Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis...

Characterization of Tumor Cell Dissemination Patterns

in Preclinical Models of Cancer Metastasis Using Flow

Cytometry and Laser Scanning Cytometry

David Goodale,1 Carolina Phay,2 Carl O. Postenka,1 Michael Keeney,2,3 Alison L. Allan1,3,4,5*

� AbstractThe inability to sensitively detect metastatic cells in preclinical models of cancer has cre-ated challenges for studying metastasis in experimental systems. We previously devel-oped a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs)in mouse models of breast cancer. We have adapted this methodology for analysis of tu-mor dissemination to bone marrow (BM) and lymph node (LN), and for analysis ofthese samples by laser scanning cytometry (LSC). Our objective was to implement thesemethodologies for characterization of tumor cell dissemination in preclinical models ofcancer metastasis. Human cancer cells were injected into mice via mammary fat pad(MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targetsbone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificedand blood, LNs, and BM were collected. Samples were immunomagnetically enrichedand labeled with human leukocytic antigen–fluorescein isothiocyanate and CD45-PEantibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis.Following MFP injection, CTCs increased over time, as did disseminated cells to theLN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2weeks postinjection. Following TV injection, CTCs were initially high but decreasedrapidly by 1 week before increasing to peak at endpoint. Combined with an observedconcurrent increase in disseminated cells to LN and BM, this suggests that tumor cellsmay shed into the circulation from lung metastases that establish following initial celldelivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumorcells in the BM (most prevalent site of metastasis after IC injection) remained at moder-ate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitivequantification of disseminated tumor cells in preclinical models of metastasis. Thesemethods will be valuable for future studies aimed at testing new therapeutics in themetastatic setting. ' 2008 International Society for Advancement of Cytometry

� Key termsbreast cancer; metastasis; preclinical animal models; tumor cell dissemination; flowcytometry; laser scanning cytometry

BREAST cancer is a leading cause of morbidity and mortality in women (1,2), pri-

marily because of the failure of effective clinical detection and management of meta-

static disease in distant sites such as lymph node (LN), bone, lung, liver, and brain

(3,4). The metastatic process is comprised of a series of sequential steps, and cancer

cells must successfully complete each step in order to give rise to a metastatic tumor.

These steps include dissemination of cancer cells from the primary tumor into the

bloodstream (intravasation), survival in the circulation, arrest and extravasation into

the secondary site, and initiation and maintenance of growth to form clinically

detectable metastases (3–7). Breast cancer cells may also disseminate from the pri-

mary tumor through the lymphatic system, although the lack of direct flow from the

lymphatic system to other organs means that tumor cells escaping via this route must

still enter the vascular system in order to be distributed to distant organs (3,4,8).

1London Regional Cancer Program,London Health Sciences Centre, London,Ontario, Canada2Special Hematology/Flow Cytometry,London Health Sciences Centre, London,Ontario, Canada3Lawson Health Research Institute,London, Ontario, Canada4Department of Oncology, Schulich Schoolof Medicine and Dentistry, University ofWestern Ontario, London, Ontario, Canada5Department of Anatomy and CellBiology, Schulich School of Medicineand Dentistry, University of WesternOntario, London, Ontario, Canada

Received 30 June 2008; RevisionReceived 26 August 2008; Accepted 14September 2008

Grant sponsor: Canadian Breast CancerResearch Alliance ‘‘Special Competition inNew Approaches to Metastatic Disease’’with special funding support from theCanadian Breast Cancer Foundation andThe Cancer Research Society; Grantnumber 016506.

Additional Supporting Information may befound in the online version of this article.

*Correspondence to: Alison L. Allan,London Regional Cancer Program, 790Commissioners Road East, London,Ontario, Canada N6A 4L6.

Email: [email protected]

Published online 14 October 2008 in WileyInterScience (www.interscience.wiley.com)

DOI: 10.1002/cyto.a.20657

© 2008 International Society forAdvancement of Cytometry

Original Article

Cytometry Part A � 75A: 344�355, 2009

Given the multistep nature of the metastatic cascade,

there should be several opportunities for early identification

and therapeutic targeting of metastatic cells before they

become a clinical problem. Indeed, the presence of circulating

tumor cells (CTCs) in the bloodstream of cancer patients has

been recognized for over a century (9), although a lack of sen-

sitive technology precluded the detailed study of these cells

until recently. However, technological advances have now

facilitated the identification, enumeration, and characteriza-

tion of individual disseminated cells in breast cancer patients

using methods such as PCR (10–12), flow cytometry (FCM)

(13–15), image-based immunologic approaches (16–19),

immunomagnetic techniques (20,21), and microchip technol-

ogy (22). In breast cancer patients with either metastatic or

apparently localized disease, there is growing evidence that the

presence of individual CTCs in the blood or disseminated tu-

mor cells (DTCs) in the bone marrow (BM) may be an impor-

tant indicator of the potential for metastatic disease and poor

prognosis [reviewed in (23–27)]. Furthermore, the histopath-

ological identification of very small metastatic tumor deposits

in the axillary LN have recently been reported to be prognosti-

cally significant (28). However, the biological implications of

individual occult tumor cells in regional and distant sites

remains poorly understood, particularly with regards to the

functional and mechanistic details of their progression to

clinically relevant metastases. Therefore, preclinical modeling

of the pattern and kinetics of CTC and DTC dissemination in

breast cancer and the relationship to endpoint metastatic dis-

ease would be extremely valuable.

There are several in vivo preclinical mouse models avail-

able for studying breast cancer metastasis, including ‘‘sponta-

neous’’ metastasis models, ‘‘experimental’’ metastasis models,

and transgenic models (29,30). Spontaneous metastasis mod-

els allow for investigation of all steps of the metastatic cascade

and involve injection of cancer cells into the correct orthotopic

site [i.e., the mammary fat pad (MFP) for breast cancer cells],

growth of a primary tumor, and eventual development of

spontaneous metastases in distant organs (most commonly in

LN and lung following MFP injection) (29,31–34). In contrast,

experimental metastasis models involve direct injection in the

blood circulation, and thus circumvent the initial steps of pri-

mary tumor growth and intravasation (29). Depending on the

route of injection, cells can be targeted for metastatic growth

in different organs using this assay. For example, one of the

most commonly used injection routes, the tail vein (TV), tar-

gets cells for delivery and growth in the lung (29,35). Other

routes of injection include the mesenteric vein to target liver,

or an intracardiac (IC) injection route which theoretically tar-

gets the entire circulation but is usually aimed toward devel-

opment of metastatic growth in the bone (29,35–38). The

occurrence and extent of metastasis in preclinical models has

typically been measured by macroscopic or histological exami-

nation at endpoint (29). However, these approaches are lim-

ited in their sensitivity and are often performed on a small

number of organ sections which may not be representative of

the true extent of metastasis. Therefore, although these precli-

nical models have been valuable for providing scientists with

the ability to test the overall effect of specific molecules or

drugs on endpoint metastasis, they are ultimately ‘‘black box’’

assays that do not allow detection and tracking of individual

DTCs during metastatic progression.

To address this problem, we previously reported the de-

velopment of a novel and sensitive multiparameter FCM assay

to quantify CTCs in preclinical mouse models of breast cancer

metastasis (39). In the present study, we have adapted this

methodology for analysis of tumor cell dissemination to BM

and LN and for analysis of these samples by laser scanning

cytometry (LSC). The overall goal of the study was to imple-

ment these cytometry methods for characterization of tumor

cell dissemination patterns and kinetics in spontaneous and

experimental models of breast cancer metastasis. Our novel

findings suggest that the combined use of FCM and LSC is

highly complimentary and allows for sensitive quantification

of CTCs and DTCs in different preclinical models of breast

cancer metastasis. These methods will allow us to gain a

greater understanding of the biology of metastasis and will be

extremely valuable for future studies aimed at preclinical test-

ing of new therapeutics in the metastatic setting.

