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Blood First Edition Paper, prepublished online June 5, 2013; DOI 10.1182/blood-2012-06-437988
Connective tissue growth factor regulates adipocyte differentiation of mesenchymal
stromal cells and facilitates leukemia bone marrow engraftment
V. Lokesh Battula,1 Ye Chen,1 Maria da Graca Cabreira,1 Vivian Ruvolo1, Zhiqiang Wang,2
Wencai Ma,2 Sergej Konoplev3, Elizabeth Shpall,4 Karen Lyons,6 Dirk Strunk,7 Carlos BuesoRamos3, Richard Eric Davis,2 Marina Konopleva,1,5 and Michael Andreeff1,5#
1
Department of Leukemia, 2Department of Lymphoma and Myeloma, 3Department of
Hematopathology, 4Department of Stem Cell Transplantation, and 5Department of Stem Cell
Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center,
Houston, Texas.
6
Department of Molecular Cell and Developmental Biology, University of California, Los
Angeles, California.
7
Institute of Experimental and Clinical Cell Therapy, Spinal Cord and Tissue Regeneration
Center, Paracelsus Medical University, Salzburg, Austria.
Running title: CTGF regulates differentiation and engraftment
# Corresponding author: The University of Texas MD Anderson Cancer Center, 1515
Holcombe Blvd. Unit 448, Houston, Texas 77030, USA, Phone: 713-792-7261, Fax: 713-5637355, E-mail: [email protected]
1
Copyright © 2013 American Society of Hematology
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Key points:
•
Connective tissue growth factor regulates adipogenic differentiation of MSCs.
•
Connective tissue growth factor regulates leukemia engraftment.
Abstract
Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM)
microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but
it’s role in the BM stroma is unknown. Therefore, we knocked-down (KD) CTGF expression in
human BM-derived MSCs by CTGF-shRNA. CTGF-KD-MSCs exhibited 5-fold lower
proliferation compared to control MSCs and had significantly fewer cells in S phase (3.5% ±
0.4% vs 14.7% ± 0.8%). CTGF-KD-MSCs differentiated into adipocytes at a 6-fold higher rate
than controls, both in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia
cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in
NOD/SCID/IL-2rγnull mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at
a 3-fold higher rate in adipocyte-rich CTGF-KD-MSC−derived EXM-BM than in control EXMBM. Leptin (adipocyte growth factor) was found to be highly expressed in CTGF-KD−EXMBM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia while
it was not expressed in normal controls. Given the established role of leptin receptor in leukemia
cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of
leptin in homing and progression of leukemia.
2
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Introduction
The bone marrow (BM) microenvironment consists of a variety of cell types, including
osteoblasts, osteoclasts, endothelial cells, perivascular reticular cells, and mesenchymal stem or
stromal cells (MSCs), all of which are critical for the regulation of hematopoietic stem cell
(HSC) maintenance and localization(1, 2). In hematological malignancies, including leukemias,
BM provides supporting niches for leukemia cell survival, proliferation, and differentiation(3, 4).
Although the mechanisms of leukemia cell homing to BM are not fully understood, recent
evidence suggests that various cytokines and chemokines secreted by components of the tumor
microenvironment facilitate this process(4-6). MSCs contribute to the leukemia BM
microenvironment by attracting leukemia cells to their BM niche by producing factors such as
angiopoietin-1 and CXCL12 (SDF-1α), and attachment to stromal cells has been shown to
activate survival signals in leukemia cells(1, 3, 6).
Mesenchymal stem/stromal cells (MSCs) are multipotent cells with self-renewal capacity(7).
They express a panel of key markers, including CD105, CD73, CD44, and CD90, but not
CD45(7, 8). Although the true nature of MSCs remains enigmatic, CD146+ MSCs were recently
reported to be self-renewing progenitors that reside on the sinusoidal surfaces and contribute to
the organization of the sinusoidal wall structure(9). They can be isolated from various adult and
fetal tissues, including BM, adipose tissue, umbilical cord blood, liver, human term placenta, and
endometrium(10, 11). MSCs differentiate into 3 major mesodermal lineages: osteoblasts,
adipocytes, and chondrocytes(7, 12).
