Biology subjects

Bennaceur Griscelli, A.

Publications and source records attributed to Bennaceur Griscelli, A..

5 recordsLinked to original sources

bioRxiv2026

EFFICIENT EXPANSION OF NK-92 CELL LINE USING A NOVEL LOW-SHEAR STRESS BIOREACTOR

BackgroundThe use of autologous or allogeneic cell therapies has now entered to the clinical practice in several fields of medicine, especially in oncology and hematology. From this regard, 2D-cell manufacturing is complex and costly and bioreactors have attracted major interest for efficient and cost-effective mass production of cells. Bioreactors have several advantages such as homogeneous repartition of nutrients and gas, control of all culture parameters and increased yield. However, the important shear stress generated by those bioreactors is an important disadvantage as it can affect cell survival or cell quality. This important shear stress is the result of the mixing method using either blades (used in stirred-tanked bioreactors) or gas bubbles (used in airlift bioreactors). Another downside of the use of bioreactors is the difficulty to scale-up. As the volume increases, the shear stress generated by blades radically increases leading to cell death and a decrease of cell quality. DescriptionIn this study, we describe a bioreactor developed using a different mixing method effectively reducing the shear stress and facilitating scale-up. This bladeless method uses an inclination of the bioreactor as well as rotation to mix fluids in a container. Here we described different steps that led to the adaptation of this bioreactor, initially developed for fragile microalgae culture, for mammalian cell culture amplification. The bioreactor was tested to amplify a natural killer (NK) cell line NK92 which is an IL-2 dependent cell line used in clinical trials for cancer therapy. We have tested the influence of 1-The number of cells seeded; 2-The influence of the rotation speed on cell growth and viability; 3-The influence of the bioreactor angle on the above parameters; 4-The duration of the culture. ResultsCells were initially seeded at 2.5.105 / ml in a volume of 380 ml. According to the rotation speed of 15, 30, 45 and 60 rpm, we have observed an increase of cell numbers at day 3 (3-fold), day 5 (7-fold) and day 7 (10-fold) compared to seeding, the best expansion being obtained at day 7 with a rotation speed of 45 rpm. The optimal angle of rotation was found to be 3 degree, with an optimal amplification at day 7 versus day 3 (p < 0.01). The viability was also found to be optimal in the latter condition. ConclusionsThese preliminary results demonstrate that NK92 cells could be amplified using this bioreactor. In the best tested condition, neither cell viability nor cell growth was impacted. These results strongly suggest the potential use of this device in future clinically applicable conditions.

bioengineering↗

bioRxiv2022

miR-495-3p Sensitizes BCR::ABL1 Expressing Leukemic cells to Tyrosine Kinase Inhibitors by Targeting Multidrug Resistance 1 Gene including in T315I Mutated cells

Chronic myeloid leukemia (CML) is a clonal hematopoietic malignancy driven by the BCR::ABL1 fusion oncoprotein. The development of tyrosine kinase inhibitors (TKIs) has deeply increased long-term survival of CML patients. Nonetheless, one patient out of four will switch TKI off owing either to drug intolerance or resistance partly due to amplification or mutations of BCR::ABL1 oncogene and alteration of ATP-binding cassette (ABC) transporters. Increasing evidence suggests an involvement of the microRNA miR-495-3p in cancer-associated chemo-resistance through multidrug resistance 1 (MDR1) gene which encodes an ATP-dependent efflux pump. Our study aimed at investigating the potential role of miR-495-3p in CML TKI chemo-sensitivity and determining the underlying molecular circuitry involved. We first observed that miR-495-3p expression was lower in BCR::ABL1 expressing cellular models in vitro. Notably, loss-of-function experiments showed increased proliferation associated with a decreased number of non-dividing cells (G0/G1) and resistance to Imatinib. Conversely, our data showed that miR-495-3p overexpression hindered leukemic cell growth and TKI resistance even in Imatinib-resistant T315I-mutant cells as well as drug efflux activity through MDR1 regulation. To further investigate the role of miR-495-3p in CML patients, we found that predicted miR-495-3p targets were upregulated in patients in blast crisis involved in protein phosphorylation and associated with the worst prognosis. Taken together, our results demonstrate that down-regulation of miR-495-3p expression is important in the malignant phenotype of CML and TKI resistance mechanisms, which could be a useful biomarker and a potential therapeutic target to eradicate CML. MeSH termsO_LIATP Binding Cassette Transporter, Subfamily B C_LIO_LIATP Binding Cassette Transporter, Subfamily B, Member 1 / drug effects C_LIO_LIATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism* C_LIO_LIBlast Crisis / pathology C_LIO_LICell Line, Tumor C_LIO_LICell Proliferation / drug effects* C_LIO_LICell Survival / drug effects C_LIO_LIDrug Resistance C_LIO_LIGenes, MDR C_LIO_LIImatinib Mesylate C_LIO_LILeukemia, Myelogenous, Chronic, BCR-ABL Positive C_LIO_LIMicroRNAs / genetics C_LIO_LIMicroRNAs / physiology* C_LI HIGHLIGHTSO_LImiR-495-3p inhibits leukemic cell growth and is downregulated in BCR::ABL1 expressing cell lines C_LIO_LImiR-495-3p modulates response to TKI treatment including in UT7 cells expressing T315I C_LIO_LIOverexpression of miR-495-3p leads to a decrease of MDR1 and drug efflux activity C_LIO_LIBioinformatics analyses reveal that MiR-495-3p target genes are upregulated in blast crisis C_LI