MATERIALS AND METHODS

Cell Culture

The human cancer cell line MDA-MB-435HAL (a kind

gift from Dr. David Griggs; Pfizer, St. Louis, MO) is a green

fluorescent protein-expressing, metastasis-derived variant of

the MDA-MB-435 cell line. This subcloned variant was iso-

lated after multiple in vivo passages and was selected for its

enhanced MFP tumor growth rate and increased metastasis to

lung (40). Cells were grown in minimal essential medium with

Earle’s salts and L-glutamine (MEM) supplemented with

25 mmol/L HEPES buffer, 1 mmol/L sodium pyruvate, 13MEM

vitamin solution, and 10% fetal bovine serum (FBS). Media,

supplements, and PBS were obtained from Invitrogen

(Carlsbad, CA). FBS was obtained from Sigma Chemical (St.

Louis, MO). It should be noted that the MDA-MB-435 cell

line was originally isolated from the pleural effusion of a

woman with metastatic breast adenocarcinoma (41). Recently,

a debate has arisen over the origins of this cell line, whether it

was derived from the M14 melanoma cell line or is in fact a

true breast cancer cell line (42,43). However, its expression of

milk proteins (44) and propensity to metastasize from MFP

but not from subcutaneous sites (29) are consistent with it

being a breast carcinoma.

Sample Collection

Fresh whole blood, LNs, and BM were collected from

female athymic NCr nude mice (nu/nu) (aged 6–15 weeks;

Harlan Sprague–Dawley, Indianapolis, IN). Blood (300 lL/mouse) was collected via terminal cardiac puncture of the

right ventricle using a 22-G needle attached to a 1-mL syringe

precoated with heparin (10,000 IU/mL; Leo Pharma, Thorn-

hill, ON). Six LNs per mouse (two brachial, two axillary, two

inguinal) were harvested into cold PBS 1 10% FBS, minced

cross-wise with scissors, passaged three times through a 16-G

ORIGINAL ARTICLE

Cytometry Part A � 75A: 344�355, 2009 345

needle and five times through an 18-G needle, filtered through

70 lM mesh, and resuspended in cold PBS 1 10% FBS. For

BM collection, one hind tibia and femur per mouse were

flushed with cold PBS 1 10% FBS, washed twice, and resus-

pended in PBS 1 10% FBS. For FCM and LSC protocol devel-

opment, samples consisting of MDA-MB-435HAL human

cancer cells ‘‘spiked’’ into mouse blood, LN homogenates, and

BM at various concentrations (10–0.001%) were prepared by

serial dilution as described previously (39). All samples were

processed within 2 h of collection.

Labeling and Immunomagnetic Enrichment Procedure

Sample labeling and immunomagnetic enrichment was

carried out essentially as described previously (39). Briefly,

blood, LN homogenates, and BM samples were subjected to

red blood cell lysis in 13 NH4Cl and washed with PBS prior

to labeling with 10 lL of mouse antihuman leukocytic antigen

(HLA) antibody (clone W6/32) conjugated to fluorescein iso-

thiocyanate (FITC) (Sigma) and 10 lL of rat antimouse pan-

leukocytic CD45 antibody (clone 30-F11) conjugated to phy-

coerythrin (PE) (Caltag Laboratories, Burlingame, CA). Sam-

ples were then immunomagnetically enriched using the Easy-

SepTM PE Selection Kit (Stem Cell Technologies, Vancouver,

BC) as per the manufacturer’s instructions, using 2 3 5 min

incubations in the EasySep magnet. This immunomagnetic

enrichment procedure has previously been established to pro-

vide �10-fold enrichment of tumor cell detection by FCM

(39). After incubation, the fraction containing the tumor cells

(supernatant) was fixed and permeabilized using the Intra-

PrepTM Fix/Perm Kit (Beckman Coulter, Fullerton, CA). At

this stage, �1/10th of the concentrated sample (10 lL; equiva-lent to a starting volume of �30 lL of whole blood) was ali-

quoted for LSC analysis. The remainder of each sample was

resuspended in 500 lL of PI (50 lg/mL) (Beckman Coulter)

and incubated for 15 min at room temperature followed by

45 min at 48C in preparation for FCM analysis.

FCM Analysis

A four color XL-MCL flow cytometer (Beckman Coulter)

was configured to detect the HLA-FITC signal in FL1 (525-nm

bandpass filter), CD45-PE in FL2 (575-nm bandpass filter),

and PI in FL3 (625-nm bandpass filter). Setup and compensa-

tion was adjusted on a �10% mixture of MDA-MB-435HAL

cells and mouse leukocytes. A threshold region was set on

DNA content based on PI fluorescence equivalent to a diploid

mouse leukocyte (cells with lowest DNA content). By plotting

integral PI signal versus the ratio of the PI peak/PI integral sig-

nal, it is possible to identify debris, cell doublets and clumps

which can be excluded from further analysis (39). A minimum

of 100,000 PI1 events were collected per sample. Gated events

which were HLA1CD452 were considered to be positive

tumor events.

LSC Analysis

Aliquots (10 lL) of concentrated samples of blood, LN

homogenate, or BM labeled with HLA-FITC and CD45-PE

were placed onto a glass slide and covered with a glass cover

slip (18 3 18 mm2; VWR International, Mississauga, ON) just

prior to analysis with an iCys LSC (CompuCyte, Cambridge,

MA). Setup and compensation was adjusted on a �10% mix-

ture of MDA-MB-435HAL cells and mouse leukocytes. The

LSC acquisition protocol was configured with primary con-

tour set on light scatter. Green fluorescence (HLA-FITC) and

orange fluorescence (CD45-PE) were excited with a 488-nm

argon ion laser and measured using standard filter settings.

The intensities of maximal pixel (pixel size: 0.5 3 0.5 lm2)

and integrated fluorescence were measured and recorded for

each event. One scan field (40 3 0.5 lm) per sample was ana-

lyzed. Cell morphology and fluorescence parameters were con-

firmed by visualizing microscopy images through the gallery

function in the iCys software. Events which were confirmed to

be HLA1CD452 were considered to be positive tumor events.

In Vivo Metastasis Assays

All animal procedures were conducted under a protocol

approved by the University of Western Ontario Council on

Animal Care. For all experiments, 6–7-week-old female athy-

mic nude (nu/nu) mice (Harlan Sprague–Dawley) were used.

MDA-MB-435HAL cells were prepared in sterile PBS and

injected in a 100 lL volume into the second thoracic MFP (2

3 106 cells/mouse), lateral TV (1 3 106 cells/mouse), or IC

via the left ventricle (2 3 105 cells/mouse) as described else-

where (32,34,37,39). At various time points postinjection

(MFP 5 1, 2, 4, and 8 weeks; TV 5 4, 24, 48 h, 1, 4, 6, and 7

weeks; IC 5 4, 24, 48 h, 1, 4, and 5 weeks), five mice/time

point were sacrificed and blood, LNs, and BM were harvested,

processed, and analyzed by FCM or LSC as described earlier.

Endpoints were chosen based on previous experience with

these model systems (our unpublished data), morbidity due

to primary tumor (mean MFP tumor size at endpoint 5 1725

mm3), and/or metastatic burden. Time points were preselected

based on the predicted endpoint and the hypothesized beha-

vior of the MDA-MB-435HAL cells following implantation via

the various injection routes.

