scholarly journals IL-6 and TNFα Drive Extensive Proliferation of Human Tregs Without Compromising Their Lineage Stability or Function

2021 ◽  
Vol 12 ◽  
Author(s):  
Nikolaos Skartsis ◽  
Yani Peng ◽  
Leonardo M. R. Ferreira ◽  
Vinh Nguyen ◽  
Emilie Ronin ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Oxidative Phosphorylation ◽  
Energy Production ◽  
Ex Vivo ◽  
Tnf Receptor ◽  
Lineage Stability ◽  
The Impact ◽  
Pro Inflammatory Cytokines

Treg therapies are being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs remains controversial. We challenged human Tregs ex-vivo with pro-inflammatory cytokines IL-6 and TNFα and observed greatly enhanced proliferation stimulated by anti-CD3 and anti-CD28 (aCD3/28) beads or CD28 superagonist (CD28SA). The cytokine-exposed Tregs maintained high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low IFNγ, IL-4, and IL-17 secretion. Blocking TNF receptor using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression. These results prompted us to consider using CD28SA together with IL-6 and TNFα without aCD3/28 beads (beadless) as an alternative protocol for therapeutic Treg manufacturing. Metabolomics profiling revealed more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential during beadless Treg expansion. Finally, beadless expanded Tregs maintained suppressive functions in vitro and in vivo. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function. This property can be harnessed for therapeutic Treg manufacturing.

scholarly journals TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

2021 ◽  
Author(s):  
Nikolaos Skartsis ◽  
Yani Peng ◽  
Leonardo M.R. Ferreira ◽  
Vinh Nguyen ◽  
Yannick Muller ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tnf Receptor ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Graft Versus Host ◽  
Lineage Stability ◽  
The Impact ◽  
Pro Inflammatory Cytokines

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNFa and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNFa may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

scholarly journals Intestinal IgA Regulates Expression of a Fructan Polysaccharide Utilization Locus in Colonizing Gut Commensal Bacteroides thetaiotaomicron

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Payal Joglekar ◽  
Hua Ding ◽  
Pablo Canales-Herrerias ◽  
Pankaj Jay Pasricha ◽  
Justin L. Sonnenburg ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ex Vivo ◽  
Microbial Colonization ◽  
Bacteroides Thetaiotaomicron ◽  
Content Type ◽  
Microbial Utilization ◽  
Polysaccharide Utilization Locus ◽  
The Impact

ABSTRACT Gut-derived immunoglobulin A (IgA) is the most abundant antibody secreted in the gut that shapes gut microbiota composition and functionality. However, most of the microbial antigens targeted by gut IgA remain unknown, and the functional effects of IgA targeting these antigens are currently understudied. This study provides a framework for identifying and characterizing gut microbiota antigens targeted by gut IgA. We developed a small intestinal ex vivo culture assay to harvest lamina propria IgA from gnotobiotic mice, with the aim of identifying antigenic targets in a model human gut commensal, Bacteroides thetaiotaomicron VPI-5482. Colonization by B. thetaiotaomicron induced a microbe-specific IgA response that was reactive against diverse antigens, including capsular polysaccharides, lipopolysaccharides, and proteins. IgA against microbial protein antigens targeted membrane and secreted proteins with diverse functionalities, including an IgA specific against proteins of the polysaccharide utilization locus (PUL) that are necessary for utilization of fructan, which is an important dietary polysaccharide. Further analyses demonstrated that the presence of dietary fructan increased the production of fructan PUL-specific IgA, which then downregulated the expression of fructan PUL in B. thetaiotaomicron, both in vivo and in vitro. Since the expression of fructan PUL has been associated with the ability of B. thetaiotaomicron to colonize the gut in the presence of dietary fructans, our work suggests a novel role for gut IgA in regulating microbial colonization by modulating their metabolism. IMPORTANCE Given the significant impact that gut microbes have on our health, it is essential to identify key host and environmental factors that shape this diverse community. While many studies have highlighted the impact of diet on gut microbiota, little is known about how the host regulates this critical diet-microbiota interaction. In our present study, we discovered that gut IgA targeted a protein complex involved in the utilization of an important dietary polysaccharide: fructan. While the presence of dietary fructans was previously thought to allow unrestricted growth of fructan-utilizing bacteria, our work shows that gut IgA, by targeting proteins responsible for fructan utilization, provides the host with tools that can restrict the microbial utilization of such polysaccharides, thereby controlling their growth.