Connective tissue growth factor (CTGF, CCN2), a member of the CCN (CYR61, CTGF, NOV)
family of proteins, regulates extracellular matrix production, chemotaxis, cell proliferation and
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differentiation, and integrin expression(13, 14), but its role in the leukemia microenvironment
has not been defined. Ctgf knockout mice die soon after birth as a result of respiratory failure
caused by abnormal skeletal growth(15). CTGF expression is tightly regulated by TGF-β in
fibroblasts(16) and recent evidence suggests that recombinant CTGF induces differentiation of
MSCs into fibroblasts and thereby inhibits their differentiation into osteoblasts, adipocytes, and
chondrocytes (17). Treatment with recombinant CTGF inhibited adipocyte differentiation of
mouse stromal cell line 3T3-L1(18). Therefore, we studied the role of CTGF in differentiation of
BM-derived MSC and leukemia-stroma interactions.
Recent reports suggest that obesity could function as a negative factor in cancer progression and
patient survival(19, 20). We previously reported that leptin produced by adipocytes derived from
MSCs, counter-acts leukemia cell death induced by chemotherapeutic agents(21). Co-culture of
acute myeloid leukemia (AML) cells with MSC-derived adipocytes prevented apoptosis after
doxorubicin treatment by activating the STAT3 and MAPK signaling pathways(21). We also
demonstrated that AML cells express higher levels of leptin receptor (OB-R) and its isoforms
(long and short) than normal cells and that leptin expression is correlated with body mass index
of leukemia patients(22).
Here we report on the role of CTGF on MSC function, including gene expression, cell
proliferation and differentiation. We also use a newly developed humanized extramedullary BM
(EXM-BM) model(23) in mice to investigate differentiation of MSCs in vivo and engraftment of
leukemia cells into CTGF-modified EXM-BM. Finally, we investigated underlying mechanism
of leukemia cell engraftment in this model and identified CTGF as a gene that regulates MSC
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differentiation into adipocytes and enhances leukemia cell engraftment in adipocyte rich EXMBM by increased production of leptin.
Methods
Isolation and culture of primary murine and human MSCs and leukemia cells lines. Pups of Ctgf
knockout or wild-type mice were collected immediately after birth. Stroma-rich body tissues,
including liver, thymus, spleen, and BM, were surgically dissected and mechanically digested
into single cells by vigorous pipetting in alpha-MEM containing 20% FBS (Gibco BRL,
Rockville, MD), L-glutamine, and penicillin–streptomycin (Flow Laboratories, Rockville, MD).
CTGF knockdown by lentiviral transduction. Lentiviral constructs expressing CTGF-shRNA
(Cat #RHS3979-962913) or empty vector (Cat #RHS4080) or were purchased from Open
Biosystems (Lafayette, CO). GFP-shRNA construct was prepared as described before(24) and
used as non-specific shRNA control. Lentiviral infections were carried out according to the
standard procedures for silencing experiments(25).
Cell proliferation and cell cycle analysis. Cells were fixed in ice-cold ethanol (70% vol/vol) and
stained with propidium iodide (PI) solution (25 μg/ml PI, 180 U/ml RNase, 0.1% Triton X-100,
and 30 mg/ml polyethylene glycol in 4 mM citrate buffer, pH 7.8; Sigma Chemical Co.). The
DNA content was determined by LSR-II flow cytometry (Becton Dickinson Immunocytometry
Systems, San Jose, CA). Cell cycle distribution was analyzed by FlowJo software (Tree Star,
Inc., Ashland, OR).
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Flow cytometry. BM MSCs in which CTGF was stably knocked down or control cells were
stained with fluorochrome-conjugated antibodies as described previously(26).
Real-time RT-PCR. Total RNA was extracted using an RNeasy ion-exchange column (Qiagen,
Valencia, CA) with on-column DNAse treatment as recommended by the manufacturer. The
yield of purified RNA was determined by a spectrophotometer (NanoDrop 2000; Thermo
Scientific, Wilmington, DE). cDNA was prepared from 1.0 μg of total RNA as described
elsewhere(25).
Gene expression profiling. Total RNA was extracted from 1 x 106 control or CTGF-KD-MSCs
using the RNAqueous kit (Ambion). After confirmation of RNA quality using a Bioanalyzer
2100 instrument (Agilent), 300 ng of total RNA was amplified and biotin-labeled through an
Eberwine procedure using an Illumina TotalPrep RNA Amplification kit (Ambion) and
hybridized to Illumina HT12 version 4 human whole-genome arrays (Illumina, San Diego, CA).
Processing of bead-level data was by methods previously described(27).
Multi-lineage differentiation. To identify osteoblast, adipocyte and chondrocyte differentiation,
MSCs expressing the empty vector or CTGF-shRNA were cultured in NH OsteoDiff- or NH
AdipoDiff- or NH ChondroDiffmedium (Miltenyi Biotec, Auburn, CA) for 21, 28, 21 days
respectively as described previosuly(26).