Additional tissues (lung, liver, spleen, ovary, adrenal

gland, brain, bone) were also collected at necropsy and fixed

in 10% neutral-buffered formalin before processing. It should

be noted that we were unable to carry out histological analysis

of LN micrometastases because all LN material was processed

for the cytometry studies. Tissues were embedded in paraffin

wax, sectioned (4 lm thick), and subjected to standard hema-

toxylin and eosin (H&E) staining. Stained slides were evalu-

ated by light microscopy in a blinded fashion by an experi-

enced pathologist in order to observe histopathological char-

acteristics and identify incidence and regions of metastatic

involvement.

Statistical Analysis

Statistical analysis was performed with GraphPad Prism

4.0� (San Diego, CA) and SigmaStat 3.5� (SYSTAT, Chicago,

IL) using ANOVA with Kruskal–Wallis test (for comparison

between more than two groups). Differences between means

were determined using the Student’s t test when groups passed

both a normality test and an equal variance test. When this

ORIGINAL ARTICLE

346 Cytometry Analysis of Tumor Cell Dissemination

was not the case, the Mann–Whitney Rank-Sum test was used.

Correlation between time and the level of DTCs was assessed

using the Pearson product moment correlation. Correlation

between methods and correlation between primary tumor size

and disseminated cells was assessed using multiple linear

regression. Unless otherwise noted, data are presented as the

mean � SEM. In all cases, P values of �0.05 were regarded as

being statistically significant.

RESULTS

FCM Analysis of DTCs

We previously developed a multiparameter FCM assay to

quantify CTCs in the blood of preclinical mouse models of

breast cancer metastasis. This method was demonstrated to

have high specificity and a detection sensitivity of 1025, or

one tumor cell in 100,000 mouse leukocytes (39). In the pres-

ent study, we have adapted this methodology for analysis of

tumor cell dissemination to LN and BM. The FCM acquisition

setup and representative analysis is illustrated in Figure 1A

threshold region (R1) was set on DNA content based on PI

fluorescence equivalent to a diploid leukocyte (cells with low-

est DNA content) (Fig. 1, left panels), and subsequent analyses

of HLA1CD452 tumor cells were gated based on this thresh-

old (Fig. 1, right panels). Representative gated analysis is

shown of �0.01% MDA-MB-435HAL cells in mouse blood

(Fig. 1A), mouse LN homogenate (Fig. 1B), or mouse BM

(Fig. 1C). Gated PI1 events which fell within region R2 were

counted as meeting the criteria for mouse leukocytes

(CD451HLA2), and those which fell within region R3 were

counted as meeting the criteria for human tumor cells

(HLA1CD452).

LSC Analysis of DTCs

LSC uniquely combines the advantages of FCM, image

analysis, and automated fluorescence microscopy such that a

large amount of multiparameter data can be simultaneously

gathered and quantified for individual events in a heterogene-

ous population of cells, including visualization of fluorescence

and morphology (45). Therefore, although our FCM method

is specific and sensitive, we were interested in using LSC as a

complimentary method to confirm our findings, particularly

because it gave us the power to analyze each HLA1CD452 tu-

mor event for its morphological properties. The LSC acquisi-

tion protocol was configured as shown in Figure 2A, with pri-

mary contour set on light scatter. Figures 2B–2D show repre-

sentative analysis of green integral (HLA-FITC) versus orange

integral (CD45-PE) of �0.01% MDA-MB-435HAL cells in

mouse blood (Fig. 2B), mouse LN homogenate (Fig. 2C), or

mouse BM (Fig. 2D). Events which fell within region R1 were

counted as meeting the criteria for mouse leukocytes if they

were CD451HLA2, and events which fell within region R3

were counted as meeting the criteria for human tumor cells if

they were HLA1CD452. Cell morphology and fluorescence

parameters of both populations were confirmed by visualiza-

tion (Figs. 2B–2D, top insets). The combined use of this LSC

assay and the FCM assay described in Figure 1 allowed us to

characterize in vivo tumor cell dissemination patterns and

kinetics in the blood, LN, and BM of mice injected with

MDA-MB-435HAL cells via MFP, TV, or IC routes (Figs. 3–6).

Tumor Cell Dissemination Patterns and Kinetics

Following MFP Injection of Human Cancer Cells

We first used our FCM and LSC assays to assess tumor

cell dissemination patterns and kinetics in an in vivo model of

spontaneous metastasis (29,31–34) (Fig. 3). Following MFP

injection of MDA-MB-435HAL cells, FCM analysis demon-

strated that CTCs in the blood increased over time [30 � 6 (1

week) to 190� 72 (endpoint) tumor cells/105 leukocytes] (Fig.

3A, solid lines), as did DTCs in the LN [3 � 1 (1 week) to 223

� 90 (endpoint) tumor cells/105 leukocytes] (Fig. 3B, solid

lines). Interestingly, tumor cells also spontaneously dissemi-

nated to the BM (unusual for this model), peaking at 268 �125 tumor cells/105 leukocytes (2 weeks) (Fig. 3C, solid lines).

Using LSC as a complementary tool for quantitative and mor-

phologic analysis, we observed that CTCs in the blood and

DTCs in LN increased over time [blood: 10 � 5 (1 week) to

101 � 26 (endpoint) tumor cells/scan field; LN: 3 � 1 (1 week)

to 52 � 9 (endpoint) tumor cells/scan field] (Figs. 3A and 3B,

dashed lines), and DTCs were again detected in the BM with a

peak of 235 � 60 cells/scan field at 2 weeks postinjection (Fig.

3C, dashed lines). Table 1 shows CTC/DTC counts obtained by

FCM and LSC in individual mice at endpoint (8 weeks postin-

jection). For all time points, the pattern and kinetics of tumor

dissemination observed was consistent between methods, and

the number of events detected by FCM was found to be signifi-

cantly correlated with those detected using LSC analysis of

blood (R2 5 0.92; P 5 0.04), LN (R2 5 0.92; P 5 0.04), and

BM (R2 5 0.95; P 5 0.03). For both methods, there was a sig-

nificant correlation between the time postinjection and the

number of disseminated cells detected in the blood (R2[ 0.91;

P� 0.05) and LN (R2[0.97; P\0.02), although no such cor-

relation was observed in the BM. Consistent with previous

clinical studies (22,46), no significant correlation was observed

between the endpoint primary tumor size of individual mice

and the level of disseminated cells in blood, LN, or BM.


Following TV Injection of Human Cancer Cells

We next assessed tumor cell dissemination patterns and

kinetics in an in vivo model of experimental metastasis target-

ing the lung (29,35) (Fig. 4). Following TV injection, FCM

analysis demonstrated that CTCs were initially high at 4 h

postinjection (336 � 112 tumor cells/105 leukocytes) but

decreased rapidly by 1 week (19 � 5 tumor cells/105 leuko-

cytes) before increasing to peak at 598 � 429 tumor cells/105

leukocytes by endpoint (Fig. 4A, solid lines). There was a

smaller but somewhat concurrent increase in DTCs observed

in the LN (peaking at endpoint; 56 � 14 tumor cells/105 leu-

kocytes) (Fig. 4B, solid lines) and BM (peaking at 6 weeks

postinjection; 37 � 5 tumor cells/105 leukocytes) (Fig. 4C,

solid lines). LSC analysis resulted in the detection of a steady

increase in levels of CTCs over time (4 � 1 tumor cells/scan

field at 4 h to 177 � 58 tumor cells/scan field at endpoint)

