A Recombinant HLA Class I-Specific Single-Chain Fv Diabody Can Target Cancer Stem Cell-Like Side Population Cells in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2799-2799
Author(s):  
Akishige Ikegame ◽  
Shuji Ozaki ◽  
Daisuke Tsuji ◽  
Takeshi Harada ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Side Population ◽  
Hla Class I ◽  
Class I ◽  
Chemotherapeutic Drugs ◽  
Single Chain ◽  
Single Chain Fv ◽  
Rpmi 8226

Abstract Abstract 2799 Poster Board II-775 Multiple myeloma (MM) remains an incurable disease despite high overall response rates induced by combination therapy of chemotherapeutic drugs and new agents such as thalidomide, lenalidomide, and bortezomib. Recently, the existence of cancer stem cells is proposed for several tumors including MM, and such cells are considered as an important target for curative therapy. The side population (SP) cells are identified by their ability to efflux Hoechst 33342 dye, which represent a small fraction with stem cell properties. Our previous studies have demonstrated that MM cells expressed HLA class I at high levels than normal hematopoietic cells, and that antibodies against HLA class I specifically induced MM cell death by Rho-mediated actin aggregation. In this study, we characterized SP fraction in MM cell lines (RPMI 8226, U266, and MM.1S) and primary MM cells (n=3) by flow cytometry, and investigated the efficacy of chemotherapeutic drugs as well as a recombinant single-chain Fv diabody specific to HLA class I (C3B3-DB, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan). MM cell lines and primary MM cells contained a distinct fraction of SP cells ranging from 0.01% to 0.6% of the gated cells, which was confirmed by disappearance after treatment with verapamil. Treatment with melphalan (10 μM, 48 hours) decreased the percentage of non-SP cells (16.7% to 3.9%) but not of SP cells (0.6% to 0.7%) in RPMI 8226. In contrast, treatment with C3B3-DB (1 μg/ml, 48 hours) caused a significant reduction in both non-SP cells (16.7% to 4.0%) and SP cells (0.6% to 0.3%) in RPMI 8226. Similar results were observed in primary MM cells enriched from patient bone marrow cells by negative selection with antibody cocktail. Next, we isolated SP cells and non-SP cells in RPMI 8226 using a cell sorter, and characterized in detail. SP cells exhibited elevated levels of ABCG2 and low levels of CD138 compared with non-SP cells, but HLA class I was expressed at high levels in both SP and non-SP cells by flow cytometry. Annexin/PI assay showed that SP cells were 1.5- and 2.0-fold more resistant to melphalan and bortezomib than non-SP cells. Whereas both SP cells and non-SP cells showed similar sensitivity to C3B3-DB. Methylcellulose colony-forming assay showed that SP cells have a higher potential for colony formation (numbers of colonies, 13.0±1.0) than non-SP cells (1.3±0.6), and the colony formation of SP cells was significantly inhibited by C3B3-DB (0.7±0.6, p<0.01). Notably, RPMI 8226 cells expressed the pluripotency-associated transcription factors including Oct3/4, Sox2, and Nanog as detected by RT-PCR, but only Sox2 mRNA expression was decreased at 6 hours after C3B3-DB treatment. Furthermore, when C3B3-DB-treated SP cells were inoculated subcutaneously in SCID mice (n=4), there was a significant decrease in a tumor volume as compared with untreated SP cells (679±148mm3 vs 3217±562 mm3, p<0.01). SP cell analysis of these tumors showed that the percentage of SP fraction of C3B3-DB-treated SP cell tumors was significantly low (0.01%) compared with that of untreated SP cell tumors (0.33%). These results indicate that C3B3-DB has a potential activity for eradicating MM cancer stem cell-like SP cells, and that the molecular targeting of such drug-resistant cells provides an important strategy for improving the efficacy of current therapies in MM. Disclosures: No relevant conflicts of interest to declare.

HLA Class I Typing of Platelets by Multi-Colour Flow Cytometry - A New Powerful Tool for Assessing Survival, Activation Status and Activation Capacity of Transfused Platelets Vs. Autologous Platelets and for Determination of Stem Cell Engraftment After Allogeneic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 22-22 ◽  
Author(s):  
Annette Vetlesen ◽  
Pål Andre Holme ◽  
Torstein Lyberg ◽  
Jens Kjeldsen-Kragh
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Allogeneic Stem Cell Transplantation ◽  
Hla Class I ◽  
Class I ◽  
Hla B27 ◽  
Cell Engraftment ◽  
Activation Capacity