In vivo extramedullary bone formation. Extramedullary bone in mice was generated as
described(23). Briefly, human BM-derived MSCs (1.5×106) were mixed with the same number
of human endothelial colony-forming cells (ECFCs) in 0.2 ml Matrigel (Millipore) and then
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immediately injected subcutaneously into the flanks of NOD/SCID/IL-2rγnull mice. Both the
MSCs and ECFCs were obtained has the large-scale expansion method described by us, with low
passages (1 to 3)(28). Eight weeks after transplantation, bone formation was visualized by
injecting the mice with OsteoSense 750. OsteoSense binding to bone was visualized using the
Xenogen IVIS bioluminescence/fluorescence optical imaging system (Caliper Life Sciences,
Hopkinton, MA).
Generation of the acute myeloid/lymphoid leukemia model. MOLM13 and Nalm6 cells were
each infected with lentivirus expressing firefly luciferase and yellow fluorescent protein (YFP)
and maintained in RPMI-1640 medium containing 10% FBS. Mice with extramedullary bones
were injected intravenously with 2×106 labeled Molm13 or Nalm6 cells suspended in100 µl of
PBS. Bioluminescence imaging was employed to monitor the tumor burden(26).
Animal study approval. NOD/SCID/IL-2rγnull mice were purchased from the Jackson Laboratory.
All animal work was done in accordance with a protocol approved by the institutional animal
care and use committee at The University of Texas MD Anderson Cancer Center.
Statistical analysis. Results are shown as the mean plus or minus the SEM from 5 independent
experiments. The Student paired t-test was used for statistical comparisons between groups. P
values less than 0.05 were considered statistically significant. All experiments were conducted at
least in triplicates.
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Results
CTGF regulates MSC development and proliferation.
MSCs are a key component of the leukemia microenvironment, supporting leukemia cell
survival by cell-to-cell contact and via paracrine mechanism. Among several growth factors
secreted by MSCs, CTGF plays an important role in the regulation of MSC function. mRNA
expression analysis of CTGF and its family of proteins in revealed that MSCs express high levels
of CTGF and its family members including Cyr61, Nov, Wisp1, Wisp2 and LRP1 compared to
leukemia cells (supplementary Figure 1). Recent evidence suggests that recombinant CTGF
induces differentiation of MSCs into fibroblasts and thereby inhibits their characteristic
differentiation into osteoblasts, adipocytes, and chondrocytes(17). Here we hypothesized that
genetic knockdown of CTGF, which regulates MSC proliferation and differentiation, would
modulate leukemic cell homing to BM.
To test this hypothesis, we used the ctgf knockout mouse model developed by Ivkovic et al.(15).
These homozygous knockout mice die soon after birth. To generate MSCs, organs enriched for
MSCs were dissected from both wild-type and homozygous knockout mice, and MSCs were
isolated from these tissues. Suspensions of these MSCs were cultured in cell culture dishes. The
Trypan blue dye exclusion method showed a >90% cell survival rate for all tissue types studied
(data not shown). Interestingly, we were able to generate CFU-F, which represent MSC colonies,
only from tissues from wild-type animals, not from ctgf knockout mice (Figure 1A). To test
whether the lack of ctgf-knockout MSC colonies was due to their inability to adhere to plastic,
the cell culture dishes were coated with 0.1% gelatin before plating the cell suspensions isolated
from ctgf knockout pup tissues. Even after 2 weeks of culture, no colonies developed from ctgf
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knockout mouse tissue suspensions (data not shown), indicating a major disruption of MSC
development in these mice.
As an alternative source of ctgf knock-out MSCs, we stably knocked down CTGF in human
MSCs by transducing the cells with a lentivirus expressing CTGF-shRNA. mRNA expression
analysis of CTGF revealed a reduction by 70% ± 5% compared to cells transduced with empty
vector (Figure 1B). Protein expression analysis indicated that CTGF expression was downregulated by more than 90% in cells transduced with CTGF-shRNA, suggesting that CTGF
expression was successfully inhibited in these cells (Figures 1C). Although no major changes in
cell morphology, adhesion or spontaneous cell death were observed between control and CTGF
knockdown MSCs (Figure 1D), we did observe that CTGF-KD-MSCs growth was inhibited 5fold compared to control MSCs, suggesting that CTGF has a major role in cell proliferation
(Figure 1E).
CTGF regulates cell cycle in MSCs.