ORIGINAL ARTICLE


(Fig. 4A, dashed lines). DTCs in the LN showed a fluctuating

increase over time, peaking at endpoint (47 � 24 tumor cells/

scan field) (Fig. 4B, dashed lines). In the BM, DTC levels

peaked at 1 week postinjection (70 � 13 tumor cells/scan

field) and dropped off slightly by endpoint (49 � 15 tumor

cells/scan field) (Fig. 4C, dashed lines). Table 1 shows CTC/

DTC counts obtained by FCM and LSC in individual mice at

endpoint (7 weeks postinjection). For all time points, the

observed pattern and kinetics of tumor dissemination in the

blood was consistent between methods, and the number of

CTCs detected by FCM was found to be significantly corre-

lated with those detected using LSC analysis (R2 5 0.96; P 5

0.01). There was also a significant correlation between the

time postinjection and the number of CTCs detected in the

Figure 1. Flow cytometry (FCM) acquisition setup and representative analysis of MDA-MB-435HAL human cancer cells in mouse blood,

lymph node (LN), and bone marrow (BM). Samples were stained with antimouse CD45-PE and antihuman HLA-FITC antibodies, enriched

using the EasySepTM-PE immunomagnetic selection kit, fixed, permeabilized, stained with propidium iodide (PI) to a final volume of

500 lL, and analyzed on a Beckman Coulter XL-MCL flow cytometer. A threshold region (R1) was first set on integral PI signal versus the ra-tio of the PI peak/PI integral to exclude debris, cell doublets, and clumps, and subsequent analyses were gated based on this threshold as

described previously (39). For all experiments, a minimum of 100,000 PI1 events were collected per sample. (A�C) Representative FCManalysis of �0.01% MDA-MB-435HAL tumor cells in (A) whole mouse blood, (B) mouse LN homogenate, and (C) mouse BM. Gated events

which fell within region R2 were counted as meeting the criteria for mouse leukocytes (CD451HLA2), while those which fell within region

R3 were counted as meeting the criteria for human tumor cells (HLA1CD452).

ORIGINAL ARTICLE


blood by both methods (R2[ 0.77; P\ 0.03). However, there

was neither a significant correlation between methods for

analysis of LN and BM, nor a correlation between time postin-

jection and the number of DTCs detected in these samples.


Following IC Injection of Human Cancer Cells

We also assessed tumor cell dissemination patterns and

kinetics in another in vivo model of experimental metastasis

that targets the entire circulation, but it is usually aimed to-

ward development of metastatic growth in the bone (29,35–

38) (Fig. 5). Following IC injection and assessment by FCM,

CTC levels in the blood were observed to increase over time,

peaking at 4297 � 1705 tumor cells/105 leukocytes (4 weeks

postinjection) before decreasing to 281 � 71 tumor cells/105

leukocytes by endpoint (Fig. 5A, solid lines). DTCs in the LN

increased by 24 h postinjection (98 � 33 tumor cells/105 leu-

kocytes) and maintained this level until endpoint (101 � 11

tumor cells/105 leukocytes) (Fig. 5B, solid lines). Levels of

DTCs in the BM (the most prevalent site of metastasis after IC

Figure 2. Laser scanning cytometry (LSC) acquisition setup and representative analysis of MDA-MB-435HAL human cancer cells in mouse

blood, lymph node (LN), and bone marrow (BM). Samples were stained with antimouse CD45-PE and antihuman HLA-FITC antibodies,

enriched using the EasySepTM-PE immunomagnetic selection kit, fixed, and �1/10th of the concentrated sample (equivalent to a startingvolume of �30 lL of whole blood) was placed on a glass slide with a cover slip for analysis on a Compucyte iCys LSC. For all experiments,one scan field (40 3 0.5 lm) per sample was scanned and analyzed. (A) Acquisition protocol. Representative LSC analysis of �0.01% tumor

cells in (B) whole mouse blood, (C) mouse LN homogenate, and (D) mouse BM. Cell morphology and fluorescence parameters for both

populations were confirmed by visualizing microscopy images through the gallery function in the iCys software (B�D, top insets). Eventswhich fell within region R1 (orange) and confirmed to be CD451HLA2 were counted as meeting the criteria for mouse leukocytes, and

events which fell within region R3 (green) and confirmed to be HLA1CD452were counted as meeting the criteria for human tumor cells.

ORIGINAL ARTICLE


injection) remained at moderate levels until endpoint, where

they peaked at 730 � 251 tumor cells/105 leukocytes (Fig. 5C,

solid lines). Using LSC, CTCs in the blood also showed a peak

at 4 weeks postinjection (1248 � 177 tumor cells/scan field)

before dropping off at endpoint (68 � 27 tumor cells/scan

field) (Fig. 5A, dashed lines). In both the LN and BM, levels of

DTCs were observed to peak at endpoint (LN: 63 � 20 tumor

cells/scan field; BM: 256 � 81 tumor cells/scan field) (Figs. 5B

and 5C, dashed lines). Table 1 shows CTC/DTC counts

Figure 3. Tumor cell dissemination patterns and kinetics follow-

ing mammary fat pad injection of human cancer cells. MDA-MB-

435HAL human cancer cells were injected into 6�7-week-oldfemale nude mice via the mammary fat pad (MFP, 2 3 106 cells/

mouse) to assess spontaneous metastasis. At several time points

postinjection (1, 2, 4, and 8 weeks), mice (n 5 5/time point) were

sacrificed and blood (300 lL), LNs (axillary, brachial, inguinal),and BM (hind femur/tibia) were collected. Samples were prepared

and analyzed by FCM or laser scanning cytometry (LSC) as

described in Figures 1 and 2. (A�C) Kinetics of tumor cell dissemi-nation in the (A) blood, (B) LN, and (C) BM following MFP injec-

tion. Data obtained by FCM are depicted by solid lines (left axis).

Data obtained by LSC are depicted by dashed lines (right axis). All

data are presented as the mean � SEM. * 5 significantly

increased relative to 1 week postinjection (FCM); d 5 significantly

increased relative to 1 week postinjection (LSC) (P\0.05).


ing tail vein injection of human cancer cells. MDA-MB-435HAL

cancer cells were injected into 6�7-week-old female nude micevia the lateral tail vein (TV, 1 3 106 cells/mouse) to assess experi-

mental metastasis. At several time points postinjection (4, 24,

48 h, 1, 4, 6, and 7 weeks), mice (n 5 5/time point) were sacrificed

and blood (300 lL), LNs (axillary, brachial, inguinal), and BM (hind

femur/tibia) were collected. Samples were prepared and analyzed

by FCM or laser scanning cytometry (LSC) as described in Figures

1 and 2. (A�C) Kinetics of tumor cell dissemination in the (A)blood, (B) LN, and (C) BM following TV injection. Data obtained

by FCM are depicted by solid lines (left axis). Data obtained by

LSC are depicted by dashed lines (right axis). All data are pre-

sented as the mean � SEM. * 5 significantly increased relative to

24 h postinjection (FCM); d 5 significantly increased relative to

24 h postinjection (LSC) (P\0.05).

ORIGINAL ARTICLE


obtained by FCM and LSC in individual mice at endpoint (5

weeks postinjection). For all time points, LSC analysis demon-

strated patterns and kinetics of dissemination in blood and

BM consistent with that observed by FCM (R2 [ 0.97; P \

0.001), although no such correlation was observed for LN. In

addition, there was no significant correlation between the time

postinjection and the number of disseminated cells detected

by either method in any of the three sample types.

Histological Incidence of Micrometastasis Following

MFP, TV, or IC Injection of Human Cancer Cells

Finally, we were interested in determining how endpoint

micrometastases in distant organs (Fig. 6) related to the

kinetics of tumor cell dissemination observed in the different

breast cancer models (Figs. 3–5). Tissues collected at necropsy

for each time point were processed, sectioned, and stained

with H&E before evaluation by an experienced pathologist in

order to observe histopathological characteristics (Fig. 6, right

panels) and identify incidence of metastatic involvement (Fig.

