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Engraftment of Acute Myeloid Leukemia in NOD/SCID Mice Is Independent of CXCR4 and Predicts Poor Patient Survival

^a Department of Human Genetics, Baylor College of Medicine, Houston, Texas, USA;
^b Department of Blood and Marrow Transplantation,
^c Department of Biostatistics, and
^d Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

Key Words. Acute myeloid leukemia • CXCR4 • NOD/SCID • Engraftment

Michael Andreeff, M.D., Ph.D., Department of Blood and Marrow Transplantation, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 448, Houston, Texas 77030, USA. Telephone: 713-792-7260; Fax: 713-794-4747, e-mail: mandreef{at}mdanderson.org

	ABSTRACT

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The aim of this study was to investigate factors influencing the engraftment potential of acute myeloid leukemia (AML) CD34⁺ cells in nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mice. We examined the relationship between engraftment, CXCR4 expression on CD34⁺ and CD34⁺CD38^- cells, and patient (Pt) clinical/laboratory characteristics in 44 samples from 11 Pts. Engraftment, evaluated by Southern blot and CD45 flow cytometric analyses, was observed in murine bone marrow of 6 of 11 Pt samples, ranging from 0.1% to 73.9% by Southern blot and from 0.1%-36.8% by flow cytometry. Poor Pt prognosis was inversely correlated with engraftment; the median overall survival was 95.9 weeks for Pts whose cells did not engraft and 26.1 weeks for those whose cells did engraft (p = 0.012, log-rank test). No other clinical/laboratory variable predicted engraftment. No correlation between the level of CXCR4 expression on AML cells and engraftment was observed. Cells with virtually absent CXCR4 expression were able to engraft, and cells from two Pts with high expression levels of CXCR4 did not engraft. Furthermore, anti-CXCR4 antibody failed to block the engraftment of AML cells into NOD/SCID mice. In conclusion, we demonstrated that CXCR4 is not critical for the engraftment of AML CD34⁺ cells in NOD/SCID mice. The model may, however, reflect the clinical course of the disease.

	INTRODUCTION

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Immunodeficient mice have been found to be suitable hosts for the evaluation of both normal and leukemic human hematopoietic cells (HHCs) in vivo. At present, the ability of HHCs to repopulate the murine bone marrow (BM) is considered the most conclusive way to identify hematopoietic stem cells [1–3]. Among different strains, nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice, characterized by a functional deficit in natural killer cells, absence of circulating complement, and defective antigen-presenting cells, are considered to be the most reliable in terms of sensitivity and consistency of the level of human engraftment achieved [4, 5]. A closer review of a substantial number of observations accumulated either with primary normal or leukemic CD34⁺ cells or with cell lines revealed the complexity of the phenomenon, leading to new studies that aim to identify steps and factors that might influence engraftment [6–10].

Several adhesion interactions are involved in the homing and engraftment of hematopoietic stem cells. Chemokines are strong candidate regulators of human stem cell chemotaxis and have been shown to influence the migration of human progenitor cells. Recently, the chemokine stromal-cell-derived factor 1 (SDF-1), as well as its receptor, CXCR4 (fusin, LESTR), have been under investigation. The importance of their roles in stem cell homing and trafficking, particularly in the BM, was suggested by the selective reduction in BM hematopoiesis observed both in SDF-1-deficient and CXCR4-deficient mice [11, 12]. SDF-1, a member of the CXC subfamily of chemokines, was first identified as a growth factor for B-cell progenitors and as a chemotactic factor for T cells and monocytes [13, 14]. This latter effect is mediated by the receptor CXCR4. CXCR4 is a G-protein family member, structurally similar to the interleukin-8 receptor, that is expressed on mononuclear leukocytes and has been implicated as a coreceptor for the entry of the human immunodeficiency virus-1 into CD4⁺ cells [15, 16].

The expression of CXCR4 on CD34⁺ hematopoietic progenitors in normal and leukemic cells has been studied by a number of laboratories. Mohle et al. [17] reported that CXCR4 was expressed at detectable levels on circulating CD34⁺ hematopoietic progenitor cells, including more primitive subsets (CD34⁺CD38^- and CD34⁺Thy-1⁺ cells). The receptor was demonstrated to be functionally active by the positive correlation between its cell-surface density and SDF-1-induced transendothelial migration. Furthermore, the observation of selective activation of SDF-1 as a result of preferential, differentiation-related expression of CXCR4 has been confirmed in different acute myeloid leukemia (AML) subtypes [18].

AML occurs as a result of genetic changes in a primitive hematopoietic cell resulting in uncontrolled growth, with egress of leukemic cells into the peripheral blood and infrequent infiltration of other tissues. One might speculate that the infiltrative, metastatic ability of leukemic cells depends on SDF-1/CXCR4 interactions. This hypothesis is also supported by the recent observation of greater CXCR4 expression and migratory response in BM-derived AML blast cells when compared with circulating cells [19]. It is notable, however, that cases of BM infiltration in AML subtypes with low or absent expression of CXCR4 have also been reported [18].

