CXCR4-overexpressing Cells Research Articles

Abstract 2738: Role of KLF8 for drug-resistant metastasis of breast cancer

Abstract Metastasis and drug resistance are intertwined processes that are responsible for the vast majority of patient deaths from breast cancer. The underlying mechanisms still need to be investigated extensively. We previously demonstrated that KLF8 activates CXCR4 transcription in metastatic breast cancer. Here, we report a novel signaling mechanism that turns a localized metastasis into multiple organ metastases in response to chemotherapy. We show that KLF8 expression in breast cancer cells can be over-induced by chemotherapeutic drugs. Data from large-cohorts of patients indicates that post-chemotherapy there is a close correlation between the aberrant high levels of KLF8 and CXCR4, and this correlation is well associated with drug resistance, metastasis, and poor prognosis. To mimic this upregulation, we overexpressed KLF8 or CXCR4 in a human breast cancer cell line known to metastasize to the lungs only after i.v. injection in the nude mice. Surprisingly, we found that these KLF8 or CXCR4 overexpressing cells metastasized all over the body after orthotopically implanted. Tube formation assay, Ki67 staining, and western blotting revealed that KLF8 or CXCR4 overexpression promoted angiogenic vascular growth involving increased expression and secretion of VEGF protein. We also performed the matrigel-on-top 3-D culture to mimic the colonization step of metastasis and found that KLF8 or CXCR4 overexpression strongly enhances the formation of filopodium-like protrusions and proliferation via CXCR4 stimulation. Taken together, these results suggest that the chemo-induction of KLF8 upregulation is critical for drug resistance and systemic metastasis through enhanced tumor angiogenesis and colonization via CXCR4 over-activation. This discovery identifies the KLF8-CXCR4 signaling axis as a potentially new therapeutic target for breast cancer. Citation Format: Jie Hao, Heng Lu, Debarati Mukherjee, Aiqin Sun, Lin Yu, Jihe Zhao. Role of KLF8 for drug-resistant metastasis of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2738.

Abstract 5162: Phosphatidylinositol 4-Kinase type IIIa (PI4KA) expression in prostate cancer

Abstract Advanced prostatic carcinoma (PC) is a progressive disease characterized by a high rate of recurrence and death even after traditional pharmacological androgen deprivation therapy. Phosphatidylinositol 4-kinases (PI4K’s) and the counteracting Sac1 phosphatase, which generates and turns over phosphoinositide phosphatidylinositol 4-phosphate [PtdIns4P], respectively, are required for maintenance and functioning of the Golgi apparatus. Intriguingly, both enzymes have been recently identified as cellular factors associated with prostate cancer. These findings support the hypothesis that modulation in PtdIns4P levels and ER-Golgi and Golgi-PM trafficking activities regulated by PI4K's and the Sac1 phosphatase, can alter the membrane trafficking flux to meet the proliferative demands of cancer-cell growth. Targeting these enzymes with drugs could potentially reduce the proliferation/invasion phenotype and prolong patient survival. The goal of our study was to demonstrate the expression of PI4KA and Sac1 in prostate cancer cells and correlate their expression with CXCR4 status. CXCR4 overexpression and knockdown were performed with PC-3 cells using lentiviral infections. Stable isotope labeling by amino acids in cell culture (SILAC) proteomics analysis was performed on lipid raft membrane micro-domains isolated from PC-3 CXCR4 knockdown and overexpressing cells. Reverse transcription, quantitative real-time PCR (RT-qPCR) was used to quantitate PI4KA mRNA; Western blotting (WB), for PI4KA and Sac1 protein expression levels and immunoprecipitation followed by lipid kinase activity assays were used to determine the PI4KA activity. SILAC analysis of lipid raft membrane microdomains in CXCR4 knockdown and overexpressing cells reveal alterations in G-protein coupled receptor signaling and endocytic vesicular traffic pathways. PI4KA is one of the proteins highly associated with CXCR4 overexpressing cells. PI4KA protein expression was 2.4 to 3.5-fold more abundant in CXCR4-overexpressing vs. control PC3 cell line. A similar finding was made for the C2-4B compared to its progenitor line LnCap, ranging from 1.2 to 2.7-fold higher. PI4KA activity measured by lipid kinase assay in CXCR4-overexpressing cells reflected these enhanced expression levels, showing increases by ∼2 fold. This upregulation may correlate with both PC cell-migration/invasion. This correlation confirmed initial proteomics analysis of CXCR4 association with PI4KA and Sac1 in lipid raft membrane microdomains. Our findings suggest that PI4KA mRNA could be used as a new molecular marker to improve established prognostic models for PC. These findings also indicate possible new lines of research for the development of innovative therapeutic approaches targeting PI4KA in PC, whereby reductions in PI4KA levels would be expected to reduce its severity/recurrence and improve patient survival. Citation Format: Diego Sbrissa, Louie Semaan, Li Yanfeng, Assia Shisheva, Sreenivasa R. Chinni. Phosphatidylinositol 4-Kinase type IIIa (PI4KA) expression in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5162. doi:10.1158/1538-7445.AM2015-5162

