Abstract
Evaluating the metastatic potential and the molecular heterogeneity of patient-derived
orthotopic xenograft models of triple-negative breast cancerVishnu C. Ramani1, Rakhi Gupta1, Gerald Quon2, Melanie Triboulet1, Clementine Lemaire3, Corinne Renier3, Kerriann Casey4, Cassandra Greene5, Chad Sanada5, Tracy Lu5, Lukasz Szpankowski5, Naveen
Ramalingam5, Ameen A. Salahudeen6, Sean de La O6, Ranjani Rajapaksa6, Shoshana Levy6, Anne A. Leyrat5, Jay A. West5, Elodie Sollier-Christen3, Calvin J. Kuo6, George W. Sledge6, Stefanie S. Jeffrey1
1. Department of Surgery, Stanford University School of Medicine, Stanford, CA; 2. Department of Molecular and Cellular Biology, University of California, Davis, CA; 3. Vortex Biosciences, Inc., Menlo Park, CA; 4. Department of Comparative
Medicine, Stanford University, Stanford, CA; 5. New Technologies Research Department, Fluidigm Corporation, South San Francisco, CA; 6. Department of Medicine, Stanford University School of Medicine, Stanford, CA.
We report an in-depth characterization of patient-derived orthotopic xenograft
(PDOX) models of triple-negative breast cancer (TNBC) regarding their molecular
profile at the single cell level, tumor heterogeneity, 3D organoid generation, and
ability to generate circulating tumor cells (CTCs). A panel of seven TNBC PDOX
tumors were grown orthotopically in NOD SCID gamma mice and used in this study.
Blood obtained via cardiac puncture from tumor bearing animals was processed on
the Vortex microfluidic platform, for label-free, size-based enrichment of circulating
tumor cells (CTCs). Enriched cell populations were stained for human-specific
cytokeratin (CK) and Vimentin (Vim), mouse-specific CD45, and DAPI; CTCs were
identified as cells that were CD45 negative and positive for CK or Vim. Bulk tumor
growing in the mammary fat pads was dissociated to single cells and characterized
using Fluidigm’s® PolarisTM platform for single cell biological experimentation and
cDNA generation within an integrated fluidic circuit (IFC). From the cell suspension,
Polaris identified single cells that were then processed for mRNA-seq. The resulting
cDNA libraries were then multiplexed using Nextera XT® (Illumina®) and sequenced
on Illumina systems. Data generated from mRNA-seq was processed to correct for
confounding factors such as cell size, cell cycle and read depth and then analyzed to
screen for heterogeneity between different populations of cells. Tumors were
analyzed by flow cytometry for both tumor and immune cells and additionally the
single cell suspension was seeded into 3-D culture to generate organoids. Finally,
organs from tumor bearing animals were analyzed for metastases. With the Vortex
platform, we detected CTCs from a majority of our PDOX tumor-bearing mice. The
total number of CTCs varied over a wide range between different PDOX tumors.
There was a clear heterogeneity in CTCs in terms of CK and Vim expression. In
CTCs from one of the PDOX tumors, we detected a small population of CTCs that
were either CK+ or Vim+ but the major fraction that was double positive (Vim+ CK+).
Probing the bulk tumor from different PDOX models revealed heterogeneity in the
levels and number of cells positive for cell surface markers like EpCAM and a
difference in the levels of infiltrating myeloid cells (CD11b+). mRNA-seq analyses of
individual tumor cells from the bulk tumor belonging to different PDOX models will be
described. Additionally, lung and brain metastases were identified. 3D organoid
cultures from our PDOX models were successfully grown and their gene expression
profiles will be analyzed. In summary, PDOX models of TNBC will help advance our
understanding of the molecular basis of this deadly cancer.
Conclusions
FundingThis work was funded by the Susan Komen Foundation SAB1500003 (GWS), the Andrew and Debra Rachleff Cancer
Research Fund (SSJ), John and Marva Warnock Research Fund (SSJ), DOD grant Awards W81XWH-14-1-0397 (SL)
and W81XWH-14-1-0398 (SSJ), and a Vortex Biosciences Industrial Research Contract to Stanford University (SSJ).
SUTI 333
SUTI 319
SUTI 215
SUTI 110
SUTI 368
SUBRTU 1
SUBRTU 2
SUBRTU1 Lung
SUBRTU2 Lung
SUTI 319 Lung
SUTI 333 M5 Lung
PCA score plot
➢ Our patient-derived orthotopic xenograft models of breast cancer generate CTCs and
metastasize to distant organs.
➢ Using label-free technology, Vortex Biosciences, CTCs were isolated in various stages of
EMT from mouse blood in our PDOX models of triple-negative breast cancer.