The exact role of SDF-1 and CXCR4 in the homing and engraftment of CD34⁺ cells in NOD/SCID mice remains to be clarified. Peled et al. [2] have recently shown that human umbilical cord blood, adult mobilized peripheral blood (PB), and BM CD34⁺ cell engraftment of NOD/SCID mice was dependent on the expression of SDF-1/CXCR4. Homing of enriched human CD34⁺ cells in NOD/SCID mice, as well as in NOD/SCID/ß₂ microglobulin (ß₂M) null mice, was inhibited by pretreatment with anti-CXCR4 antibodies. On the basis of these observations, the authors recharacterized the SCID repopulating cells (SRCs) with major stem cell properties as CD34⁺CD38^-/low CXCR4⁺. However, two subsets of cells with equivalent engraftment abilities in NOD/SCID mice have recently been described as either CD34⁺CD38^- Lin^-CXR4⁺ or CD34⁺CD38^-Lin^-CXCR4^- cells, suggesting that cells that demonstrate the potential to repopulate murine BM may be heterogeneous with respect to the expression of CXCR4 [20]. Therefore, CXCR4 expression may not be indicative of a human stem cell phenotype or of reconstituting capability. Since the NOD/SCID transplant model represents the best model for the evaluation of the trafficking capabilities of the malignant leukemic cells, the analysis of expression and function of the SDF-1 receptor in this leukemia model could be useful to further elucidate mechanisms involved in the dissemination of the disease. In the present study, we investigated the functional role of CXCR4 expression on AML CD34⁺ progenitor cells in engraftment of NOD/SCID mice. We also evaluated the correlation between engraftment in NOD/SCID mice and clinical characteristics of the AML samples, such as patient (Pt) prognosis, cytogenetics, white blood cell count (WBC), and French-American-British (FAB) leukemia classification. The results demonstrate a correlation between CXCR4 expression and engraftment but also confirm that efficient engraftment may take place even when cell surface CXCR4 expression is very low or absent. The latter result suggests a CXCR4-independent homing pathway. However, an intriguing correlation was found between engraftment in NOD/SCID mice and survival of the AML Pts from whom the cells were derived, suggesting that the NOD/SCID model reflects the basic biology of AML.

	MATERIALS AND METHODS

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AML and Progenitor Samples
AML samples from BM (n = 4) or PB apheresis (n = 7) were obtained through the M.D. Anderson Cancer Center and Baylor Affiliated Hospitals following informed consent, according to institutional policies. The clinical and laboratory characteristics of the AML Pts are shown in Table 1. For further analysis of cytogenetic data, cytogenetic abnormalities were grouped according to published criteria adopted by the Southwest Oncology Group [21]. The favorable risk category included Pt 1 with t(8;21), the intermediate risk category included Pts with normal karyotypes (Pts 2, 5, 6, 9), and the unfavorable risk category encompassed all other Pts (Pt 3, 4, 8, 10, 11). Pts received combinations of cytosine arabinoside (Ara-C) with fludarabine, idarubicin, or cyclophosphamide and topotecan [22] (Table 1). Treatment outcome was comparable for Pts on these regimens. Cord blood samples were obtained from discarded placentas by venipuncture of one of the placental veins after delivery.

Immunomagnetic Selection of CD34⁺ Cells
For in vivo experiments, only AML samples expressing CD34 were used. All samples were magnetically enriched for CD34⁺ leukemic cells. The MNCs were incubated with anti-CD34 paramagnetic particles (Miltenyi Biotech Inc.; Auburn, CA; http://www.miltenyibiotec.com) and separated using the magnetic-activated cell sorting system (MACS; Miltenyi Biotech) according to the manufacturer’s recommendations. Using this technique, the purity of the selected population is >90%, as demonstrated by flow cytometry following staining with monoclonal anti-CD34 (Anti-HPCA-2; Becton Dickinson; San Jose, CA; http://www.bd.com).

Transplantation of Cells into NOD/SCID Mice
The NOD/SCID mice were bred and maintained under specific pathogen-free conditions at the Barrier Facility of the Center for Comparative Medicine Baylor College of Medicine (Houston, TX). The animals were housed in microisolator cages in laminar flow cage racks. Four- to 6-week-old mice were sublethally irradiated with 250 cGy (approximately 100 cGy/minute) using a GammaCell40 137Cs source (Nordion; Ottawa, Canada; http://www.mds.nordion.com) immediately before intravenous injection via the lateral tail vein of CD34⁺ AML cells, as previously reported [23].