Because of the major decrease in cell growth in CTGF-KD-MSCs, we analyzed cell cycle
progression using Propidium Idodide (PI) staining. CTGF-KD-MSCs displayed a significant
decrease in the number of cells in S phase (from 14.7% ± 0.8% to 3.5% ± 0.4%) and a
concomitant increase in the number of G0/1 cells (from 68.8% ± 1.8% to 82.4% ± 1.3%), (Figure
2A). To investigate whether CTGF also affects expression of MSC surface proteins, we analyzed
standard MSC markers, including CD105, CD90, CD73, CD44, CD140b, CD166, and CD45 (as
a negative marker). Surprisingly, expression of these markers in CTGF-KD-MSCs and control
MSCs did not differ significantly (supplementary Figure 2).
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Cell cycle–related genes are down-regulated in CTGF-KD-MSCs.
To investigate gene expression, we employed microarray analysis using Illumina arrays (GEO
accession number: GSE47575). Gene set enrichment analysis (GSEA) suggested that genes
involved in cell cycle progression (supplementary Figure 3A), especially genes related to the M
phase of the cell cycle (supplementary Figure 3B), were down-regulated in CTGF-KD-MSCs
compared to control MSCs. Differentially expressed probes (DEPs) were determined using a t
test and an FDR q statistic < 0.1, with no fold-change threshold. We imposed an arbitrary fold
change (FC) threshold of 2, in which case 302/383 DEPs were higher and 261/272 DEPs were
lower in CTGF-KD MSC (independent experiments 1and 2, respectively). The top 20 Gene
Ontology (GO) biological process (BP) categories most significantly enriched by the
hypergeometric distribution test among genes down-regulated in CTGF-KD-MSCs are listed in
supplementary Table 1. Cell proliferation–related genes such as CDC2, CDC20, HMGA2, and
PRMT were down-regulated in CTGF-KD-MSCs compared to control MSCs (n=2,
supplementary Figure 3C), suggesting that CTGF-mediated signaling regulates the expression of
a multitude of cell cycle–related genes (supplementary Table 2 lists cell proliferation related
genes down-regulated in CTGF-KD-MSCs).
CTGF-KD-MSCs are primed to differentiate into adipocytes.
MSCs are known to differentiate into 3 mesodermal lineages: osteoblasts, adipocytes, and
chondrocytes. To test the differentiation potential of CTGF-KD-MSCs, cells were cultured in 3
different media designed to promote differentiation to osteoblasts, adipocytes, or chondrocytes.
Staining by alkaline phosphatase and Alizarin-Red-S revealed that both control and CTGF-KDMSCs displayed equal potential to differentiate into osteoblasts (supplementary Figure 4A).
Similarly, Alcian Blue staining revealed that controls and CTGF-KD-MSCs displayed equal
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potential to differentiate into chondrocytes (supplementary Figure 4B). Interestingly, however,
CTGF-KD-MSCs differentiated into mature adipocytes at a 6-fold higher rate than control cells,
as revealed by Oil Red O staining, indicating that knockdown of CTGF primed MSCs to undergo
adipocyte differentiation (Figures 3A and B; supplementary Figure 5).
CTGF-KD-MSCs differentiate into adipocytes in humanized extramedullary BM model in vivo.
To test the differentiation potential of CTGF-KD-MSCs in vivo, we used the extramedullary BM
(EXM-BM) model developed by our group(29). This model is represented schematically in
Figure 5A. Briefly, MSC transduced with lentivirus expressing CTGF-shRNA or non-specific
shRNA (GFP) or empty vector (EV) together with EPCs and Matrigel were transplanted
subcutaneously into NOD/SCID/IL-2rγnull mice to generate extramedullary bone (Figure 3C).
Eight weeks later, H&E staining of EXM-BM sections revealed more cortical bone formation in
control (EV and non-specific) EXM-BM, whereas CTGF-KD-MSCs generated a completely
different morphology comprised of less cortical bone and more adipose-like tissue (Figure 3D).
As there was no significant difference between EV and non-specific shRNA transduction
controls (Figure 3D), we chose to use EV as control for the following experiments. To analyze
the bone differentiation, the mice were injected with OsteoSense, which binds to newly formed
bone and can be detected by fluorescence imaging. As expected, EXM-BM derived from control
MSCs displayed bright fluorescence, indicating new bone formation. In contrast, EXM-BM
derived from CTGF-KD-MSC showed only weak fluorescence (supplementary figure 6),
suggesting that these cells differentiate poorly into the osteoblast lineage in vivo. This
morphology of CTGF-KD-MSC derived bones resembles the reported bone morphology of
CTGF deficient mice(15).
