Identification of CXCR4 Expression By Targeted PET/MR Imaging in Patients with Acute Myeloid Leukemia (AML)

Abstract Introduction AML is an aggressive hematological neoplasm originating from hematopoietic precursor or stem cells and is rapidly fatal if untreated. Although rates of complete remission (CR) approach 80% in patients with favorable risk profile according to European Leukemia Net classification, CR rates drop dramatically within the intermediate or adverse prognostic groups, and most patients relapse in spite of CR. The origin of refractory or relapsed disease is - according to several studies - the bone marrow (BM) niche that protects the leukemia initiating cells (LIC) from cytotoxic drugs.The chemokine receptor CXCR4 is a key factor mediating the crosstalk between the BM niche and LICs. CXCR4 is a G-protein coupled chemokine receptor with Stromal cell-derived factor-1α (SDF-1α) as its single known chemokine-ligand, and high expression is associated with poor prognosis in AML. Targeting the CXCR4/SDF-1αaxis with small peptide inhibitors or monoclonal antibodies has shown promising results in preclinical and clinical studies, thus rendering it an attractive target for future therapeutic strategies (Peled & Tavor, Theranostics, 2014). In this observational study we analyzed whether the new CXCR4-targeted positron emission tomography (PET)-probe [68Ga]-Pentixafor (Philipp-Abbrederis et al, EMBO Mol Med, 2015; Wester et al, Theranostics, 2015) is applicable for molecular imaging of patients with AML with the goal to develop a CXCR4- targeted peptide-receptor-radiotherapy (PRRT). Methods Using myeloid malignancies (MDS, active AML) as an exemplary CXCR4-expressing cancer entity we imaged 10 patients with the CXCR4 specific PET-probe [68Ga]-Pentixafor by whole body PET/magnetic resonance imaging (PET/MR) after they signed informed consent for this observational assessment. Maximum standardized uptake values (SUVmax) of the involved BM areas were compared to BM from patients without BM malignancy assessed in a different study. CXCR4 surface expression of leukemic BM cells of an unselected cohort of 67 patients with myeloid malignancies was analyzed by flow cytometry. CXCR4 expression in 14 established AML cell lines was measured by flow cytometry and quantitative PCR. Results  Out of 10 patients who were imaged with [68Ga]-Pentixafor-PET/MR, 4 - all of them with the diagnosis of an AML - showed visually positive PET signal of the BM. Those areas correlated well with disease infiltration as determined by MR imaging and the meanSUVmax was significantly higher as compared to visually CXCR4 negative patients (meanSUVmax 8.23 ± 5.23 vs. 2.26 ± 0.47; p=0.036). One of the 10 patients presenting with proven extramedullary relapse showed a CXCR4 positive PET signal within the lesion (SUVmax 5.12), whereas the cytological non-involved BM was PET negative (meanSUVmax 1.82). CXCR4 surface expression in established AML cell lines was variable and ranged from low to high, and correlated with mRNA levels. These data reflect the different expression values we found in the cohort of patients with myeloid malignancies. There was a trend to higher CXCR4 surface expression on blasts when AML patients were compared to MDS patients (median fluorescent intensity CXCR4/isotype 1.73 ± 0.25 vs. 0.96 ± 0.06; p=0.063) that became significant when blast frequency exceeds 30% (≤30% vs. >30% blasts: 1.07 ± 0.11 vs. 2.08 ± 0.38; p=0.006). In this patient cohort we did not observe any correlation between CXCR4 expression and factors such as diagnosis (secondary vs. de novo AML), disease status (refractory or relapse vs. no pretreatment), molecular subtypes or cytogenetic aberrations. Discussion Imaging of CXCR4 by PET/MR with [68Ga]-Pentixafor is feasible in patients with AML. Due to the higher uptake in leukemic BM compared to control BM and its low uptake in other organs (e.g. liver, brain and gut), CXCR4 could serve as an attractive new PRRT-target in selected patients with AML. Such PRRT approaches are of special interest in the setting of allogeneic BM transplantation as conventional myeloablative conditioning regimens - although they are associated with a better relapse free survival - show substantial rates of transplant-related mortality and are therefore not applicable in many AML patients due to their age. In particular, therapeutic targeting of CXCR4 by PRRT provides a unique opportunity of integrating a LIC/niche-directed treatment into the conditioning regimen. Such clinical studies are in development. Disclosures Wester: Scintomics GmbH: Other: General Manager. Keller:Roche: Consultancy, Honoraria; Pfizer: Consultancy.

