224 CHARACTERIZATION OF EQUINE ENDOMETRIAL-DERIVED MESENCHYMAL STROMAL CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 243
Author(s):  
E. Rink ◽  
H. French ◽  
E. Watson ◽  
C. Aurich ◽  
F. X. Donadeu
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Magnetic Beads ◽  
Type I ◽  
Trilineage Differentiation ◽  
Significant Difference ◽  
Cell Fractions

Equine mesenchymal stromal cells (MSC) are mainly harvested from bone marrow and adipose tissue, requiring surgical procedures. Although human endometrium is known to harbor mesenchymal precursor cells, the presence of MSC in equine endometrium, a dynamic tissue, has not been investigated. This study reports for the first time the culture and characterisation of MSC from equine endometrium compared with equine bone marrow (BM)-derived MSC. Samples of equine endometrium (n = 6) and BM (n = 3) were collected postmortem. Endometrial tissue was digested using a dissociation medium containing collagenase I and DNase type I, and CD227 (mucin-1)-bound magnetic beads were utilised to separate epithelial (CD227+) from stromal (CD227–) cell fractions. Red blood cells from BM samples were excluded using a density gradient. All cell fractions were cultured in DMEM/F-12 containing 10% fetal bovine serum. After expansion, colony-forming unit (CFU) assay at passage 2, trilineage differentiation (adipogenic, chondrogenic, osteogenic), and flow cytometry analysis at passage 3/4 were performed for CD227– fractions and BM-MSC. Descriptive statistical analysis and 2-tailed t-test was performed with IBM SPSS Statistics 22 (SPSS Inc./IBM, Chicago, IL, USA). Both isolated cell fractions were plastic adherent and grew well under standard MSC culture conditions, although endometrial CD227– cells attached quicker to culture plasticware than did BM-MSC. The CFU assay at passage 2 showed no significant difference in cloning efficiency (CE) between BM-MSC (20.78 ± 2.86%) and CD227– (24.89 ± 3.04%) cell lines (P = 0.36). Flow cytometry showed the expression of MSC markers (CD29, CD44, CD90, CD105) and perivascular markers (CD146, NG2) but almost no expression of haematopoietic markers (CD34, CD45) in both cell lines (Table 1). No statistically relevant difference was seen except for the higher expression of NG2 in BM-MSC (P = 0.054). Trilineage differentiation was successfully induced in both cell lines. In conclusion, we showed the presence of putative MSC in equine endometrium. We successfully isolated and cultured these cells, which display comparable characteristics in MSC criteria as well-established BM-derived MSC. These endometrial-derived MSC may provide a convenient source for veterinary regenerative therapies in equine reproduction. Table 1.Flow cytometry marker expression (mean ± standard error) at passage 4

Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

2018 ◽  
Vol 205 (4) ◽  
pp. 226-239 ◽  
Author(s):  
Marijana Skific ◽  
Mirna Golemovic ◽  
Kristina Crkvenac-Gornik ◽  
Radovan Vrhovac ◽  
Branka Golubic Cepulic
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Immunological Tolerance ◽  
Biological Properties ◽  
Platelet Lysate ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Significant Difference

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

scholarly journals Dual Activation of NRF2 in Multiple Myeloma and Bone Marrow Mesenchymal Stromal Cells Regulates Chemotherapy Resistance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3287-3287 ◽  
Author(s):  
Yu Sun ◽  
Lyubov Zaitseva ◽  
Manar S Shafat ◽  
Kristian M Bowles ◽  
Stuart A Rushworth
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Transcriptional Activation ◽  
Proteasome Inhibitors ◽  
Heme Oxygenase 1 ◽  
Antioxidant Genes ◽  
Pharmacological Inhibition