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To confirm morphological evidence of adipocyte differentiation of these cells, the sections were
immunostained with adipocyte differentiation markers, including PPARγ and cEBPα. We
observed strong positive staining for these markers in the nuclear region of large cells,
suggesting differentiation of CTGF-KD-MSCs into adipocytes in vivo (n=4, Figure 3E). In
contrast, PPARγ and cEBPα expression was significantly down-regulated in control cells (Figure
3E). No positive staining was observed in the stromal/endothelial compartment of CTGF-KDMSC–derived EXM-BM or control EXM-BM. These findings suggest that inhibition of CTGF
expression in MSCs is sufficient to induce adipocyte differentiation in vivo.
Leukemia cells specifically engraft into CTGF-KD-MSC–derived EXM-BM.
MSCs secrete several factors, including stroma-derived factor1-α (SDF-1α), which promotes
leukemia cell homing to BM. Therefore, we tested engraftment of leukemia cells into EXM-BM
derived from control MSCs or CTGF-KD-MSCs. Molm13 AML cells (Figure 4A-C) and Nalm6
ALL cells (Figure 4D-F) stably expressing the firefly luciferase gene were transplanted into
NOD/SCID/IL-2rγnull mice harboring EXM-BM derived from control-MSCs (left side) or CTGFKD-MSCs (right side, n=4). Two weeks later, the mice were imaged for leukemia engraftment in
EXM-BM. Although there was identical engraftment in the sites of murine BM (spine), there
were significantly higher luminescence signals in CTGF-KD–EXM-BM than in control EXMBM, indicating preferential leukemia engraftment (Figure 4A,B and 4D,E) in CTGF-KD bones.
To confirm the engraftment of leukemia cells, sections derived from EXM-BM were stained with
antibody against firefly luciferase and showed significantly higher numbers of luciferase
expressing cells in CTGF-KD–EXM-BM than in controls (n=4, Figure 4C and 4F p<0.001).
These findings suggested that CTGF-KD–EXM-BM favors leukemia cell engraftment.
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Higher levels of SDF1α and adipocyte growth factor leptin may favor leukemia cell engraftment
in CTGF-KD-MSC–derived EXM-BM.
To understand the mechanisms behind leukemia homing, CTGF-KD- and contol-MSCs were
tested for mRNA expression of SDF1α and leptin before and after adipocyte differentiation.
Interestingly, knock-down of CTGF induced SDF1α mRNA expresion by ~2fold in MSCs before
and after adipocyte differentiation compared to control MSCs (Fig 5A and B). In addition, leptin
mRNA expression was more than 100-fold higher after adipocyte differentiation of CTGF-KD
MSCs compared to undifferentiated cells (Fig 5B). Immuno-histochemical analysis of
extramedullary BM niches revealed that expression of leptin was significantly higher in CTGFKD-MSC–derived EXM-BM than in control EXM-BM (Figure 5C). These findings suggest that
knockdown of CTGF in MSCs facilitated adipocyte differentiation in vivo, resulting in increased
expression of leptin growth factor (Figure 5D). Consequently, leukemia cells which express
CXCR4 and leptin receptor (OB-R) engrafted into BM niches rich in SDF1 and leptin (Figure
5D).
Human AML and ALL bone marrow cells express significantly higher leptin levels compared to
normal bone marrows.
To investigate leptin expression in human bone marrows, we examined leptin expression by
immuno-histochemistry. The selected cases included 3 normal bone marrow specimens (these
samples were obtained from adult patients with breast cancer as a part of staging procedure and
did not show any metastatic involvement), 5 adult patients with AML, and 5 adult patients with
B-ALL. Leptin expression was detected in all AML and all ALL cases. Leptin was strongly
expressed in all AML cases and in 4 of 5 ALL cases; one ALL case showed a moderate
expression of Leptin (Fig 6). In contrast, all three normal BM were negative for leptin showing
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only moderate leptin expression in about 5-10 % of all hematopoietic cells (Fig 6). These
findings indicate that leptin is highly expressed in human AML and ALL BMs and may facilitate
expansion of the leukemia clone.
Recent findings suggest that acute leukemia cells express higher CTGF levels compared to their
normal counterparts (30). Therefore, in order to test the paracrine effect of CTGF, MSCs derived
from bone marrows of leukemia patients and normal donors were compared for their ability to
differentiate into adipocytes. Leukemia BM-derived MSCs generated 15-18 fold fewer
adipocytes compared to normal BM-derived MSCs (supplementary Figure 7), once again
indicating the regulatory role of CTGF in MSC differentiation into adipocytes.