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CXCR7 Regulates SDF-1 Induced Adhesion and Homing in Multiple Myeloma.

Blood ◽

10.1182/blood.v112.11.1674.1674 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1674-1674 ◽

Cited By ~ 2

Author(s):

Nicholas Burwick ◽

Anne-Sophie Moreau ◽

Xiaoying Jia ◽

Xavier Leleu ◽

Judith Runnels ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Adhesion ◽

Cell Lines ◽

Cxcr4 Expression ◽

Rt Pcr ◽

Growth And Survival ◽

Tumor Types

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

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Bone Marrow Interaction in Multiple Myeloma Pathogenesis: Phenotypical Analysis, Kinetics and Novel Therapy Approaches Based On CXCR4 Inhibition.

Blood ◽

10.1182/blood.v120.21.2450.2450 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2450-2450

Author(s):

Johannes Waldschmidt ◽

Dagmar Wider ◽

Marie Follo ◽

Josefina Udi ◽

Martina Kleber ◽

...

Keyword(s):

Flow Cytometry ◽

Adhesion Molecules ◽

Cell Lines ◽

Cxcr4 Expression ◽

Cell Trafficking ◽

Newly Diagnosed ◽

Infiltration Rates ◽

Dependent Cell ◽

And Migration

Abstract Abstract 2450 Introduction: The interaction between malignant plasma cells and their microenvironment is central in multiple myeloma (MM) pathogenesis. Binding of MM cells to bone marrow (BM) stroma cells triggers the expression of adhesion molecules and secretion of chemo- and cytokines, promoting MM cell growth, drug resistance and migration. Stromal-derived factor-1 (SDF-1) and its receptor CXCR4 are essential for normal hematopoietic progenitor cell movement and adherence within the BM microenvironment. In leukemia and lymphoma, oncoproteins may inhibit SDF-1-dependent cell trafficking within the BM through a mechanism that is not fully understood. For that reason, understanding SDF-1-dependent cell trafficking within the BM and targeting MM-cell - host-BM interactions display a promising approach for the development of novel therapeutic strategies. Methods: BM samples of MM patients (n=59) were analysed using flow cytometry and compared to MGUS patients (n=3) and healthy volunteers (n=7). We compared patient samples with low BM infiltration (≤5%; n=13) intermediate (5–30%; n=29) and high infiltration rates (≥30%; n=17). We also assessed expression of adhesion molecules in MM patients with long-term disease control (n=20) vs. both newly diagnosed (n=16) and symptomatic MM patients (n=23) as previously grouped by San Miguel et al. (Haematologica July 6,2012). We also sought to elucidate in vitro, whether specific anti-MM agents (bortezomib, vorinostat, pomalidomide, EGCG), with and without M210B4 stroma support, and with and without the CXCR4 inhibitor AMD3100, target the interaction of MM cells. Experiments were performed using MM cell lines (U266, RPMI8226, L363, NCI-H929), the control T-cell line MOLT-4 and MM-patient BM samples. Cell viability was assessed via Trypan Blue- and AnnexinV/PI-staining. CD138, CXCR4 (SDF1-receptor), CD49d (VLA-4), CD11a (LFA-1) and CD44 (HERMES antigen) expression was evaluated by flow cytometry and ScanR microscopy. Results: In BM samples of MM patients as compared to MGUS and healthy volunteers, the CXCR4/CD138- (p=.036), CD49d/CD138- (p=.0013) and CD44/CD138-expression (p=.0072) was significantly amplified and correlated with increasing BM infiltration rates (p=.001). Both newly diagnosed and symptomatic MM patients confirmed significantly increased CXCR4/CD138-, CD49d/CD138- (p=.0013) and CD44/CD138-expression as compared to patients with long-term disease control. Of note, in newly diagnosed patients, the expression of adhesion molecules was even more enhanced than in symptomatic myeloma patients, underlining their critical and future potential role as targets for novel therapeutics. Comparison of MM cell lines' adhesion and migration markers with that of MM-patient BM specimens revealed U266 as the cell line most closely resembling human specimens. Cytotoxic effects with use of MM cell lines and bortezomib, vorinostat and pomalidomide confirmed prior cytotoxic concentrations. Cocultivation with stroma substantially reduced apoptosis and induced tumor protective effects. Additional AMD3100 treatment restored sensitivity to bortezomib, vorinostat and pomalidomide. CXCR4 expression was substantially reduced after AMD3100 treatment, while that of CD49d, CD44 and CD11a remained widely unchanged. Toxic or therapeutic effects of AMD3100 monotherapy were excluded for used doses of 50μM. Additional use of ScanR microscopy visualized co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. ScanR microscopy results correlated with flow cytometry-determined CXCR4 expression. Ongoing analyses of both ScanR microscopy and flow cytometry will allow the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Conclusions: Our findings underline the critical role of adhesion and migration molecules in MM and may pave the way for novel therapeutic approaches targeting these microenvironmental mediators. Disclosures: No relevant conflicts of interest to declare.

