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A Simple Flow Cytometry-Based Barcode for
Routine Authentication of Multiple Myeloma
and Mantle Cell Lymphoma Cell Lines
To the Editor:
CELL lines are widely used in laboratories for in vitro experi-
ments, especially for investigating abnormal hallmarks in can-
cer cells and identifying therapeutic targets. Human cell lines
are typically derived in academic laboratories from a wide
range of cancer samples. To achieve a representation of intra-
cancer heterogeneity, several laboratories, including ours, have
established cell line collections. However, the establishment
and maintenance of such collections significantly increase the
risk of cross-contaminations and misidentification of cell
lines, leading to the publication of false data/interpretation
(1). In addition to the risk of cross-contamination, widely
used cell lines can be described in contrasting manners for a
particular feature (e.g., the JJN3 myeloma cell line appears
either TP53
or TP53
, depending on the article), suggest-
ing that cell lines may have been misidentified. ICLAC, the
international cell line authentication committee, recommends
cell lines authentication using single tandem repeat (STR)
profiles that are usually performed by suppliers (2). Neverthe-
less, while cell lines are frozen and thawed at least four times
per year, STR profile is assessed upon receipt of cell lines but
not for routine assessment of cell lines identity. Because cross-
contaminations might happen, a rapid and low-cost method
for re-identification after each thawing of cells is required, as
it is for mycoplasma detection.
In this letter, we describe a simple and low cost method
involving human leukocyte antigen (HLA) typing and pheno-
typing that could be used for routine re-authentication using
flow cytometry. HLA typing is an international worldwide
nomenclature dedicated to blood transfusion and organ trans-
plant that identifies the HLA alleles carried by an individual
(3,4). Initially performed for research projects in immunology,
HLA Class I typing appears to be very useful for identification
of cell lines (5,6). The genomic typing is performed at the
generic (e.g., HLA-A*02) or specific (e.g., HLA-A*02:01) level
with the generic typing usually being sufficient to identify cell
lines within a dedicated collection. This genomic identifica-
tion is particularly useful upon the inclusion of new cell lines
within a collection and also for the establishment of deriva-
tives such as drug-resistant cell lines, which could indicate the
emergence of cryptic contaminating resistant cells within the
parental cell lines. Our laboratory currently uses a large num-
ber of both human multiple myeloma cell lines (HMCLs,
n517) and mantle cell lymphoma cell lines (MCLCLs, n58)
that we have collected from ATCC, DSMZ, or from academic
laboratories (7–9). Multiple myeloma (MM) and mantle cell
lymphoma (MCL) are plasma cell and B cell malignancies,
respectively. Independently of the global characterization of
cell lines (karyotype, gene expression profile, characterization
of TP53, and RAS mutations), the HLA Class I typing shown
in Supporting Information Table S1 confirms that the cell
lines examined were derived from independent individuals
(7). To routinely check their identity using flow cytometry, we
generated an algorithm that is based on HLA-A*02 expression
and on the mutually exclusive expression of the kappa or
lambda light chain of immunoglobulin. We used HLA-A*02
expression because HLA-A*02 is the most frequent allele in
the population and because a specific mAb is commercially
available. Expression of HLA-A*02, either positive or negative,
segregated cell lines into two groups: kappa or lambda expres-
sion then segregated the cell lines into 2–3 subgroups (Tables
1 and 2). A minimum of markers with a global, stable, and
selective expression (absent/present or low/bright expression)
was then defined within each subgroup. As shown in Table 1
Grant sponsor: Actions Cancer 44.
Additional Supporting Information may be found in the online version
of this article.
*Correspondence to: Catherine Pellat-Deceunynck, INSERM,
UMR892, Nantes, F-44000, France.
Sophie Ma
ıga, Carole Brosseau, G
eraldine Descamps, Christelle Dousset,
and Patricia Gomez-Bougie contributed equally to this work.
Published online 00 Month 2015 in Wiley Online Library
DOI: 10.1002/cyto.a.22643
C2015 International Society for Advancement of Cytometry
Cytometry Part A 00: 0000, 2015
Communication to the Editor
and Figure 1A, the HLA-A*02-cytoplasmic kappa/lambda
algorithm segregates HMCLs into five groups of 1–7 cell lines.
To further identify HMCLs within each group, we looked for
surface markers differentially expressed across cell lines. We
used gene expression profile to select molecules either
acquired or lost by malignant plasma cells and thus heteroge-
neously expressed by myeloma cells across both patients and
cell lines (7,10,11). We found nine markers (CD9, CD27,
CD33, CD45, CD81, CD106, CD117, CD137, FGFR3) that
were usually absent or present in an entire HMCL population
and efficient in segregating cell lines within the groups (Fig.