Discussion
In this report, we show that CTGF knockout mice were not able to generate MSCs in vitro and
that knockdown of CTGF by stable shRNA expression inhibited MSC proliferation. The CTGFKD-MSC cell cycle was inhibited at the G0/G1 phase. Knockdown of CTGF induced adipocyte
differentiation in vitro and in vivo. In the humanized extramedullary BM model, CTGF-KDMSCs generated less cortical bone but more adipose-like tissue than controls. Positive staining
for adipocyte-specific markers including, PPARγ and C/EBPα, indicated adipocyte
differentiation of CTGF-KD-MSCs in EXM-BM. When transplanted, luciferase-labeled
leukemia cells engrafted preferentially into CTGF-KD-MSC–derived EXM-BM. Analysis of the
mechanisms behind this engraftment revealed higher leptin expression in CTGF-KD-MSCs than
in control MSCs.
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CTGF (CCN2) belongs to the CCN family of proteins and is involved in embryonic development
and extracellular matrix production. CTGF knockout mice die soon after birth of respiratory
failure due to abnormal skeletal growth. In our attempt to isolate MSCs from CTGF knockout
mice, none of the stroma-rich organs, including liver, spleen, thymus, or BM, gave rise to MSCs
in our in vitro cultures. In addition, gene expression data indicate that genes involved in cell
proliferation are down-regulated in CTGF-KD-MSC, suggesting that CTGF is an important
factor during MSC development. Other members of the CCN family, including cysteine-rich
protein 61 (CyR 61, CCN1) and neuroblastoma-overexpressing protein (Nov, CCN3), were also
highly expressed in the MSC compartment (supplementary Figure 1), suggesting a role in MSC
biology. CCN3 expression was shown to regulate differentiation of early myeloid cells into more
committed progenitors, whereby CCN3 levels increase with myeloid cell commitment and loss
of “stemness”(31). In MSCs, CCN3 promotes differentiation into chondrocytes(32), suggesting
that CCN family members regulate differentiation of stem or progenitor cells.
The expression of CTGF is regulated mainly by TGF-β(33). Recent reports suggest that overexpression of constitutively active TGF-β receptor-1 induces CTGF expression in fibroblasts and
postnatally recapitulates major clinical, biochemical, and histologic features of fibrotic pathology
in the skin and small blood vessels(33). Selective over-expression of CTGF in fibroblasts
promotes systemic fibrosis in vivo, suggesting that both TGF-β and CTGF may have important
roles in a sustained chronic fibrotic outcome(34). CTGF expression is also known to be elevated
in certain types of cancers. In colon cancer, high CTGF expression confers poor prognosis and is
associated with inferior survival(35). Moreover, over-expression of CTGF was observed in Bcell acute lymphocytic leukemia (ALL) and correlated with poor prognosis and survival(30, 36).
AML bone marrow derived MSCs demonstrate reduced ability to differentiate into adipocytes.
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This may be due to CTGF over-expression in leukemia cells. Therefore, CTGF may act on
MSCs in a paracrine fashion and inhibit their ability to differentiate into adipocytes. These
reports suggest that autocrine CTGF production induces cancer growth and that targeting this
growth factor might be useful in therapy of several cancer types. In most cancer types, including
solid tumors and leukemias, impaired differentiation of the cancer cell is a key defect. Our data
suggest that knock-down of CTGF in MSCs not only inhibits cell proliferation but also induces
differentiation in vivo. These findings suggest that inhibition of CTGF could decrease cancer cell
proliferation and induce differentiation.
Our finding of high CTGF production by BM-MSC prompted us to investigate the role of
paracrine, MSC-secreted CTGF, in leukemia homing and survival. Surprisingly, knock down of
CTGF in MSC resulted in consistently higher leukemia cell engraftment in our humanized
extramedullary bone model. The composition of this extramedullary environment was strikingly
different in that it displayed features of fat-abundant bone marrow. We have further confirmed
that CTGF knockdown results in adipogenic differentiation of MSCs in these marrows. Adipose
tissue content and factors secreted by adipocytes including leptin and SDF1α may result in
higher leukemia engraftment into CTGF-KD-EXM-BM compared to control EXM-BM. The fact
that leukemia frequency is increased in older individuals(37) and adipose content of BM
increases with age(38, 39), suggests that factors associated with adipocytes including leptin and
SDF1α could play an important role in leukemogenesis and actively promote leukemia
progression. Leptin over-expression in AML and ALL bone marrows also supports the notion of
increased adipose tissue content in leukemic bone marrows. We have previously shown that
leptin receptor (OB-R) is over-expressed in leukemia cells which supports engraftment of
leukemia cells to adipocyte rich bone marrows. Although not tested in this study, increased
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adipocyte content of the BM and age are likely associated with overall obesity (19, 20). In a
recent report, rate of relapse after monotherapy with vincristine was higher in obese mice than in
mice of normal weight injected with syngeneic ALL cells (40). Co-culture of the leukemia cells
with 3T3-L1 adipocytes before injection, significantly impaired the anti-leukemia efficacy of
vincristine, and of 3 other chemotherapeutic agents (40). Interestingly, this protection was
independent of cell-cell contact, and it extended to human leukemia cell lines as well. In another
report, diet-induced obesity accelerated ALL progression in two murine models (41). Obesity is
known to associate with increased risk for numerous types of cancers in adults (42). Moreover,
obese cancer patients have poorer outcomes than their leaner counterparts (43).