Flow Cytometry
For evaluation of CXCR4 expression, 1–2 x 10⁵ AML MNCs were incubated for 30 minutes at 4°C with phycoerythrin (PE)-conjugated monoclonal antibody (mAb) to CXCR4 (Clone 12G5; Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen). An isotype-identical mAb served as control. The cells were analyzed using a fluorescence-activated cell sorting (FACS)can flow cytometer (Becton Dickinson). Coexpression analysis of PE-labeled CXCR4, fluorescein isothiocyanate (FITC)-labeled CD34, and peridinin chlorophyll protein-labeled CD38 antibodies (Becton Dickinson) was used to selectively analyze CXCR4 expression on leukemic progenitor cells. The percentage of positive cells was calculated by subtracting the percentage of cells with a fluorescence intensity greater than the set marker using the isotype control (background) from the percentage of cells with a fluorescence intensity greater than the control intensity using the specific antibody. As controls, normal peripheral blood lymphocytes were brightly positive for CXCR4.

In order to assess leukemic cell engraftment in transplanted animals, two-color flow cytometry was performed on a FACScan. Before analysis, the recipient mouse BM erythrocytes were lysed with isotonic NH₄Cl (10^-4 mol/l EDTA, 10^-3 mol/l KHCO₃, 0.17 mol/l NH₄Cl in water, pH 7.3). Single-cell suspensions of recipient BM cells were washed with 1% fetal calf serum (FCS) in phosphate-buffered saline and preincubated for 20 minutes at 4°C in staining medium (0.01 M Hepes containing 0.15 M NaCl, 0.1% NaN₃, and 4% FCS) containing 5% human serum to block human Fc receptors. Cells were labeled with human-specific mAbs then washed and resuspended in 200 µl of staining medium. BM of nontransplanted animals stained with anti-human CD45 were used as negative controls. To identify human cells, we used anti-human CD45 PE, anti-human CD33-FITC, and CD34-FITC for dual color labeling (Becton Dickinson). Dead cells were excluded from the analysis by propidium iodide (Sigma; St. Louis, MO; http://www.sigmaaldrich.com) uptake. Samples were analyzed on a FACScan flow cytometer (10,000–20,000 events). Positive cells were defined as those exhibiting a level of fluorescence that exceeded 99% of that obtained with isotype-matched control antibodies.

Southern Blotting
Quantitative Southern blotting was also used to analyze human cell engraftment. High molecular-weight DNA was isolated from BM cells of the transplanted mice and digested (2.5 µg) with EcoR1. Serial dilutions of control human DNA mixed with mouse DNA were included in each experiment as a control. After blotting, the filter was probed with a ³²P-labeled plasmid probe corresponding to human chromosome 17 alpha-satellite sequences (p17H8). After high stringency washing, the total lane signal was compared with the controls to estimate the percent human DNA by visual comparison or by phosphorimager analysis [24]. The limit of sensitivity of this analysis technique for detection of human cells is 0.1%.

Fluorescence In Situ Hybridization Analysis
Fluorescence in situ hybridization (FISH) analysis was performed on CD45⁺ human cells FACS sorted from the mouse BM. Slides were fixed in methanol/acetic acid (1:1) for 30 minutes and air dried. FISH was performed according to Vysis protocol (Downers Grove, IL; http://www.vysis.com). Briefly, slides were denatured in 70% formamide/2x SSC at 72°C ± 2°C for 5 minutes, dehydrated in 70%, 90%, and 100% ethanol, and then probed for chromosome X (alpha-satellite DNA/DXZ1-SpectrumOrange; Vysis), which was denatured at 70°C for 5 minutes and placed on slides. After incubation at 37°C overnight, slides were washed in 0.4% 2x SSC/0.1% NP40 at 72°C for 1 minute and then counterstained with 4,6-diamidino-2-phenyl-indole (0.1 mg/ml). FISH signals were analyzed under a Nikon fluorescence microscope with a triple-band pass filter. The image was captured in an image analysis system.

Real-Rime Quantitative Polymerase Chain Reaction
RNA was isolated according to the single-step acid guanidinium thiocyanate-phenol-chloroform method. A reverse-transcription kit was used to synthesize cDNA according to manufacturer’s instructions (Boehringer-Mannheim; Indianapolis, IN). One microgram of the total RNA template was used per 10 µl of reverse transcriptase reaction. Duplicate samples of cDNA were used as templates in the polymerase chain reaction (PCR) by using the ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems; Foster City, CA; http://home.appliedbiosystems.com). In brief, the primers CXCR4-forward 5'-GCC GTGGCAAACTGGTACTT-3' and CXCR4-reverse 5'-TT GGCCTCTGACTGTTGGTG-3' amplified a 150-bp fragment from the CXCR4 cDNA that was detected by the FAM-labeled TaqMan probe CXCR4-p 5'-CTGGACCGC TACCTGGCCATCG-3'. ß₂-microglobulin primers were: forward 5'-AGCTGTGCTCGCGCTACTCT-3' and reverse 5'-TTGACTTTCCATTCTCTGCTGG-3'; fluorogenic TaqMan probe (FAM-labeled 5'-TCTTTCTGGCCTGG AGGGCATCC-3'). Amplification conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds. ß₂-microglobulin coamplified with CXCR4 was included as an internal control for normalization of the variable content of human cDNA in each sample. For each of the two reactions, the PCR cycle number that generated the first fluorescence signal above a threshold (the threshold cycle [C_T]) was determined. A comparative C_T method detected relative gene expression [25]. The following formula was used to calculate the relative amount of CXCR4 in the sample (X), normalized to the endogenous reference (ß₂M) and relative to the amount of CXCR4 in the calibrator sample (Y): 2^-CT, where C_T is the difference in threshold cycles for CXCR4 and ß₂M, and C_T for the sample X = C_T,X-C_T,Y. CXCR4 expression of normal BM CD34⁺ cells from one of the samples analyzed was designated as a calibrator (=1); using this reference, expression in CD34⁺ cells from other normal BM samples was 1.43 ± 0.36 (n = 6).