1A). Of note, some of them (CD27, CD33, CD45, CD117, or
FGFR3) are well-known myeloma-related deregulated
markers. The use of clonally related markers, such as HLA-
A*02 and kappa/lambda, may also help to identify cross-
contaminations that may occur after initial genomic authenti-
cation. As shown in Table 2 and Figure 1B, the HLA-A*02-
surface kappa/lambda algorithm in association with the differ-
ential expression of CD28, CD40, or CD5 discriminates the
eight MCLCLs (the MAVER-1 and MINO cell lines are discri-
minated using the differential levels in lambda and CD5
expression). Thus, HMCLs and MCLCLs are identified using
at least two (e.g., KARPAS620 or JEKO-1) and at most five
markers (ANBL-6).
Our authentication procedure of cell lines can be per-
formed in one half-day and does not require DNA. Moreover,
flow cytometry is powerful for the detection of very low cross-
contamination, which might increase during culture time and
repetitive freezing and thawing of cells. This algorithm can
also be used for STR-certified cell lines for which HLA Class I
Table 1. Algorithm for HMCLs.
KARPAS620 111 11 2 11 2 2 2 1 2 11 2 2 2
JIM3 1112 11 2 22 2 2 2 2 22 3
U266 1112 11 1 12 2 2 2 2 22 3
KMM1 11 2 11 11 2 1 2 2 2 2 2 2 3
L363 111 2 11 11 2 2 2 2 2 2 2 2 4
KMS11 2 11 2 2 2 2 111 2 2 111 2 2 3
NCIH929 2112 1221221
22 3
AMO1 2 11 2 11 11 2 2 2 2 2 2 2 3
JJN3 2 11 2 111 2 2 2 1
2 111
22 3
SKMM2 2 11 2 111 2 2 2 2 11
OPM2 2 2 11 111 2 2 111 2 2 2 2 2 3
LP1 2 2 11 2 2 2 1 2 2 2 2 2 3
RPMI8226 2 2 11 11 2 2 2 2 11 11 2 2 3
MM1S 2 2 11 2 2 2 2 2 2 111 2 2 3
ANBL6 221 2222221
22 5
KMS12BM 222
1 2 2 2 2 2 111 11 2 4
KMS12PE 222
11122 2 2 2 2 21 4
Expressed by a subpopulation.
KMS12BM and KMS12PE are non-secreting cell lines derived from the same patient and express no detectable level of lambda pro-
tein (both are weakly lambda positive at the mRNA level).
“Total” indicates the minimum number of markers required for cell line identification (the required markers are indicated by gray
shaded areas). Expression was determined using flow cytometry. The monoclonal antibodies (mAbs) used were PE-conjugated, except
for CD117 and FGFR3 mAbs (APC conjugated) and for CD45 mAb (FITC conjugated). The level of expression was defined by calculating
the ratio of fluorescence (specific staining over matched-conjugated isotype staining).
Ratio <2: 2.
2<ratio <10: 1.
10<ratio <50: 11.
Ratio>50: 111.
Table 2. Algorithm for MCLCLs
JEKO-1 11 11 2 2 1 1 2
GRANTA-519 111 2 11 1 1 2 3
JVM2 11 2 1 212 3
REC-1 2 112 212 3
UPN-1 2 111 2 2 2 2 3
Z138 2 2 111 2 1 2 3
MAVER-1 2 2 111 2 1 1 3
MINO 2 2 11 21113
“Total” indicates the minimum number of markers required
for cell line identification (the required markers are indicated by
gray shaded areas). Expression was determined using flow
cytometry. The monoclonal antibodies (mAbs) used were PE-
conjugated, except for CD40 mAb (FITC conjugated). The level of
expression was defined by calculating the ratio of fluorescence
(specific staining over matched-conjugated isotype staining).
Ratio <2: 2.
2<ratio <10: 1.
10<ratio <50: 11.
Ratio>50: 111.
Communication to the Editor
typing is unknown because flow cytometry directly assesses
HLA-A*02 expression. This HLA-A*02-based algorithm is
applicable not only to other types of B-cell malignancies but
also to other types of cell line collections if both type-specific
(such as kappa/lambda for B cells) and selective markers are
Figure 1. Histograms represent the overlay of specific staining (thick line) over control staining (thin line) in HMCLs (A) or MCLCLs (B).
The monoclonal antibodies (mAbs) used were purchased from Beckman Coulter or Becton Dickinson: they were PE-conjugated, except
for CD117 and FGFR3 mAbs (APC conjugated) and for CD40 and CD45 mAbs (FITC conjugated). Cytoplasmic kappa (c-kappa) and c-
lambda staining was performed after the permeabilization of cells using the Intraprep Permeabilization Reagent Kit (Beckman Coulter). A
single color staining was performed for all markers. Fluorescence acquisition (20,000 events were acquired) and analysis were performed
using FACsCalibur (Becton Dickinson) and Cell Quest software (PT Cytocell, SFR Bonamy, Nantes, France).