➢ Altering the immune microenvironment does not significantly impact PDOX tumor growth,
CTC generation, and metastases, but does affect myeloid cell recruitment.
➢ Single cell suspensions of PDOX tumors are easily processed by the MagSweeper to
isolate specific subpopulations of cells from PDOX tumor models.
➢ Tumor cells from PDOX models generate 3D organoid structures ex vivo.
➢ RNA-seq analyses at a single cell resolution reveals very low intra-mice heterogeneity and
a high level of intra-tumor heterogeneity in our PDOX models.
CTCs and metastases in PDOX models of breast cancer
SUTI 151 NSG lung SUTI 151 NSG liver
151 SCID lung 151 SCID liver
Impact of immune cells on CTCs and metastases
in PDOX models of breast cancer
Mesenchymal(Vimentin pos)
Epithelial(Cytokeratin pos)
0
2 0
4 0
6 0
8 0
% o
f C
TC
s
C K + V im + C K + , V im +
Epithelial to mesenchymal transition in CTCs from PDOX models
Workflow
30 - 210days
Surgically excised primary breast
tumor
Cardiac blood
NSGmouse
A.
C. D.
Single cell tumor
suspension
PDOX tumor
POLARISTM
Chamber bearing stained
tumor cell inside PolarisTM
B.
Single Cell RNA Seq, Analyses
3D Organoid Cultures
Circulating Tumor Cells
Flow Cytometry and H&E Staining for Metastases
A) Images of CTCs from PDOX tumors. Live CTCs were isolated from individual PDOX tumor bearing animals by label-free Vortex technology.
Cells captured were then stained for cytokeratin, vimentin, DAPI, and mouse CD45. CTCs were identified as cells positive for either cytokeratin or
vimentin and negative for CD45. B) Animals bearing PDOX tumors were euthanized and individual organs were collected, fixed in formalin
followed by 70% ethanol, sectioned and stained with hematoxylin and eosin (H&E) and identified by an expert veterinary pathologist. We
successfully characterized the presence of CTCs in TNBC PDOX models. Not surprisingly, CTC numbers varied between different PDOX tumors.
In addition, we detected the presence of metastases in lung and liver by H&E staining in many of our PDOX models.
Representative growth curves for different PDOX models growing in NOD SCID gamma animals. For all the models
shown here, small pieces of patient-derived tumor were implanted orthotopically into the 4th mammary fat pad of
female NSG mice, monitored and tumors were measured weekly twice.
PC
2
PC1
SUTI 151 SUBRTU2 10/19 SUBRTU2 10/26
SUBRTU2 Brain
MagSweeper
PDOX tumor
suspension
EpCAM+
tumor cells
A. B.
Violin Plot of Gene Expression By the Order of PCA Gene Scores (1 of 4)
Exp
ressio
n (
log
2)
051015
CALCA COL1A2 IGFBP4 NDUFA4L2 TFPI2 SPHK1 SPINK6 GNG11 GPX3
051015
CBR1
051015
CLU RAMP1 CD24 ITM2B CTSL1 RFX4 NUCB2 FGFR1 MBOAT1
051015
PTGS2
051015
PON3 BHLHE40 SLC10A4 LOXL2 ARMCX2 GPX2 PDE11A KRT19 CRABP2
051015
AARD
051015
TKT MAFF DAPP1 PROCR CLDN4 UCHL1 KRT81 CLCF1 RNH1
051015
B4GALT1
051015
CD320 ILVBL MGP MUC16 ACOT7 PRDX2 PPIC PIR HSPB1
051015
TNFRSF11B
051015
ISG15 AZGP1 SOX11 CALML5 COL6A1 TECR BCAP31 PADI2 IFI6
051015
LOC650226
051015
GABRP SLC25A6 NQO1 RRM1 KRT15 APOA1BP KRT86 IGF1R S100A16
051015
MDM2
051015
LDHB TEKT3 ACTG2 TACSTD2 EBP PKM DLD GAPDH EDN1
051015
COL2A1
051015
ID4 DNAJA1 TALDO1 NDUFA13 GLT25D1 TFAP2C LDHA ITGB8 ENO1
051015
SLC25A5
051015
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
ID2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
MFAP2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
C10orf10
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
MIA
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
KRT23
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
SH3BGRL3
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
FGD2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
TXN
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
GSTP1
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
051015
KRT17
Violin Plot of Gene Expression By the Order of PCA Gene Scores (2 of 4)
Exp
ressio
n (
log
2)
051015
MFGE8 F3 ARHGAP29 RPPH1 MDH1 NGFRAP1 CDH1 ALDOA ERBB3
051015
KRTCAP2
