Engraftment of Acute Myeloid Leukemia in NOD/SCID Mice Is Independent of CXCR4 a - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

a Department of Human Genetics, Baylor College of Medicine, Houston, Texas, USA;

b Department of Blood and Marrow Transplantation,2 M3 ~' M! L4 d# n& e: m

c Department of Biostatistics, and

d Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA
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Key Words. Acute myeloid leukemia ? CXCR4 ? NOD/SCID ? Engraftment* e6 f; _1 O. |) C$ j& R3 q. l
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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@mdanderson.org7 B/ j& N5 m- E
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ABSTRACT

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 . 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 . 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 .

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 . 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 . 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 .
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The expression of CXCR4 on CD34  hematopoietic progenitors in normal and leukemic cells has been studied by a number of laboratories. Mohle et al.  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 ., G1 j6 C& }5 \" [2 W/ E) L

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 . It is notable, however, that cases of BM infiltration in AML subtypes with low or absent expression of CXCR4 have also been reported .

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.  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/?2 microglobulin (?2M) 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 . 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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Engraftment and Clinical Characteristics
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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 7 a/ V0 `" F7 i1 K* g+ ]

Table 2. CXCR4 expression on CD34  and CD34 CD38- primary AML cells7 c, ?# o% c6 C6 C* a; m* Q0 @5 t8 z( ]
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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).

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 ). Three cells have monosomy X, one cell is diploid, and one cell does not have a signal (mouse cell)., z8 u# \9 d8 L9 ^  Q# f) S# N
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To further explore the biological variables influencing the engraftment potential of primary AML cells in NOD/SCID mice, we investigated whether the ability to engraft the murine BM correlated with various clinical characteristics of the AML Pts. Poor prognosis, as estimated by overall survival from time of diagnosis, was found to be associated with a greater ability to engraft the NOD/SCID BM. Kaplan-Meier estimates for Pts whose cells engrafted and for those whose cells did not engraft are shown in Figure 3. The log-rank statistic was used to assess differences between these two groups with respect to overall survival. The five Pts whose cells did not engraft in any mouse had a median overall survival of 95.9 weeks, while the six Pts whose cells engrafted had a median survival of 26.1 weeks (p = 0.012, log-rank test). We also observed a longer time to relapse in Pts whose cells did not engraft (52.6 weeks), compared with those whose cells engrafted (22.9 weeks), even though the difference did not reach statistical significance (p = 0.13, log-rank test, Table 3). Of importance, the numbers of newly diagnosed and relapsed AML Pts were similar for Pts whose cells engrafted in NOD/SCID mice and those who did not. Specifically, cells from two of five newly diagnosed and four of six relapsed/resistant AML Pts engrafted (p = 0.5, Fisher’s exact test). Of note, Lumkul et al. reported no difference in engraftment between cells from newly diagnosed and relapsed AML Pts in NOD/SCID mice .. o2 Z1 `+ f, }0 m% G% E% A

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).9 s4 |* f7 y' @& k# E$ U

Table 3. Median overall survival for CXCR4 data0 r0 o0 D; K/ A- M2 Q7 D

Additional clinical parameters, including WBC count at diagnosis, cytogenetic classification, and clinical response to chemotherapy were also evaluated (Table 4). The mean WBC for the group without engraftment was 57 ± 32.4 x 109/l and was 44.2 ± 48.2 x 109/l for the group whose cells engrafted (Wilcoxon rank-sum test, p = 0.329), suggesting that the two groups did not differ significantly. Fisher’s exact test was used to compare these two groups with respect to cytogenetics and response to therapy, with p values of 0.061 and 1.000, respectively; again no statistically significant difference was found although cytogenetic status approached significance (Table 3), as previously reported . Correlation with FAB classification was not investigated due to the low number of AML Pts included in this study and the five FAB types represented.
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Table 4. Classification of the AML Pts according to cytogenetic characteristics and response to chemotherapy; s4 ^& Z, v% x" m8 G7 Y$ x2 g9 }

Engraftment and CXCR45 O5 ^+ f- L8 ?: R7 t2 z6 @; b
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In order to assess whether the level of CXCR4 expression of AML CD34  cells correlated with their ability to engraft the BM of NOD/SCID mice, CXCR4 expression on normal and AML CD34  and CD34 CD38- cells was determined. In normal BM CD34  cells, 46.9% ± 6.2% of cells were CXCR4 positive (n = 12), while CXCR4 expression was significantly lower on normal PB CD34  cells (13.7% ± 2.4%, n = 8, p 6 s4 P3 W: c0 `9 A- [6 i

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.

Logistic regression analysis was used to model the association between engraftment of human leukemic cells in the BM of NOD/SCID mice and the level of CXCR4 expression. The results are shown in Table 5 and Table 6. No statistically significant association was observed between the level of CXCR4 expression on either CD34  (p = 0.494) or CD34 CD38- cells (p = 0.402) and engraftment as determined by Southern blot. Similar negative results were found when the percentage of either CD34 CXCR4  (p = 0.931) or CD34 CD38-CXCR4  cells (p = 0.726) was modeled with the engraftment determined by CD45 positivity (Table 5).

Table 5. Logistic regression analysis of human engraftment assessed by Southern blot and CD45 positivity

Table 6. Summary statistics for CXCR4 expression (%)7 c( F) u4 o' C8 R* G- U* r! x

Summary statistics for each assay are shown in Table 6. Although the mean CXCR4 expression was higher for those Pts whose cells engrafted, the standard deviation was so large that it was impossible to detect a difference between those whose cells engrafted and those whose cells did not.

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.5 T4 e; T8 J# I4 J1 W( S4 n

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 . Consistent with the results reported by Aiuti et al. , 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).
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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.
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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).

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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.# }3 C) s; v* _5 Y, U; w& b. w
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