Communication to the Editor
Cytometry Part A 00: 0000, 2015 3
Sophie Ma
Carole Brosseau,
eraldine Descamps,
Christelle Dousset,
Patricia Gomez-Bougie,
David Chiron,
Emmanuelle M
Charlotte Kervoelen,
Henri Vi
Anne Cesbron,
`s Moreau-Aubry,
Martine Amiot,
Catherine Pellat-Deceunynck
INSERM, UMR892, Nantes F-44000, France
e de Nantes, Nantes F-44000, France
CNRS, UMR 6299, Nantes F-44000, France
CHU Nantes, Nantes
F-44000, France
Centre d’investigation Clinique, CHU de
Nantes, Nantes F-44000, France
Myelomax SAS, Nantes, France
Laboratoire d’Histocompatibiliteet
etique, Etablissement Franc¸ais du
Sang Pays de la Loire, Nantes F-44000, France
1. Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RA,
Masters JR, Nakamura Y, Reid YA, Reddel RR, et al. Check your cultures! A list of
cross-contaminated or misidentified cell lines. Int J Cancer 2010;127:1–8.
2. Dirks WG, MacLeod RA, Nakamura Y, Kohara A, Reid Y, Milch H, Drexler HG,
Mizusawa H. Cell line cross-contamination initiative: An interactive reference data-
base of STR profiles covering common cancer cell lines. Int J Cancer 2010;126:303–
3. Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Fernandez-
Vina M, Geraghty DE, Holdsworth R, Hurley CK, et al. Nomenclature for factors of
the HLA system, 2010. Tissue Antigens 2010;75:291–455.
4. Marsh SG, Albert ED, Bodmer WF, Bontrop RE, Dupont B, Erlich HA, Fernandez-
Vina M, Geraghty DE, Holdsworth R, Hurley CK, et al. An update to HLA nomen-
clature, 2010. Bone Marrow Transplant 2010;45:846–848.
5. Pellat-Deceunynck C, Jego G, Harousseau JL, Vie H, Bataille R. Isolation of human
lymphocyte antigens class I-restricted cytotoxic T lymphocytes against autologous
myeloma cells. Clin Cancer Res 1999;5:705–709.
6. Adams S, Robbins FM, Chen D, Wagage D, Holbeck SL, Morse HC III, Stroncek D,
Marincola FM. HLA class I and II genotype of the NCI-60 cell lines. J Transl Med
7. Moreaux J, Klein B, Bataille R, Descamps G, Maiga S, Hose D, Goldschmidt H,
Jauch A, Reme T, Jourdan M, et al. A high-risk signature for patients with multiple
myeloma established from the molecular classification of human myeloma cell lines.
Haematologica 2011;96:574–582.
8. Brosseau C, Dousset C, Touzeau C, Maiga S, Moreau P, Amiot M, Le Gouill S, Pellat-
Deceunynck C. Combination of lenalidomide with vitamin D3 induces apoptosis in
mantle cell lymphoma via demethylation of BIK. Cell Death Dis 2014;5:e1389.
9. Touzeau C, Dousset C, Bodet L, Gomez-Bougie P, Bonnaud S, Moreau A, Moreau P,
Pellat-Deceunynk C, Amiot M, Le Gouill S. ABT-737 induces apoptosis in mantle
cell lymphoma cells with a Bcl-2high/Mcl-1low profile and synergizes with other
antineoplastic agents. Clin Cancer Res 2011;17:5973–5981.
10. Bataille R, Jego G, Robillard N, Barille-Nion S, Harousseau JL, Moreau P, Amiot M,
Pellat-Deceunynck C. The phenotype of normal, reactive and malignant plasma cells.
Identification of “many and multiple myelomas” and of new targets for myeloma
therapy. Haematologica 2006;91:1234–1240.
11. Rawstron AC, Orfao A, Beksac M, Bezdickova L, Brooimans RA, Bumbea H, Dalva
K, Fuhler G, Gratama J, Hose D, et al. Report of the european myeloma network on
multiparametric flow cytometry in multiple myeloma and related disorders. Haema-
tologica 2008;93:431–438.
Communication to the Editor
... Granta-519 cells express high levels of cyclin D1 due to the chromosomal translocation of t(11;14)(q13;q32), but are deficient for ATM. 11,[13][14][15] In contrast to Granta-519 cells, the cell line Z-138 was derived from the bone marrow of a blastoid mantle cell lymphoma patient 16 Z-138 cells express normal levels of ATM and relatively higher levels of cyclin D. 16 In terms of cell surface markers, Granta-519 and Z-138 cell lines both lack CD5 expression and express lambda light chain and CD19 surface proteins. 14-16 Unlike Granta-519, Z-138 cells do not express HLA-A2 on their surface. ...