051015
COX8A S100A6 CLDN11 SPARC SSR4 PRDX4 BAMBI COX6A1 PLP2
051015
ATP5C1
051015
CHD7 EIF4G2 CCPG1 TMED9 HMGA1 PRDX5 SORBS2 TMEM59 PGK1
051015
SOX4
051015
ANXA5 ATP6V0B PLK2 SERPINB6 TM4SF1 FBLN2 RAN UQCRH H1F0
051015
VDAC2
051015
PARK7 AIF1L CRABP1 PROM1 TUBA1B GADD45B SHC4 CTGF AMOTL2
051015
PRDX1
051015
MARCKSL1 B2M BST2 ANAPC11HIST1H2BG TPI1 PSMD4 CCT3 ATP5B
051015
ELF3
051015
CD63 SOX10 ATP5J CTTN RAB25 MORF4L2 COTL1 HIST1H2AE DNAJB6
051015
COX7C
051015
PDGFA TUBB ANPEP ARPC4 TAGLN2 NEDD9 AP1G2 COX7B ISLR
051015
NUDT5
051015
CLDN3 SSR2 NDUFB2 MYO5B RND3 FDPS TNF PPAP2C IFITM2
051015
CAPS
051015
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
ROPN1B
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
C11orf10
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
LAMP2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
S100A14
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
JARID2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
FXYD6
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
HBEGF
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
ACTG1
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
NDUFV2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
051015
ARPC2Violin Plot of Gene Expression By the Order of PCA Gene Scores (3 of 4)
Exp
ressio
n (
log
2)
051015
BRK1 NDUFA12 DUSP10 ST6GAL1 SLIRP NDRG2 PART1 CSRP2 HAPLN3
051015
MDH2
051015
ABCF2 ART3 MAL2 FAU SPCS1 PPIB DYNLL1 CYR61 TRMT112
051015
FAM96A
051015
HSPA8 RIPK2 BTNL8 STEAP4 CLIC1 RPLP0 FAM60A TUBG1 PSMB4
051015
NDUFA1
051015
ANXA2 PXDC1 GNG5 MYL6 CLDN7 PMAIP1 PEBP1 KRT18 S100P
051015
GDI2
051015
ETV6 OAZ1 MIF MEA1 SLC29A1 EEF1G MYL12B HIST1H2BD C9orf3
051015
B3GNT7
051015
NDUFB8 TPM1 CRISPLD1 TP53 HIST1H2BCPDZK1IP1 CLIC4 FMO2 ATP5A1
051015
RASSF3
051015
TAGLN UQCRC1 ZNFX1-AS1 S100A11 HINT1 HIST1H3D TUBA1A CALM2 TPT1
051015
THBS1
051015
TRPS1 LIF TRIM16 FXYD3 COX4I1 RPL38 ARF1 YWHAE COX5B
051015
PTTG1IP
051015
AK1 H2AFZ NDUFB4 RPL24 AEN RPL8 LGALS3 PTPRF ATG12
051015
PSMB3
051015
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RPL5
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
C6orf62
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RAD21
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
PDCD4
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
PPP1CB
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
EPCAM
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
TPD52
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
NET1
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
SUN1
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
051015
MORF4L1
Violin Plot of Gene Expression By the Order of PCA Gene Scores (4 of 4)
Exp
ressio
n (
log
2)
051015
DEGS1 MYL12A SRP9 NDUFB9 KRT8 HNRNPH1 TFRC HIST1H2ACHIST1H2BK
051015
RPS13
051015
SUMO3 NFIB PLAUR FEZ1 SNHG8 COX7A2 SNX27 HMGN1C17orf76-AS1
051015
DDX39B
051015
RPL35A LINC00152 CDC42 MAGI2-AS3 FOS PSMB6 PSMB5 FTL TMSB10
051015
TRA2A
051015
PMP22 RPL30 PTMA NPM1 FXYD5 WTAP LY6E GADD45A PAICS
051015
RPS25
051015
DBI KRT7 RPS14 TNFRSF12A C2orf18 CHCHD2 CAPG GABARAP MRPL14
051015
RPL27
051015
RPS27 RPL21 TMEM33 RAP2B ANKRD10 RPS5 SNHG6 SAR1A RPL23A
051015
MSMO1
051015
SLFN13 RPS19 CNN3 UBB TSTD1 TOMM22 ATP5F1 MRPL49 RPS3
051015
RPS16
051015
MICA RPS11 TMSB4X GNB2L1 RPS12 RPLP1 SNRPD2 ATP5O RPL7
051015
LOC100507246
051015
ANXA1 UQCR10 ZNF562 MORC2-AS1 RPL29 EIF3H RPL37A CDC6 HIGD1A
051015
MPC2
051015
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RPS18
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
IFITM3
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RPL3
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
SLC48A1
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
SARS
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RPL41
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
RPL23
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
ASPH
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
SKA2
PD
X1
51
No
v3
0S
UB
R1
01