Abstract 1656: CXCR4 antagonist-expressing liposomes reduce lung metastases and deliver drugs to CXCR4 expressing cells: a new drug-targeting device

Abstract Introduction. CXCR4 is overexpressed in multiple tumors regulating metastatic dissemination. A new class of cyclic peptides antagonist for CXCR4 receptors was recently developed1. To improve peptide efficacy and increase its delivery delivery to target cancer cells the most active antagonist, Pep R, was coupled to PEGylated liposomes (PL). Experimental Procedures. PL conjugated to the liposomes (Lip-PepR) were prepared starting by athiolated derivative of antiCXCR4 peptides coupled to the pre-formed PL. Doxorubicin (DOX) was then encapsulated by remote loading method. Lip-PepR was evaluated through migration assay in A498 human renal cancer cell line in vitro and in an experimental animal model of pulmonary metastasis development in vivo (C57/BL mice injected with B16-CXCR4 cells and treated with 0.1 mg/kg Lipo-PepR and lipo-unconjucated i.v. twice a week for two weeks). DOX-encapsulating Lipo-PepR was evaluated in CXCR4 positive cells A498 and HT29 (human renal and colon cancer cell lines, respectivally) versus negative CXCR4 expressing cells FB-1(human anaplastic thyroid cell line), as mean cellular fluorescence. Finally, the cytotoxic effect of the lipo-DOX-PepR was examined, in A498, and HT29 cells. Results. To evaluate the antagonistic CXCR4 function of Lipo-PepR, migration assays were conducted in A498, CXCR4 expressing cells. A498 cells were treated with the PepR or with liposome preparation (Lipo-PepR) and induced to migrate toward the CXCR4 ligand CXCL12. PepR alone inhibited cell migration such as the CXCR4 antagonist, AMD3100 while the functionalized peptide, Lipo-PepR (10µM) more efficiently inhibited migration CXCL12-induced compared. To validate the Lipo-PepR efficacy in vivo, metastases development assays were conducted. C57/BL mice were injected with murine B16-hCXCR4 melanoma cells and treated twice a week iv with Lipo-PepR versus Peptide R alone. A significant reduction in lung metastases was detected in mice treated with Lipo-Pep R even with lower dose of the Lipo-PepR (0.1mg/kg) compared to the usually used (2mg/kg). Moreover, to allow a target selective drug delivery, Lipo-PepR liposomes were loaded with doxorubicin (DOX). A CXCR4 dependent higher DOX accumulation was registered in CXCR4 positive cells, A498 and HT29 resulting in a specific higher cytotoxicity. Conclusions. Liposomes conjugated- rationally designed CXCR4 antagonist were more efficient in inhibiting CXCR4 in vitro and in vivo. Moreover Lip-PepR loaded with a chemotherapeutic drug, such as DOX, demonstrated an enhanced drug accumulation into CXCR4 overexpressing cells. 1. Portella L et al. PLoS One. 2013 Sep 13;8(9):e74548 Citation Format: Caterina Ierano, Sara Lusa, Crescenzo D'Alterio, Giuseppina Salzano, Maria Napolitano, Maria Buoncervello, Massimo Spada, Daniele Macchia, Antonio Barbieri, Antonio Luciano, Lucia Gabriele, Giuseppe De Rosa, Stefania Scala. CXCR4 antagonist-expressing liposomes reduce lung metastases and deliver drugs to CXCR4 expressing cells: a new drug-targeting device. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2014-1656

A Novel Activating Mutation Of CXCR4 Plays a Crucial Role In Waldenstrom Macroglobulinemia Biology