Migration Assay
All assays were performed in duplicate using 5-mm pore filters (Transwell, 24-well cell clusters; Costar; Boston, MA; http://www.corning.com). One hundred microliters of chemotaxis buffer (RPMI 1640, 0.5% bovine serum albumin) containing 2 x 10⁵ CD34⁺ AML cells were added to the upper chamber of a Transwell, and 0.6 ml of chemotaxis buffer alone or containing 125 ng/ml of SDF-1 (R&D Systems; Minneapolis, MN; http://www.rndsystems.com) was added to the lower chamber. Ten percent of the input population (2 x 10⁴) was also seeded in duplicate wells in 600 ml of serum-free media for the same period of time (but not subjected to any chemotaxis assay) to be used as a control for FACS counting. Chambers were incubated at 37°C at 5% CO₂ for 4 hours. Cells migrating into the lower chamber were counted using a FACScan, with appropriate gating, for 20 seconds with a high flow rate. Data were expressed as the percentage of input population or as a chemotactic index. For blocking experiments, CD34⁺ AML cells were treated with anti-CXCR4 antibody (12g5 mAb, 250 mg/ml; Pharmingen) or with control IgG2a antibody for 1 hour, washed twice, and transplanted into NOD/SCID mice. An aliquot was taken for the migration assay. Due to the detection limit of this assay, migration of >1% could be quantified reliably.

Statistical Methods
Logistic regression analysis was used to model the association between an assay (Southern blot or CD45 analysis) testing positive and the percentage of a particular cell phenotype (CD34⁺CXCR4⁺, CD34⁺CD38^-CXCR4⁺) in the AML Pts. The analysis was performed using each Pt as the experimental unit since we observed that different mice transplanted with identical aliquots of leukemic cells from each AML Pt responded in a consistent way regarding BM engraftment.

The analysis was done in a univariate fashion for each cell phenotype and assay. Statistical significance was determined if the p value from the test of significance for a cell dose was less than 0.05. Odds ratios (ORs) and their associated 95% confidence intervals (CIs) are provided to illustrate the association of engraftment with each phenotype.

The log-rank test with Kaplan-Meier analysis was used to analyze the relation between the presence of human engraftment in the NOD/SCID mice and overall survival, time to relapse, and progression-free survival of AML Pts. The Wilcoxon rank-sum test and the Fisher’s exact test were used for the analysis of other characteristics such as WBC, cytogenetics, and response to therapy. The Student’s t-test was used to compare the expression levels of CXCR4 in different cell populations.

	RESULTS

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Engraftment and Clinical Characteristics
To examine the in vivo role of CXCR4 in migration and repopulation of human progenitor cells, CD34⁺-enriched cells obtained from 11 AML Pts were intravenously injected (5–10 x 10⁶ cells/mouse) into 2–6 NOD/SCID mice/Pt samples, where the variation in the number of mice reflected differences in the available number of cells from each sample. Baseline expression of CD34 in primary leukemic blasts ranged from 14.9%-97.3% (median 74.45%, Table 1); among CD34⁺ cells, a subpopulation comprised early CD34⁺CD38^- cells (from 0.27%-87%, median 4.6%). Five weeks after transplantation, the mice were sacrificed, and the presence and level of human engraftment were assayed by Southern blot or CD45⁺ flow cytometric analysis. Engraftment of human leukemic cells was observed in the BM of NOD/SCID mice for 6 of the 11 Pt samples, as shown in Table 2 and Figure 1. The mice appeared healthy and showed no apparent clinical features of illness. The proportion of human cells ranged from 0.1%-73.9% by Southern blot analysis and from 0.1%-36.8% by flow cytometric analysis of CD45. The concordance of results obtained by both assays was 85.7% (36/42, p < 0.01, Fisher’s exact test). Mice transplanted with cells from a particular AML Pt behaved in a consistent way regarding the BM engraftment. Only mice transplanted with cells from Pt 6 showed discordant results when two different methods were used (low-level engraftment by Southern blot analysis, no engraftment by flow cytometry), perhaps due to higher sensitivity of Southern blot analysis or due to antigen masking. The immunophenotype of the leukemic cells engrafted in the murine BM was found to be similar to that observed in the primary leukemic cells used for transplantation (Fig. 2A). FISH analysis on CD45⁺ FACS-sorted cells from murine BM using centromeric probes demonstrated 38%-68% clonal cells (Fig. 2B). These results confirm the ability of CD34⁺-enriched AML cells to engraft in murine BM, as previously reported [9, 26, 27].

