Just transportation to the hotel and some walking in the proximity

Non-invasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a

strategy for pre-clinical modeling

Running title: Bioluminescent imaging of primary ALL

David M. Barrett1, Alix E. Seif1, Carmine Carpenito2, David T. Teachey1, Jonathan D. Fish1, Carl

H. June2, Stephan A. Grupp1,2 and Gregor S.D. Reid1,3

1Department of Pediatrics, Division of Oncology, Center for Childhood Cancer Research,

Children’s Hospital of Philadelphia, Philadelphia, PA; 2 Abramson Family Cancer Research

Institute, University of Pennsylvania Cancer Center, Philadelphia, PA 19104: 3Child and Family

Research Institute and Department of Pediatrics, University of British Columbia, Vancouver, BC

V5Z 4H4.

Corresponding author: Gregor Reid

A5-162 CFRI Translational Research Bldg

950 West 28th Avenue

Vancouver

BC V5Z 4H4

Canada

Telephone: 604-875-2000 ext4692

Fax: 604-875-3120

Email: [email protected]

Key words: Optical Imaging, acute lymphoblastic leukemia, patient samples

Blood First Edition Paper, prepublished online August 19, 2011; DOI 10.1182/blood-2011-04-346528

Copyright © 2011 American Society of Hematology

For personal use only.on April 15, 2019. by guest www.bloodjournal.orgFrom

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Abstract

The efficient engraftment in immune-deficient mice achieved with both acute lymphoblastic

leukemia (ALL) cell lines and primary samples has facilitated identification of the anti-leukemia

activity of a wide variety of agents. Despite widespread usage, however, little is known about

the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs

primarily to survival and end-point analysis at high disease burden. In this study, we report that

bioluminescent imaging can be reproducibly achieved with primary human ALL samples. This

approach provides a noninvasive, longitudinal measure of leukemia burden and localization that

enhances the sensitivity of treatment response detection and provides greater insight into the

mechanism of action of anti-leukemia agents. In addition, this study reveals significant cell line-

and species-related differences in leukemia migration, especially early in expansion, which may

confound observations between various leukemia models. Overall this study demonstrates that

the use of bioluminescent primary ALL allows the detection and quantitation of treatment effects

at earlier, previously unquantifiable disease burdens and thus provides the means to

standardize and expedite the evaluation of anti-ALL activity in pre-clinical xenograft studies.




Introduction

Murine leukemia models have been useful and versatile tools for identifying and targeting

underlying disease mechanisms.1 However, pre-clinical studies involving human samples are

more immediately applicable when investigating new drugs or therapies. Initial studies with

human leukemia in immune-deficient mice such as the NOD/SCID (NOD.CB17-Prkdcscid/J) were

a great step forward and remain valuable for rapid iterations in the study of novel therapies at

the pre-clinical level.2,3 More recently, use of severely immune-deficient NOD/SCID/γc-/- (NSG;

NOD.Cg-Prkdcscid/IL2rgtm1Wjl/SzJ and NOG; NOD.Cg-Prkdcscid/Il2rgtm1Sug/Jic) mice, which lack

functional B, T and NK cells, has allowed for more consistent and reproducible engraftment of

normal human cells and primary leukemias.4-6

Primary acute lymphoblastic leukemia (ALL) xenografts in immune-deficient mice accurately

represent human disease and gene expression and may have predictive value for clinical

variables such as drug resistance and time to relapse.7-11 While this model is a powerful and

widely used tool for the ongoing development of new drugs and biological therapeutics for

ALL,12-19 the kinetics of every primary patient sample are different, some engrafting in four

weeks and others taking as long as a year, when measured by appearance of human ALL

blasts in peripheral blood.5,10,20 The endpoints of these studies are typically limited to survival

analysis and time to grossly detectable disease. Monitoring the peripheral blood compartment

for blasts is the primary method of disease detection, leaving the early engraftment kinetics and

localization, therefore, largely unknown.21-24 Furthermore, the time from appearance of

peripheral blasts to death is also highly variable in our experience, taking from as little as a few

days to as long as several months. Animals may show no outward signs of engraftment, and

samples are occasionally lost when mice die before displaying evidence of disease. Non-

invasive measures, including magnetic resonance imaging, ultrasound and positron emission

tomography/computed tomograpy (PET/CT) scans rely on specific organ changes as a




surrogate marker of disease. For individual samples this may correlate well to disease

development, such as with leukemia that reliably produces splenic enlargement, but as the

library of primary patient samples grows, it is clear that not all human leukemias behave the

same when engrafted in a mouse. The early migration and anatomic niche population of each

leukemia may be different, and current standard endpoint analyses (organ size, peripheral

blood) do not give a complete picture of disease expansion or treatment response.

