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j.leukres.2018.08.012

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Leukemia Research 72 (2018) 105–112
Contents lists available at ScienceDirect
Leukemia Research
journal homepage: www.elsevier.com/locate/leukres
Research paper
A novel extracellular matrix-based leukemia model supports leukemia cells
with stem cell-like characteristics
T
Dandan Lia, Tara L. Linb, Brea Lipeb, Richard A. Hopkinsc, Heather Shinogled,
⁎
Omar S. Aljitawia,b,e,
a
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, United States
Division of Hematology/Oncology and Blood and Marrow Transplantation Program, 2330 Shawnee Mission Parkway, University of Kansas Medical Center, Kansas City,
KS, United States
c
Cardiac Surgery Research Laboratories, Children's Mercy Hospital and Clinics, Kansas City, Missouri, United States
d
Microscopy and Analytical Imaging Laboratory, University of Kansas, Lawrence, KS, United States
e
Department of Medicine, Hematology/Oncology and Bone Marrow Transplant Program, University of Rochester Medical Center, Rochester, NY, 14642, United States
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Extracellular matrix
In vitro 3D model
Leukemia
Leukemia stem cell-like
Decellularized Wharton's jelly matrix
Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and
extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We
used decellularized Wharton’s jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in
DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in
DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM
demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and
secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow
cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell
behavior and chemotherapy sensitivity.
1. Introduction
Acute myeloid leukemia (AML) is a heterogeneous hematopoietic
malignancy characterized by an aberrant clonal expansion of undifferentiated myeloid blasts. Studies have shown that leukemia stem
cells (LSCs) contribute to relapse after chemotherapeutic treatment. [1]
Like normal hematopoietic stem cells (HSCs), LSCs maintain their selfrenewal ability while generating clonogenic leukemic progenitors
capable of producing leukemic cells [2]. Anti-proliferative chemotherapeutic agents commonly target the rapidly cycling leukemic cells, but
they generally are ineffective against the quiescent LSCs, partly because
of enhanced drug efflux in LSCs [3]. Therefore, it is important to develop therapeutic strategies which eliminate the LSCs in the bone
marrow, where they share the “hematopoietic niche” along with normal
HSCs [4]. The LSC niche, similar to the hematopoietic niche, is a 3D
⁎
microenvironment composed of bone marrow stromal cells and ECM
components like collagen, fibronectin and tenascin [4]. These components create compartments that not only provide structural support to
the cells in the bone marrow, but also provide chemokines and cytokines that are important in regulating LSC self-renewal, trafficking,
proliferation and differentiation [5].
Currently, most leukemia in vitro studies are based on conventional
two-dimensional (2D) cultures in tissue culture polystyrene (TCP)
dishes/ flasks and stromal co-cultures. These models are useful in elucidating some of the molecular mechanisms of leukemia initiation and
progression. However, 2D culture systems lack the leukemia-microenvironment interaction present in the 3D bone marrow microenvironment. Therefore, the LSCs in 2D culture frequently differentiate
and lose their “stem-ness”. The development of a 3D model that replicates the in vivo mechanical and biochemical properties of bone
Corresponding author at: 601 Elmwood Avenue, Rochester, NY, 14642, United States.
E-mail address: omar_aljitawi@urmc.rochester.edu (O.S. Aljitawi).
https://doi.org/10.1016/j.leukres.2018.08.012
Received 15 March 2018; Received in revised form 12 August 2018; Accepted 13 August 2018
Available online 16 August 2018
0145-2126/ © 2018 Elsevier Ltd. All rights reserved.
Leukemia Research 72 (2018) 105–112
D. Li et al.
times, 4 h prior to proliferation assessment. 10% alamarBlue
(Biocentric) was added into each well. After 4 h, 100 μl of supernatant
from each well was aspirated to a new well of a 96 well plate and
fluorescence was measured by micro-plate reader, with excitation wavelength at 530 nm and emission wavelength at 590 nm.
marrow could allow maintenance of a true LSC-state.
Our laboratory has been focused on characterizing decellularized
Wharton’s jelly matrix (DWJM) and examining its potential for regenerative medicine applications. [6–8], We hypothesized that DWJM
would provide a similar environment to the bone marrow ECM, because
its components, such as collagen, fibronectin, hyaluronic acid, and
sulphated proteoglycan [8], also exist in the bone marrow hematopoietic niche. Moreover, because cell-ECM interactions play an important role in chemoresistance in leukemia cells [9], the environment
provided by DWJM could support the maintenance of LSCs.