View larger version (90K): [in this window] [in a new window]   Figure 1. Southern blot analysis of human engraftment in murine BM (lanes 1–10). BM DNA analysis from mice transplanted with AML CD34+ cells, control or pretreated with anti-CXCR4 antibody (A), of the two representative Pts (Pt 3, lanes 4–7; Pt 8, lanes 8–10). High molecular-weight DNA was isolated from BM cells of the transplanted mice, and EcoR1 digested (2.5 mg). The arrow points at the characteristic human 2.7-kb band. Serial dilutions of control human DNA mixed with murine DNA were included in each experiment as controls (%; lanes 8–14). After blotting, filters were probed with 32P-labeled plasmid probe corresponding to human chromosome 17 alpha-satellite sequences (p17H8).

View larger version (28K): [in this window] [in a new window]   Figure 2. A) Flow cytometric analysis of human cells from Pt 9 present in murine BM. The numbers indicate the percentages of CD34+ and CD33+ cells, respectively, after gating on CD45+ cells. B) FISH analysis of CD45+ FACS-sorted cells from mouse BM (cells from Pt 8, probe for chromosome X [alpha-satellite DNA/DXZ1]). Three cells have monosomy X, one cell is diploid, and one cell does not have a signal (mouse cell).

View larger version (20K): [in this window] [in a new window]   Figure 3. Kaplan-Meier estimates of survival from sample date (time to death) of Pts whose cells did or did not engraft. A) based on Southern blot data (six Pts whose leukemic cells engrafted, five nonengrafted); B) based on flow cytometry CD45 analysis (five Pts whose leukemic cells engrafted, six nonengrafted). The median survival for Pts whose leukemic cells engrafted was 26.1 weeks and was 95.1 weeks for those without engraftment (p = 0.012).

View larger version (40K): [in this window] [in a new window]   Figure 4. Real-time Taqman RT-PCR amplification plots for CXCR4 in AML CD34+ (Pts 3, 2, and 4). A) Comparative CT method to compare relative gene expressions was employed as described in Materials and Methods, and results are expressed as relative numbers compared with the calibrator sample. CXCR4 expression on CD34+ cells from a normal BM sample was designated as a calibrator (=1). B) Flow cytometric measurement of CXCR4 in AML CD34+ cells.

Furthermore, we observed engraftment, detected both by CD45 positivity and Southern blot analysis, in NOD/SCID mice transplanted with AML CD34⁺ cells with virtually absent CXCR4 expression (Pt 5, Table 2). On the other hand, two AML samples (Pt 7 and Pt 11, Table 2) with high expression levels of CXCR4 on their CD34⁺ cells failed to engraft the murine BM.

It has been previously reported that pretreatment of cord blood CD34⁺ cells with anti-CXCR4 antibody decreased their ability to engraft NOD/SCID mice BM and to migrate in an in vitro transwell assay [2]. Consistent with the results reported by Aiuti et al. [14], we observed that 20%-25% of normal cord blood CD34⁺ cells migrated in response to a chemotactic gradient of SDF-1 when tested in the transwell assay, and that this migration was efficiently abrogated by anti-CXCR4 antibody (n = 2).

In four AML experiments (Pt 3, Pt 6, Pt 8, and Pt 11, Table 2) in which there were sufficient numbers of cells available, aliquots of AML CD34⁺ cells were pretreated with anti-CXCR4 antibody prior to transplantation in NOD/SCID mice. The presence of human engraftment was demonstrated by Southern blot analysis in two of four AML Pt samples (Fig. 1, Pt 3 and Pt 8). Surprisingly, in both cases, the level of human engraftment was not decreased by the antibody treatment (Fig. 1, Pt 3: lanes 4–7; Pt 8: lanes 8–10). Flow cytometry data correlated with Southern blot results (Fig. 5). For the other two Pts (Pt 6 and Pt 11), the levels of engraftment of untreated cells in NOD/SCID mice were too low to allow conclusions regarding inhibition by CXCR4 antibody pretreatment. Of importance, anti-CXCR4 antibody effectively reduced spontaneous (from 6.06% ± 2.25% to 1.54% ± 0.01%) and SDF-1-mediated in vitro migration in Pt 3 (from 17.32% ± 1.27% to 2.92% ± 0.52%). Thus, anti-CXCR4 antibody successfully blocked spontaneous and SDF-1-induced migration of BM CD34⁺ cells, but did not prevent their engraftment in NOD/SCID mice.

View larger version (40K): [in this window] [in a new window]   Figure 5. Anti-CXCR4 antibody 12G2a did not block the engraftment of AML cells in NOD/SCID mice. CD34+ cells from Pt 3 were incubated with anti-CXCR4 antibody (12g5 monoclonal antibody) or with control IgG2a antibody and transplanted into NOD/ SCID mice. The engraftment was analyzed by CD45 PE/CD34 FITC double-staining of mouse BM as described; the percentage of double-positive cells is indicated in the upper right corner. The corresponding Southern blot data are presented in Figure 1 (controls, lanes 6 and 7; anti-CXCR4 antibody, lanes 4 and 5).