It has been demonstrated that growth factors, including leptin, insulin, and interleukin-6, are
highly expressed in the serum of obese compared to lean mice(41). In another report, leptin was
shown to revert the proapoptotic and anti-proliferative effects of α-linoleic acids in BCR-ABL–
positive leukemic cells, suggesting a role for the PI3K pathway in this process(44). In this report,
we demonstrated leptin over-expression in AML and ALL bone marrows. We have previously
shown that the adipocyte growth factor leptin is essential for leukemia growth(21, 22) and that
blocking the leptin receptor could help prevent leukemia growth(21). Our findings suggest that
CTGF regulates MSC differentiation into adipocytes which in turn produce leptin in bone
marrows and promote leukemia cell engraftment and growth within BM niches. Therefore, leptin
could be one of the key mediators of leukemia progression within adipocyte-enriched bone
marrow microenvironment and targeted therapy against leptin may interfere with leukemia
progression.
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Taken together, findings reported here suggest a previously unrecognized role of bone marrow
adipogenesis in leukemia progression. Our data indicate that CTGF is a key negative regulator
of adipocytic differentiation of BM-derived MSC. Hence, targeting CTGF in hematologic
malignancies should be considered with caution and possibly be directed at diseases with high
pathologic fibrotic stromal component, such as myeloproliferative disorders. Furthermore, future
strategies targeting leptin-producing adipocytes may prove beneficial in generating a bone
marrow micro-environment inhospitable for leukemic cells. Although adipocytes are not readily
detectable at the stage of full leukemia-infiltrating marrows, they may play an important role in
the setting of aplastic anemias or hypoplastic myelodysplastic syndromes, nurturing preleukemic malignant clones and hence facilitating leukemia development. Future studies aimed at
characterization of adipocyte component of pre-leukemic bone marrow microenvironment are
warranted.
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Acknowledgments
We thank Dr. Juliana Benito, Dr. Peter Ruvolo, and Dr. Rodrigo Jacamo for their invaluable help
and discussion, and Teresa McQueen for her technical assistance. We also thank Dr. Shonali
Majumdar, Kathryn Hale, and Dr. Numsen Hail, Jr. for their critical review of the manuscript.
This work was supported in part by the NIH/NCI grants CA044164, CA016672, CA100632,
R01 FD003733, R21 CA143805, CA049639, and CA153019 (to M.A.).
Authorship
Contribution: V.L.B. performed experiments, analyzed data, wrote the paper, and conceived the
study; Y.C., M.G.C., Z.W., V.R., S.K. and W.M. performed experiments; E.S., K.L. and D.S.
designed research and provided reagents; C.B.R. and R.E.D. designed research, performed
experiments and analyzed data; M.K. designed research, analyzed data, wrote the paper and
conceived the study; and M.A. designed research, analyzed data, wrote the paper, conceived the
study and provided final approval of the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
19
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Figure legends
Figure 1. Knockdown of CTGF expression inhibits proliferation of MSCs isolated from
BM. (A) CTGF knockout or control pups were euthanized and their stroma-rich body organs,
including liver, thymus, spleen and BM, were surgically dissected. Cell suspensions of these
organs were cultured in alpha-MEM containing 20% FBS Fibroblast-like CFUs were observed 710 days after plating in controls but not in CTGF knockout cells. (B) Normal MSCs derived from
human BM were transduced with a lentivirus expressing CTGF-shRNA. Data represent relative
expression of CTGF to GAPDH in control and CTGF-KD-MSCs (C) CTGF protein expression
was analyzed in cell lysates from control and CTGF-KD-MSCs by western blotting with antiCTGF antibody. α-tubulin served as a loading control. (D) Morphology of control- and CTGFKD-MSCs. (E) Cell proliferation was analyzed by counting absolute cell numbers with a Vi-Cell
XR cell counter. Control or CTGF-KD-MSCs (2×104) were cultured in 6-well dishes in triplicate
and counted on days 2, 3, 4, and 5.