We therefore used DWJM as an ECM to examine leukemia cell-ECM
interactions, hypothesizing that DWJM would support leukemia cells
with LSC-like characteristics. In this model, we investigated the growth
pattern of 3 human leukemia cell lines (HL60, Kasumi-1, and MV411),
with a focus on proliferation, viability, morphology and myeloid differentiation. We also studied the drug resistance and stem cell characteristics of leukemia cells cultured in this model, compared to leukemia cells cultured in suspension. We found that leukemia cells
cultured in our DWJM-based ECM model had LSC characteristics, suggesting that DWJM may prove useful in LSC characterization and in
developing therapeutic interventions that target LSCs.
2.5. CellTrace proliferation assay
To monitor the cell division of leukemia cells in suspension and in
DWJM, cells were labeled with CellTrace Violet (Life Technology) before seeding. Briefly, cells were washed and resuspended with PBS at
the concentration of 106cells/ml, and CellTrace Violet stock solution
was added in a final concentration of 1 μl/ml. After incubation at room
temperature for 20 min, 5 ml of PBS with 10% FBS were added and
incubated for 5 min, followed by centrifugation to obtain pellets. Cells
were resuspended in culture medium and cultured in either suspension
or DWJM. Cell division was measured by flow cytometry soon after
seeding and after 7 days of culture. To isolate cells from DWJM, we
washed wafers in PBS, and then used collagenase II (0.05 g collagenase
II in 50 ml DMEM for 1–2 hours) to digest DWJM at 37 °C.
2.6. Cell viability
2. Materials and methods
Cell survival in DWJM was measured by Vi-CELL Series Cell
Viability Analyzer (Beckman Coulter), which is based on Trypan Blue
dye exclusion. Cells in each DWJM wafer were released by treating with
1 ml 0.002 g/ml collagenase II (Worthington) for about 2 h at 37 °C. The
released cells were assessed for viability according to the manufacturer’s recommendations.
2.1. Cell culture
Human AML cell lines HL60, Kasumi I and MV 411 (ATCC,
Manassas, VA) were maintained in T 75 tissue culture flasks with
Advanced Roswell Park Memorial Institute (RPMI) 1640 Medium
(Gibco), supplemented with 5% fetal bovine serum (Sigma-Aldrich) and
1% penicillin/streptomycin (pen/strep) (Life Technologies). Cells were
maintained at 37 °C in a fully humidified 5% CO2 incubator.
2.7. Histology and immunohistochemistry
For morphological analysis, wafers were washed with PBS three
times, fixed in 4% PFA, embedded in paraffin, sectioned and stained
with hematoxylin and eosin, and visualized under the microscope using
an Olympus BX40 microscope; pictures were obtained using a DP72
digital camera.
2.2. DWJM scaffold preparation
The preparation of DWJM was previously described. [8,10], Briefly,
to prepare DWJM scaffolds, fresh human umbilical cords were dissected
after removing surrounding membranes and blood vessels. Then they
were subjected to two cycles of osmotic shock in hypertonic and hypotonic solutions, followed by immersion in a non-ionic detergent
Triton-x, an anionic detergent sodium lauryl succinate; finally, they
underwent enzymatic digestion with recombinant endonuclease. The
resulting DWJM pieces were cut into thin wafers (3 mm thick) as previously described [10] (Supplementary Figure-01A). Cartoon depiction
of leukemia cells interacting with DWJM fibers (Supplementary Figure01B).
2.8. Treatment with chemotherapeutic agents
After 7 days, cells cultured in suspension and cells in DWJM were
treated with 50μM of doxorubicin hydrochloride (Sigma-Aldrich) for
48 h. For cells in suspension, culture medium was removed, and chemotherapeutic agents were added in fresh medium. For cells in DWJM,
scaffolds were transferred into new 24-well plates and washed with PBS
three times; then a chemotherapeutic agent was added to the culture
medium.
2.3. Seeding DWJM wafers with AML cell lines
2.9. Apoptosis assay
Before seeding, cryopreserved DWJM wafers were thawed, washed
three times in phosphate buffered saline (PBS), and pre-incubated with
Advanced RPMI overnight. AML cells (2*105 cells/well) were seeded
into DWJM wafers in 24-well non-tissue culture treated plates with 60%
area of each well covered by DWJM. Culture plates were then placed in
an incubator at 37 °C with 5% CO2 and maintained in Advanced RPMI
with 5% FBS for 7 days; half of the medium was changed every other
day. AML cells in suspension (2*105 cells/well), cultured under the
same conditions, were used as controls. In AlamarBlue assay, CellTrace
proliferation and Ki67 immunohistology sample preparation, cells were
maintained in RPMI 1640 (Sigma-Aldrich) with 10% FBS for HL60 and
MV411 cells, and 20% FBS for Kasumi I cells.