	DISCUSSION

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In our series, engraftment in NOD/SCID mice was observed in 54% of AML samples studied. In two recent reports, the variability in engraftment was attributed to cytogenetic abnormalities [27] or to secondary AML [31]. Moreover, Rombouts et al. reported greater engraftment of primary AML samples from Pts that failed to achieve complete remissions, but this finding did not reach statistical significance [31]. In order to explain our findings, we first investigated the correlation between the ability to engraft the murine BM and clinical characteristics of the Pts. To our knowledge, our results are the first to show that AML with poor prognosis, as estimated by survival from time of sample collection, exhibits a greater ability to engraft NOD/SCID BM. This finding could not be attributed to an imbalance introduced by samples from relapsed/resistant Pts as compared with newly diagnosed Pts, and likely reflects an intrinsic propensity of AML blasts. When we analyzed other clinical characteristics, such as WBC count at diagnosis, cytogenetic classification, and response to chemotherapy, no statistically significant correlation with human engraftment was found, as reported by other groups, perhaps related to different therapeutic strategies or to the limited number of Pts studied [31], although cytogenetics reached borderline significance (p = 0.06).

The exact mechanisms underlying the association between poor prognosis AML and higher engraftment potential in the NOD/SCID BM still remains to be elucidated. Recently, the expression of Flt3/internal tandem duplication mutation, which activates the Flt3 receptor tyrosine, has been described as a new important diagnostic marker for AML Pts with poor prognosis [32]. Subsequently, Lumkul et al. [28] analyzed factors that could influence the engraftment potential of AML cells in NOD/SCID mice and reported that cases with Flt3 mutations tended to engraft the murine BM more efficiently, while AML cells obtained from Pts at relapse did not engraft more efficiently than cells obtained from the same Pts at initial diagnosis. Since we did not analyze our samples for the Flt3 mutation, its presence could have contributed to our findings.

We then investigated the role of SDF-1/CXCR4 interactions for the ability of primary AML CD34⁺ cells to engraft in NOD/SCID mice. SDF-1 is the major chemoattractant released by BM stromal cells that acts on hematopoietic cells such as lymphocytes and progenitors [14, 33]. Dramatically reduced BM hematopoiesis in SDF-1/CXCR4-deficient mice further supports the notion that the interaction of SDF-1 and CXCR4 is critical for stem cell homing [11, 12]. Therefore, it is conceivable that expression of CXCR4 on leukemic progenitor cells contributes to the homing to the BM microenvironment [11, 12]. Recently, chronic lymphocytic leukemia (CLL) [34] and other lymphoproliferative disorders, such as acute lymphoblastic leukemia [35], were reported to express functional CXCR4 levels that exceeded those of normal B cells. Moreover, the autocrine secretion of SDF-1 by blood-derived adherent nurse-like cells in CLL has been reported, capable of protecting leukemic B cells from spontaneous apoptosis [36]. Those data strongly suggest that SDF-1/ CXCR4 interactions are involved in the microenvironmental regulation of CLL cells. The role of CXCR4 in primary AML progenitor cells is less defined. The receptor is detected at variable degrees on leukemic blasts, with the highest levels observed on myelomonocytic cells. Of note, CXCR4 surface protein is virtually absent in myeloid leukemic cell lines with few exceptions, while it is highly expressed in the majority of lymphocytic cell lines [15, 17]. In a recent report, ex vivo culture of AML cells with cytokines failed to increase CXCR4 expression and did not influence engraftment [28]. However, the number of observations was limited (n = 3), and no formal analysis of baseline CXCR4 status and engraftment was attempted.

In this study, we demonstrate that the expression of CXCR4 does not correlate with the ability of AML leukemic cells to engraft in NOD/SCID mice. To reduce the contamination with nonleukemic cells, AML samples were enriched by magnetic bead separation, and CXCR4 was selectively determined on CD34⁺ and CD34⁺CD38^- leukemic cells by simultaneous correlated flow cytometric analysis. However, unlike normal progenitor cells, primary AML blasts expressed variable amounts of CXCR4 on the surface, and cells with virtually absent CXCR4 expression were still able to engraft in NOD/SCID mice (Pt 5, Table 2). This finding, together with the observation that CD34⁺CD38^-Lin^-CXCR4^- cells can engraft into the BM of NOD/SCID mice, as previously reported, suggests the presence of alternative mechanisms critical for engraftment [37]. In support of this notion, cases of human BM infiltration in AML subtypes with low or absent expression of CXCR4 have been reported [17]. Altogether, these data indicate that other factors, such as adhesion molecules, might play an important role in the dissemination of leukemic cells. Bone marrow stromal and endothelial cells constitutively express integrin ligands (e.g., vascular cell-adhesion molecule), while the corresponding integrins (e.g., very late antigen) are expressed on leukemic blasts [38, 39]. Adhesion molecule-mediated homing to the BM might, therefore, account for marrow infiltration of AML cells with low CXCR4 expression. Recent evidence suggests that CD34⁺CXCR4^- cells can express intracellular CXCR4, which can be induced to be expressed on the cell surface and to mediate SDF-1-dependent homing and repopulation [40]. In our study, Pt 5 expressed CXCR4 by PCR analysis only, albeit at 10-fold lower levels than normal BM CD34⁺ cells (median, 0.14 ± 0.06). Whether this phenomenon could explain engraftment of AML in the absence of surface CXCR4 remains to be determined.