6, left panels). Interestingly, for MFP (Fig. 6A), TV (Fig. 6B),

and IC (Fig. 6C) injection routes, the kinetics of micrometa-

static incidence (% of mice with metastases) was consistent

but slightly delayed relative to the kinetics of tumor dissemi-

nation observed in Figures 3–5 (i.e., DTCs were detected at 1

week postinjection or earlier in all models (Figs. 3–5), while

micrometastases were only observed starting at 4 weeks post-

injection (Fig. 6). Micrometastases were only observed in the

lung of mice injected via the MFP (Fig. 6A) or TV (Fig. 6B),

but were observed in multiple organs (ovary, adrenal gland,

lung, and bone) following IC injection (Fig. 6C). Table 1

shows a comparison between the levels of CTC/DTCs detected

by FCM and LSC and the development of micrometastases in

individual animals at endpoint. Although the number of ani-

mals in each group (n 5 5) does not provide appropriate

power to draw any statistical conclusions, the data do suggest

some interesting trends. For example, following MFP injec-

tion, mice that had the highest levels of CTC/DTCs in the

blood, LN and BM were also the mice that developed micro-

metastases. Similarly, following TV injection, mice that had

the highest levels of CTCs in the blood were again the mice

that developed micrometastases. Finally, although all mice

injected via the IC route developed micrometastases in the

bone and lung, the two mice with the highest levels of CTCs

in the blood also developed micrometastases in the adrenal

gland and the ovary.

DISCUSSION

There is growing evidence that the presence of individual

disseminated cells in the blood, LN, or BM of breast cancer

patients may be an important indicator of the potential for

metastatic disease and poor prognosis (23–28). In the clinical

setting, the immunomagnetic-based CellSearchTM assay

(21,47) has resulted in a number of promising studies relating

to clinical outcome and prognostic value of CTCs in breast

cancer (20,46,48–54). In addition, a well-established immuno-

cytochemical method has provided valuable data regarding

the prognostic significance of DTCs in the BM (16,55). How-

ever, current American Society of Clinical Oncology guidelines

do not yet support the use of CTC or DTC assays for clinical

management decisions in breast cancer, mainly because of the

wide range of methodologies being used and the need for fur-


ing intracardiac injection of human cancer cells. MDA-MB-435HAL

human cancer cells were injected into 6�7-week-old female nudemice via the left ventricle (IC, 2 3 105 cells/mouse) to assess ex-

perimental metastasis. At several time points postinjection (4, 24,

48 h, 1, 4, and 5 weeks), mice (n 5 5/time point) were sacrificed

and blood (300 lL), LNs (axillary, brachial, inguinal), and BM (hind

femur/tibia) were collected. Samples were prepared and analyzed

by FCM or laser scanning cytometry (LSC) as described in Figures

1 and 2. (A�C) Kinetics of tumor cell dissemination in the (A)blood, (B) LN, and (C) BM following IC injection. Data obtained by

FCM are depicted by solid lines (left axis). Data obtained by LSC

are depicted by dashed lines (right axis). All data are presented as

the mean � SEM. *5 significantly increased relative to 4 h postin-

jection (FCM); d 5 significantly increased relative to 4 h postinjec-

tion (LSC) (P\0.05).

ORIGINAL ARTICLE


ther clinical validation of such tests (56). Furthermore,

although the presence of CTCs and DTCs in the blood of

breast cancer patients is hypothesized to reflect the presence of

micrometastases and/or aggressive disease, a causative biologi-

cal link between these cells and metastasis has not yet been

demonstrated (25).

Figure 6. Histological incidence of metastasis following mammary fat pad, tail vein, or intracardiac injection of human cancer cells. MDA-

MB-435HAL human cancer cells were injected into 6�7-week-old female nude mice via mammary fat pad (MFP, 2 3 106 cells/mouse), tail

vain (TV, 1 3 106 cells/mouse) or intracardiac (IC, 2 3 105 cells/mouse) routes to assess spontaneous or experimental metastasis to distant

organs. At several time points postinjection (4 h to 8 weeks), mice (n 5 5/time point) were sacrificed and blood, lymph nodes, and bone

marrow were harvested, processed, and analyzed by FCM or laser scanning cytometry (LSC) as described in Figures 1�5. Additional tis-sues collected at necropsy (lung, liver, spleen, ovary, adrenal gland, brain, bone) were fixed, stained with H&E, and analyzed by light mi-

croscopy for histological features by a trained pathologist. (A�C) Incidence of metastasis in various organs (% of mice at each time point

with metastases; left panels) as detected by H&E staining and pathohistological analysis following (A) mammary fat pad injection, (B) tail

vein injection, or (C) intracardiac injection. Right panels show representative H&E sections demonstrating metastases in various organs at

different time points.

ORIGINAL ARTICLE


Experimental challenges in detecting and quantifying rare

metastatic tumor cells in mouse models of human breast can-

cer has hindered the ability to use preclinical animal models

to their full capacity for understanding the metastatic process,

particularly with regards to determining the timing and loca-

tion of CTC and DTC dissemination, quantifying early steps

in metastasis, and determining how disseminated cells contri-

bute to endpoint metastases. In the present study, we describe

the development and implementation of novel FCM and LSC

assays for characterizing tumor cell dissemination patterns

and kinetics in three different in vivo models of breast cancer

metastasis, including evidence of CTC/DTC contribution to

the development of metastatic disease.

We believe that the findings of the current study have a

number of important biological and technical implications.

From a biological perspective, for the first time we were able

to quantify individual CTCs and DTCs in our preclinical ani-

mal models at very early time points following tumor cell

injection and during progression to metastatic disease. In our

spontaneous metastasis model, the steady increase in CTCs in

the blood and DTCs in the LN over time was perhaps not

unexpected and is reflective of the clinical situation in which

the lymphatic system is considered to be a primary route for

early dissemination of breast cancer (57–59). Furthermore, we

observed that the timing of micrometastatic incidence to dis-

tant organs such as lung was consistent but slightly delayed

relative to CTC and DTC kinetics. For example, DTCs were

detected in the blood, LN, and BM as early as 1 week postin-

jection, while lung micrometastases were only observed start-

ing at 4 weeks postinjection. Interestingly, this timing corre-

sponded to detection of increasing levels of CTCs in the blood

and DTCs in the LN, suggesting that, in addition to shedding

from the primary tumor, CTCs and DTCs may also be disse-

minating from secondary metastatic tumors in the lung and

possibly the LN. We also observed DTCs in the BM, an unex-

pected finding given that bone metastases have not been

reported to develop spontaneously following orthotopic injec-

tion of human breast cell lines (29,60). However, since there

was no corresponding histological evidence of bone metastases

in this model, these findings suggest [as other studies have

(16,61–64)] that the BM may act as a tumor cell reservoir and/

or a passive filter interacting with the blood circulation.

Following TV injection, we observed a similar increase in

CTCs and DTCs over time. Although the blood seemed to be

the major route of tumor dissemination in this experimental

metastasis model, the observed concurrent increase in DTCs in

the LN and BM and the confirmed histological presence of

micrometastases in the lung supports the idea that tumor cells

may be shed into the circulation from metastases that establish

following initial cell delivery. Following IC injection, we

observed the highest levels of CTCs in the blood of any of the

three metastasis models. High CTC levels were observed even as

late as 2–4 weeks postinjection, when it might be expected that

most tumor cells would have been eliminated from the circula-

Table 1. Comparison of CTC/DTC levels obtained via flow cytometry (FCM) or laser scanning cytometry (LSC) and the development of

micrometastases in individual mice at endpoint

MOUSE ID

NO. OF CTCS IN

BLOOD (FCMa/LSCb)

NO. OF DTCS

IN LN (FCMa/LSCb)

NO. OF DTCS IN

BM (FCMa/LSCb)

MICROMETASTASES AT

ENDPOINT (YES OR NO; SITE)c

MFP injection (8 weeksd)

Mouse 1 180/130 187/51 149/161 Yes; lung

Mouse 2 100/60 104/44 54/118 No

Mouse 3 39/33 85/26 34/80 No

Mouse 4 169/100 166/60 102/139 Yes; lung

Mouse 5 461/180 578/80 75/124 Yes; lung

TV injection (7 weeksd)