Apoptosis in leukemia cells was measured by flow cytometry using
Annexin V-Alexa 568 (Invitrogen, USA) and DAPI (Invitrogen, USA)
staining. Prior to flow cytometry analysis, cells in DWJM wafers were
released as described previously and 105 released cells as well as cells in
suspension were stained with DAPI and Annexin V according to manufacturer’s recommendations. Data were acquired within 1 h, using LSR
II (BD Biosciences), and analyzed by FlowJo software.
2.10. Aldefluor assay
Aldehyde dehydrogenase (ALDH) activity was examined by using
Aldefluor reagent (Stem Cell Technologies) according to the manufacturer’s protocol, followed by flow cytometry. Cells negative for
propidium iodide (PI) staining were considered positive for ALDH,
based on a negative control using the ALDH inhibitor diethylaminobenzaldehyde (DEAB). Data were analyzed within 1 h, using LSR II (BD
2.4. AlamarBlue assay
To assess the proliferation of AML cells, DWJM wafers with cells
were transferred to new 24 well plates and washed with PBS three
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Fig. 1. Leukemia cells cultured in DWJM demonstrate reduced proliferation while maintaining cell viability and undifferentiated state.
(A) HL60, Kasumi 1 and MV411 cells were
seeded onto DWJM wafers and allowed to adhere to DWJM for 2 days. Then DWJM wafers
with attached cells were replated, and
AlamarBlue assay was used to assess cell proliferation on day 2, 4, and 6. Data are normalized to the day 2 fluorescence reading. (B)
Collagenase II was used to release cells embedded in DWJM, and cell viability was assessed by Vi-CELL, based on Trypan blue exclusion on days 2, 4, and 6. All values represent
means ± SEM. (C) Violet intensity of HL60
and MV411 in DWJM and suspension before
and 7 days after seeding. (D) Apoptosis and
necrosis of HL60 and MV411 cultured in suspension or in DWJM, as measured by flow cytometry following co-staining with AnnexinV/
PI. Results are shown as density plots of one
representative experiment (upper) and as
summary of results of multiple experiments
(bottom). Data represent means ± SEM.
Experiments done in triplicate. (E) CD11b expression in HL60 and Kasumi 1 cultured in
suspension and DWJM.
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Biosciences).
statistical threshold of p < 0.05.
2.11. Analysis of differentiation marker
3. Results
Expression of CD11b was measured by flow cytometry. 105 cells,
cultured in suspension or in DWJM, were harvested and incubated with
FITC-conjugated anti-human CD11b (Abcam) for 10 min at room temperature. After staining, cells were washed and resuspended in 400 μl
PBS and analyzed by flow cytometry by using LSR II (BD Biosciences).
3.1. Leukemia cells cultured in DWJM demonstrated reduced proliferation
while maintaining cell viability and undifferentiated state
Since LSCs are quiescent, we first examined leukemia cell proliferation over time in our model, hypothesizing that leukemia-DWJM
interactions result in decreased leukemia cell proliferation over time.
Using AlamarBlue assay on days 2, 4, and 6 after leukemia cell seeding,
we demonstrated decreased proliferation in all three leukemia cell lines
(HL 60, Kasumi I and MV411) (Fig. 2A). Despite reduced proliferation,
all three cell lines maintained nearly unchanged viability at the three
time points, as measured by Trypan Blue dye exclusion (Fig. 1B).
Next, we studied leukemia cell proliferation and viability in our
model, compared to suspension culture systems. We found that leukemia cells cultured in DWJM demonstrated reduced proliferation
measured by CellTrace proliferation assay (Fig. 1C), but no significant
differences in apoptosis between the two culture conditions when assessed by Annexin V/PI flow cytometry (Fig. 1D). LSCs are expected to
maintain an undifferentiated state. We next examined DWJM culture
effects on leukemia cell differentiation by examining expression of
CD11b, a common myeloid marker [11], using flow cytometry. Our
experiments showed no increase in CD11b expression in either suspension or DWJM-culture conditions, indicating that both conditions
maintain leukemia cells in an undifferentiated state (Fig. 1E). Taken
together, our data suggest that leukemia cells cultured in DWJM are
more quiescent than leukemia cells in suspension.