In addition, we observed that the anti-CXCR4-blocking antibody failed to reduce the engraftment in NOD/SCID mice in two cases even though the in vitro migration of AML blasts in response to recombinant SDF-1 was reduced in one case. Dissociation between in vitro and in vivo cellular results regarding the SDF-1/CXCR4 pathway has already been reported. Pertussin toxin (PT), an inhibitor of signaling by many Gi protein-coupled chemokine receptors, almost completely abrogated in vitro migration of CD34⁺ cells toward a gradient of SDF-1 in transwells [14]. In contrast, in vivo transplant experiments performed with PT-pretreated murine stem and progenitor cells showed only delayed engraftment of the spleen and no change in BM repopulation [41]. Furthermore, it has been reported that anti-CXCR4-blocking antibody treatment does not inhibit homing of human CD34⁺ cells in the lungs of immunodeficient mice [1].

We also observed two AML Pts (Pt 7 and Pt 11, Table 2) with high expression levels of CXCR4 on their CD34⁺ cells, which failed to engraft the murine BM, suggesting the presence of a nonfunctional CXCR4 pathway in AML.

In conclusion, we demonstrated that the AML-NOD/SCID model is a valuable tool for studies of CD34⁺ AML cells. The expression of CXCR4 on the surface of AML cells does not correlate with engraftment in NOD/SCID mice. Moreover, absence or minimal expression of CXCR4 (<1%) did not preclude engraftment, not all samples with high expression engrafted, and inhibition of CXCR4 with anti-CXCR4 antibody diminished migration in vitro, but not engraftment in vivo. Regardless of CXCR4 expression, engraftment of primary AML cells was found to be correlated with survival of the Pts from which they were obtained, suggesting the ability of the NOD/SCID model to reflect the clinical course of AML.

	ACKNOWLEDGMENT

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This work was supported in part by grants from the National Institutes of Health (CA55164, CA49639, and CA16654) and the Stringer Professorship for Cancer Treatment and Research to M.A.

	FOOTNOTES

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^* G.M. and M.K. contributed equally to the results.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kollet O, Spiegel A, Peled A et al. Rapid and efficient homing of human CD34(+)CD38(-/low) CXCR4(+) stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2m(null) mice. Blood 2001;97:3283–3291.[Abstract/Free Full Text]

2. Peled A, Grabovsky V, Habler L et al. The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest 1999;104:1199–1211.[Medline]

3. Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329–1337.[CrossRef][Medline]

4. Shultz LD, Schweitzer PA, Christianson SW et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol 1995;154:180–191.[Abstract]

5. van der Loo JC, Hanenberg H, Cooper RJ et al. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells. Blood 1998;92:2556–2570.[Abstract/Free Full Text]

6. Danet GH, Lee HW, Luongo JL et al. Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34(+) cells after ex vivo expansion. Exp Hematol 2001;29:1465–1473.[CrossRef][Medline]

7. Bonnet D, Bhatia M, Wang JC et al. Cytokine treatment or accessory cells are required to initiate engraftment of purified primitive human hematopoietic cells transplanted at limiting doses into NOD/SCID mice. Bone Marrow Transplant 1999;23:203–209.[CrossRef][Medline]

8. Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.[CrossRef][Medline]

9. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730–737.[CrossRef][Medline]

10. Cashman JD, Eaves CJ. Human growth factor-enhanced regeneration of transplantable human hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient mice. Blood 1999;93:481–487.[Abstract/Free Full Text]

11. Ma Q, Jones D, Borghesani PR et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4^- and SDF-1-deficient mice. Proc Natl Acad Sci USA 1998;95:9448–9453.[Abstract/Free Full Text]

12. Nagasawa T, Hirota S, Tachibana K et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996;382:635–638.[CrossRef][Medline]

13. Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(⁺) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289–3296.[Abstract/Free Full Text]

14. Aiuti A, Webb IJ, Bleul C et al. The chemokine SDF-1 is a chemoattractant for human CD34⁺ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34⁺ progenitors to peripheral blood. J Exp Med 1997;185:111–120.[Abstract/Free Full Text]

15. Majka M, Rozmyslowicz T, Honczarenko M et al. Biological significance of the expression of HIV-related chemokine coreceptors (CCR5 and CXCR4) and their ligands by human hematopoietic cell lines. Leukemia 2000;14:1821–1832.[CrossRef][Medline]