... 14-16 Unlike Granta-519, Z-138 cells do not express HLA-A2 on their surface. Thus, this marker can serve to distinguish between the two cell lines 14,16 We confirmed these differences by flow cytometry in our cultures (Data not shown). ...
Full-text available
Mantle cell lymphoma (MCL) presents a therapeutic challenge. The B cell targeting agent, ibrutinib, is currently one of the most effective second‐line therapies for MCL, but frequently leads to development of drug resistance, and short overall survival time upon relapse. Olaparib targets tumor cells with deficiencies in single‐strand DNA break repair and thus may slow the development of genetic drug resistance. We found that the olaparib‐ibrutinib combination significantly inhibits cell culture growth compared to either drug alone in two genetically distinct MCL cell lines. Moreover, these inhibitory effects are either additive or synergistic, depending on genetic background. Culture growth is inhibited due to increases in apoptosis, cell death, and cell cycle arrest, and the magnitude of each is cell line dependent. The additive and synergistic inhibition of this combination additionally supports a therapeutic strategy involving lower dosing of each drug to reduce potential side effects.
... HMCLs were previously characterized [1,6,7]. HMCLs were cultured in RPMI-5% fetal calf serum with or without 3 ng/ml of IL6 [1,6,7]. ...
... HMCLs were previously characterized [1,6,7]. HMCLs were cultured in RPMI-5% fetal calf serum with or without 3 ng/ml of IL6 [1,6,7]. Gene expression profile of HMCLs has been previously published [1]. ...
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Background: Human myeloma cell lines (HMCLs) are widely used for their representation of primary myeloma cells because they cover patient diversity, although not fully. Their genetic background is mostly undiscovered, and no comprehensive study has ever been conducted in order to reveal those details. Methods: We performed whole-exon sequencing of 33 HMCLs, which were established over the last 50 years in 12 laboratories. Gene expression profiling and drug testing for the 33 HMCLs are also provided and correlated to exon-sequencing findings. Results: Missense mutations were the most frequent hits in genes (92%). HMCLs harbored between 307 and 916 mutations per sample, with TP53 being the most mutated gene (67%). Recurrent bi-allelic losses were found in genes involved in cell cycle regulation (RB1, CDKN2C), the NFκB pathway (TRAF3, BIRC2), and the p53 pathway (TP53, CDKN2A). Frequency of mutations/deletions in HMCLs were either similar to that of patients (e.g., DIS3, PRDM1, KRAS) or highly increased (e.g., TP53, CDKN2C, NRAS, PRKD2). MAPK was the most altered pathway (82% of HMCLs), mainly by RAS mutants. Surprisingly, HMCLs displayed alterations in epigenetic (73%) and Fanconi anemia (54%) and few alterations in apoptotic machinery. We further identified mutually exclusive and associated mutations/deletions in genes involved in the MAPK and p53 pathways as well as in chromatin regulator/modifier genes. Finally, by combining the gene expression profile, gene mutation, gene deletion, and drug response, we demonstrated that several targeted drugs overcome or bypass some mutations. Conclusions: With this work, we retrieved genomic alterations of HMCLs, highlighting that they display numerous and unprecedented abnormalities, especially in DNA regulation and repair pathways. Furthermore, we demonstrate that HMCLs are a reliable model for drug screening for refractory patients at diagnosis or at relapse.
... Human myeloma cell lines (HMCLs) (n = 26) and MM1S Dexamethasone resistant cell line (MM1SDR) were characterized as previously described 27,28 ...
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Multiple myeloma is a plasma cell malignancy that escapes from apoptosis by heterogeneously over-expressing anti-apoptotic BCL2 proteins. Myeloma cells with a t(11;14) translocation present a particular vulnerability to BCL2 inhibition while a majority of myeloma cells relies on MCL1 for survival. The present study aimed to determine whether the combination of BCL2 and MCL1 inhibitors at low doses could be of benefit for myeloma cells beyond the single selective inhibition of BCL2 or MCL1. We identified that half of patients were not efficiently targeted neither by BCL2 inhibitor nor MCL1 inhibitor. Seventy percent of these myeloma samples, either from patients at diagnosis or relapse, presented a marked increase of apoptosis upon low dose combination of both inhibitors. Interestingly, primary cells from a patient in progression under venetoclax treatment were not sensitive ex vivo to neither venetoclax nor to MCL1 inhibitor, whereas the combination of both efficiently induced cell death. This finding suggests that the combination could overcome venetoclax resistance. The efficacy of the combination was also confirmed in U266 xenograft model resistant to BCL2 and MCL1 inhibitors. Mechanistically, we demonstrated that the combination of both inhibitors favors apoptosis in a BAX/BAK dependent manner. We showed that activated BAX was readily increased upon the inhibitor combination leading to the formation of BAK/BAX hetero-complexes. We found that BCLXL remains a major resistant factor of cell death induced by this combination. The present study supports a rational for the clinical use of venetoclax/S63845 combination in myeloma patients with the potential to elicit significant clinical activity when both single inhibitors would not be effective but also to overcome developed in vivo venetoclax resistance.