The application of bioluminescent reporters has enabled the non-invasive, longitudinal

monitoring of hematological malignant cells for in vivo studies of cellular migration and

expansion, as well evaluation of responses to cytotoxic therapy.25-30 Luciferase reporters provide

great sensitivity due to the extremely low background luminescence in wild type animals and

represent a broadly applicable approach to non-invasive disease monitoring.31-33 Firefly

luciferase is particularly versatile as a bioluminescent reporter, as a significant portion of its

emission spectrum is greater than 600 nm, wavelengths that achieve good tissue penetrance.34

While these reporters require introduction of a novel substrate to achieve luminescence, the two

most common substrates for bioluminescent reporters, luciferin and colenterazine, are well

tolerated by animals. The kinetics following intraperitoneal administration of D-luciferin are

reliable and reproducible with peak emission times exceeding several minutes.34

Lentiviral transduction has been shown to be an effective method for gene transfer into primary

human ALL blasts.35-37 In this study, we use lentivirus-mediated gene transfer of luciferase to

generate a panel of bioluminescent primary ALL samples and perform a systematic study of

early engraftment kinetics using multiple ALL cell lines and primary samples. By enabling

quantification of early disease burdens and identification of specific organ involvement, the

ability to generate bioluminescent primary ALL samples reproducibly will provide the means for

standardization and more accurate comparison of pre-clinical of ALL treatment responses.




Methods

Cells and Patient Samples

The human BCP ALL lines Nalm-6 and RS4-11 were obtained from American Type Culture

Collection (ATCC, Manassas, VA), and 380 was kindly provided by Dr. Dario Campana (St

Jude, Memphis, TN). Mouse BCP ALL cell lines 289 and t309 were previously derived from

primary leukemias developing in Eμ-Ret fusion protein transgenic mice on a BALB/cJ

background.38-40 Mouse cell lines were cultured in RPMI medium with 20% fetal bovine serum

(FBS) and 3% interleukin-7 supernatant, as previously described.40 Human lines were cultured

in RPMI medium with 10% fetal bovine serum and 1% penicillin/streptomycin. Human ALL

samples were obtained as de-identified samples from the Leukemia Bank at the University of

Pennsylvania and the ALL Cell Bank maintained by the Children's Oncology Group. The

samples were collected for both cell banks under the Children's Hospital of Philadelphia IRB-

approved protocols and distributed under standard material transfer agreements. ALL-1 was

obtained from peripheral blood of an adult ALL patient; ALL-2 and ALL-3 were obtained from

bone marrow of pediatric ALL patients at diagnosis.

Mouse Xenograft and Allograft Studies

Briefly, 6-10 week old NOD.Cg-Prkdcscid/IL2rgtm1Wjl/SzJ (NSG) and NOD.CB17-Prkdcscid/J (NS)

mice were obtained from the Jackson Laboratory (Bar Harbor, ME) or bred in-house and

maintained under specific pathogen-free conditions. Animals were injected via tail vein with 106

viable murine or human leukemia cells in 0.2 mL sterile PBS unless otherwise indicated.

Peripheral blood was obtained by retro-orbital bleeding, and presence of ALL blasts was

determined by flow cytometry. Human CD19 and CD45 and mouse B220 and BP-1 expression

were detected by staining with fluorescently-conjugated monoclonal antibodies (BD

Biosciences, San Jose, CA). All experiments were conducted under the supervision of the

facility’s Institutional Animal Care and Use Committee (IACUC) according to an IACUC-

approved protocol.




Lentiviral Transduction of Primary Leukemia Samples

The self-inactivating lentiviral construct encoding firefly luciferase (ffluc) and green fluorescent

protein (GFP), utilizing the EF-1α promoter and a 2A ribosomal skip sequence, used in these

experiments is described elsewhere.41,42 Primary human BCP ALL samples were expanded in

NSG mice. Human ALL cells (2.5-5x104), isolated from the spleens of moribund recipient NSG

mice, were incubated directly ex vivo in RPMI 1640 media supplemented with 10% FBS and 1%

penicillin/streptomycin containing the lentivirus at a multiplicity of infection of 50:1 for 16 hours in

polystyrene 5 mL tubes. Viable, unsorted cells were washed in excess volumes of sterile PBS

four times and injected via tail vein into a single NSG mouse for expansion. The recipient mice

were evaluated weekly by bioluminescence imaging for the presence of luciferase activity.