BackgroundThe C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating the biology of B-cell lymphoproliferative disorders. Recent whole genome sequencing studies have identified unique CXCR4 mutations in 29% of the 55 evaluated patients with Waldenstrom Macroglobulinemia (WM). In this study, we sought to better define the mutation status of CXCR4 in B-cell malignancies and define the functional role of this mutation in the progression of WM in vivo. MethodsAllele-specific(AS) PCR has been performed on bone marrow (BM)-derived tumor cells of patients with WM (n: 131); IgM monoclonal gammopathy of undetermined significance (MGUS; n: 40); as well as in patients with diffuse large cell lymphomas (DLBCL; n: 75), splenic marginal zone lymphoma (SMZL; n: 14), B-chronic lymphocytic leukemia (B-CLL; n: 37), hairy cell leukemia (HCL; n: 35), multiple myeloma (MM; n: 36), IgA/IgG MGUS (n: 22), lymphoplasmacytic lymphoma without WM criteria (n: 13), and amyloidosis (n: 6). CXCR4-loss and -gain of function studies have been performed on WM cells stably expressing either shRNA-CXCR4, CXCR4-ORF-GFP-tagged or scramble-RFP-tagged (generated via lentivirus-based infection). A mutagenesis kit has been used to generate the C1013GCXCR4 mutant protein (C1013GCXCR4) in WM cells, via lentivirus-based infection. CXCR4-knock-in or C1013GCXCR4-mutated cells and the corresponding controls have been injected i.v. into SCID/Bg mice and tumor dissemination has been evaluated ex vivo by immunohistochemistry IHC (human-CD20; -CXCR4). C1013GCXCR4-mutated cells have been characterized at mRNA levels (U133 plus2) using GSEA. A novel human anti-CXCR4 mAb (BMS-936564/MDX-1338; Bristol Myers Squibb, NY) has been tested in vitro (cell proliferation, MTT, adhesion and migration to primary WM BM mesenchymal stromal cells) and in vivo (10mg/kg i.p. x3-4/week). Tumor growth has been evaluated by IHC ex vivo (hCD20; hCXCR4) and by immunofluorescence. ResultsWe examined the mutational status of C1013GCXCR4 and confirmed the presence of this specific mutation in 28% of the 131 cases evaluated. The mutation was also detected at the stage of IgM-MGUS (20%); while it was present in a minority of patients with DLBCL (1%) and SMZL (7%). Remarkably, it was absent in all MM (n=36) and IgA/IgG MGUS patients (n=22), and it was not detected in healthy subjects (n=32). The functional relevance of the C1013G-CXCR4 variant was next examined in vivo. Mice injected with C1013GCXCR4-cells presented with a significant dissemination of tumor cells, demonstrating involvement of liver, bone marrow, lymph nodes, kidney and lung. IHC showed the presence of CXCR4+ and CD20+ cells in all the tissues examined; and quantification of CXCR4 and CD20 positivity was higher in C1013GCXCR4-cells-, compared to parental(p)-WM cell-injected mice (NIS Elements software, Nikon, Melville, NY; P<0.05). In addition, C1013GCXCR4-cells were further characterized in vitro, showing increased adhesion and cell proliferation in the presence of primary WM BM-MSCs. These findings were also confirmed using CXCR4-overexpressing cells. In contrast CXCR4-knock-down cells presented the opposite behavior, where reduced adhesion and proliferation in presence of primary WM BM-MSCs were observed. By performing GSEA we demonstrated that genes related to invasiveness, cell proliferation, anti-apoptosis, and oncogenesis were all enriched in C1013GCXCR4-cells compared to the parental-WM cells. These findings let us hypothesize that C1013GCXCR4 may act as an activating mutation in WM cells. Indeed, in a different mouse model, CXCR4 over-expressing cells and scramble infected cells were injected into mice, showing similar phenotype to the one observed upon C1013GCXCR4-WM cell-injected-mice. Finally, the novel antibody BMS-936564/MDX-1338 exerted anti-WM activity both in vitro and in vivo, with anti-tumor effects observed also against the mutated variant. This was supported by inhibition of pro-survival pathways (p-ERK; pAKT); induction of pro-apototic proteins (cleaved-PARP and -caspase-9); up-regulation of p-GSK3beta, p-beta catenin and relative beta catenin degradation. ConclusionThese findings demonstrate that C1013GCXCR4 acts as an activating mutation in WM; and it is targetable by using MDX936564/1338 thus providing the basis for translating these observations into clinical trials for WM patients. Disclosures:Kuhne:BMS: Employment. Cardarelli:BMS: Employment. Ghobrial:BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other; Noxxon: Research Funding; Sanofi: Research Funding.