... Методы определения подлинности КЛ постоянно совершенствуются. На протяжении многих лет для аутентификации клеток применялись такие методы, как изоферментный анализ, кариотипирование, HLA-типирование и иммунофенотипирование [5,6]. Однако в последнее десятилетие наиболее широкое распространение получили молекулярно-биологические методы определения подлинности КЛ. ...
Full-text available
Short tandem repeat analysis (STR) is a well-established international method of authentication and genetic stability testing of cell lines (CLs). Therefore, the development and introduction of this method into routine practice of cell banks and cell culture collections is a pressing concern. In addition, the expansion of the field of cell-line based biomedical cell products (BСPs) necessitates the implementation of STR as a tool of identification testing during quality control. The State Pharmacopoeia of the Russian Federation does not require mandatory use of STR for cell line identification, while other countries have been using this method for cell line quality control for about a decade. The use of identified CLs in medical practice will ensure the efficacy and safety of BCPs. The aim of the study was to assess the possibility of using STR analysis for authentication and genetic stability testing of CLs using U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs as examples. Materials and me­thods : the following human CLs were used in the study: U937 (ECACC), WISH (ATCC), WIL2S (ATCC), NK-92 (ATCC), and Jurkat Clone E6-1 (ATCC). The CL allelic profiles were determined by STR using the COrDIS Plus kit (Gordiz, Russia). The electrophoretic separation was performed using a Genetic Analyzer 3500 Series instrument. The data provided on the websites of the European Collection of Authenticated Cell Cultures and American Type Culture Collection were used to compare the CL profiles. Results : the AuthentiFiler PCR Amplification Kit (Thermo Fisher Scientific, USA) and the GenePrint 10 System (Promega Corporation, USA) intended for CL authentication by STR were compared with the characteristics of the COrDIS plus kit (Gordiz, Russia). The results of the comparison demonstrated that the COrDIS plus kit includes all the loci found in the foreign kits, as well as the loci recommended by the International Cell Line Authentication Committee. The U-937, WIL2S, and NK-92 CLs demonstrated genetic identity with the reference profiles available on the websites of the international collections. The Jurkat Clone E6-1 CL was found to be genetically instable due to the loss of the amelogenin gene. Conclusions : it was demonstrated by the examples of U937, WISH, WIL2-S, NK-92, and Jurkat Clone E6-1 CLs that STR and the COrDIS plus kit could be used for authentication and genetic stability testing. The obtained results suggest the feasibility of using the COrDIS plus kit for the analysis of CLs used in BCPs, for BCP quality control, and biomedical research.
... However, routine controls, such as cell authentication and contamination testing, are required to minimize behavioral changes due to cross-contamination with other cell lines, mycoplasma contamination, or phenotypic modifications caused by extensive culturing [30,31]. Therefore, a rapid and low-cost method for cell identity confirmation after each thawing is required, as currently done for mycoplasma detection [32]. A variety of methods are available for authentication testing. ...
Full-text available
Multiparameter flow cytometry (MFC) is a fast and cost-effective technique to evaluate the expression of many lymphoid markers in mature B-cell neoplasms, including diffuse large B cell lymphoma (DLBCL), which is the most frequent non-Hodgkin lymphoma. In this study, we first characterized by MFC the expression of 27 lymphoid markers in 16 DLBCL-derived cell lines to establish a robust algorithm for their authentication. Then, using the expression profile in DLBCL samples of the genes encoding B lymphoid markers that are routinely investigated by MFC, we built a gene expression-based risk score, based on the expression level of BCL2, BCL6, CD11c, and LAIR1, to predict the outcome of patients with DLBCL. This risk score allowed splitting patients in four risk groups, and was an independent predictor factor of overall survival when compared with the previously published prognostic factors. Lastly, to investigate the potential correlation between BCL2, BCL6, CD11c, and LAIR1 protein level and resistance to treatment, we investigated the response of the 16 DLBCL cell lines to cyclophosphamide, etoposide, doxorubicin, and gemcitabine. We found a correlation between BCL6 overexpression and resistance to etoposide. These results show the interest of MFC for the routine characterization of DLBCL cells and tumors samples for research and diagnostic/prognostic purposes.