Moribund recipient mice were euthanized and human CD45, CD19 and GFP triple-positive cells

were purified from spleen and bone marrow by flow cytometric sorting. Purified cells were then

injected via tail vein into secondary recipient mice for further expansion and purification. This

protocol is summarized in Figure 1. The secondary expansion cells were then employed in the

treatment assays described. All human cell lines and samples were subjected to genomic

fingerprinting via a combination of short tandem repeat (STR) and single nucleotide

polymorphism (SNP) analysis to verify the identity of the cells before and after luciferase

transduction and in vivo expansion.43,44

Bioluminescent Imaging

Anesthetized mice were imaged using a Xenogen Spectrum system and Living Image v4.0

software. Mice were given an intraperitoneal injection of 150 mg/kg D-luciferin (Caliper Life

Sciences, Hopkinton, MA). Previous titration indicated time to peak of photon emission to be

five minutes, with peak emission lasting for 6-10 minutes. Each animal was imaged alone (for

photon quantitation) or in groups of up to 5 mice (for display purposes) in the anterior-posterior

prone position at the same relative time point after luciferin injection (6 minutes). Data were




collected until the mid range of the linear scale was reached (600 to 60000 counts) or maximal

exposure settings reached (f stop 1, large binning and 120 seconds), and then converted to

photons/second/cm2/steradian (p/s/cm2/sr) to normalize each image for exposure time, f stop,

binning and animal size. This approach avoids collection of saturated images. Mice without

luciferase-containing cells were imaged at maximal settings and a mean value of 3.6 x105

p/s/cm2/sr was obtained. Mice with luciferase-expressing leukemia reliably became moribund

when photon flux approached 2x1011 p/s/cm2/sr. For anatomic localization, a pseudocolor map

representing light intensity was superimposed over the grayscale body-surface reference image.

For confirmation of organ involvement, moribund animals were imaged as above then

euthanized and organs removed, including vertebral body, skull base (calvarium), brain, liver,

spleen, thymus, sternum, kidney with adrenal, gonads and femur. Organs were then imaged

immediately for luciferase signal (within the 10 minute peak emission) followed by

homogenization or perfusion to isolate cells for flow cytometry. Diffuse Luminescence

Tomography (DLIT) was performed and analyzed as per the manufacturer’s instructions.45,46

This algorithm reconstructs the depth of light sources based on the known properties of the

emitted light from the luciferase, the index of attenuation of the tissues involved, and an iterative

algorithm based on spectral filters acquired in sequence.

Treatment Response Monitoring

Primary ALL cells from the second passage mice were purified to >99% ffluc/GFP+ and injected

via tail vein into NSG or NOD/SCID mice as indicated (106 cells/mouse). Mice with equivalent

levels of bioluminescent disease at Day 4 were randomized to either vehicle control or

experimental groups. NSG mice were treated by oral gavage with 5 mg/kg sirolimus (SIR) in 0.5

ml 5% dextrose once daily for 24 days where indicated. NOD/SCID mice were treated with

immunostimulatory DNA containing unmethylated CpG dinucleotides (CpG ODN) 100 mcg in

0.2 mL sterile PBS intraperitoneally every 4 days for 3 doses where indicated.




Statistical Considerations

Analysis was performed Prism 4 (Graphpad Software, La Jolla, CA). In vitro data represent

means of duplicates, and comparisons of means were made via Mann-Whitney test. For

comparison among multiple groups, Kruskal-Wallis analysis was performed with Dunn Multiple

Comparison tests to compare individual groups. Survival curves were compared using the log-

rank test with a Bonferroni correction for comparing multiple datasets.