Figure 2. Knockdown of CTGF inhibits cell cycle of MSCs (A) Control or CTGF-KD-MSCs
(1×106) were stained with propidium iodide. The cells were analyzed on an LSR-II flow
cytometer. There is an accumulation of cell cycle in G0/G1 phase was observed in CTGF-KDMSCs. The data were analyzed on FlowJo software.
Figure 3. CTGF-KD-MSCs differentiate into adipocytes in-vitro and in-vivo. (A) To
examine the differentiation potential of control or CTGF-KD-MSCs, cells (5×104) were cultured
in adipocyte differentiation medium for 28 days. After incubation, the cells were stained by Oli
Red O dye or LipidTox fluorescent dye to observe adipocyte differentiation. (B) Quantitative
representation of data showed in supplementary Figure 4. (C) To examine the differentiation
27
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potential of CTGF-KD-MSCs in vivo, a model of extramedullary bone marrow (EXM-BM) was
developed by injecting human MSCs (1.5×106) mixed with human endothelial progenitor
cells(1.5×106) in 0.2 ml Matrigel subcutaneously into the flanks of NOD/SCID/IL-2rγnull mice.
Control cells (Empty vector and non-specific shRNA controls) were transplanted on the left and
knockdown CTGF-KD-MSCs were transplanted on the right. (D) Eight weeks after
transplantation, the bone pellets were dissected and fixed in 4% PFA. Tissue sections were then
stained with H&E to observe tissue architecture. Scale bar represents 200μm. (E) To investigate
adipocyte differentiation, immunohistochemical (IHC) analysis was performed on EXM-BM
using a PPARγ or C/EBPα antibody.
Figure 4. Leukemia cells specifically engraft into EXM-BM derived from CTGF-KDMSCs. To investigate leukemia engraftment in EXM-BM derived from control (left) or CTGFKD-MSCs (right), the corresponding cells, in combination with EPC and Matrigel, were
transplanted subcutaneously into NOD/SCID/IL-2rγnull mice. Eight weeks later, Molm13 (A-C)
or Nalm6 (D-F)cells (2×106) stably expressing firefly (FF) luciferase were transplanted
intravenously into the mice harboring EXM-BM. Two weeks after transplantation, the mice were
imaged via the IVIS bioluminescence imager after injection of the luciferase substrate (A and D).
The signal intensities were measured by the IVIS live imaging software package. (B and E). As
an alternative, bone pellets were dissected and fixed in 4% PFA, and the tissue sections were
stained for immunohistochemical analysis with the anti-firefly luciferase antibody. The brown
color indicates positive luciferase staining (C and F).
28
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Figure 5. Adipocyte growth factor leptin may be involved in leukemia cell engraftment in
EXM-BM derived from CTGF-KD-MSCs. (A-B) mRNA was isolated from control and
CTGF-KD-MSCs before and after differentiation into adipocytes, and expression of SDF1α (A)
and leptin (B) was analyzed by qRT-PCR. (C) Immunohistochemical analysis was performed on
EXM-BM generated by control or CTGF-KD-MSCs. The sections were stained with anti-leptin
antibody and later developed with DAB. (C) Schematic representation of a possible mechanism
behind leukemia engraftment into CTGF-KD–EXM-BM.
Figure 6. Leptin is highly expressed in AML and ALL bone marrows. To analyze leptin
expression on leukemia bone marrows, paraffin-embedded bone marrow biopsy specimens were
formalin-fixed and formic acid-decalcified. Leptin expression was assessed using a rabbit-antihuman Leptin polyclonal antibody (Cone Ab16227) by immunohistochemistry.
29
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Prepublished online June 5, 2013;
doi:10.1182/blood-2012-06-437988
Connective tissue growth factor regulates adipocyte differentiation of
mesenchymal stromal cells and facilitates leukemia bone marrow
engraftment
V. Lokesh Battula, Ye Chen, Maria da Graca Cabreira, Vivian Ruvolo, Zhiqiang Wang, Wencai Ma, Sergej
Konoplev, Elizabeth Shpall, Karen Lyons, Dirk Strunk, Carlos Bueso-Ramos, Richard Eric Davis, Marina
Konopleva and Michael Andreeff
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