... NTS-3 and NTS-4 have been generated in our laboratory (CRCINA) [12]. Cell lines are routinely identified using a flow cytometry-based barcode as previously described [28], as well as MHC class I sequencing and are tested for mycoplasma contamination. Values for MCL cell lines are the mean of at least three independent experiments. ...
Full-text available
The microenvironment strongly influences mantle cell lymphoma (MCL) survival, proliferation, and chemoresistance. However, little is known regarding the molecular characterization of lymphoma niches. Here, we focused on the interplay between MCL cells and the associated monocytes/macrophages. Using circulating MCL cells (n = 58), we showed that, through the secretion of CSF1 and, to a lesser extent, IL-10, MCL polarized monocytes into specific CD163+ M2-like macrophages (MϕMCL). In turn, MϕMCL favored lymphoma survival and proliferation ex vivo. We next demonstrated that BTK inhibition abrogated CSF1 and IL-10 production in MCL cells, leading to the inhibition of macrophage polarization and consequently resulting in the suppression of microenvironment-dependent MCL expansion. In vivo, we showed that CSF1 and IL-10 plasma concentrations were higher in MCL patients than in healthy donors, and that monocytes from MCL patients overexpressed CD163. Further analyses of serial samples from ibrutinib-treated patients (n = 8) highlighted a rapid decrease of CSF1, IL-10, and CD163 in responsive patients. Finally, we showed that targeting the CSF1R abrogated MϕMCL-dependent MCL survival, irrespective of their sensitivity to ibrutinib. These data reinforced the role of the microenvironment in lymphoma and suggested that macrophages are a potential target for developing novel therapeutic strategies in MCL. Access :
... We showed that activity of auranofin and Prima-1 Met correlated in myeloma cells and that both drugs induced a Bax/Bak-independent cell death. extensively characterized (10,15,16). TP53 status was performed by direct sequencing of RT-PCR products (16) and by whole exon sequencing (17). ...
Full-text available
Prima-1Met (APR-246) was previously shown to be dependent on glutathione inhibition and on ROS induction in cancer cells with mutated or deleted TP53. Because this ROS induction was, at least in part, due to a direct interference with the thioredoxin reductase enzyme, we investigated whether activity of Prima-1Met could be mimicked by auranofin, an inhibitor of the thioredoxin reductase. We thus compared the activity of auranofin and Prima-1Met in 18 myeloma cell lines and in 10 samples from patients with multiple myeloma or plasma cell leukemia. We showed that, similar to Prima-1Met, the activity of auranofin was not dependent on either TP53 status or p53 expression; was inhibited by N-acetyl-L-cysteine, a ROS scavenger; displayed a dramatic synergy with L-buthionine sulfoximine, an irreversible inhibitor of glutathione synthesis; and induced cell death that was not dependent on Bax/Bak expression. These data showed that auranofin and Prima-1Met similarly overcome cell death resistance in myeloma cells due to either p53 deficiency or to mitochondrial dysfunction.
... All cell lines used in this study have been extensively characterized. [27][28][29][30][31] TP53 and RAS mutations were performed by whole-exon sequencing 32 and confirmed by direct sequencing of reverse transcription polymerase chain reaction (RT-PCR) products. 29 p53 deficiency was confirmed by resistance to nutlin3a. ...
Full-text available
In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53 abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53 wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53wt cell lines, but not the TP53 abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.
... Human myeloma cell lines (HMCLs) were characterized as previously described. 16 After informed consent, MM bone marrow/blood samples were collected at University Hospital of Nantes. ...
Full-text available
BH3 mimetics are promising drugs for hematologic malignancies that trigger cell death by promoting the release of proapoptotic BCL2 family members from antiapoptotic proteins. Multiple myeloma is considered to be a disease dependent mainly on MCL1 for survival, based mostly on studies using cell lines. We used a BH3-mimetic toolkit to study the dependency on BCL2, BCLXL, or MCL1 in malignant plasma cells from 60 patients. Dependencies were analyzed using an unbiased BH3 mimetics cell-death clustering by k-means. In the whole cohort of patients, BCL2 dependency was mostly found in the CCND1 subgroup (83%). Of note, MCL1 dependence significantly increased from 33% at diagnosis to 69% at relapse, suggesting a plasticity of the cellular dependency favoring MCL1 dependencies at relapse. In addition, 35% of overall patient samples showed codependencies on either BCL2/MCL1 or BCLXL/MCL1. Finally, we identified a group of patients not targeted by any of the BH3 mimetics, predominantly at diagnosis in patients not presenting the common recurrent translocations. Mechanistically, we demonstrated that BAK is crucial for cell death induced by MCL1 mimetic A1210477, according to the protection from cell death observed by BAK knock-down, as well as the complete and early disruption of MCL1/BAK complexes on A1210477 treatment. Interestingly, this complex was also dissociated in A1210477-resistant cells, but free BAK was simultaneously recaptured by BCLXL, supporting the role of BCLXL in A1210477 resistance. In conclusion, our study opens the way to rationally use venetoclax and/or MCL1 BH3 mimetics for clinical evaluation in myeloma at both diagnosis and relapse.