16. Tachibana K, Hirota S, Iizasa H et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 1998;393:591–594.[CrossRef][Medline]

17. Mohle R, Bautz F, Rafii S et al. The chemokine receptor CXCR-4 is expressed on CD34⁺ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1. Blood 1998;91:4523–4530.[Abstract/Free Full Text]

18. Mohle R, Shittenhelm M, Faienschmid C et al. Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukemia. Br J Haematol 2000;110:563–572.[CrossRef][Medline]

19. Mohle R, Failenschmid C, Bautz F et al. Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1). Leukemia 1999;13:1954–1959.[CrossRef][Medline]

20. Rosu-Myles M, Gallacher L, Murdoch B et al. The human hematopoietic stem cell compartment is heterogeneous for CXCR4 expression. Proc Natl Acad Sci USA 2000;97:14626–14631.[Abstract/Free Full Text]

21. Slovak ML, Kopecky KJ, Cassileth PA et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group study. Blood 2000;96:4075–4083.[Abstract/Free Full Text]

22. Estey EH, Thall PF, Cortes JE et al. Comparison of idarubicin + ara-C-, fludarabine + ara-C-, and topotecan + ara-C-based regimens in treatment of newly diagnosed acute myeloid leukemia, refractory anemia with excess blasts in transformation, or refractory anemia with excess blasts. Blood 2001;98:3575–3583.[Abstract/Free Full Text]

23. Lin F, Monaco G, Sun T et al. BCR gene expression blocks Bcr-Abl induced pathogenicity in a mouse model. Oncogene 2001;20:1873–1881.[CrossRef][Medline]

24. Lapidot T, Fajerman Y, Kollet O. Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis. J Mol Med 1997;75:664–673.[CrossRef][Medline]

25. Livak KJ. Comparative Ct method. ABI Prism 7700 Sequence Detection System. User Bulletin no. 2. Foster City, CA: PE Applied Biosystems, 1997.

26. Ailles LE, Humphries RK, Thomas TE et al. Retroviral marking of acute myelogenous leukemia progenitors that initiate long-term culture and growth in immunodeficient mice. Exp Hematol 1999;27:1609–1620.[CrossRef][Medline]

27. Ailles LE, Gerhard B, Kawagoe H et al. Growth characteristics of acute myelogenous leukemia progenitors that initiate malignant hematopoiesis in nonobese diabetic/severe combined immunodeficient mice. Blood 1999;94:1761–1772.[Abstract/Free Full Text]

28. Lumkul R, Gorin NC, Malehorn MT et al. Human AML cells in NOD/SCID mice: engraftment potential and gene expression. Leukemia 2002;16:1818–1826.[CrossRef][Medline]

29. Blair A, Hogge DE, Ailles LE et al. Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo. Blood 1997;89:3104–3112.[Abstract/Free Full Text]

30. Blair A, Hogge DE, Sutherland HJ. Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71 (-)/HLA-DR^-. Blood 1998;92:4325–4335.[Abstract/Free Full Text]

31. Rombouts WJ, Martens AC, Ploemacher RE. Identification of variables determining the engraftment potential of human acute myeloid leukemia in the immunodeficient NOD/SCID human chimera model. Leukemia 2000;14:889–897.[CrossRef][Medline]

32. Rombouts WJ, Blokland I, Lowenberg B et al. Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the Flt3 gene. Leukemia 2000;14:675–683.[CrossRef][Medline]

33. Bleul CC, Farzan M, Choe H et al. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 1996;382:829–833.[CrossRef][Medline]

34. Durig J, Schmucker U, Duhrsen U. Differential expression of chemokine receptors in B cell malignancies. Leukemia 2001;15:752–756.[CrossRef][Medline]

35. Crazzolara R, Kreczy A, Mann G et al. High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia. Br J Haematol 2001;115:545–553.[CrossRef][Medline]

36. Burger JA, Tsukada N, Burger M et al. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 2000;96:2655–2663.[Abstract/Free Full Text]

37. Rosu-Myles M, Khandaker M, Wu DM et al. Characterization of chemokine receptors expressed in primitive blood cells during human hematopoietic ontogeny. STEM CELLS 2000;18:374–381.[Abstract/Free Full Text]

38. Bendall LJ, Kortlepel K, Gottlieb DJ. Human acute myeloid leukemia cells bind to bone marrow stroma via a combination of beta-1 and beta-2 integrin mechanisms. Blood 1993;82:3125–3132.[Abstract/Free Full Text]

39. Liesveld JL, Winslow JM, Frediani KE et al. Expression of integrins and examination of their adhesive function in normal and leukemic hematopoietic cells. Blood 1993;81:112–121.[Abstract/Free Full Text]

40. Kollet O, Petit I, Kahn J et al. Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation. Blood 2002;100:2778–2786.[Abstract/Free Full Text]

41. Wiesmann A, Spangrude GJ. Marrow engraftment of hematopoietic stem and progenitor cells is independent of Galphai-coupled chemokine receptors. Exp Hematol 1999;27:946–955.[CrossRef][Medline]

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