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Extended Exposure To The CXCR4 Inhibitor Plerixafor May Lead To Enhanced Microenvironment Interactions in Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood.v122.21.1295.1295 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1295-1295

Author(s):

Edward Allan R. Sison ◽

Daniel Magoon ◽

Patrick Brown

Keyword(s):

Bone Marrow ◽

Adhesion Molecules ◽

Cell Lines ◽

Prolonged Exposure ◽

Surface Expression ◽

Cxcr4 Expression ◽

Antibody Binding ◽

Drug Binding ◽

Dose Dependent ◽

Extended Exposure

Abstract Background We have previously demonstrated that inhibition of CXCR4 in ALL decreases CXCR4 antibody binding, inhibits SDF-1α-(CXCL12)-induced chemotaxis, and overcomes chemotherapy resistance conferred by the bone marrow microenvironment. Specifically, we found that treatment with plerixafor and araC significantly decreased leukemic burden in a xenograft model of infant ALL, compared to treatment with araC alone. In those experiments, we treated mice on 3 consecutive days per week for 2 weeks with plerixafor and araC. However, the combination did not eradicate the leukemia in our model. We hypothesized that extended exposure to plerixafor may have led to increased interactions between surviving leukemic blasts and the bone marrow microenvironment. In our current experiments, we sought to characterize the effects of prolonged exposure to plerixafor in ALL. Methods/Results We treated pre-B (HB-1119, Nalm-6) and T (CCRF-CEM-1301, Jurkat) ALL cell lines with a dose range of plerixafor and harvested cells for FACS over an extended time course. We measured surface CXCR4 (s-CXCR4) expression using 3 antibodies: 12G5, which attaches to the SDF-1α and drug-binding site of CXCR4, and 1D9 and 2B11, which do not compete with SDF-1α or drug binding. 12G5 binding was decreased by plerixafor even at 1 hour and this effect was concentration-dependent. Interestingly, we found a time and dose-dependent increase in 1D9 and 2B11 antibody binding, suggesting that plerixafor caused an actual increase in s-CXCR4 over time. Increases in 1D9 and 2B11 binding were inversely proportional to decreases in 12G5 binding. We also measured surface expression of CD49d (VLA-4), which binds to fibronectin and VCAM-1; CXCR7, which binds to SDF-1α and CXCL11; and CXCR3, which binds to CXCL9, 10, and 11. We hypothesized that CXCR4 inhibition would lead to upregulation of parallel pathways of leukemia-stroma interactions. CD49d was highly expressed at baseline, while CXCR7 and CXCR3 were expressed to a lesser degree. Treatment with plerixafor led to dose-dependent increases in CXCR7 and variable changes in CD49d and CXCR3 surface expression, suggesting that plerixafor can modulate surface expression of adhesion molecules other than CXCR4. Next, we treated ALL cell lines with plerixafor (0, 10, 100 nM) for 72 hours, washed with PBS, and resuspended the cells in fresh medium to determine the effects of extended exposure to plerixafor and subsequent withdrawal. First, we measured surface expression of s-CXCR4 after 72 hours of treatment with plerixafor and found that 12G5 binding was decreased, while 1D9/2B11 binding was increased in an inversely proportional manner. After withdrawal, 12G5 binding increased to untreated levels between 4 and 24 hours, while 1D9/2B11 binding decreased to untreated levels between 4 and 72 hours. We also measured surface expression of CD49d, CXCR7, and CXCR3 and found that the effects of plerixafor treatment and withdrawal were variable by cell line. For example, after plerixafor treatment, surface expression of CD49d and CXCR7 was increased in Nalm-6 and surface expression of CXCR7 and CXCR3 was increased in CCRF-CEM-1301. Interestingly, 4 hours after plerixafor withdrawal, CD49d expression was increased in Jurkat and Nalm-6, and CXCR7 expression was increased in CCRF-CEM-1301, HB-1119, and Jurkat. Finally, we measured migration of washed cells from each treatment condition through a permeable membrane toward medium containing SDF-1α or medium alone. Despite CXCR4 inhibition for 72 hours, all plerixafor-treated cells migrated in response to SDF-1α. In addition, some plerixafor-treated cells exhibited significantly increased SDF-1α-induced chemotaxis compared to control-treated cells. These findings imply that increases in s-CXCR4 induced by 72 hours of treatment with plerixafor are functional. Conclusions Treatment of ALL cell lines with plerixafor led to a dose-dependent decrease in 12G5 antibody binding with a simultaneous overall increase in s-CXCR4 expression. Prolonged exposure to plerixafor led to increased s-CXCR4 expression that persisted for up to 72 hours after drug withdrawal, modulated surface expression of additional adhesion molecules, and enhanced SDF-1α-induced chemotaxis. Therefore, additional careful studies of CXCR4 inhibitors and other microenvironment-targeted agents must be performed in order to determine their optimal use in ALL. Disclosures: No relevant conflicts of interest to declare.