Mouse 1 163/113 59/20 17/6 No

Mouse 2 264/180 32/22 38/72 Yes; lung

Mouse 3 191/122 91/11 6/18 Yes; lung

Mouse 4 2313/400 16/140 6/78 Yes; lung

Mouse 5 62/70 80/40 16/73 No

IC injection (5 weeksd)

Mouse 1 108/30 120/134 299/100 Yes; bone, lung



Mouse 4 358/70 94/40 1474/540 Yes; bone, lung, adrenal

Mouse 5 514/170 63/46 855/300 Yes; bone, lung, adrenal, ovary

a Flow cytometry analysis was carried out as described in the ‘‘Materials and Methods’’ section and Figure 1. CTC/DTC numbers

obtained by FCM are presented as number of tumor cells/105 leukocytes.b Laser scanning cytometry analysis was carried out as described in the ‘‘Materials and Methods’’ section and Figure 2. CTC/DTC num-

bers obtained by LSC are presented as number of tumor cells/scan field.c The presence or absence and location of micrometastases were determined by pathohistological analysis of H&E-stained tissue sec-

tions as described in the ‘‘Materials and Methods’’ section.d Respective final time point for each injection route.

ORIGINAL ARTICLE


tion either by cell death and/or by seeding in distant organs.

Indeed, micrometastases were observed in multiple organs

including bone, and these metastases may have been contribut-

ing to the high CTC levels observed in the blood. At endpoint,

the highest level of disseminated cells was observed in the BM, a

logical finding considering that bone is the most prevalent site

of metastases following IC injection (29,60), and all mice had

histologically detectable bone metastases at endpoint.

From a technical perspective, our results demonstrate

that the combined use of FCM and LSC allows for sensitive

quantification of DTCs in preclinical models of breast cancer

metastasis. Although FCM is a proven and reliable high-

throughput technology, its major disadvantages for rare event

analysis are that high numbers of cells are needed for accurate

quantitation, and there is no capacity for morphological anal-

ysis (65,66). It has also been suggested that FCM may have

limited sensitivity for CTC analysis in human blood samples

(67). However, we have found that it has sufficient sensitivity

(1025) for use in our animal models when combined with

immunomagnetic enrichment, perhaps because of the much

smaller overall blood volume of a mouse relative to a human

(39). Comparatively, LSC has the distinct ability to confirm

the true positive nature of identified rare cells by visualization,

has been demonstrated to be effective and sensitive for rare

event analysis, and can be used for relatively small samples

sizes. Disadvantages of LSC include relatively high infrastruc-

ture costs, moderate analysis speed, and manual sample han-

dling (17,45,67,68).

Given that both technologies have specific strengths and

weaknesses, our goal in the present study was to expand the

capacity of our previously validated FCM assay and use LSC as

a complimentary (rather than a comparative) approach. As a

consequence, we did not perform direct comparisons of abso-

lute CTC/DTC counts between methods. Instead, we were

interested in determining whether FCM and LSC demon-

strated consistency with regards to the patterns and kinetics of

tumor cell dissemination in different sample types and in dif-

ferent metastasis models. In all cases, we observed a significant

correlation between methods for analysis of blood samples.

Combined with the fact that the LSC assay required only a

very small sample size (equivalent to �30 lL of whole mouse

blood), this suggests that LSC may be particularly useful for

future studies aimed a longitudinal sampling of mice over

time to assess real-time CTC kinetics and the relationship with

metastasis and/or response to therapy. Interestingly, there was

more variability between FCM and LSC results when analyzing

BM and LN samples, and in many cases we detected greater

than expected cell numbers by LSC relative to those events

detected by FCM in a much larger volume of the same sample.

One possible explanation is that both sample types (and BM,

in particular) tended to be more ‘‘cellular’’ and contain more

cell aggregates than blood, and in the FCM assay these aggre-

gates were lost from analysis based on our gating strategy. In

contrast, LSC allowed for visual confirmation of fluorescence

and morphological parameters, and thus permitted inclusion

of positive events within cell aggregates which may have other-

wise been excluded from FCM analysis.

In summary, the ability to quantify CTCs and DTCs fol-

lowing injection of human cancer cells via different routes pro-

vides valuable information with regards to the pattern and

kinetics of MDA-MB-435HAL tumor cell dissemination in our

spontaneous and experimental metastasis models. Further-

more, since our selection and labeling strategy is designed to

specifically identify human cancer cells derived from a variety

of sources (39), these methods will have broad application for

characterizing the tumor cell dissemination patterns and meta-

static ability of other human breast cancer cell lines. Although

a number of quantitative tools have been previously developed

to study in vivo metastasis (69–71), the detection and quantifi-

cation of rare metastatic events has remained challenging. The

novel methods presented in the current study will begin to

address this need and have future potential for helping to eluci-

date the mechanistic details of early steps in metastasis and

how these steps relate to the development of life-threatening

macrometastases. In addition, these methods will be extremely

valuable for the future identification, development, and testing

of new therapeutic strategies to combat breast cancer.

ACKNOWLEDGMENTS

The authors thank Dr. Waleed Al-Katib for his assistance

with pathohistological analysis. A.L.A. receives salary support

from the Imperial Oil Foundation.

LITERATURE CITED

1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer statistics, 2008.CA Cancer J Clin 2008;58:71–96.

2. Canadian Cancer Statistics 2008. Toronto: National Cancer Institute of Canada; 2008.Available at www.cancer.ca.

3. Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cellsin metastatic sites. Nat Rev Cancer 2002;2:563–572.

4. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev Cancer2004;4:448–456.

5. Chambers AF, Naumov GN, Varghese HJ, Nadkarni KV, MacDonald IC, Groom AC.Critical steps in hematogenous metastasis: An overview. Surg Oncol Clin N Am2001;10:243–55,vii.

6. Fidler IJ. 7th Jan Waldenstrom Lecture. The biology of human cancer metastasis.Acta Oncol 1991;30:668–675.

7. Woodhouse EC, Chuaqui RF, Liotta LA. General mechanisms of metastasis. Cancer1997;80(8,Suppl):1529–1537.

8. Swartz MA, Skobe M. Lymphatic function, lymphangiogenesis, and cancer metasta-sis. Microsc Res Tech 2001;55:92–99.

9. Ashworth TR. A case of cancer in which cells similar to those in the tumours wereseen in the blood after death. Aust Med J 1869;14:146–149.

10. Bossolasco P, Ricci C, Farina G, Soligo D, Pedretti D, Scanni A, Deliliers GL. Detec-tion of micrometastatic cells in breast cancer by RT-pCR for the mammaglobin gene.Cancer Detect Prev 2002;26:60–63.

11. Iakovlev VV, Goswami RS, Vecchiarelli J, Arneson NC, Done SJ. Quantitative detec-tion of circulating epithelial cells by Q-RT-PCR. Breast Cancer Res Treat 2008;107:145–154.

12. Xenidis N, Perraki M, Kafousi M, Apostolaki S, Bolonaki I, Stathopoulou A, Kalbakis K,Androulakis N, Kouroussis C, Pallis T, Christophylakis C, Argyraki K, Lianidou ES,Stathopoulos S, Georgoulias V, Mavroudis D. Predictive and prognostic value of periph-eral blood cytokeratin-19 mRNA-positive cells detected by real-time polymerase chainreaction in node-negative breast cancer patients. J Clin Oncol 2006;24:3756–3762.

13. Cabioglu N, Igci A, Yildirim EO, Aktas E, Bilgic S, Yavuz E, Muslumanoglu M, Bozfa-kioglu Y, Kecer M, Ozmen V, Deniz G. An ultrasensitive tumor enriched flow-cyto-metric assay for detection of isolated tumor cells in bone marrow of patients withbreast cancer. Am J Surg 2002;184:414–417.