Abstract Abstract 22 Studies of in vivo survival of transfused platelets (PLTs) are usually performed by tracing PLTs labelled with radioactive isotopes. The aim of the present work was to develop a flow cytometry-based method without involving manipulation of PLTs before transfusion, where differences in HLA class I molecules between donor and recipient might be used to trace transfused PLTs. Of 14 fluorochrome-conjugated HLA class I monoclonal antibodies (mAbs) from 7 suppliers only 3 (anti-HLA A2, anti-HLA A9 and anti-HLA B27) were found satisfactory for HLA class I typing of PLTs. As earlier studies have claimed the existence of a considerable exchange of HLA class I antigens between plasma and PLTs, a series of experiments were conducted to examine the exchange of HLA A2, A9 and B27 class I antigens during storage for 4 days. Three pairs of HLA +ve and HLA −ve PLT aphaeresis products were collected in autologous plasma for each of the specificities HLA A2, HLA A9 and HLA B27. Cell-free plasma was prepared from an equal number of HLA +ve and HLA −ve whole blood units with the same specificities. Plasma-free PLTs and cell-free plasma were mixed and stored in PLT storage bags at 22°C in the following combinations: HLA +ve PLTs in HLA −ve plasma, HLA +ve PLTs in HLA +ve plasma, HLA −ve PLTs in HLA −ve plasma and HLA −ve PLTs in HLA +ve plasma. Samples from each PLT concentrate-mix were transferred to sterile non-treated culture plates and stored on a flatbed mixer at 37°C in an atmosphere of 5% CO2 in humidified air. HLA class I surface expression was tested daily during storage. The difference in % between pos PLTs in neg plasma and pos PLTs in pos plasma was in the range of 0.8 – 1.2 at 22°C and 0.8 – 3.3 at 37°C for HLA A2; 3.3 – 1.7 at 22°C and 3.5 – 0 at 37°C for HLA A9, and 3.9 – 1.9 at 22°C and 0 – 2.1 at 37°C for HLA B27. Percent-wise difference between neg PLTs in pos plasma and neg PLTs in neg plasma was in the range of 6.9 – 10 at 22°C and 7.7 – 12.5 at 37°C for HLA A2; 4.5 – 0 at 22°C and 13.6 – 6.5 at 37°C for HLA A9, and 5.3 – 5.3 at 22°C and 5.6 – 4.8 at 37°C for HLA B27. These results indicate that the amounts of eluted vs. adsorbed HLA class I antigens are negligible at both 22°C and 37°C. Hence, this method was applied in a clinical setting to study in vivo survival of transfused PLTs. A patient was transfused with PLTs of non-self HLA class I types and tracing and testing of the transfused PLTs were performed by daily measuring of HLA class I surface expression by multi-colour flow cytometry. PLTs were identified by light scatter properties and expression of CD41. The anti-HLA class I mAbs were used to distinguish transfused PLTs from autologous PLTs. The activation status of transfused PLTs was determined by using anti-CD63. PLT activation capacity was further determined by examining the expression of CD63 on different PLT populations before and after stimulation with thrombin receptor agonist peptide (SFLLRN). In a 52 years old patient with AML undergoing allogeneic stem cell transplantation early stem cell engraftment could be detected by following the PLT production (see figure). After the transplantation the number of autologous HLA A2 +ve PLTs decreased gradually. From day 8 till day 10 there was a 50% decrease in total PLT count, while the number of HLA A2 +ve PLTs was low but stable. From day 10 and onwards a gradual increase of HLA A2 +ve PLTs was seen independently of transfusion. These results were interpreted as engraftment from day 8. The standard criteria for stem cell engraftment - a neutrophile count of > 0, 1 × 109/L - occurred 5 days later in this patient. It was also found that the transfused PLTs in the patient's circulation were not activated, evaluated by CD63 expression, and that the residual activation capacity of the transfused PLTs were reduced compared with the capacity of the autologous PLTs (40% up regulation of CD63 vs. 70%; as a comparison there was a 90% up regulation of CD63 in a healthy individual). In conclusion, HLA class I typing by flow cytometry represents a powerful tool for studies of transfused PLTs. The method can 1) be used to study the survival of 3 different populations of transfused PLTs individually, 2) give early evidence of stem cell engraftment after allogeneic stem cell transplantation, 3) be used to assess in vivo activation of transfused PLTs and 4) be used for functional studies of transfused vs. autologous PLTs, which to our knowledge, is the first time this has been possible. Disclosures: No relevant conflicts of interest to declare.

