26 research outputs found
The Route of the Malignant Plasma Cell in Its Survival Niche: Exploring “Multiple Myelomas”
No full textSIMPLE SUMMARY: Multiple Myeloma is a hematological neoplasia originating from malignant plasma cells in the bone marrow. Despite improved therapies, this cancer is still considered incurable due to the occurrence of relapse and the development of drug resistance over time. Apart from cell-intrinsic oncogenic features such as genetic alterations and clonal evolution that promote tumor growth and survival, increasing evidence demonstrates that the bone marrow microenvironment plays a pivotal role in disease relapse and immune evasion by providing protected bone marrow niches in which dormant myeloma cells are able to reside and survive. This review summarizes the ways of the bone marrow micromilieu to nurture and interact with the malignant plasma cells, and provides insights into the vicious cycles arising from the interplay between myeloma cells and their surrounding tumoral stroma. Knowledge about these mechanisms and how to disrupt them may provide novel approaches to targeting and tackling multiple myeloma. ABSTRACT: Growing evidence points to multiple myeloma (MM) and its stromal microenvironment using several mechanisms to subvert effective immune and anti-tumor responses. Recent advances have uncovered the tumor-stromal cell influence in regulating the immune-microenvironment and have envisioned targeting these suppressive pathways to improve therapeutic outcomes. Nevertheless, some subgroups of patients include those with particularly unfavorable prognoses. Biological stratification can be used to categorize patient-, disease- or therapy-related factors, or alternatively, these biological determinants can be included in a dynamic model that customizes a given treatment to a specific patient. Genetic heterogeneity and current knowledge enforce a systematic and comprehensive bench-to-bedside approach. Given the increasing role of cancer stem cells (CSCs) in better characterizing the pathogenesis of solid and hematological malignancies, disease relapse, and drug resistance, identifying and describing CSCs is of paramount importance in the management of MM. Even though the function of CSCs is well-known in other cancer types, their role in MM remains elusive. With this review, we aim to provide an update on MM homing and resilience in the bone marrow micro milieu. These data are particularly interesting for clinicians facing unmet medical needs while designing novel treatment approaches for MM
RAL GTPases mediate multiple myeloma cell survival and are activated independently of oncogenic RAS
No full textOncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right
Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation - An Appraisal
No full textCell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in "hard-to-transfect" MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs
Parameters for MM cell line electroporation.
No full text<p>Rule-of-thumb voltage settings for different HMCLs as deduced from electroporation with an expression plasmid for enhanced green fluorescent protein (pEGFP-N3). All transfections were carried out with a Gene Pulser (Bio-Rad) with a capacity setting of 960 µF. The whole unpurified cell culture was measured (FACS) at day one post-electroporation. The percentage ranges given are to be considered as guidelines for standard results based on between 10 (cell lines not regularly used in our experiments) and hundreds of electroporations (the regularly used “well-transfectable” MM lines). The best electroporations achieved have yielded up to 40% EGFP-positive cells, but suboptimal conditions - for example due to overly high cell densities in the preceding cell culture - may result in lower efficiencies than indicated.</p
Knockdown efficiency in MM cells.
No full text<p>Knockdown of ERK2 in different MM cell lines after transfection with either a short-hairpin expression vector (pSU-ERK2) or the “corresponding” target sequence synthesised as 25 bp stealth siRNA (stERK2; see Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097443#pone.0097443-Chatterjee1" target="_blank">[21]</a>). At day 1 post-electroporation half of the transfected culture was subjected to the column purification method (also see Fig. 1b)–e)) (resp. cell sorting for AMO-1 cells) and the other half to debris removal only (also see Fig. 1f)). Cells were harvested for Western blotting at the times indicated. Empty pSUPER vector (pSU) transfected cells served as controls. The blots show that the ERK2 knockdown efficiency for stealth siRNA is virtually identical between cells that only underwent debris removal and those that were subjected to the column purification procedure. ERK2 knockdown using the short-hairpin expression vector was less efficient in debris-removal-only samples compared with their cognate column purification complements (see JJN-3, L-363). Representative experiments (JJN-3: n = 3; L-363: n = 2, AMO-1: n = 2) are shown. Anti-ERK1/2 antibody: CST.</p
Electroporation of AMO-1 cells with a 6-FAM-labelled siRNA oligonucleotide.
No full text<p>Left column: Fluorescence of AMO-1 cells electroporated with the siERK2-6-FAM oligonucleotide (green curve) in relation to mock transfected cells (blue curve) at different time points post-electroporation. Right: Western analysis for ERK2 knockdown at days 3, 5, 7 post-electroporation. One representative experiment from a total of three is shown. Anti-ERK1/2 antibody: CST.</p
Electroporation and knockdown efficiencies in “easy-to-transfect” vs. “hard-to-transfect” MM cell lines.
