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

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R1

01

9S

UB

RT

U2

Oct2

6

FGD2

PD

X1

51

No

v3

0S

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01

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RT

U2

Oct2

6

TXN

PD

X1

51

No

v3

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01

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Oct2

6

GSTP1

PD

X1

51

No

v3

0S

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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

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9

SU

BR

TU

2 1

0/2

6

SU

TI 15

1

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2 1

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6

SU

TI 15

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BR

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2 1

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6S

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I 15

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9

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BR

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2 1

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UT

I 15

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2 1

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U2 1

0/2

6

SU

TI 15

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BR

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2 1

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9

SU

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TU

2 1

0/2

6S

UT

I 15

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0/2

6

SU

TI 15

1

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BR

TU

2 1

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6

SU

TI 15

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2 1

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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.