9S
UB
RT
U2
Oct2
6
051015
CDKN1A
SU
BR
TU
2 1
0/1
9
SU
TI 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/2
6
SU
BR
TU
2 1
0/1
9
SU
TI 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9
SU
BR
TU
2 1
0/2
6S
UT
I 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/1
9S
UB
RT
U2 1
0/2
6
SU
TI 15
1
SU
BR
TU
2 1
0/2
6
0 2 0 4 0 6 0 8 0 1 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
S U T I 1 1 0
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
0 5 0 1 0 0 1 5 0 2 0 0
0
5 0 0
1 0 0 0
1 5 0 0
S U B R T U 2 P 2
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
M 4
0 5 0 1 0 0 1 5 0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S U B R T U 1 P 2
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
M 4
M 5
0 3 0 6 0 9 0 1 2 0 1 5 0
0
1 0 0 0
2 0 0 0
3 0 0 0
S U T I3 3 3
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
M 4
M 5
0 3 0 6 0 9 0 1 2 0 1 5 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
S U T I 3 6 8
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
M 4
M 5
0 2 0 4 0 6 0 8 0 1 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
S U T I3 1 9
D a y s
Tu
mo
r v
olu
me
(m
m3
) M 1
M 2
M 3
M 4
M 5
1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0
0
2 0 0
4 0 0
6 0 0
S U T I 2 1 5
D a y s
Tu
mo
r v
olu
me
(m
m3
)
Growth curves of PDOX tumors
Single cells RNA-seq analyses of PDOX tumors
A) CTCs from an individual mouse bearing PDOX tumor SUTI 151 were captured using Vortex and probed epithelial markers (cytokeratin cocktail,
CK) and a mesenchymal marker (Vimentin, Vim). CTCs were identified as expressing either CK and/or Vim and negative for CD45. B) Percentage
of CTCs staining positive for CK and/or Vim from two individual mice bearing PDOX SUTI 151 tumors. A major fraction of CTCs expressed both
markers, showing epithelial to mesenchymal transition in CTCs isolated from PDOX models of TNBC.
A.
B.
SUTI 151 SCID
SUTI 151 NSG
#1847
Single cell suspensions of PDOX tumors
# Mouse 1 # Mouse 2
A. B.
A. B.
14
18
28
32
35
38
41
44
49
54
0
5 0 0
1 0 0 0
1 5 0 0
D a y s a f t e r i m p la n t a t io n
Tu
mo
r v
olu
me
(m
m3
)
S U T I 1 5 1 S C I D S U T I 1 5 1 N S G
SUTI 151 SCID - SpleenSUTI 151 SCID - Tumor
SUTI 151 NSG - Tumor SUTI 151 NSG - Spleen
A) Single cell suspension of PDOX tumor cells purified with the MagSweeper using magnetic beads coated with anti-human
EpCAM. Image shows tumor cells bearing the EpCAM magnetic beads after enrichment. B) Images of 3D organoids generated
from single cell suspension of PDOX tumors SUBRTU2 (top panel) and SUTI 151 (bottom panel).
A) Single cell RNA-seq analyses of tumor cells generated from PDOX tumors, SUBRTU2 (isolated on 10/19 and 10/26) and
SUTI 151. B) Principal component analysis of RNA-seq data from the PDOX tumors SUBRTU2 (10/19, 10/26) and SUTI151
reveals that the same tumor growing in two different animals (SUBRTU2) and excised at different times demonstrates a high level
of transcriptional similarity, as expected, and is distinct from another PDOX tumor, SUTI 151.
A. B.
C. D.
A) Growth curves for SUTI I51 PDOX tumors implanted in either SCID mice, which lack functional T and B cells, but express natural killer (NK)
cells, or in NOD SCID gamma (NSG) mice, which lack functional T cells, B cells, and NK cells. B) Examples of images of CTCs captured using
Vortex from SCID and NSG mice bearing SUTI 151 PDOX tumors. C) FACS images of cells from primary tumors and spleens obtained from SUTI
151 PDOX models implanted in SCID or NSG mice and probed for different human and mouse cell surface tumor markers. Note similarity in levels
of tumor markers between SCID and NSG mice, but differences in myeloid cell levels (CD11b), especially in the spleen, between tumor-bearing
SCID and NSG mice. Red curves = stained with specific antibodies; blue curves = cells stained by isotype control (representing non-specific
antibody binding). D) H&E staining of metastases to distant organs present in both SUTI 151 tumor-bearing SCID and NSG mice.