Results

Primary patient ALL can be made bioluminescent with a self-inactivating lentiviral vector

encoding firefly luciferase and GFP

The engraftment of primary ALL in immune-deficient mice has been shown to be a selective

event.8,47,48 To optimize the transduction of ALL cells with xenografting potential, we transduced

primary ALL cells harvested after an initial passage through NSG mice. We have applied the

methodology depicted in Figure 1 to three separate primary BCP ALL samples (ALL-1, ALL-2

and ALL-3). Over several weeks, a distinct bioluminescent population of each transduced,

unsorted sample emerged, though it remained a fraction of the total body disease as indicated

by peripheral blood monitoring once tumor appeared. Animals were selected for harvest when

the peripheral disease burden (human blasts detected by flow cytometery) approached

moribund levels, and human GFP+ ALL cells were isolated from bone marrow and spleen. A

small percentage of harvested cells were GFPhigh (0.5%-5%), and 5 x104 viable GFP+ cells were

sorted and reinjected into a second set of NSG mice, this time at >99% purity for GFP/ffluc

expressing cells. These animals developed detectable bioluminescent disease more quickly

than the first-passage mice, giving a true bioluminescent picture of the early development of

disease in a xenograft model of primary patient leukemia (Figure 1). When moribund, the

second passage mice of ALL-1 were sacrificed, and the spleen and marrow harvested and

combined. Human leukemia was 85% GFPhigh, and this population was divided and re-injected,




either unsorted at 85% purity or after flow sorting to >99% GFPhigh. The third passage mice were

followed for natural history and the populations from spleen and marrow at terminal disease

burdens later showed stable GFPhigh expression (85% or 99%, respectively). This indicates that

the transduced population is stable through repeated passages, and that the appearance of

peripheral blasts and time to death are unchanged from the non-transduced population. In

addition, genomic fingerprinting via a combination of STR and SNP analyses revealed no

changes in the cells after transduction, or after 3 passages in mice (data not shown).

Bioluminescence can be used to infer organ localization of ALL cells

Two dimensional pseudocolor intensity maps can approximate organ localization but may be

dependent on mouse orientation (prone, supine, decubitus) and sensitivity of the charge-

coupled device (CCD) camera. We combined information from the two-dimensional intensity

maps of mice engrafted with ffluc+ primary ALL with three-dimensional reconstruction using the

DLIT technology. We were able to isolate areas that corresponded to involved organs on the

two-dimensional intensity maps in combination with DLIT. A representative map generated

using primary ALL-1 is shown in Figure 2A. To confirm organ involvement and avoid false

positive assumptions, we imaged isolated organs from sacrificed mice (Figure 2A). For each of

the ALL samples, the primary sites of early engraftment were the bone marrow and liver, with

minor variations seen in other organs (Supplemental Figure 1).

Having validated the identification of involved organs, we then compared the 3 primary ALL

samples to 5 similarly labeled BCP ALL lines (3 human, 2 mouse) for early engraftment kinetics

and localization over the first 7 days. The cells are not detectable in the peripheral blood in the

first 7 days and are only detectable at minimal levels in femoral bone marrow (0.1-1% of total

cells) by flow cytometry on Day 7 (data not shown). While all human-derived ALL showed

preferential early migration to the bone marrow and liver, we observed differences depending




both on species of origin as well between cell lines (Figure 2B). The human cell lines (RS4-11,

380, Nalm-6) exhibited more variability than primary ALL samples. Nalm-6 and RS4-11 rapidly

populated the liver and bone marrow but were not detectable in the spleen, while 380 rapidly

populated the bone marrow but spared the liver and spleen in these early time points. In

contrast, the two mouse BCP ALL lines (t309 and 289) homed to the bone marrow and spleen,

but not liver, within the first 72 hours. Over several weeks, all samples eventually populated all

target organs including liver, bone marrow and spleen but the preference and timing of each

varied considerably.

Despite variation between ALL samples and cell lines in the rates of engraftment in different

target organs, we observed a clear reproducibility in the impact of systemic disease burden on

the appearance of blasts in peripheral blood and terminal morbidity (Figure 2C). Detection of

>1% ALL blasts in peripheral blood by flow cytometry did not occur consistently until

bioluminescent leukemia burdens of >1x1010 p/s/cm2/sr were reached. Notably, while the level

of maximal peripheral disease varies among samples, a whole-body photon flux of 1-2x1011

p/s/cm2/sr is consistently close to terminal disease burden in all samples. As mice without

luciferase-containing cells yielded a mean value of 3.6 x105 p/s/cm2/sr, a detection range of 6

orders of magnitude is achieved with this approach, greatly expanding the window for treatment

response evaluation.