No full text<p>Left-hand panel: MM cell lines were electroporated with an expression vector for EGFP (pEGFP-N3; 10 µg/ml) and stealth siRNAs against either ERK2 (stERK2; 3 µM) or against no specific target (control; 3 µM). The FACS-measurements represent the cell cultures at day 1 post-electroporation after debris removal with OptiPrep. Right-hand panel: Knockdown of ERK2 and intrinsic levels of phospho-ERK2 (cells from the cultures represented on the left were harvested at day 3 post-electroporation for Western blotting). Good knockdown of ERK2 and lowered levels of phospho-ERK2 were found for all four MM cell lines tested. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: Santa Cruz Biotechnology.</p
Electroporation of INA-6 cells stably expressing enhanced green fluorescent protein with an siRNA oligonucleotide against EGFP.
No full text<p>INA-6-EGFP cells were electroporated with a solution containing a stealth siRNA targeting EGFP as well as an expression plasmid for CD4Δ. One day post-electroporation one half of the cell culture was purified according to the column procedure (red curves, also see Fig. 1b)–e)), whereas the other half only underwent debris removal with OptiPrep (blue curves, also see Fig. 1f)). Purified cells were further cultured and FACS-analysed for EGFP expression at the times indicated. Only the live cell fraction (as demarcated in the forward/sideward scatter) was analysed and plotted against similarly treated INA-6-EGFP cells (green curves) transfected with a non-EGFP targeting siRNA. Knockdown efficiency was essentially identical in strength and over time between both purification approaches. One representative experiment from a total of three is shown.</p
Voltage dependence of electroporation and knockdown efficiency in AMO-1 cells.
No full text<p>Transfection of AMO-1 cells across a range of voltages using an expression vector for EGFP (pEGFP-N3) and a stealth siRNA against ERK2 (stERK2) in the electroporation mixture. Top panel: increases of the fractions of EGFP-expressing as well as of dead cells with higher voltages (top row). Cells taken in culture after OptiPrep-mediated debris removal reflect only the increase in transfection efficiency for the EGFP expression plasmid (bottom row). Middle panel: purified AMO-1 cells (those shown in the upper panel, bottom row) after culture for another 4 days. Top row: EGFP expression. Bottom row: annexin V-PromoFluor 647/PI staining. Even for the highest voltage used (320 V) the purified live cell fraction did not fare worse in subsequent culture than cells electroporated under milder conditions. Bottom panel: Western analysis of ERK2 knockdown at days 3 and 5 post-electroporation from the same cultures from which the FACS panels were derived. Efficient siRNA-mediated ERK2 knockdown was achieved at voltages significantly lower than required for the best levels of plasmid electroporation. However, a lower limit for successful knockdown was reached between the settings for 160 and 200 V. Shown is a representative experiment of two complete sets (Western blotting included). Anti-ERK1/2 antibody: CST.</p
Electroporation of MM cell lines and subsequent purification of transfected cells.
No full text<p>Shown is a representative example of the procedure using the well-transfectable MM cell line JJN-3. This standard column purification has now been performed hundreds of times in our laboratory and is also easily applicable for MM cell lines INA-6, KMS-11, L-363, MM.1S and U-266 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097443#pone-0097443-t001" target="_blank">Table 1</a>). a) Cell culture one day after electroporation with expression plasmids for enhanced green fluorescent protein (EGFP) and CD4Δ, showing about a quarter of cells strongly positive for EGFP. b)-e) Enrichment of strongly transfected cells by selection for CD4 surface expression (CD4 MicroBead column selection). b) Column runthrough of cell culture shown in a). Of note is the similar look with a), but with depletion of the strongest transfected cells in b). c) Column eluate of the cell culture shown in a). Untransfected cells (EGFP- and CD4Δ-negative) have effectively been removed, but the column procedure tends to retain significant amounts of dead cells (EGFP-negative, PI-positive). d) Floating fraction of the column eluate as shown in c) after “density gradient” (more properly: density step) treatment using OptiPrep, consisting mostly of viable and strongly transfected cells. e) Pelleted fraction of the column eluate as shown in c) after “density gradient” treatment using OptiPrep, consisting mostly of debris. f) Removal of debris by OptiPrep treatment from the cell culture as shown in a) without prior column separation, leaving two main fractions which are either EGFP-negative, or distinctly EGFP-positive. See Methods section for further details.</p