cancer biology↗

bioRxiv2020

Evidence of Increased Hemangioblastic and Early Hematopoietic Potential in Chronic Myeloid Leukemia (CML)-derived Induced Pluripotent Stem Cells (iPSC)

Hemangioblasts derived from mesodermal lineage are the earliest precursors of hematopoietic stem cells and endothelial cells. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are the only experimental systems in which these cells can be assayed and quantified. We show here using CML-derived iPSC and blast-cell colony forming (Bl-CFC) assays that hemangioblasts are highly expanded in CML derived iPSC as compared to human H1-ESC-derived hemangioblasts. BCR-ABL signaling pathway is intact in these cells with evidence of CRK-L phosphorylation which is reduced by the use of Imatinib. Hematopoietic progenitor assays generated using blast-CFC demonstrates also a highly increased hematopoietic progenitor potential of these cells as compared to H1-ESC. The same results were also obtained using hematopoietic progenitor assays via embryoid body formation. In CML iPSC, we have also found a significant reduction of Aryl Hydrocarbon Receptor (AHR) expression which is involved in hematopoietic quiescence. Further inhibition of AHR using StemRegenin (SR1), an AHR antagonist, led to an increase of blast-cell colonies in CML iPSC whereas the use of an AHR agonist inhibited blast cell colonies. Thus, our results show for the first time, the possibility of establishment of a myeloproliferative phenotype using patient-derived iPSC and the presence of a major expansion hemangioblast compartment and derived hematopoietic progenitors in this context. They also suggest that the AHR signaling pathway could represent a novel druggable target in CML.

cancer biology↗

bioRxiv2019

Evidence of massive leukemic progenitor expansion in blood during molecular recurrence after TKI discontinuation in chronic myeloid leukemia (CML)

Chronic myeloid leukemia (CML) represents one of the major success stories of targeted therapies of the 21st century with the use of tyrosine kinase inhibitors (TKIs). Following discontinuation of TKIs in deep molecular remission context, at least half of the patients experience molecular relapse. Cellular events occurring during this phase are not yet established. In this work, we show a massive amplification of clonogenic progenitors or CFCs (colony-forming cells) in the peripheral blood of patients in molecular recurrence after a TKI discontinuation. We demonstrate, by qRT-PCR analysis on individual and pooled CFCs, that leukemic clonogenic progenitor expansion in the peripheral blood represents the first cellular event before any evidence of cytological change. These findings also suggest that the amplification of the leukemic clone during the first stages of CML takes place not only in the bone marrow but also in the peripheral blood.

cancer biology↗

bioRxiv2019

iPSC-derived Cancer Organoids Recapitulate Genomic and Phenotypic Alterations of c-met-mutated Hereditary Kidney Cancer

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis as a driving oncogenic event is present in germline, exposing the healthy member of a family to the occurrence of cancer. The study of the secondary events in a tissue-specific manner is now possible by the induced pluripotent stem cell (iPSC) technology offering the possibility to generate an unlimited source of cells that can be induced to differentiate towards a tissue at risk of malignant transformation. We report here for the first time, the generation of a c-met-mutated iPSC lines from the somatic cells of a patient with type 1 papillary renal cell carcinoma (PRCC). We demonstrate the feasibility of kidney differentiation with iPSC-derived organoids expressing markers of kidney progenitors with presence of tight junctions and brush borders in tubular structures at transmission electron microscopy. Importantly, c-met-mutated kidney organoids expressed PRCC markers both in vitro and in vivo in NSG mice. Gene expression profiling of c-met-mutated iPSC-derived organoid structures showed striking molecular similarities with signatures found in a large cohort of PRCC patient samples and identified 11 common genes. Among these, BHLHE40 and KDM4C, well-known factors involved in PRCC pathogenesis, were expressed in c-met-mutated kidney organoids. This analysis applied to primary cancers with and without c-met mutation showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated organoid transcriptome. These data represent therefore the first proof of concept of the generation of \"renal carcinoma in a dish\" model using c-met-mutated iPSC-derived organoids, opening new perspectives for discovery of novel potentially predictive disease markers and novel drugs for future precision medicine strategies.

cancer biology↗