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell “dormancy.” Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from “sanctuary” niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda“protective niche” for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.

Genetically modified adipose tissue−derived mesenchymal stem cells overexpressing CXCR4 display increased motility, invasiveness, and homing to bone marrow of NOD/SCID mice

This study evaluates usefulness of CXCR4 overexpression via retroviral transduction in adipose tissue-derived mesenchymal stem cells (AT-MSCs) as a strategy to increase their migration and engraftment ability. AT-MSCs were isolated from lipoaspirates from human healthy donors with liberase 3. Cells were transduced with retroviral vector carrying either CXCR4 or green fluorescent protein (GFP) complementary DNA, and neo-resistant colonies were selected and used in experiments. Chemotaxis, invasion through Matrigel, motor activity, gene expression, osteodifferentiation potential, and engraftment into bone marrow of nonobese diabetic/severe combined immunodeficient mice were analyzed for CXCR4-overexpressing cells and GFP-control cells. Approximately 90% of retrovirus-transduced AT-MSCs expressed CXCR4 or GFP and maintained their ability to differentiate into osteocytes. CXCR4-transduced AT-MSCs displayed enhanced migration and higher invasiveness toward SDF-1 gradient. The upregulation of CXCR4 led to phosphorylation of mitogen-activated protein and AKT kinases and an increase in metalloproteinase expression after SDF-1 stimulation. The transplantation of CXCR4-transduced AT-MSCs into nonobese diabetic/severe combined immunodeficient mice led to increased engraftment into bone marrow in comparison to GFP-transduced AT-MSCs. Adipose tissue is one of the alternative sources of MSCs to bone marrow. We showed that AT-MSCs overexpressing CXCR4 preserve their ability for osteodifferentiation. Enhanced migration and engraftment of the transduced AT-MSCs into bone marrow indicate the usefulness of this strategy in overcoming low engraftment of MSCs in clinical approaches of cellular therapies for bone disorders and can represent a powerful tool in regenerative medicine and gene therapies. Thus, these cells may be used as an alternative to bone marrow-derived MSCs.

436 Functional analysis of oncogenic YB-1 in the cytoplasm of multiple myeloma reveals that survival is accomplished by stabilizing the mRNA translation of Mcl-1 and TCTP

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell “dormancy.” Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from “sanctuary” niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda“protective niche” for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.

Tissue Microenvironment Modulates CXCR4 Expression and Tumor Metastasis in Neuroblastoma

Neuroblastoma (NB) is derived from intrinsic migratory neural crest cells and has a high potential for distant metastasis. Growing evidence has implicated chemokine receptors, especially CXCR4, which normally control immune and inflammatory cell migration, as having important roles in tumor progression. In this study, we investigated the expression of CXCR4 in eight different NB cell lines and found that CXCR4 expression is dynamically regulated in NB and can be modulated by different tissue stromata. In addition, we demonstrate that IL-5 and IFN-γ are released from stromal cells and act as differential mediators for CXCR4 expression. We also overexpressed CXCR4 in two NB cell lines, NUB-7 and SK-N-BE(2), and studied the role of CXCR4 in NB metastasis both in vitro and in vivo. In vitro transwell invasion assay showed that CXCR4 overexpression promoted NB cell migration preferentially toward a bone marrow stromal cell-conditioned medium. Using an in vivo xenograft model, CXCR4-overexpressing cells showed an increased incidence of metastasis, most notably bone marrow metastasis. Our studies reveal critical roles for CXCR4 in NB metastasis and provide insights into the regulatory mechanism of chemokine receptors in NB and the importance of the tissue microenvironment in modulating tumor cell behavior.

Overexpression of CXCR4 Increases Migration and Proliferation of Human Adipose Tissue Stromal Cells

Stromal-derived factor-1 (SDF-1)-mediated CXCR4 signaling plays important roles in migration, engraftment, and proliferation of stem cells. We report here that CXCR4 overexpression on human adipose tissue stromal cells (hADSCs) using a lentiviral gene transfer technique helped navigate these cells to the injured tissues in response to SDF-1 signaling. Transduced hADSCs, expressing high levels of CXCR4, displayed an increased capacity for cellular growth and protection against etoposide-induced cell death. CXCR4-overexpressed cells showed higher ERK activity than that of vector-transduced cells. U0126, an ERK inhibitor, and AMD3100, a CXCR4 antagonist, inhibited the proliferation of CXCR4 overexpression-induced proliferation and ERK phosphorylation. CXCR4-overexpressing cells showed increased level of beta-catenin and luciferase activity driven by the Tcf promoter. Our results suggest CXCR4 overexpression for improved hADSC motility, retention, and proliferation could be beneficial for in vivo navigation and expansion of stem cells.