14. Cruz I, Ciudad J, Cruz JJ, Ramos M, Gomez-Alonso A, Adansa JC, Rodriguez C,Orfao A. Evaluation of multiparameter flow cytometry for the detection of breastcancer tumor cells in blood samples. Am J Clin Pathol 2005;123:66–74.

15. Simpson SJ, Vachula M, Kennedy MJ, Kaizer H, Coon JS, Ghalie R, Williams S, Van EppsD. Detection of tumor cells in the bone marrow, peripheral blood, and apheresis pro-ducts of breast cancer patients using flow cytometry. Exp Hematol 1995;23:1062–1068.

16. Braun S, Vogl FD, Naume B, Janni W, Osborne MP, Coombes RC, Schlimok G, DielIJ, Gerber B, Gebauer G, Pierga JY, Marth C, Oruzio D, Wiedswang G, Solomayer EF,Kundt G, Strobl B, Fehm T, Wong GY, Bliss J, Vincent-Salomon A, Pantel K. A pooledanalysis of bone marrow micrometastasis in breast cancer. N Engl J Med 2005;353:793–802.

ORIGINAL ARTICLE


17. Pachmann K, Camara O, Kavallaris A, Krauspe S, Malarski N, Gajda M, Kroll T, JorkeC, Hammer U, Altendorf-Hofmann A, Rabenstein C, Pachmann U, Runnebaum I,Hoffken K. Monitoring the response of circulating epithelial tumor cells to adjuvantchemotherapy in breast cancer allows detection of patients at risk of early relapse. JClin Oncol 2008;26:1208–1215.

18. Ring AE, Zabaglo L, Ormerod MG, Smith IE, Dowsett M. Detection of circulatingepithelial cells in the blood of patients with breast cancer: Comparison of three tech-niques. Br J Cancer 2005;92:906–912.

19. Zabaglo L, Ormerod MG, Parton M, Ring A, Smith IE, Dowsett M. Cell filtration-laser scanning cytometry for the characterisation of circulating breast cancer cells.Cytometry Part A 2003;55A:102–108.

20. Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM,Doyle GV, Allard WJ, Terstappen LW, Hayes DF. Circulating tumor cells, disease pro-gression, and survival in metastatic breast cancer. N Engl J Med 2004;351:781–791.

21. Riethdorf S, Fritsche H, Muller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C,Beck K, Janicke F, Jackson S, Gornet T, Cristofanilli M, Pantel K. Detection of circu-lating tumor cells in peripheral blood of patients with metastatic breast cancer: A val-idation study of the CellSearch system. Clin Cancer Res 2007;13:920–928.

22. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, KwakEL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, TonerM. Isolation of rare circulating tumour cells in cancer patients by microchip technol-ogy. Nature 2007;450:1235–1239.

23. Alix-Panabieres C, Muller V, Pantel K. Current status in human breast cancer micro-metastasis. Curr Opin Oncol 2007;19:558–563.

24. Dawood S, Cristofanilli M. Integrating circulating tumor cell assays into the manage-ment of breast cancer. Curr Treat Options Oncol 2007;8:89–95.

25. Pantel K, Brakenhoff RH, Brandt B. Detection, clinical relevance and specific biologi-cal properties of disseminating tumour cells. Nat Rev Cancer 2008;8:329–340.

26. Riethdorf S, Pantel K. Disseminated tumor cells in bone marrow and circulatingtumor cells in blood of breast cancer patients: Current state of detection and charac-terization. Pathobiology 2008;75:140–148.

27. Smerage JB, Hayes DF. The prognostic implications of circulating tumor cells inpatients with breast cancer. Cancer Invest 2008;26:109–114.

28. Tan LK, Giri D, Hummer AJ, Panageas KS, Brogi E, Norton L, Hudis C, Borgen PI,Cody HS III. Occult axillary node metastases in breast cancer are prognosticallysignificant: Results in 368 node-negative patients with 20-year follow-up. J ClinOncol 2008;26:1803–1809.

29. Welch DR. Technical considerations for studying cancer metastasis in vivo. Clin ExpMetastasis 1997;15:272–306.

30. Webster MA, Muller WJ. Mammary tumorigenesis and metastasis in transgenic mice.Semin Cancer Biol 1994;5:69–76.

31. Allan AL, George R, Vantyghem SA, Lee MW, Hodgson NC, Engel CJ, Holliday RL,Girvan DP, Scott LA, Postenka CO, Al-Katib W, Stitt LW, Uede T, Chambers AF, TuckAB. Role of the integrin-binding protein osteopontin in lymphatic metastasis ofbreast cancer. Am J Pathol 2006;169:233–246.

32. Schulze EB, Hedley BD, Goodale D, Postenka CO, Al-Katib W, Tuck AB, ChambersAF, Allan AL. The thrombin inhibitor Argatroban reduces breast cancer malignancyand metastasis via osteopontin-dependent and osteopontin-independent mechan-isms. Breast Cancer Res Treat 2007; Dec 21. [Epub ahead of print] PMID: 18097747.

33. Vantyghem SA, Allan AL, Postenka CO, Al-Katib W, Keeney M, Tuck AB, ChambersAF. A new model for lymphatic metastasis: Development of a variant of the MDA-MB-468 human breast cancer cell line that aggressively metastasizes to lymph nodes.Clin Exp Metastasis 2005;22:351–361.

34. Vantyghem SA, Wilson SM, Postenka CO, Al-Katib W, Tuck AB, Chambers AF. Die-tary genistein reduces metastasis in a postsurgical orthotopic breast cancer model.Cancer Res 2005;65:3396–3403.

35. Hedley BD, Vaidya KS, Phadke P, Mackenzie L, Dales DW, Postenka CO, MacdonaldIC, Chambers AF. BRMS1 suppresses breast cancer metastasis in multiple experimen-tal models of metastasis by reducing solitary cell survival and inhibiting growthinitiation. Clin Exp Metastasis 2008; Jun 10. [Epub ahead of print] PMID: 18543067.

36. Mastro AM, Gay CV, Welch DR. The skeleton as a unique environment for breastcancer cells. Clin Exp Metastasis 2003;20:275–284.

37. Phadke PA, Vaidya KS, Nash KT, Hurst DR, Welch DR. BRMS1 suppresses breastcancer experimental metastasis to multiple organs by inhibiting several steps of themetastatic process. Am J Pathol 2008;172:809–817.

38. Naumov GN, Townson JL, MacDonald IC, Wilson SM, Bramwell VH, Groom AC,Chambers AF. Ineffectiveness of doxorubicin treatment on solitary dormant mammarycarcinoma cells or late-developing metastases. Breast Cancer Res Treat 2003;82:199–206.

39. Allan AL, Vantyghem SA, Tuck AB, Chambers AF, Chin-Yee IH, Keeney M. Detectionand quantification of circulating tumor cells in mouse models of human breastcancer using immunomagnetic enrichment and multiparameter flow cytometry.Cytometry Part A 2005;65A:4–14.

40. Schmidt CM, Settle SL, Keene JL, Westlin WF, Nickols GA, Griggs DW. Characteriza-tion of spontaneous metastasis in an aggressive breast carcinoma model using flowcytometry. Clin Exp Metastasis 1999;17:537–544.

41. Price JE, Polyzos A, Zhang RD, Daniels LM. Tumorigenicity and metastasis of humanbreast carcinoma cell lines in nude mice. Cancer Res 1990;50:717–721.

42. Rae JM, Creighton CJ, Meck JM, Haddad BR, Johnson MD. MDA-MB-435 cells arederived from M14 melanoma cells–a loss for breast cancer, but a boon for melanomaresearch. Breast Cancer Res Treat 2007;104:13–19.