Multi-Drug Resistant Leukemic Cells Highly Express HLA Class I Molecules and Single-Chain Fv Diabody Specific to HLA-A Overcomes Drug Resistance in These Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2376-2376
Author(s):  
Ali Jalili ◽  
Shuji Ozaki ◽  
Naoki Kimura ◽  
Masahiro Abe ◽  
Toshio Matsumoto
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Hla Class I ◽  
Resistant Cell ◽  
Class I ◽  
Single Chain ◽  
Expression Levels ◽  
Hla Class I Molecules ◽  
Single Chain Fv ◽  
Mdr 1

Abstract Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters. Specifically, p-glycoprotein (MDR-1) is frequently up-regulated in chemotherapy-resistant tumor cells, which is associated with poor prognosis. On the other hand, human leukocyte antigen (HLA) class I molecules are known to be significantly down-regulated in advanced tumor cells to escape from immune surveillance. However, the relationship between MDR-1 expression and HLA expression is not fully understood. Recently, we have developed a recombinant single-chain Fv diabody specific to HLA-A and demonstrated that this agent mediates cell death in HLA-overexpressing lymphoid tumor cells but not in normal cells (Cancer Res2007; 67:1184). Here, we investigated the expression levels of HLA class I in chemo-resistant leukemic cells and evaluated the therapeutic potential of single-chain Fv diabody specific to HLA-A, C3B3-DB (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan). Chemotherapy-resistant cells were established by subculturing of myeloid leukemia cell line HL60 and Burkitt’s lymphoma cell line BL-TH in increasing doses of vincristine (VCR), and named HL60/VCR and BL-TH/VCR, respectively. MDR-1 is strongly expressed in HL60/VCR and BL-TH/VCR cells both at the mRNA and protein levels, but not in the parental cells. Interestingly, expression levels of HLA class I molecules are 8 times higher in HL60/VCR and BL-TH/VCR cells than in the parental cells, suggesting that MDR-1 modulates cell surface expression of HLA by its transporter function. Next, we examined the cytotoxic activity of C3B3-DB on these chemo-resistant cell lines. C3B3-DB induced apoptosis in HL60/VCR and BL-TH/VCR cells and these chemo-resistant cell lines were more sensitive to C3B3-DB than the parent cells. Combination of C3B3-DB with chemotherapeutic agents such as VCR and daunorubicin (DNR) resulted in enhanced cytotoxicity against HL60/VCR and BL-TH/VCR cells. Importantly, pretreatment of these chemo-resistant cell lines with C3B3-DB reduced expression levels of MDR-1 and increased drug retention in these cells as detected by flow cytometry and confocal microscopy. Furthermore, combination of C3B3-DB with VCR significantly blocked the cell cycle at the G2 phase compared with VCR alone. Similar results were obtained with primary acute myeloid leukemia cells from 2 patients, resulting in up-regulation of both HLA class I and MDR-1 molecules at relapse phase compared at diagnosis. These results suggest that C3B3-DB enhances cytotoxicity of chemotherapeutic agents and provides a novel approach for overcoming drug resistance in hematological malignancies.

scholarly journals PIWIL1 Drives Chemoresistance in Multiple Myeloma by Modulating Mitophagy and the Myeloma Stem Cell Population

2022 ◽  
Vol 11 ◽  
Author(s):  
Yajun Wang ◽  
Lan Yao ◽  
Yao Teng ◽  
Hua Yin ◽  
Qiuling Wu
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Stem Cell ◽  
Cell Population ◽  
Side Population ◽  
Chemotherapeutic Agents ◽  
Stem Cell Population ◽  
Exact Function

As an important member of the Argonaute protein family, PIWI-like protein 1 (PIWIL1) plays a key role in tumor cell viability. However, the exact function of PIWIL1 in multiple myeloma (MM) and the underlying mechanism remain unclear. Here, we revealed that PIWIL1 was highly expressed in myeloma cell lines and newly diagnosed MM patients, and that its expression was notably higher in refractory/relapsed MM patients. PIWIL1 promoted the proliferation of MM cells and conferred resistance to chemotherapeutic agents both in vitro and in vivo. More importantly, PIWIL1 enhanced the formation of autophagosomes, especially mitophagosomes, by disrupting mitochondrial calcium signaling and modulating mitophagy-related canonical PINK1/Parkin pathway protein components. Mitophagy/autophagy inhibitors overcome PIWIL1-induced chemoresistance. In addition, PIWIL1 overexpression increased the proportion of side population (SP) cells and upregulated the expression of the stem cell-associated genes Nanog, OCT4, and SOX2, while its inhibition resulted in opposite effects. Taken together, our findings demonstrated that PIWIL1 induced drug resistance by activating mitophagy and regulating the MM stem cell population. PIWIL1 depletion significantly overcame drug resistance and could be used as a novel therapeutic target for reversing resistance in MM patients.

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