Bioluminescent primary patient leukemia samples enable earlier and more sensitive

disease response monitoring

To evaluate the ability to detect treatment response provided by the use of bioluminescent

primary ALL and to validate that the transduction process did not significantly alter the overall

outcomes, we used two treatment models with which we have considerable experience. First,

we measured the in vivo response of bioluminescent primary ALL to treatment with sirolimus, an

mTOR inhibitor that we have previously demonstrated significantly prolongs survival of mice




with xenografted leukemia.13,40 While untreated animals had continual progression of their

disease, the bioluminescent burden of disease in sirolimus-treated animals stabilized at a low

level (Figures 3A and B). Over time, this represented a significantly reduced leukemia burden

(P < 0.0001), delayed appearance of peripheral blasts, and resulted in extension of overall

survival. Once therapy is discontinued, leukemia burden immediately begins to rise, as indicated

by bioluminescence levels even though peripheral blasts are still not detected. Time to

appearance of peripheral blasts and moribund disease was not altered from the parent, non-

transduced sample (data not shown). Second, in the NOD/SCID model, we applied our

published protocol for CpG ODN-induced anti-ALL immune activity.14 In this model, NOD/SCID

mice treated with CpG ODN demonstrated a rapid significant reduction in disease burden (P =

0.0001), while control animals had unchecked, progressive disease (Figures 3A and B). Again,

following the cessation of treatment ongoing imaging of the treated mice reveals a steady

increase in disease burden.

In both experimental settings significant treatment effects were clearly apparent weeks before

they are detectable by non-bioluminescence methods (Figure 3C). The contrast between the

early disease stabilization achieved with sirolimus and the rapid reduction of disease burden by

CpG ODN treatment is clearly revealed by the use of bioluminescent primary ALL blasts. In

addition, the occurrence of spontaneous regression of established bioluminescent disease in

one untreated NOD/SCID mouse was observed (data not shown). While it has long been

observed that some NOD/SCID animals will ‘fail to engraft’ human samples, one assumption

has been that the cells died initially. We observed that in some cases the disease can engraft

but is then spontaneously cleared after 7 days rather than failing to engraft at all; as we

observed this only in NOD/SCID and never in NSG mice, the presence of NK cells may be

responsible for this disease clearance.




Discussion

In this study, we report that bioluminescent disease monitoring, an approach used widely in

other cancer models,49 is achievable with primary human ALL samples. Lentivirus constructs in

which Firefly luciferase is co-expressed with GFP provide a sortable marker to isolate cells

expressing the bioluminescent reporter. Using this strategy, we demonstrate the reproducible

generation of primary ALL samples containing >99%, stable ffluc-expressing cells. While there

is concern that the in vitro modification of primary leukemia cells could impair engraftment

potential,50 the luciferase tagging protocol used here does not appear to significantly alter

leukemia propagation; we achieve reliable and stable secondary and tertiary passage rates

comparable to those of non-transduced cells from the same original primary ALL sample. In

addition, ffluc+ leukemia samples result in disease that is genetically and phenotypically identical

to the non-transduced leukemia samples based on STR and SNP analysis, disease kinetics and

response to therapy in our established models.

Our comparison of ALL responses following administration of two well-described treatments

reveals the ability of the bioluminescence approach to provide more mechanistic and

quantitative detail than current evaluation protocols. The use of luciferase-expressing ALL cells

clearly and rapidly distinguishes between the apparent global disease stabilizing effects of

sirolimus and the cytotoxic activity of CpG ODN that underlie their superficially comparable

inhibition of ALL progression by survival analysis. In addition, the information gained by learning

about early anatomic niche localization may also open new avenues of study into the

importance of organ specific homing molecules or drug penetration into sites of leukemic

harbor. As certain therapies, whether pharmacologic or immunologic, may or may not treat

various anatomic niches (eg liver, CNS, gonads), understanding if or when those areas are

infiltrated by the leukemia and whether disease in those areas responds to treatment would

contribute significantly to results from pre-clinical studies.




The non-invasive, longitudinal monitoring of primary ALL xenografts we describe will expedite

the investigation and discovery of novel therapies by allowing early detection at minimal disease

levels, earlier response measurements, and identification of sites of leukemic relapse or

resistance. By enabling 1) the identification of mice with similar organ involvement and equal

disease burdens; 2) the precise quantification of pre- and post-treatment disease levels; and 3)

the selection of more appropriate endpoints, bioluminescent imaging will provide a method for

standardization of treatment response monitoring in primary ALL xenograft models. This

approach may prevent mischaracterization of therapies as ineffective based on current endpoint

analysis (overall survival and peripheral blood blast counts). Finally, and not insignificantly, the

ability to eliminate non-engrafted mice prior to the onset of experimental treatments, combined

with the more accurate quantification of responses, will reduce the number of animals required

to achieve statistical significance in pre-clinical testing.