Abstract Background The cornerstone treatments of multiple myeloma (MM) are proteasome inhibitors bortezomib (BZ) and carfilzomib (CFZ). However, MM still remains incurable for that MM cells rapidly develop resistance to chemotherapy. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathways have been shown to contribute to the malignant phenotypes of several cancers through effects on proliferation and drug sensitivity. NRF2 functions to rapidly change the sensitivity of the cells environment to oxidants and electrophiles by stimulating the transcriptional activation of drug metabolism and antioxidant genes. NRF2 is negatively regulated by proteasome degradation through its inhibitor KEAP1. The aim of this study was to determine if proteasome inhibitor induced NRF2 signalling orchestrates survival of MM in the bone marrow (BM) microenvironment. Methods To investigate the role of NRF2 in the MM microenvironment primary human MM and BM mesenchymal stromal cells (MSC) were obtained under UK ethical approval (LREC ref 07/H0310/146). NRF2 activity in MM and BM-MSC was measured by NRF2 protein expression, target genes expression and using promoter assays. Lentiviral mediated shRNA knockdown of NRF2 in the MM and BM-MSC. The NRF2 inhibitor, brusatol was used to verify the knockdown experiments. Results Results show that primary MM and MM cell lines have increased NRF2 activity in response to the proteasome inhibitors BZ and CFZ as measured by increased nuclear NRF2, increased NRF2 regulated genes and increased ARE activity in the promoter of heme oxygenase-1. Expression of basal NRF2 was high in the majority of primary MM cells and cell lines tested. Pharmacological inhibition and shRNA mediated knock-down of NRF2 showed a significant reduction in survival of MM cells, when treated alone and in combination with BZ or CFZ. Investigations also revealed that BM-MSC had increased NRF2 activity in response to BZ and CFZ. Moreover, knockdown of NRF2 in BM-MSC or pharmacological inhibition of NRF2 in BM-MSC/MM co-cultures reverses the protection conferred to MM by BM-MSC in response to BZ and CFZ. Conclusion: Here we show the first description of NRF2 driven cytoprotective responses in MM. We show that NRF2 in MM is activated by both BZ and CFZ which subsequently activates pro-survival mechanisms in response to proteasome inhibition. Furthermore, NRF2 is also activated in the BM microenvironment by BZ and CFZ, which also confers protection to MM. This highlights the importance of NRF2 in regulating MM drug resistance within the BM microenvironment through independent actions in both the tumour and the non-malignant BM-MSC which support it. Disclosures Rushworth: Infinity Pharmaceuticals: Research Funding.

scholarly journals Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2228-2228
Author(s):  
Xin Long ◽  
Laszlo Perlaky ◽  
Tsz-Kwong Chris Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clumping and Viability of Bone Marrow Derived Mesenchymal Stromal Cells under Different Preparation Procedures: A Flow Cytometry-Based In Vitro Study

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Li-li Cui ◽  
Tuure Kinnunen ◽  
Johannes Boltze ◽  
Johanna Nystedt ◽  
Jukka Jolkkonen
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Normal Saline ◽  
Stromal Cells ◽  
Pulse Width ◽  
In Vitro Study ◽  
High Viability ◽  
Storage Solutions

Complications of microocclusions have been reported after intra-arterial delivery of mesenchymal stromal cells. Hence, quantification and efficient limitation of cell clumps in suspension before transplantation is important to reduce the risk. We used a flow cytometry-based pulse-width assay to assess the effects of different cell suspension concentrations (0.2–2.0 × 106/mL), storage solutions (complete growth medium, Dulbecco’s phosphate-buffered saline, and normal saline), storage time in suspension (0–9 h), and freeze-thawing procedure on the clumping of rat bone marrow derived mesenchymal stromal cells (BMMSCs) and also evaluated cell viability at the same time. Surprisingly, increasing the cell concentration did not result in more cell clumps in vitro. Freshly harvested (fresh) cells in normal saline had significantly fewer cell clumps and also displayed high viability (>90%). A time-dependent reduction in viability was observed for cells in all three storage solutions, without any significant change in the clumping tendency except for cells in medium. Fresh cells were more viable than their frozen-thawed counterparts, and fresh cells in normal saline had fewer cell clumps. In conclusion, cell clumping and viability could be affected by different cell preparation procedures, and quantification of cell clumping can be conducted using the flow cytometry-based pulse-width assay before intra-arterial cell delivery.

scholarly journals Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Xue Luo ◽  
Hongxiao Li ◽  
Xiaolin Sun ◽  
Qisheng Zuo ◽  
Bichun Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blot ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ionic Current ◽  
Immunofluorescence Staining ◽  
Patch Clamp Technique ◽  
Marker Proteins ◽  
Myocardial Marker

Aim. Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation. Methods. Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins α-actin and cTnT were detected by immunofluorescence staining, while α-actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents. Results. Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, α-actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties. Conclusion. HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.

