vivo validation

                                                           Reimplantation

                                               Dasatinib

                                                                    Est. number of CSC/10,000 cells
Tumor volume (mm3)

                                               Control

                                               Docetaxel

                     Days after implantation
Clinical validation

                                           PERMED1045
  PERMED1045
                                          Post‐dasatinib 1
   (skin mets)

                     Dasatinib, 1 month

 pre TRT Biopsy

       9.4%

       ALDH
CD44

         79.9%

        CD24

  implantation
in serial dilution
Clinical validation

                                                  PERMED1045
  PERMED1045
                                                 Post‐dasatinib 1
   (skin mets)

                     Dasatinib, 1 month

 pre TRT Biopsy                                  Post TRT Biopsy 1

                                                   1.8%

       9.4%

       ALDH                                         ALDH

                                                     50,1%
CD44

                                          CD44

         79.9%

        CD24                                        CD24

  implantation                                 implantation
in serial dilution                           in serial dilution
Clinical validation

                                                  PERMED1045
  PERMED1045
                                                 Post‐dasatinib 1
   (skin mets)

                     Dasatinib, 1 month

                                                                                          1000,

                                                                                                       pre TRT (Biopsy 1)

                                                                     Tumor volume (mm3)
                                                                                           750,

 pre TRT Biopsy                                  Post TRT Biopsy 1                         500,
                                                                                                                            post TRT (Biopsy 2)
                                                   1.8%                                    250,

       9.4%                                                                                  0,
                                                    ALDH                                          0      50     100     150       200      250
       ALDH
                                                     50,1%
                                                                                                      Days after reimplantation
CD44

                                          CD44

         79.9%

        CD24                                        CD24

  implantation                                 implantation
in serial dilution                           in serial dilution
CSCs assays for personalized therapy: « predictive Medicine »

                                                                    PDX

CSC personalized
clinical trials

                                                               Chemogram

                                                            Bulk drug screening

                                                            CSC drug screening

        From « personalized medicine » to « predictive medicine »
Conclusion

                                                            Tam et al. Cancer Cell, 2013

• CSCs drug sensitivity follow their own rule
• CSC drug sensitivity profile is INDEPENDANT of CSC genomic background
• CSCs drug sensitivity profile is DEPENDANT of CSC intrinsic signaling pathways
Molecular Oncology
                                      Daniel Birnbaum
Epithelial stem cell and cancer Lab   Francois BERTUCCI
                                      Max CHAFFANET
                                      Pascal FINETTI
Emmanuelle CHARAFE-JAUFFRET           Arnaud GUILLE
Christophe GINESTIER                  Jose ADELAIDE
Geraldine GUASCH-GRANGEON             Nadine CARBUCCIA
Véronique CHEVRIER                                                      University of Michigan,
                                      Severine GARNIER
Julien WICINSKI                                                         Ann Arbor, MI, USA
                                      Marc LOPEZ
Violaine FORISSIER                    Olivier CABAUD
Violette AZZONI                                                         Max WICHA
                                      Emilie MAMESSIER
Abir ARFAOUI                                                            Suling LIU
Mary-loup PICOD
                                      Medical Oncology                  Hasan KORKAYA
Lhoucine MITOYAN                      Anthony GONCALVES
                                      Patrice VIENS
Former members                        Oncology Development
Marion SALVADOR                       J-Fred SAUNIERE
Rita EL HELOU                                                           Cancer Research Center of
Simon LAUNAY                          CiBi platform                     Lyon,
                                      Ghislain BIDAUT                   Lyon, France
                                      Claire RIOUALEN
                                      Quentin DA COSTA
                                                                        Alain PUISIEUX
                                                                        Anne-Pierre MOREL
                                      TrGET platform                    Arnaud VIGNERON
                                      Yves COLLETTE
                                      Remy CASTELLANO
                                      Yves TOIRON

                                      Flow Cytometry platform           PARi platform,
                                                                        CEA, Saclay, France
                                      Manon RICHAUD
                                      Françoise MALLET
                                                                        Annick HAREL-BELLAN
                                                                        Guillaume PINNA
                                      ICEP
                                      Experimental Pathology platform   iRTSV, Grenoble, France
                                      Anne FARINA
                                      Emilie COUQUIAUD                  Laurent Guyon
Acknowledgements

  CiBi platform
  Ghislain BIDAUT
  Claire RIOUALEN
  Quentin DA COSTA

  TrGET platform
  Yves COLLETTE
  Remy CASTELLANO
  Yves TOIRON

  Flow Cytometry platform
  Marie-Laure THIBULT
  Françoise MALLET

  ICEP
  Experimental Pathology platform

  Anne FARINA
  Emilie COUQUIAUD
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