Enhanced Engraftment and Maintenance of CXCR4 Expressing CD34+ Cells In Vivo Is Related to Microenvironmental Interactions, Rather Than Direct Receptor Signaling to Hematopoietic Stem Cells.

Interaction of the chemokine receptor CXCR4 with its ligand SDF-1α (SDF) has been reported to play an important role in engraftment of hematopoietic stem cells (HSC) in the bone marrow (BM). However a critical requirement for CXCR4 in HSC engraftment is still controversial. It also remains unclear whether the effects that CXCR4 has on hematopoietic cell engraftment are related to enhanced homing of HSC to the bone marrow cavity, increased retention in marrow microenvironment or direct and indirect effects of CXCR4 stimulation on stem and progenitor cell proliferation, self-renewal and survival. To address these questions we have overexpressed CXCR4 in human cord blood CD34+ cells by transduction with an MSCV retroviral vector containing CXCR4 and eGFP (MIG-CXCR4). CXCR4 overexpressing cells were compared with control cells transduced with vectors expressing eGFP alone (MIG). CD34+eGFP+ cells were selected after transduction by flow cytometry sorting. We confirmed that CD34+ cells transduced with the MIG-CXCR4 vector demonstrated increased CXCR4 expression compared with MIG vector transduced controls (mean channel fluorescence for CXCR4 was 340±77.8 for MIG-CXCR4 transduced CD34+ cells compared with 142±37.1 for MIG transduced cells, n=8). MIG-CXCR4 transduced CD34+ cells demonstrated significantly enhanced chemotaxis to SDF in transwell migration assays (36±2% migration for MIG-CXCR4 vs. 20±4% migration for MIG transduced CD34+ cells to 100nM SDF-1, n=4, p=0.05). CD34+ cells transduced with MIG-CXCR4 demonstrated a 1.52±0.4 fold increase in expansion of total cell number compared with controls after 1 week of in vitro culture with growth factors (GF) [SCF (50ng/ml), TPO (100ng/ml), FL (100ng/ml), SDF (60ng/ml), n=3]. However, enhanced cellular expansion was not sustained on further GF culture. To evaluate the effect of CXCR4 overexpression on in vivo engraftment, CD34+ cells transduced with MIG-CXCR4 and MIG vectors were injected intravenously into sublethally irradiated NOD/SCID mice and human hematopoietic cell engraftment was evaluated after 6–8 weeks. MIG-CXCR4 transduced cells demonstrated significantly higher levels of engraftment with human CD45+ cells compared with MIG transduced cells (8±4.8% vs. 0.22±0.07% CD45+ cells in bone marrow, 1.3±0.9% vs. 0.2±0.09% CD45+ cells in spleen, and 1.8±1.0% vs. 0.3±0.25% CD45+ cells in peripheral blood for MIG-CXCR4 vs. MIG transduced cells, respectively, n=5). In addition, markedly higher levels of CD34+ cell engraftment was observed in the bone marrow of animals receiving MIG-CXCR4 vs. MIG transduced cells (1.7±1.0% vs. 0.06±0.03% CD34+ cells respectively, n=5). Consistent with this, the human CFC frequency in bone marrow of mice receiving MIG-CXCR4 transduced CD34+ cells was increased compared to mice receiving MIG transduced cells (31±0.5 CFC/100,000 cells vs. 5±3.2 CFC/100,000 cells, n=2,3, respectively). In conclusion, our results indicate that ectopic expression of CXCR4 in CD34+ cells results in enhanced engraftment of human hematopoietic cells and increased maintenance of hematopoietic stem and progenitor cells in the NOD/SCID mouse model. The effects of CXCR4 overexpression are considerably more prominent in vivo than in direct in vitro assays. It therefore appears that altered stem and progenitor cell homing and microenvironmental interaction, rather than direct signaling to HSC, may be responsible for enhanced CD34+ cell engraftment and maintenance following CXCR4 receptor overexpression.