GD2+ Selected Mesenchymal Stromal Cells (MSCs) Demonstrate a More Robust Proliferation and Differentiation Potential Compared to Unselected Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1919-1919
Author(s):  
Caridad Martinez ◽  
Ted J. Hofmann ◽  
Roberta Marino ◽  
Massimo Dominici ◽  
Edwin M. Horwitz
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Doubling Time ◽  
Blue Staining ◽  
Differentiation Potential ◽  
Proliferation And Differentiation

Abstract Human mesenchymal stromal cells (MSCs) are spindle-shape, plastic-adherent cells with capacity to differentiate to bone, cartilage, and fat. MSCs express fibroblast, endothelial, and lymphocyte antigens, e.g. CD105, CD73, CD90, and CD166 which are the cornerstone of phenotypic characterization of these cells. We recently showed that MSCs are the only bone marrow cell to express GD2, a neural ganglioside. Now, for the first time we show that GD2 may serve as the single, unique, and definitive marker of marrow and adipose derived MSCs that can be used to isolate GD2+ MSCs, which possess important biologic properties justifying prospective isolation. MSCs expression of GD2 is uniformly high on freshly isolated and culture-expanded cells. Using the Miltenyi AutoMACS® device and a monoclonal antibody recognizing GD2 (clone 14.G2A) we prospectively isolated a highly enriched MSC population from bone marrow MNCs. The selected fraction was &gt;98% pure for GD2+ cells determined by flow cytometry. Light microscopy showed that the GD2-selected cells were smaller, thinner, and more spindle-like when attached to plastic compared to unselected MSCs which spread wider along the surface of the culture flask, the so-called “fried egg” appearance. The doubling time of GD2-selected MSCs was 30 hrs compared to 90 hrs for unselected cells representing a 3-fold greater growth rate. Cell cycle analysis by flow cytometry showed ∼80% of cells were in G0/G1 and ∼20% were in S/G2/M phases of the cell cycle in both populations. With the shorter doubling time, this data indicates that GD2-selected MSCs move through the cell cycle more rapidly than unselected cells. In accordance with this finding, electron microscopy showed few organelles in the GD2-selected cells, but increase lamellar bodies indicating overall less complexity, but consistent with a greater membrane turnover rate (cell division) than unselected MSCs. Moreover, flow cytometric analysis revealed an increased expression of receptors for bFGF and EFG, known mitogenic factor receptors for MSCs, compared to unselected MSCs. In vitro differentiation of GD2-selected MSCs showed a more robust osteoid matrix formation (osteoblast) and proteoglycan formation (chondroblast) assayed by semi-quantitative Alizarin Red and Alcian blue staining, respectively. Additionally, more GD2-selected MSCs differentiated to adipocytes than among unselected cells. Surprisingly, GD2 expression persisted on the in vitro human MSC-differentiated osteoblasts, chondroblasts, and adipocytes, in contrast to human bone-derived osteoblasts, adipose tissue, and cartilage which lacked GD2 expression. We conclude that GD2 is a unique, stably expressed surface MSC marker which can be used to prospectively isolate MSCs from marrow, GD2-selcted cells have a more robust in vitro proliferation and differentiation potential which may be valuable for cell therapy, and biologically, in vitro isolated MSCs may not represent the in vivo progenitor for bone, fat, or cartilage.

scholarly journals Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli

2019 ◽  
Vol 20 (14) ◽  
pp. 3607 ◽  
Author(s):  
Merel Pool ◽  
Tim Eertman ◽  
Jesus Sierra Parraga ◽  
Nils ’t Hart ◽  
Marieke Roemeling-van Rhijn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Adipose Tissue ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Human Adipose Tissue ◽  
Machine Perfusion ◽  
Normothermic Machine Perfusion ◽  
Glomerular Capillaries

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

scholarly journals Molecular and Functional Phenotypes of Human Bone Marrow-Derived Mesenchymal Stromal Cells Depend on Harvesting Techniques

2020 ◽  
Vol 21 (12) ◽  
pp. 4382 ◽  
Author(s):  
Sebastian Walter ◽  
Thomas Randau ◽  
Cäcilia Hilgers ◽  
El-Mustapha Haddouti ◽  
Werner Masson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cellular Therapy ◽  
Donor Site ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Differentiation Potential ◽  
Significant Difference ◽  
Harvesting Techniques

Mesenchymal stromal cells (MSC) harvested in different tissues from the same donor exhibit different phenotypes. Each phenotype is not only characterized by a certain pattern of cell surface markers, but also different cellular functionalities. Only recently were different harvesting and processing techniques found to contribute to this phenomenon as well. This study was therefore set up to investigate proteomic and functional properties of human bone marrow-derived MSCs (hBM-MSC). These were taken from the same tissue and donor site but harvested either as aspirate or bone chip cultures. Both MSC populations were profiled for MSC markers defined by the International Society for Cellular Therapy (ISCT), MSC markers currently under discussion and markers of particular interest. While classic ISCT MSC markers did not show any significant difference between aspirate and outgrowth hBM-MSCs, our additional characterization panel revealed distinct patterns of differentially expressed markers. Furthermore, hBM-MSCs from aspirate cultures demonstrated a significantly higher osteogenic differentiation potential than outgrowth MSCs, which could be confirmed using a transcriptional approach. Our comparison of MSC phenotypes obtained by different harvesting techniques suggests the need of future standardized harvesting, processing and phenotyping procedures in order to gain better comparability in the MSC field.