scholarly journals EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lyess Allas ◽  
Sybille Brochard ◽  
Quitterie Rochoux ◽  
Jules Ribet ◽  
Cleo Dujarrier ◽  
...  
Keyword(s):  
Functional Disability ◽  
Ex Vivo ◽  
Cartilage Degradation ◽  
Motor Functions ◽  
Methyl Transferase ◽  
Expression Of Genes ◽  
Medial Meniscectomy ◽  
The Impact

Abstract Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1β. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1β in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1β effects. Ex vivo, EZH2 inhibition decreased IL-1β-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

scholarly journals MR imaging of model drug distribution in simulated vitreous

2015 ◽  
Vol 1 (1) ◽  
pp. 236-239 ◽  
Author(s):  
Sandra Stein ◽  
Christian Simroth-Loch ◽  
Sönke Langner ◽  
Stefan Hadlich ◽  
Oliver Stachs ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ex Vivo ◽  
Drug Distribution ◽  
Injection Technique ◽  
Innovative Therapy ◽  
Age Related ◽  
The Impact

AbstractThe in vitro and in vivo characterization of intravitreal injections plays an important role in developing innovative therapy approaches. Using the established vitreous model (VM) and eye movement system (EyeMoS) the distribution of contrast agents with different molecular weight was studied in vitro. The impact of the simulated age-related vitreal liquefaction (VL) on drug distribution in VM was examined either with injection through the gel phase or through the liquid phase. For comparison the distribution was studied ex vivo in the porcine vitreous. The studies were performed in a magnetic resonance (MR) scanner. As expected, with increasing molecular weight the diffusion velocity and the visual distribution of the injected substances decreased. Similar drug distribution was observed in VM and in porcine eye. VL causes enhanced convective flow and faster distribution in VM. Confirming the importance of the injection technique in progress of VL, injection through gelatinous phase caused faster distribution into peripheral regions of the VM than following injection through liquefied phase. VM and MR scanner in combination present a new approach for the in vitro characterization of drug release and distribution of intravitreal dosage forms.

scholarly journals Factors Important to the Prioritization and Development of Successful Topical Microbicides for HIV-1

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Karen W. Buckheit ◽  
Robert W. Buckheit
Keyword(s):  
Clinical Trials ◽  
Ex Vivo ◽  
Sexual Transmission ◽  
Virus Transmission ◽  
Preclinical Development ◽  
Sexual Transmission Of Hiv ◽  
The Right ◽  
Hiv 1

Significant advancements in topical microbicide development have occurred since the prevention strategy was first described as a means to inhibit the sexual transmission of HIV-1. The lack of clinical efficacy of the first generation microbicide products has focused development attention on specific antiretroviral agents, and these agents have proven partially successful in human clinical trials. With greater understanding of vaginal and rectal virus infection, replication, and dissemination, better microbicide products and delivery strategies should result in products with enhanced potency. However, a variety of development gaps exist which relate to product dosing, formulation and delivery, and pharmacokinetics and pharmacodynamics which must be better understood in order to prioritize microbicide products for clinical development. In vitro, ex vivo, and in vivo models must be optimized with regard to these development gaps in order to put the right product at the right place, at the right time, and at the right concentration for effective inhibition of virus transmission. As the microbicide field continues to evolve, we must harness the knowledge gained from unsuccessful and successful clinical trials and development programs to continuously enhance our preclinical development algorithms.

scholarly journals Novel Rotational Combination Regimen of Skin Topicals Improves Facial Photoaging: Efficacy Demonstrated in Double-Blinded Clinical Trials and Laboratory Validation

2021 ◽  
Vol 8 ◽  
Author(s):  
Lisa DiNatale ◽  
Jolanta Idkowiak-Baldys ◽  
Young Zhuang ◽  
Anthony Gonzalez ◽  
Thomas J. Stephens ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Ex Vivo ◽  
Dermal Fibroblasts ◽  
Combination Regimen ◽  
Crow’S Feet ◽  
Skin Explants ◽  
Study Methodology ◽  
Double Blinded