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Tryptophan Deprivation Promotes an Adaptive Response and Contributes to Bioenergetics in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-116447 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4511-4511 ◽

Cited By ~ 1

Author(s):

Alessandra Romano ◽

Nunziatina Laura Parrinello ◽

Katia Todoerti ◽

Piera La Cava ◽

Fabrizio Puglisi ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Adaptive Response ◽

Plasma Cells ◽

Synthetic Lethality ◽

Surface Expression ◽

Advisory Committees

Abstract Background and Objective : There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy.In multiple myeloma (MM) disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth. Our previous work showed that i) MM cells have metabolic plasticity due to availability of external nutrients; ii) aminoacid shortage due to the tryptohan (trp) degrading enzyme IDO-1 was associated to clinical MM progression. Thus, we tested bio-energetic changes in MM upon trp deprivation. Experimental design : We evaluated the trascriptome changes in the adaptive response in four human MM cell lines (MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c-myc and primary BM samples of MGUS and MM to trp shortage. The pathway was confirmed looked at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293). Results Trp deprivation induced an adaptive response through increased expression, time and dose-dependent, of ATF4-ASNS-CHOP-GADD34, part of GCN2 signaling, associated to increased expression of p62 (the main autophagic receptor in MM) followed by autophagy flux induction, as shown by flow cytometry and immunofluorescence evaluations. In MM cell lines, trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation, inducing in vitro increase of lactate dehydrogenase (LDH) and intracellular lactate and NAD/NADH with down-regulation of surface expression of the NAD-ase CD38. Similarly, short-term trp deprivation in CD138+ plasma cells isolated by magnetic sorting from MM patients, downregulated surface expression of CD38 and intra-cellular amount of c-myc, as detected by flow cytometry. This phenomenon was associated to increased isocitrate dehydrogenase (IDH-1), enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production. In the GSE66293 MM progression was associated to increased expression of ENO-1, PGK-1, and DLD, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant. Conclusion Overall, our study reveals that trp deprivation promotes an adaptive response of MM offering new putative targets in the bio-energetic pathway for synthetic lethality. Disclosures Palumbo: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.

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Monoclonal Antibody Against Sortilin Induces Apoptosis in Human Breast Cancer Cells

Avicenna Journal of Medical Biotechnology ◽

10.18502/ajmb.v14i1.8168 ◽

2022 ◽

Author(s):

Miganoosh Simonian ◽

Mozhan Haji Ghaffari ◽

Ali Salimi ◽

Ebrahim Mirzadegan ◽

Niloufar Sadeghi ◽

...