Role Of Folate Receptor Targeted Therapy In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4436-4436
Author(s):  
Sonali Panchabhai ◽  
Katalin Kelemen ◽  
Sinto Sebastian Chirackal ◽  
Rafael Fonseca
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Targeted Therapy ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Newly Diagnosed ◽  
Tumoricidal Activity ◽  
Significant Difference

In multiple myeloma (MM) the interaction of plasma cells, bone marrow stromal cells and tumor associated macrophages (TAMs) plays a significant role in conferring resistance to therapy and in the maintenance of residual disease. Folate receptors (FR) are specifically expressed on metabolically active malignant cells and TAMs and their expression in normal tissues and resting macrophages is limited. FR mediates folate uptake by receptor-mediated endocytosis. This qualifies the receptor to be exploited for drug delivery of folate conjugated cancer therapeutics. Our primary goal in this study is to evaluate the expression of functional FR in MM cells and TAMs with a view to exploit this for folate conjugated targeted therapy for MM. First we evaluated the presence of TAMs in paraffin embedded bone marrow slides of newly diagnosed MM patients with CD68 (pan-Macrophage marker) and CD163 antibodies (specific marker of TAMs) and found extensive infiltration of macrophages in bone marrow from MM patients. Next to evaluate expression of FR in MM cells, we employed an FR antibody and evaluated MM cell lines with immunoblot, flow cytometry and confocal microscopy. In a panel of ten MM cell lines, we found that out of the three FR isoforms (α, β and γ), FR beta (FR-β) is expressed by all of them. Next to test whether this expressed receptor is indeed functional, we incubated the cells with folate deficient media, added different concentrations of EC17 (folate conjugated to FITC, Endocyte Inc.) to the medium and looked for the uptake by flow cytometry after washing off the drug at different time points (after 10, 20, 40 and 60 min).We observed that the uptake begins in 10 min and is saturated at 1 hour with 100nM Folate-FITC. With confocal imaging, Folate-FITC was found in the cytoplasm of MM cells suggesting internalization of Folate-FITC and localization in the cytoplasm. In addition to myeloma cell lines, we also confirmed the uptake of Folate-FITC in CD138+ plasma cells of a newly diagnosed myeloma patient by flow cytometry. This strongly suggests that MM cells express functional FR-β. To test the specificity of this FR mediated uptake, we pre-incubated cells with 0.1mM folic acid in medium for 30 min and then added EC17. This maneuver blocked the activity mediated by FR and no uptake was observed , which proves that the Folate-FITC is internalized only through the FR. To evaluate the expression of FR in-vivo samples, we stained paraffin embedded bone marrow slides of newly diagnosed MM patients with FR-β antibody and TAM specific markers. We observe that FR is expressed on both MM cells as well as TAMs. To assess the endurance of cytotoxic effect of folate conjugated chemotherapeutic agents, we treated MM cell lines with folate conjugated vinka alkaloids and compared them to unconjugated drug and found no significant difference in their action suggesting conjugation with folate does not alter its efficacy. To assess potential toxicity of folate conjugated therapeutics, we obtained CD34+ cells and looked for the uptake of Folate-FITC with flow cytometry. We found no uptake and this is in line with previous reports suggesting that CD34 positive cells express nonfunctional FR. So we propose that FR qualify as potential targets for cancer treatment. Folate targeted therapy using folate-conjugated drugs which can selectively act against both MM cells and supporting TAMs has the potential of specific anti-MM tumoricidal activity. This therapeutic approach would broaden the use of drugs that could be conjugated with folate for MM therapy. Additionally assessment of TAMs in bone marrow sections of MM patients would add another feature for grading, classifying and prognosticating MM. Disclosures: Fonseca: Cylene: Research Funding; AMGEN: Consultancy; Millennium: Consultancy; Binding Site: Consultancy; Onyx: Consultancy, Research Funding; Lilly: Consultancy; BMS: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Medtronic: Consultancy; Otsuka: Consultancy; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.

Sign in / Sign up

Export Citation Format

Share Document