2.12. Colony forming unit (CFU) assay
Cells were plated on 35 mm dishes (500 cells/dish for Kasumi I, and
300 cells/dish for HL60 and MV411) in triplicate in MethoCult® H4434
Classic methylcellulose (Stemcell Technologies, Vancouver, Canada).
Cells were washed and resuspended in RPMI 1640 medium. After
measuring cell number and viability by Trypan blue, cell densities were
adjusted at a concentration of 10 cells/μl, and cells were added into
methylcellulose, followed by addition of 1 ml cell-methylcellulose mix
into each dish. After 12–14 days of incubation at 37 °C in 5% CO2,
colonies consisting of > 30 cells were counted, then harvested and replated in methylcellulose. After another 12–14 days, colonies were
counted.
2.13. Confocal microscopy
Samples of Wharton’s Jelly matrix with incorporated MV-4-11 cells
were fixed with 4% paraformaldehyde in Hanks Balanced Saline
Solution (HBSS), and then cleared using pancreatin solution (0.01 g
sucrose, 0.1 g pancreatin, 0.05 g saponin, 0.05% Triton-X100 diluted in
HBSS) and placing it into an oven at 34 °C for seven hours. Next,
samples were immersed in HBSS overnight at room temperature (RT).
After permeabilization with HBSS and 0.1 g saponin (HBSS:S) for ten
minutes, samples were immersed in blocking solution (3% normal goat
serum and 0.05% Triton-X100 diluted in HBSS:S) and placed on a rotator for two hours at RT. Then, samples were immersed overnight in
primary antibody solution of mouse monoclonal anti-Actin α-smooth
muscle antibody (Sigma Aldrich) at 4 °C. Samples were rinsed with
HBSS:S five times (10 min each) and then incubated with secondary
antibody solution using Alexa Fluor 488 goat anti-mouse polyclonal
antibody (Life Technologies, ThermoFisher Scientific). All samples were
placed in a rotator during four hours of incubation at RT, and then
rinsed with HBSS:S two times (ten minutes each), followed by two
rinses with HSSS (ten minutes each). 10 μM of the nuclear counterstain
DAPI (Life Technologies, ThermoFisher Scientific) was added to each
sample for one hour at RT. After three rinses with HBSS, samples were
immersed in Vectashield (Vector Laboratories, CA) mounting medium
for fluorescence overnight and mounted on coverslips with silicon
isolators (Grace Bio-labs, PC1R-2.5). For negative control, samples were
processed following the same procedure, but without the primary antibody.
Images were collected on a customized spinning disc Olympus IX-81
inverted microscope, equipped with CSU-10 (Yokogawa Electric
Company); 405 nm, 488 nm, 642 nm (Coherent, Inc) and 561 nm
(CrystaLaser) lasers; a Prior (Prior Scientific) stage; a Sutter (Sutter
Instruments) emission filter wheel; and UPlanSApo 20 × 0.75NA air
and a UPlanFL N 40 × 1.3NA oil Olympus objectives. Images were
acquired using SlideBook 6 (Intelligent Imaging Innovations, Inc.), and
data was deconvolved using SlideBook’s constrained iterative deconvolution algorithm.
3.2. Leukemia cells in DWJM developed spindle cell-shaped morphology
Since leukemia cell interactions with ECM matrix resulted in reduced cell proliferation, we wondered if leukemia cell-matrix interactions might have caused morphologic changes in leukemia cells cultured in DWJM. By examining histology sections, we noticed that
leukemia cells (HL 60 and Kasumi I) cultured in DWJM tended to
change their morphology, switching from round cells, which is the
morphology of AML cells in suspension, to spindle-shaped cells (Fig. 2A
and B). In general, there were more spindle-shaped cells than round
cells per high power field (HPF) (Fig. 2C). Round cells are mostly seen
in the open spaces within the matrix, while the spindle shaped cells are
mostly embedded inside the matrix. These morphologic changes were
also visualized by confocal microscopy in actin-stained cells in fixed
tissue (Fig. 2D).