43. Rae JM, Ramus SJ, Waltham M, Armes JE, Campbell IG, Clarke R, Barndt RJ, JohnsonMD, Thompson EW. Common origins of MDA-MB-435 cells from various sources withthose shown to have melanoma properties. Clin Exp Metastasis 2004;21:543–552.

44. Sellappan S, Grijalva R, Zhou X, Yang W, Eli MB, Mills GB, Yu D. Lineage infidelityof MDA-MB-435 cells: Expression of melanocyte proteins in a breast cancer cell line.Cancer Res 2004;64:3479–3485.

45. Pozarowski P, Holden E, Darzynkiewicz Z. Laser scanning cytometry: Principles andapplications. Methods Mol Biol 2006;319:165–192.

46. Lang JE, Mosalpuria K, Cristofanilli M, Krishnamurthy S, Reuben J, Singh B, Bedro-sian I, Meric-Bernstam F, Lucci A. HER2 status predicts the presence of circulatingtumor cells in patients with operable breast cancer. Breast Cancer Res Treat 2008;Mar 9. [Epub ahead of print] PMID: 18327638.

47. http://www.veridex.com/CellSearch/Default.aspx.

48. Budd GT, Cristofanilli M, Ellis MJ, Stopeck A, Borden E, Miller MC, Matera J, Repol-let M, Doyle GV, Terstappen LW, Hayes DF. Circulating tumor cells versus imaging–predicting overall survival in metastatic breast cancer. Clin Cancer Res 2006;12:6403–6409.

49. Cristofanilli M, Broglio KR, Guarneri V, Jackson S, Fritsche HA, Islam R, Dawood S,Reuben JM, Kau SW, Lara JM, Krishnamurthy S, Ueno NT, Hortobagyi GN, ValeroV. Circulating tumor cells in metastatic breast cancer: Biologic staging beyond tumorburden. Clin Breast Cancer 2007;7:471–479.

50. Cristofanilli M, Hayes DF, Budd GT, Ellis MJ, Stopeck A, Reuben JM, Doyle GV,Matera J, Allard WJ, Miller MC, Fritsche HA, Hortobagyi GN, Terstappen LW. Circu-lating tumor cells: A novel prognostic factor for newly diagnosed metastatic breastcancer. J Clin Oncol 2005;23:1420–1430.

51. Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, Matera J, AllardWJ, Doyle GV, Terstappen LW. Circulating tumor cells at each follow-up time pointduring therapy of metastatic breast cancer patients predict progression-free and over-all survival. Clin Cancer Res 2006;12(14, Part 1):4218–4224.

52. Nole F, Munzone E, Zorzino L, Minchella I, Salvatici M, Botteri E, Medici M, Verri E,Adamoli L, Rotmensz N, Goldhirsch A, Sandri MT. Variation of circulating tumorcell levels during treatment of metastatic breast cancer: Prognostic and therapeuticimplications. Ann Oncol 2008;19:891–897.

53. Stojadinovic A, Mittendorf EA, Holmes JP, Amin A, Hueman MT, Ponniah S, PeoplesGE. Quantification and phenotypic characterization of circulating tumor cells formonitoring response to a preventive HER2/neu vaccine-based immunotherapy forbreast cancer: A pilot study. Ann Surg Oncol 2007;14:3359–3368.

54. Yagata H, Nakamura S, Toi M, Bando H, Ohno S, Kataoka A. Evaluation of circulat-ing tumor cells in patients with breast cancer: Multi-institutional clinical trial inJapan. Int J Clin Oncol 2008;13:252–256.

55. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S, WischnikA, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G. Cytokeratin-positive cellsin the bone marrow and survival of patients with stage I, II, or III breast cancer. NEngl J Med 2000;342:525–533.

56. Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, Somerfield MR, HayesDF, Bast RC Jr. American Society of Clinical Oncology 2007 update of recommenda-tions for the use of tumor markers in breast cancer. J Clin Oncol 2007;25:5287–5312.

57. Fisher B, Bauer M, Wickerham DL, Redmond CK, Fisher ER, Cruz AB, Foster R,Gardner B, Lerner H, Margolese R, et al. Relation of number of positive axillarynodes to the prognosis of patients with primary breast cancer. An NSABP update.Cancer 1983;52:1551–1557.

58. Foster RS Jr. The biologic and clinical significance of lymphatic metastases in breastcancer. Surg Oncol Clin N Am 1996;5:79–104.

59. McGuire WL. Prognostic factors for recurrence and survival in human breast cancer.Breast Cancer Res Treat 1987;10:5–9.

60. Rosol TJ, Tannehill-Gregg SH, LeRoy BE, Mandl S, Contag CH. Animal models ofbone metastasis. Cancer 2003;97(3,Suppl):748–757.

61. Braun S, Kentenich C, Janni W, Hepp F, de Waal J, Willgeroth F, Sommer H, Pantel K.Lack of effect of adjuvant chemotherapy on the elimination of single dormant tumorcells in bone marrow of high-risk breast cancer patients. J Clin Oncol 2000;18:80–86.

62. Muller V, Stahmann N, Riethdorf S, Rau T, Zabel T, Goetz A, Janicke F, Pantel K. Cir-culating tumor cells in breast cancer: Correlation to bone marrow micrometastases,heterogeneous response to systemic therapy and low proliferative activity. Clin Can-cer Res 2005;11:3678–3685.

63. Pantel K, Schlimok G, Braun S, Kutter D, Lindemann F, Schaller G, Funke I,Izbicki JR, Riethmuller G. Differential expression of proliferation-associated moleculesin individual micrometastatic carcinoma cells. J Natl Cancer Inst 1993;85:1419–1424.

64. Scatton O, Chiappini F, Riou P, Marconi A, Saffroy R, Bralet MP, Azoulay D, Bou-cheix C, Debuire B, Uzan G, Lemoine A. Fate and characterization of circulating tu-mor cells in a NOD/SCID mouse model of human hepatocellular carcinoma. Onco-gene 2006;25:4067–4075.

65. Kuckuck FW, Edwards BS, Sklar LA. High throughput flow cytometry. CytometryPart A 2001;44A:83–90.

66. Terstappen LWMM. Detection of infrequent cells in blood and bone marrow by flowcytometry. In: Ho A, Haas R, Champlin RE, editors. Hematopoietic Stem Cell Trans-plantation. New York: Marcel Dekker; 2000. pp137–152.

67. Bocsi J, Varga VS, Molnar B, Sipos F, Tulassay Z, Tarnok A. Scanning fluorescent mi-croscopy analysis is applicable for absolute and relative cell frequency determinations.Cytometry Part A 2004;61A:1–8.

68. Darzynkiewicz Z, Bedner E, Li X, Gorczyca W, Melamed MR. Laser-scanning cytome-try: A new instrumentation with many applications. Exp Cell Res 1999;249:1–12.

69. Jenkins DE, Oei Y, Hornig YS, Yu SF, Dusich J, Purchio T, Contag PR. Bioluminescentimaging (BLI) to improve and refine traditional murine models of tumor growth andmetastasis. Clin Exp Metastasis 2003;20:733–744.

70. Li X, Wang J, An Z, Yang M, Baranov E, Jiang P, Sun F, Moossa AR, Hoffman RM.Optically imageable metastatic model of human breast cancer. Clin Exp Metastasis2002;19:347–350.

71. Naumov GN, Wilson SM, MacDonald IC, Schmidt EE, Morris VL, Groom AC, Hoff-man RM, Chambers AF. Cellular expression of green fluorescent protein, coupledwith high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.J Cell Sci 1999;112 (Part 12):1835–1842.

ORIGINAL ARTICLE


Date post:	24-Nov-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis...

Documents