Topical antiaging products are often a first-line intervention to counter visible signs of facial photoaging, aiming for sustained cosmetic improvement. However, prolonged application of a single active topical compound was observed clinically to lead to a plateau effect in improving facial photoaging. In view of this, we set out to reduce this effect systematically using a multi-tiered approach with laboratory evidence and clinical trials. The objective of the study was to evaluate the effects of active topical ingredients applied either alone, in combination, or in a rotational manner on modulation of facial photoaging. The study methodology included in vitro, organotypic, and ex vivo skin explants; in vivo biopsy study; as well as clinical trials. We demonstrate for the first time that a pair of known antiaging ingredients applied rotationally, on human dermal fibroblasts, maximized pro-collagen I production. Indeed, rotational treatment with retinol and phytol/glycolic acid (PGA) resulted in better efficacy than application of each active ingredient alone as shown by explants and in vivo biopsy study, with penetration of active ingredients confirmed by Raman spectroscopy. Furthermore, two split-face, randomized, double-blinded clinical trials were conducted, one for 12 months to compare treated vs. untreated and the other for 6 months followed by a 2-month regression to compare treated vs. commercially marketed products. In both studies, rotational regimen showed superior results to its matching comparison as assessed by clinical grading and image analysis of crow's feet wrinkles. In conclusion, rotational regimen using retinol and PGA is effective in treating facial photoaging signs with long-lasting benefits.

Trastuzumab versus MYL-1401O (a proposed trastuzumab biosimilar) in a phase I bioequivalence study: In vivo and in vitro immunomodulation.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 10-10
Author(s):  
Régine Audran ◽  
Haithem Chtioui ◽  
Anne-Christine Thierry ◽  
Carole Mayor ◽  
Laure Vallotton ◽  
...  
Keyword(s):  
Phase I ◽  
Mononuclear Cell ◽  
Ex Vivo ◽  
Reference Drug ◽  
Gm Csf ◽  
Significant Difference ◽  
Ifn Γ ◽  
The Impact

10 Background: Trastuzumab is a humanized monoclonal antibody targeting breast cancer cells overexpressing the HER2-oncoprotein. During a Phase-I single centre, single dose, randomized, double-blind, cross-over study assessing the bioequivalence of a proposed trastuzumab biosimilar (MYL-1401O) versus the initially marketed drug (Herceptin), we investigated in addition a large panel of pharmacodynamics parameters comparing the immunomodulatory activity of both drugs. Methods: 22 healthy males were included, 19 subjects receiving randomly a single intravenous infusion of MYL-1401O and 22 of Herceptin, separated by 16 to 22 week wash-out. Blood samples drawn pre- and post- infusion were assessed for in vivo serum cytokines induction (IL-1β, IL-2, IL-6, IL-10, IL-12, TNF-α, GM-CSF and IFN-γ) whereas the impact of treatment on mononuclear cell subsets and their level of activation was tested ex vivo. Volunteers’ PBMC (peripheral blood monocnuclear cells) were stimulated in vitro with recall antigens and mitogen for cytokine production. At baseline, we performed in addition a cytokine release assay on PBMC upon stimulation with trastuzumab as a preclinical safety test. Results: Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6h, and a modulation of mononuclear cell subset profile and level of activation. Notably CD16+ cells frequency decreased at 3h and peaked at 48h. Except for CD8+ T cells, there were no significant differences between Herceptin and its proposed biosimilar ex vivo. PBMC stimulated in vitro with trastuzumab secreted IL-6, TNF-a, IL-1β, GM-CSF, IFN-γ, and IL-10, but no IL-2. There was no significant difference between the two mAbs. Conclusions: Based on these in vivo, ex vivo and in vitro experiments, there is a strong assumption that MYL-1401O is biosimilar to the reference drug Herceptin for its immunomodulation properties as already proven for its bioequivalence. Clinical trial information: 2011-001406-94.