Keyword(s):

Flow Cytometry ◽

Cancer Cells ◽

Cell Lines ◽

Surface Expression ◽

Surface Flow ◽

Human Breast ◽

Promising Target ◽

Apoptosis Assay ◽

Mb231 Cell

Background: Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells. Methods: In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. Results: Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines. Conclusion: Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies.

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Chemokine Receptor CXCR4 Expression in Colorectal Cancer Patients Increases the Risk for Recurrence and for Poor Survival

Journal of Clinical Oncology ◽

10.1200/jco.2005.07.078 ◽

2005 ◽

Vol 23 (12) ◽

pp. 2744-2753 ◽

Cited By ~ 261

Author(s):

Joseph Kim ◽

Hiroya Takeuchi ◽

Stella T. Lam ◽

Roderick R. Turner ◽

He-Jing Wang ◽

...

Keyword(s):

Colorectal Cancer ◽

Liver Metastasis ◽

Cell Lines ◽

Chemokine Receptor ◽

Cancer Metastasis ◽

Cxcr4 Expression ◽

Signaling Mechanism ◽

Chemokine Receptor Cxcr4 ◽

Ajcc Stage ◽

Increased Risk

Purpose Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome. Methods CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival. Results High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001). Conclusion CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.

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CXCL12/CXCR4 Axis Drives Mitochondrial Trafficking in Tumor Myeloma Microenvironment

Blood ◽

10.1182/blood-2021-152175 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2663-2663

Author(s):

Cesarina Giallongo ◽

Ilaria Dulcamare ◽

Daniele Tibullo ◽

Vittorio Del Fabro ◽

Nunzio Vicario ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Selective Inhibitor ◽

Cxcr4 Expression ◽

Cell Contact ◽

Cxcl12 Expression ◽

Critical Pathways ◽

Mitochondrial Transfer ◽

Mitochondrial Trafficking ◽

Cxcr4 Axis

Abstract Mesenchymal stromal cells (MSCs) within the protective microenvironment of multiple myeloma (MM) promote tumor growth, confer chemoresistance and support metabolic needs of plasma cells (PCs) also transferring mitochondria. In this scenario, heterocellular communication and dysregulation of critical signaling axes are among the major contributors to progression and treatment failure. As metabolic rewiring is involved in the regulation of MSC phenotype, we first analyzed metabolic profile of healthy control (HC-) and MM-MSCs. NAD +/NADH ratio was decreased in MM-MSCs (n=8) as compared with HC-MSCs (n=4, p<0.05), meanwhile ATP/ADP ratio was not significantly different between the two groups. This led us to analyze whether MM-MSCs were much prone in transferring mitochondria than HC-MSCs. We first labeled HC- and MM-MSCs with Mitotracker Red CMXRos before co-culture with MM cells. After 24h of coculture, we quantified mitochondria transfer by flow cytometry. The obtained values were significantly higher in MM cells co-cultured with MM-MSCs (n=10) as compared to PCs co-cultured with HC-MSCs (n=5, p<0.01). In the cell-to-cell contact the gap junction-forming protein CX43 has been found critical for mitochondria uptake in lung and brain injury and it also can regulate CXCL12 secretion by MSCs. We found that MM-MSCs showed a significantly up-regulated CXCL12 expression as compared to HC-MSCs (p<0.001). Therefore, we co-cultured HS-5 cells with myeloma cell lines and observed that significantly increased CXCL12-CX43 colocalization in healthy MSCs. To evaluate the selective PC-induced activation of CXCL12 expression via CX43 in MSCs, we co-cultured HS-5 cells with MM cell lines and exposed cocultures to ioxynil octanoate (IO), a selective inhibitor of CX43-based gap junctions. We found that the up-regulation of CXCL12 induced by MM cells was reverted by exposition to the CX43 inhibitor, thereby indicating that CX43 activated by PCs regulates CXCL12 production in MSCs. Given that CX43 is involved in mitochondria trafficking, we subsequently cocultured MM cells with HS-5 in presence or not of IO. Our data showed that mitochondrial transfer was abolished by CX43 inhibitor. Given that MM PCs induced increased CX43 and CXCL12 colocalization in HS-5 cells, we supposed that CXCL12/CXCR4 signaling could regulate mitochondria trafficking throughout this axis. For this reason, we analyzed the kinetic of mitochondria uptake of several HMCLs and related their CXCR4 expression with the percentage of transferred mitochondria. Our data demonstrated that HMCLs with higher expression of CXCR4 had also higher percentage of transferred mitochondria both in time lapse and flow cytometry. The correlation between CXCR4 expression and the percentage of mitochondria uptake in HMCLs was also confirmed in primary myeloma PCs. Furthermore, plerixafor, a selective inhibitor of CXCR4, significantly reduced mitochondrial transfer from MSCs to myeloma PCs further establishing mechanistically that CXCR4/CXCL12 is directly involved in mitochondrial trafficking. Next, we investigated whether combination of plerixafor with bortezomib or carfilzomib interferes with mitochondrial transfer from MSCs to PCs. Interestingly, we found that the proteasome inhibitors promoted mitochondrial transfer while their combination with plerixafor inhibited mitochondria trafficking. Moreover, intracellular expression of CXCR4 in myeloma PCs from BM biopsy specimens demonstrated higher CXCR4 colocalization with CD138+ cells of non-responder patients to bortezomib compared with responder patients, suggesting that CXCR4 mediated chemoresistance in MM. In conclusion, we have shown that MM-MSCs are relatively low dependent on mitochondria metabolism and are inclined to transfer mitochondria to MM tumor cells. Furthermore, tumor PCs increase the expression of CX43 in MSCs leading to an increased levels of CXCL12 and stimulation of its corresponding receptor expressed on MM cells. The resulting CX43/CXCL12/CXCR4 interplay enhances mitochondrial trafficking from MSCs to myeloma PCs and can protect cancer cells against anti-myeloma agents. Understanding pro-tumorigenic phenotype of MSCs and mechanisms of adhesion and heterocellular communication favoring their interaction with cancer PCs, will allow to manipulate critical pathways, including CXCL12/CXCR4 axis, thus improving disease outcome. Disclosures Di Raimondo: Pfizer: Honoraria; AbbVie: Honoraria; Bristol Myers Squibb: Honoraria; Jazz Pharmaceutical: Honoraria; Janssen Pharmaceuticals: Honoraria; Amgen: Honoraria.