3.3. Leukemia cells cultured in DWJM demonstrated an increase in the
ALDH positive population
Data suggest that AML cells with ALDH positivity are associated
with quiescent state and resistance to chemotherapy treatment. [12]
Since our data suggested that leukemia cells cultured in DWJM were
more quiescent than leukemia cells cultured in suspension, we examined ALDH expression in our model, hypothesizing that leukemia
cells cultured in DWJM were associated with increased ALDH expression compared to leukemia cells cultured in suspension. Indeed, significantly increased ALDH expression was found in both Kasumi I (∼7
fold, p < 0.05) and MV411 (∼2 fold, p < 0.05) cells cultured in
DWJM compared with cells in suspension (Fig. 3A), while no differences were seen in HL60 cells (data not shown).
2.14. Statistical analysis
3.4. Leukemia cells cultured in DWJM demonstrated an increase in
leukemia cell clonogenic ability
All data analyses were performed with Graphpad Prism 6
(GraphPad Software, Inc.) and presented as means ± standard deviation (SEM). Significance was determined using Student’s t-test, with a
Our data indicated that leukemia cells cultured in DWJM exhibited
some stem and progenitor cell characteristics. Accordingly, we next
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Fig. 2. Leukemia cell morphologic changes during culture in
DWJM. (A) Representative H&E stained histology sections of
HL60 (left), Kasumi 1 (middle) and MV411 (right) cells cultured in DWJM for one week (40x). (B) Kasumi 1 cells change
from round to spindle-shaped. Left: round; Middle: roundspindle; Right: spindle (100x). (C) Number of different leukemia cell shapes per high power field (HPF). Data represent
means ± SEM. (D) Confocal microscopy images of leukemia
cells cultured in DWJM following actin-staining (green) and
Dapi nuclear staining (blue) (40×).(For interpretation of the
references to colour in this figure legend, the reader is referred
to the web version of this article).
the majority of the cultured cells. As expected, doxorubicin induced cell
death mainly through apoptosis. We observed that the survival rate of
cells cultured in DWJM was significantly higher than that of suspension
cells in all three cell lines HL60 (∼2.5 fold, p < 0.5), Kasumi I (∼8
fold, p < 0.05), and MV411 (∼5 fold, p < 0.1) (Fig. 5 A and B),
suggesting that leukemia cells cultured in DWJM are more resistant to
cytotoxicity of anti-cancer drugs.
To examine whether reduced uptake or increased efflux of chemotherapeutic drugs in DWJM-cultured cells might explain their relative resistance to chemotherapy, we analyzed doxorubicin fluorescence intensity in leukemia cells. We found that the accumulation of
doxorubicin was significantly lower in leukemia cells cultured in DWJM
compared to cells in suspension (Fig. 5C).
Because N-cadherin has been reported to be a LSC marker and to be
associated with stem cell drug resistance [13], we assessed N-cadherin
expression in leukemia cells under DWJM and suspension conditions.
We found that leukemia cells cultured in DWJM had increased N-cadherin expression compared to cells cultured in suspension (Fig. 5D).
examined the clonogenic ability of leukemia cells (which correlates
with self-renewal ability) comparing cells cultured in DWJM to cells in
suspension, using CFU assay with secondary replating. In all three cell
lines, colony number from both primary and secondary plating showed
significantly increased colony numbers in DWJM-cultured leukemia
cells compared to suspension cells (Fig. 4A–C). In two cases, the increase approached statistical significance in Kasumi 1 after primary
plating (Fig. 4C, p = 0.08) and MV411 after secondary plating (Fig. 4B,
p = 0.051). These findings suggest that leukemia cells cultured in
DWJM gain the long-term ability to self-renew.
3.5. Leukemia cells cultured in DWJM demonstrated increased drug
resistance
Since leukemia cells cultured in DWJM demonstrated LSC-like
characteristics and since LSCs are chemoresistant, we next investigated
doxorubicin’s effects on leukemia cells cultured in DWJM versus cells in
suspension, using a very high dose of doxorubicin to cause apoptosis in
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Fig. 3. Leukemia cells cultured in DWJM demonstrate an increase in ALDH positive population. Representative density plots (A) and quantification (B) of ALDH
positive MV411 and Kasumi 1 cells after 7 day culture in DWJM vs suspension. Data represent means ± SEM. Experiments done in at least duplicate.
Fig. 4. Leukemia cells cultured in DWJM demonstrate an increase in leukemia cell clonogenic ability. HL60 (A), MV411 (B), and Kasumi 1 (C) were cultured in
DWJM or in suspension for 7 days. An equal number of cells were plated in methylcellulose for 10–14 days. Cells were replated in methylcellulose for another 10–14
days. The number of colony forming units (CFUs) after primary (upper) and secondary plating (lower) of HL60 (A), MV411 (B), and Kasumi 1 (C) cells were
measured. Data represent means ± SEM. Experiments done in triplicate.*P < 0.05; **P < 0.01.