Full-text available
Mantle cell lymphoma (MCL) accumulates in lymphoid organs but disseminates early on in extranodal tissues. Although proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem, few studies have assessed MCL microenvironment. We therefore cocultured primary circulating MCL cells from 21 patients several weeks ex vivo with stromal or lymphoid-like (CD40L) cells to determine which interactions could support their proliferation. We showed that coculture with lymphoid-like cells, but not stromal cells, induced cell-cycle progression, which was amplified by MCL-specific cytokines (IGF-1, BAFF, IL-6, IL-10). Of interest, we showed that our model recapitulated the MCL in situ molecular signatures i.e., proliferation, NFkB and survival signatures. We further demonstrated that proliferating MCL harbored an imbalance in Bcl-2 family expression leading to a consequent loss of mitochondrial priming. Interestingly, this loss of priming was overcome by the Type II anti-CD20 antibody obinutuzumab, which counteracted Bcl-xL induction through NFkB inhibition. Finally, we showed that the mitochondrial priming directly correlated with the sensitivity toward venetoclax and alkylating drugs. By identifying the microenvironment as the major support for proliferation and drug resistance in MCL, our results highlight a selective approach to target the lymphoma niche.
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Mantle cell lymphoma (MCL) is a currently incurable B-cell malignancy. Lenalidomide (Len) has been demonstrated to be one of the most efficient new treatment options. Because Len and 1α,25-dihydroxyvitamin (VD3) synergize to kill breast cancer cells, we investigated whether VD3 could increase the ability of Len to induce MCL cell death. While MCL cells were weakly sensitive to Len (1 μM), the addition of VD3 at physiological dose (100 nM) strongly increased cell death, accompanied by slowdown in cell cycle progression in MCL cell lines (n=4 out of 6) and primary samples (n=5 out of 7). The Len/VD3 treatment markedly increased the expression of the BH3-only BCL2-interacting killer (Bik) without affecting the expression of other Bcl-2 molecules. Immunoprecipitation assays demonstrated that Bik was free from anti-apoptotic partners, Bcl-2 and Bcl-xL, in treated cells. Moreover, silencing of BIK prevented apoptosis induced by Len/VD3, confirming the direct involvement of Bik in cell death. Bik accumulation induced by Len/VD3 was related to an increase in BIK mRNA levels, which resulted from a demethylation of BIK CpG islands. The sensitivity of MCL cells to Len/VD3 was similar to the response to 5-azacytidine, which also induced demethylation of BIK CpG islands. These preclinical data provide the rationale to investigate the role of VD3 in vivo in the response to Len.
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Mantle cell lymphoma (MCL) is considered to be incurable. ABT-737 is a BH3 mimetic that targets Bcl-2, which is overexpressed in MCL and implicated in drug resistance. The present work investigated the antitumor effect of ABT-737. Six MCL cell lines and primary MCL cells (n = 13) were used. Sensitivity to ABT-737 was assessed, and expression levels of Bcl-2 and Mcl-1 were analyzed. Finally, ABT-737 was combined with other cytotoxic agents to promote tailored therapy. MINO and GRANTA-519 cell lines were highly sensitive to ABT-737 [the median lethal dose (LD₅₀) = 20 and 80 nmol/L, respectively], whereas other cell lines were resistant. In primary MCL cells, 46% of patients' samples were sensitive to ABT-737. The analysis of protein expression levels revealed that both sensitive cell lines and primary MCL cells could be characterized by a Bcl-2(high)/Mcl-1(low) profile, whereas resistant MCL cells contained high levels of Mcl-1. ABT-737 induced a rapid disruption of both Bcl-2/Bax and Bcl-2/Bik complexes. In addition, silencing of Mcl-1 by siRNA sensitized MCL cell lines to ABT-737. Similarly, flavopiridol, which induces Mcl-1 downregulation, in combination with ABT-737 led to a synergistic anti-MCL effect in ABT-737-resistant cell lines. This synergy was also observed when ABT-737 was combined with either bortezomib or cytarabine. The present work shows that ABT-737 induces strong apoptosis in MCL cells expressing a Bcl-2(high)/Mcl-1(low) profile. In ABT-737-resistant MCL cells, downregulation of Mcl-1 overcomes Mcl-1-induced resistance and synergizes ABT-737 effects. Our results strongly support the use of ABT-737 according to the Bcl-2/Mcl-1 tumor cell profiles in the treatment of MCL.