Acknowledgments

Supported in part by NIH K12CA076931 (DMB), R01CA102646, R01CA116660 and support

from the Weinberg Foundation (SAG), an ASBMT/Robert A. Good New Investigator Award

(AES), awards from The Prevent Cancer Foundation and When Everyone Survives Foundation

(GSDR).

Authorship

D.M.B., A.E.S. and G.S.D.R. designed the research, performed the experiments, interpreted the

data, and wrote the paper.

C.C., C.H.J, J.D.F. and D.T.T. provided critical reagents and designed the research.

S.A.G. provided critical reagents, interpreted the data, and wrote the paper.

Conflict of Interest Statements

All authors: no conflict of interest.




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Figure legends

Figure 1: Our strategy to generate stable ffluc+ primary ALL samples. Bulk transduction of

primary patient ALL, freshly obtained from NSG mouse bone marrow, via lentivirus results in low

level GFP/ffluc expression which can be purified by flow sorting. As per methods, 3 primary

patient ALL samples (ALL-1, ALL-2 and ALL-3) were harvested from moribund NSG mice,

transduced overnight and viable cells injected into primary recipients via tail vein. All 3 samples

engrafted with a small GFP positive population that was then purified using the gating strategy

shown (sample ALL-1 sorting is depicted). This enrichment results in stable, >99%

bioluminescent primary ALL with resultant early detection and evenly disseminated disease in

secondary (shown in lower panels) and later recipient mice.

Figure 2: Bioluminescence can be used to distinguish patterns and timing of organ involvement

for both human and mouse leukemia. A) Prone, supine and organ images of a NSG mouse

injected with 1x106 ffluc+ primary ALL-1 cells. Outlined 2D regions of interest correspond to

organ involvement validated at day 3 of engraftment by isolated imaging. DLIT image of mouse

internal anatomy, with skin and gut removed for clarity, reconstructed from images is shown

(right panel). Green voxels represent point sources of light within putative target organs,

verified by ex vivo imaging and flow cytometry. B) Day 3 intensity maps of NSG mice injected

with 1x106 ffluc+ leukemia cells as indicated. These representative mice show the differing

patterns of organ involvement at this early time point. Human ALL cells establish within the liver

and bone marrow (primary ALL, RS4-11 and Nalm-6) or bone marrow only (380), while both

mouse cell lines studied rapidly home to the spleen and bone marrow (t309 and 289). C) Time

course of leukemia engraftment reveals consistent bioluminescent correlation with appearance

of peripheral blasts and maximal moribund disease burden. Nalm-6 (20-22 days), primary ALL-1




(40-50 days), primary ALL-2 (80-100 days) and primary ALL-3 (35-40 days) all become

moribund at 2x1011 photons/s/cm2. Appearance of peripheral blasts (>1%) happens consistently

at systemic bioluminescent tumor burdens of greater than 1x1010 photons/s/cm2. In ALL-2, the

stable bioluminescent burden from 46-75 days corresponds with stable but high peripheral blast

burden (30-50%) before a progression to end stage between 80-100 days. Results presented as

mean +/- SE (n=5 for primary ALL samples, n=10 for Nalm-6).

Figure 3: Bioluminescence enables the visualization and quantification of therapeutic responses

of primary ALL significantly earlier than other measurable parameters. A) Intensity maps of total

body disease developing over time in NSG mice treated with sirolimus (SIR) for 24 days (upper

panels) or in NOD/SCID mice treated with CpG ODN (CpG) over 8 days (lower panels).

Untreated (PBS) controls for both strains are shown. Results obtained with patient sample ALL-

1 are depicted. B) Graphs with quantification of the bioluminescent ALL responses over time

shown in Panel A to treatment with sirolimus (upper panel) or CpG ODN (lower panel). C) Flow

cytometric evaluation of peripheral blood ALL burden at day 45 in the control, sirolimus- and

CpG ODN-treated mice shown in panels A and B. Overall, this experiment is not designed to

compare the treatment strategies but rather to demonstrate the additional detail of ALL

treatment response revealed by bioluminescent imaging.




Figure 1




Figure 2




Figure 3




doi:10.1182/blood-2011-04-346528Prepublished online August 19, 2011;

Stephan A. Grupp and Gregor S.D. ReidDavid M. Barrett, Alix E. Seif, Carmine Carpenito, David T. Teachey, Jonathan D. Fish, Carl H. June, lymphoblastic leukemia: a strategy for pre-clinical modelingNon-invasive bioluminescent imaging of primary patient acute

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