Quantitative Assessment of the Impact of Ex Vivo Manipulation on the In Vivo Functionality of Human T Cells in RAG2−/− γc−/− mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 450-450
Author(s):  
Rozemarijn S. van Rijn ◽  
Elles R. Simonetti ◽  
Gert Storm ◽  
Mark Bonyhadi ◽  
Anton Hagenbeek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Critical Parameters ◽  
In Vivo Evaluation ◽  
Human T Cells ◽  
The Impact

Abstract T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P<0.0001). Additional ligation of 4-1BB did not increase engraftment potential. In addition, different T cell subsets (naïve, memory, effector) were monitored based on the combined expression of CD45RA, CD27 and CCR7. For all primary T cells and variably cultured T cells, a strikingly similar pattern was observed in vivo. After 3 weeks mainly effector and memory effector T cells (both CD4+ and CD8+) could be detected, suggesting a (xeno-)antigen-driven survival and expansion. This was a very consistent observation independent of donor, culture condition, engraftment level or severity of disease. In conclusion, in vitro costimulation preserves in vivo functionality of human T cells and should therefore be included in future clinical protocols for ex vivo manipulation of T cells. These data show the feasibility to use the huPBMC-RAG2−/−γc−/− model for in vivo evaluation of in vitro effects on human T cells. This model is the most sensitive to date for in vivo evaluation of human T cells and will be a promising new tool for the study of human T cells in, for instance, autoimmune disease, cancer and infectious diseases like AIDS.

CXCR4/SDF-1 Is a Key Regulator for Leukemia Migration and Homing to the BM: Impact of AMD3100 on In Vivo Response to Chemotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 569-569 ◽  
Author(s):  
Bruno Nervi ◽  
Pablo Ramirez ◽  
Matthew Holt ◽  
Michael P. Rettig ◽  
Julie K. Ritchey ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Survival Benefit ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Dual Function ◽  
Hematopoietic Stem ◽  
Response To Chemotherapy ◽  
The Impact

Abstract Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and interact with stroma cells and extracellular matrix. CXCR4/SDF-1 axis regulates the trafficking of HSC to and from the BM. We utilized a PML-RARα knock-in mouse model of human acute promyelocytic leukemia (APL) to study APL interaction with the normal BM. We have previously shown there is a rapid mobilization of APL cells from the BM into peripheral blood (PB) after administration of AMD3100, a competitive inhibitor of CXCR4. We hypothesize that we can sensitize these tumor cells to chemotherapy by interrupting the interaction between APL and the BM stroma. We transduced banked APL cells with a dual function reporter gene that encodes a fusion protein comprised of Click Beetle Red luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (Luc/EGFP). Upon iv injection into genetically compatible recipients (F1 129/B6 mice), APL rapidly migrated to the BM with increased BLI signal in the femurs, spine, ribs, and skull, at 4 days after injection, followed by spleen infiltration and death due to leukostasis by day 15. 129/B6 F1 mice (n=28) were injected iv with 106 APL cells. By day 12 all mice had ±5% APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12 and 13, and another 8 mice received AraC+AMD (5mg/kg/sq) 1 hour before and 3 hours after each AraC injection. 6 mice received only AMD and 6 control mice were observed. Total body BLI signal, WBC, and blasts per μl of blood on days 19 and 23 were higher in AraC versus AraC+AMD (p<0.004). Median survival for control, AMD, AraC and AraC+AMD groups were 18, 19, 23 and 30 days respectively (p<0.0006). Hemoglobin, platelet and granulocyte recovery post-chemotherapy was similar in both groups. We developed an in vitro mouse stroma system to study engraftment, ex vivo mobilization and sensitivity to chemotherapy. In vitro culture of APL cells showed no difference in APL survival between AraC versus AraC+AMD as measured by flow cytometry or BLI. Stroma offered a survival benefit versus no stroma (p<0.0001). We injected 4 genetically compatible mice with 106 APL cells iv and after 14 days mice were sacrificed. Blast percentage in blood, spleen and BM was 47, 58 and 40% respectively. We cultured cells from all three compartments ex vivo with AraC (25ng/ml). After 24 hours APL survival was 25, 80 and 60% respectively (p<0.006). We repeated the same experiment, but we did, in addition, a positive selection for CD34 to purify APL cells away from surrounding cells in the BM and spleen. Survival after ex vivo AraC incubation was 32, 30, 34% respectively (p=NS). In summary, CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. The interaction of APL cells with the BM and splenic microenvironments provides a survival benefit. Rapid mobilization of APL cells in vivo by AMD3100 interrupts APL-stromal interactions and sensitizes APL to chemotherapy. The impact of additional mobilizing agents on APL mobilization on sensitizing APL to chemo and radiotherapy will be presented. Finally, preliminary RNA profiling studies will be presented in an attempt to identify genes in APL cell that are differentially expressed when bound to and released from the BM.

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