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Multiple myeloma is a plasma-cell tumor with heterogeneity in molecular abnormalities and treatment response. We have assessed whether human myeloma cell lines have kept patients' heterogeneity using Affymetrix gene expression profiling of 40 human myeloma cell lines obtained with or without IL6 addition and could provide a signature for stratification of patient risk. Human myeloma cell lines, especially those derived in the presence of IL6, displayed a heterogeneity that overlaps that of the patients with multiple myeloma. Human myeloma cell lines segregated into 6 groups marked by overexpression of MAF, MMSET, CCND1, FRZB with or without overexpression of cancer testis antigens (CTA). Cell lines of CTA/MAF and MAF groups have a translocation involving C-MAF or MAFB, cell lines of groups CCND1-1 and CCND1-2like have a t(11;14) and cell lines of group MMSET have a t(4;14). The CTA/FRZB group comprises cell lines that had no or no recurrent 14q32 translocation. Expression of 248 genes accounted for human myeloma cell line molecular heterogeneity. Human myeloma cell line heterogeneity genes comprise genes with prognostic value for survival of patients making it possible to build a powerful prognostic score involving a total of 13 genes. Human myeloma cell lines derived in the presence of IL6 recapitulate the molecular diversity of multiple myeloma that made it possible to design, using human myeloma cell line heterogeneity genes, a high-risk signature for patients at diagnosis. We propose this classification to be used when addressing the physiopathology of multiple myeloma with human myeloma cell lines.
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The WHO Nomenclature Committee for Factors of the HLA System met following the 14th International HLA and Immunogenetics Workshop in Melbourne, Australia in December 2005 and Buzios, Brazil during the 15th International HLA and Immunogenetics Workshop in September 2008. This report documents the additions and revisions to the nomenclature of HLA specificities following the principles established in previous reports (1-18).
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The WHO Nomenclature Committee for Factors of the HLA System met during the 15th International Histocompatibility and Immunogenetics Workshop in Buzios, Brazil in September 2008. This update is an extract of the main report that documents the additions and revisions to the nomenclature of human leukocyte antigen (HLA) specificities following the principles established in previous reports. Bone Marrow Transplantation (2010) 45, 846-848; doi: 10.1038/bmt.2010.79; published online 29 March 2010 HLA; Nomenclature; update
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Peripheral blood T cells from a patient with multiple myeloma in complete remission were selected in vitro against an autologous myeloma cell line (SBN-1), using a protocol designed for the selection of relatively rare precursor cytotoxic T cells (pCTL). Delayed addition (2 weeks) of interleukin 2 induced T-cell proliferation, and a bulk culture (T-cell line) was obtained 2 days later. This T-cell line displayed cytotoxicity against SBN-1. A CD8+ CD4- cytotoxic T-cell clone (CT5) was then obtained that recognized SBN-1 but not autologous EBV+ B-lymphoblastoid cells, autologous T PHA-blasts, or Daudi, Raji, K562, and 11 allogeneic myeloma cell lines. Moreover, CT5 cytotoxic activity against SBN-1 was blocked by monoclonal antibodies recognizing human lymphocyte antigen class I molecules. This seems to be the first demonstration of myeloma-specific pCTL in peripheral blood T cells of patients with multiple myeloma.
Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.
Dear Sir, Recent reports 1–4 demonstrate the growing perception in the scientific community that cross contamination (CC) of mammalian cell lines represents a major risk for generating false scientific data. The level to which research has been compromised by the use of contaminated or misidentified cell lines has become a major concern for scientists, granting agencies, and, increasingly, scientific journals. In 2007, a group of cell biologists led by Roland M. Nardone petitioned the United States Secretary of Health and Human Services to develop an active program for cell line authentication. 5 They stressed that research and teaching tools in diverse fields of science and industry would be unimaginable without cell cultures. Despite the key importance of cell cultures, only little consensus exists regarding the technical means by which cell line identity can be controlled and how to follow through the results of any such testing. The key problems of CC are known and chronic in nature: neglecting guidelines for quality control and disregarding adequate cell culture techniques are the main reasons why cell lines have been misidentified or cross contaminated. The incidence of CC in directly and indirectly provenanced cell lines alike 1,3 implies that the majority of false cell lines are perpetrated in originators’ own laboratories, presumably by failures during the establishment of new cell lines. A plethora of reports unmasking bogus cancer cell lines, including members of the NCI-60 panel used to generate reference baseline transcriptional drug responses has triggered calls for remedial action. 5,6 Nevertheless, standard authentication procedures for testing cell line identity have yet to be defined. Short tandem repeat (STR) microsatellite sequences are highly polymorphic in human populations, and their stability throughout the lifespan of individuals renders STR profiling (typing) ideal for forensic use. STR typing has served as a reference technique for identity control of human cell lines at Biological Resource Centers (BRCs) since the turn of the millennium. 7 Ideally, authentication involves coincident STR