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Fig. 5. Leukemia cells cultured in DWJM demonstrate increased drug resistance. (A) Representative density plots and (B) quantification of HL-60, Kasumi I and
MV411 cells undergoing apoptosis and necrosis in either DWJM or suspension, measured after 50μM doxorubicin treatment for 48 h. Data represent means ± SEM.
Experiments done in triplicate. *P < 0.05; **P < 0.01. (C) Doxorubicin uptake in HL60 (left) and MV411 (right) cells after doxorubicin treatment. (D) N-cadherin
expression in HL60 (left) and MV411 (right) after 7 days culture in suspension (blue) and DWJM (red).(For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article).
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4. Discussion
help with confocal microscopy studies. This work was partly supported
by the Robert K. Dempski Cord Blood Research Fund.
In this study, we examined DWJM as a 3D ECM-based model to
learn about leukemia cell behavior. Our findings support the hypothesis
that an in vitro matrix modeling the in vivo ECM maintains leukemia cell
quiescence and LSC traits. In support of this interpretation, we found
that leukemia cells maintained in DWJM showed an increase in ALDH+
population in both Kasumi 1 and MV411 cell lines. Since ALDH activity
has been reported to be increased in LSCs in bone marrow samples of
AML patients [14], our findings suggest that our ECM model system
favored the LSC-like phenotype. In addition, we used serial CFU assays
after both primary plating and secondary replating to demonstrate increased clonogenic ability of leukemia cells cultured in DWJM, which is
a feature of LSCs required for the long-term disease maintenance.
Chemoresistance is a major characteristic of cancer stem cells, including LSCs, and is also an significant obstacle to successful chemotherapy [15]. Prior work has demonstrated that different components in the microenvironment play an important role in inducing
cancer cell drug resistance [16]. For example, previous studies found
that different types of cancer cells demonstrated increased drug resistance when cultured in 3D collagen gels [17,18]. Others have shown
that lymphoma cell adhesion to fibronectin resulted in acquired resistance to mitoxantrone [19]. Hyaluronan is also associated with leukemia drug resistance [20]. Since collagen, fibronectin, and hylauronan
are present in DWJM, we partially attribute the drug resistance phenotype in DWJM-cultured leukemia cells to interaction of leukemia
cells with these components of DWJM. It is possible that this interaction
induces a drug resistant phenotype in leukemia cells cultured in DWJM
by decreasing the concentration of doxorubicin in leukemia cells, which
could be related to decreased uptake or increased efflux of doxorubicin.
In addition, these interactions may have resulted in enhanced N-cadherin expression by leukemia cells cultured in DWJM, a property also
associated with adhesion-induced chemoresistance [13. Finally, others
have shown that spindle-shaped cells in leukemia are associated with
chemoresistence [21]. Our observation that our model enriched for
spindle-shaped leukemia cells is consistent with this finding.
In our experimental design, we compared our ECM-based culture
system to traditional suspension culture conditions. However, other
collagen-based 3-dimensional culture systems are available. These include collagen gel [22] and Histoculture [23,24], in which 3D tissue
pieces are put in growth medium with collagen gel support or freely
floating without support. Because of its ability to maintain the original
tissue phenotype, Histoculture has been used in drug screening for
different types of cancer in clinical trials. One advantage to our system
compared to collagen-only based systems is that DWJM has other
components that are present in the bone marrowECM, including fibronectin, hyaluronic acid, and sulphated proteoglycan. In addition,
our DWJM-based model recapitulates some of the features of the LSC.
Since currently many drugs targeting LSCs are under development, our
DWJM-based model will potentially provide useful for screening of LSCtargeted therapeutics. Future studies will focus on examining DWJM vs
other commercially available collagen-based 3D culture systems.
In conclusion, our DWJM-based ECM model maintains leukemia
cells with stem cell-like characteristics. DWJM should be further examined as a platform for leukemia research.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.leukres.2018.08.012.
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Acknowledgments
We thank Constance D. Baldwin, PhD for reviewing the manuscript
for clarity. We thank Dr. Linheng Li for his advice in studying N-cadherin expression in our model. We also thank Dr. Fariba Behbod for her
help with ALDH-related studies and Dr. Eduardo Rosa-Molinar for his
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