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Mimicking Myeloma Niche Ex Vivo

Blood ◽

10.1182/blood.v124.21.2076.2076 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2076-2076

Author(s):

Jana Jakubikova ◽

Danka Cholujova ◽

Richard W Groen ◽

Jungnam Joo ◽

Sun-Young Kong ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Tumor Cells ◽

Cell Lines ◽

Plasma Cells ◽

Ex Vivo ◽

3 Dimensional ◽

Bm Niche ◽

Hla Dr ◽

D Model

Abstract Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed. Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system. Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p<0.001) in 3-D vs. 2-D MM-MSCs. Similarly, activation of integrins including VLA-2, VLA-4 and VLA-5 on the MSCs surface was also increased in 3-D MM-MSCs, as determined by confocal microscopy and flow cytometry analysis. Importantly, MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche. Gene expression analyses of 3-D MM-MSCs revealed upregulation of BMP-2, MGP, PTGIS, COL14A1 and other genes and down-regulation of DKK1, ADAM9, OPCML genes and others compared to 2-D MM-MSCs. We also measured significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p<0.001) in 3-D vs. 2-D MM-MSCs, by multiplex luminex analysis. Next, we co-cultured tumor cells from MM patients (12 MM patients) with either autologous or allogeneic MM-MSCs in 3-D vs. 2-D model. Plasma (CD38/CD138+) cells in 3-D co-culture were increased in 8/12 MM patients and equivalent to 2-D in 4/12 patients. By co-culturing MM cell lines (OPM1, RPMI-S, OCIMY5 and KMS11) labeled with CFSE fluorescent dye with various MSCs, we evaluated expression of side population (SP) cells, identified by Hoechst staining, and gating on CFSE positive MM cells, as low Hoechst stained cells. Our results showed that the SP fraction was significantly lower in 3-D compared to 2-D in co-cultures of various MM-MSCs with all 4 MM cell lines. Finally, we validated drug resistance to melphalan, bortezomib, lenalidomide, and carfilzomib in 3-D co-cultures of CFSE labeled primary tumor cells with various MM-MSCs. Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. Disclosures No relevant conflicts of interest to declare.

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ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

10.1101/2020.01.31.926972 ◽

2020 ◽

Author(s):

Olivia J. Kelada ◽

Nicholas T. Gutsche ◽

Meghan Bell ◽

Rose M. Berman ◽

Kwamena E. Baidoo ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

Liver Cancer ◽

Cell Lines ◽

Human Liver ◽

Surface Expression ◽

Cell Surface Expression ◽

Human Liver Cancer ◽

Gpc3 Expression

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3−(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

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Mechanisms Regulating CXCR4 Expression in Myeloma Cells.

Blood ◽

10.1182/blood.v110.11.4748.4748 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4748-4748

Author(s):

Seong-Woo Kim ◽

Ha-Yeon Kim ◽

Hyo-Jin Lee ◽

Hwan-Jung Yun ◽

Samyong Kim ◽

...

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Surface Expression ◽

Myeloma Cell ◽

Cxcr4 Expression ◽

Cell Surface Expression ◽

Apoptotic Cells ◽

Myeloma Cells ◽

Hypoxic Conditions ◽

Tnf Α

Abstract The chemokine receptor CXCR4 plays roles in the homing of myeloma cells to the bone marrow (BM) and in the progression of the disease. However, the regulation of CXCR4 expression in myeloma cells is poorly defined. This study investigated the mechanisms regulating CXCR4 expression in myeloma cells. RPMI8226 and U266 myeloma cells strongly expressed CXCR4 on the cell surface, whereas ARH77 myeloma cells expressed minimal CXCR4 on the cell surface, as determined by flow cytometry using three different monoclonal antibodies to CXCR4. However, Western blot analysis, flow cytometry after permeabilization, and immunofluorescence staining reveled that ARH77 cells have abundant CXCR4 in the cytoplasm, in amounts similar to those in RPMI8226 and U266 cells. The cell surface expression of CXCR4 in primary CD138+ cells obtained from the BM of multiple myeloma patients differed among different specimens. Similar to myeloma cell lines, the primary myeloma cells that expressed minimal CXCR4 on the cell surface had abundant CXCR4 in the cytoplasm. The transmigration of the cells induced by stromal cell-derived factor-1 (SDF-1) was correlated with the cell surface expression of CXCR4, indicating that only the CXCR4 on the cell surface is functional. These findings suggest that myeloma cells have their own intrinsic mechanisms for regulating CXCR4 expression on the cell surface. In all three myeloma cell lines and in some primary BM CD138+ cells, dexamethasone (Dex) enhanced CXCR4 expression both in the cytoplasm and on the cell surface while downregulating SDF-1; this led to enhanced cell migration in response to SDF-1. Cell surface CXCR4 expression was more prominent in annexin V-positive apoptotic cells. VEGF and proinflammatory cytokines, including TNF-α and TGF-β1, also upregulated the cell surface expression of CXCR4 in RPMI8226 and some primary BM CD138+ cells, but not in U266 and ARH77 cells. Myeloma cells, including all the three cell lines and some primary BM CD138+ cells, incubated under hypoxic conditions (1% O2) exhibited upregulation of CXCR4 both in the cytoplasm and on the cell surface. Again, surface CXCR4 expression was stronger in apoptotic cells than in non-apoptotic cells, suggesting that the upregulation of CXCR4 is a counter-regulatory phenomenon for stimuli causing cell damage. As proven previously for other cell types, hypoxia induced the accumulation of HIF-1α in myeloma cells. Topotecan, which inhibits HIF-1α, attenuated the hypoxia-induced upregulation of CXCR4, whereas the proteasome inhibitor bortezomib slightly enhanced it. Dex, VEGF, TNF-α, and TGF-β1 all induced the accumulation of HIF-1α in RPMI8226 and some primary BM CD138+ cells. In addition, the effects of topotecan and bortezomib under hypoxic conditions were observed in the change of CXCR4 expression mediated by Dex and cytokines. These results indicate that complex intrinsic and extrinsic mechanisms regulate CXCR4 expression in myeloma cells and suggest that HIF-1α is a common regulatory molecule, at least in the extrinsic mechanisms.

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