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Background:
Glioblastoma (GB) is considered one of the most lethal tumors. Extensive research at the molecular level may enable to gain more profound insight into its biology and thus, facilitate development and testing of new therapeutic approaches. Unfortunately, stable glioblastoma cell lines do not reflect highly heterogeneous nature of this t...
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... analysis was also conducted to evaluate whether the observed phenomenon may have an impact on stabilization success. To make the analysis more profound, not only the percentage of SA-β-Gal positive cells, but also the percentage of apoptotic (using caspase 3/7 substrate; Additional file 5: Figure S5), proliferating (BrdU-positive) and other cells in these cultures was calculated (Fig. 2g-k, Additional file 4: Table S4). In case of cancer cells, the significant increase in senescent and decrease in proliferating cells during culture course was observed, irrespective to culture conditions applied (as e.g. for GB9, in monolayer with p = 0.032 and p = 0.008, for senescent and proliferating cells, respectively, and in NSC-like ones for senescent and proliferating cells with p = 0.028 and p = 0.045, respectively), while in case of normal cells, BrdU-positive cells remained on a relatively stable level in monolayer conditions (with an exception for GB9 with p = 0.037; Additional file 4: Table S4), while in later passages of NSC-like conditions population of normal cells was so marginal that it was not a subject of any analyses. ...
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... to initiate tumor formation, we carried out subcutaneous transplantation into immunocompromised mice. Three out of ten GB specimens were analyzed in this approach (GB6, 8 and 9) and each time tumor tissue was extracted, minced just like tissue specimen, and either reinjected the same way into another mice (next transfer) or cultured in vitro (Fig. 5a). Preliminary in vivo analyses indicated that for efficient tumor growth cells need to be mixed with Matrigel prior to injection (Fig. 5b). Importantly, injected cells were analyzed in terms of phenotypic ( Fig. 5c-g) and genotypic ( Fig. 5f-i) tumor characteristics in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd ...
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... analyzed in this approach (GB6, 8 and 9) and each time tumor tissue was extracted, minced just like tissue specimen, and either reinjected the same way into another mice (next transfer) or cultured in vitro (Fig. 5a). Preliminary in vivo analyses indicated that for efficient tumor growth cells need to be mixed with Matrigel prior to injection (Fig. 5b). Importantly, injected cells were analyzed in terms of phenotypic ( Fig. 5c-g) and genotypic ( Fig. 5f-i) tumor characteristics in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd resection (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses ...
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... minced just like tissue specimen, and either reinjected the same way into another mice (next transfer) or cultured in vitro (Fig. 5a). Preliminary in vivo analyses indicated that for efficient tumor growth cells need to be mixed with Matrigel prior to injection (Fig. 5b). Importantly, injected cells were analyzed in terms of phenotypic ( Fig. 5c-g) and genotypic ( Fig. 5f-i) tumor characteristics in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd resection (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses (mostly MLPA and Real-time PCR; Fig. 5h-i), but also the decrease in ...
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... specimen, and either reinjected the same way into another mice (next transfer) or cultured in vitro (Fig. 5a). Preliminary in vivo analyses indicated that for efficient tumor growth cells need to be mixed with Matrigel prior to injection (Fig. 5b). Importantly, injected cells were analyzed in terms of phenotypic ( Fig. 5c-g) and genotypic ( Fig. 5f-i) tumor characteristics in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd resection (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses (mostly MLPA and Real-time PCR; Fig. 5h-i), but also the decrease in tumorcharacteristic phenotype (loss ...
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... cells were analyzed in terms of phenotypic ( Fig. 5c-g) and genotypic ( Fig. 5f-i) tumor characteristics in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd resection (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses (mostly MLPA and Real-time PCR; Fig. 5h-i), but also the decrease in tumorcharacteristic phenotype (loss of GFAP expression; Fig. 5d) was observed with the fraction of remaining proliferating cells (BrdU-positive; Fig. 5e). Therefore, such conditions enabled to maintain up to 3 subsequent transfers of GB cells in mice, while maintaining their original genotype and phenotype as ...
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... in each step of the procedure. Following 2 nd (in case of GB8) or 3 rd resection (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses (mostly MLPA and Real-time PCR; Fig. 5h-i), but also the decrease in tumorcharacteristic phenotype (loss of GFAP expression; Fig. 5d) was observed with the fraction of remaining proliferating cells (BrdU-positive; Fig. 5e). Therefore, such conditions enabled to maintain up to 3 subsequent transfers of GB cells in mice, while maintaining their original genotype and phenotype as well as their proliferation capacity. This approach can be basically considered an "in ...
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... (in case of GB6 and GB9) not only the original mutational profile of analyzed cells was lost, as monitored by molecular analyses (mostly MLPA and Real-time PCR; Fig. 5h-i), but also the decrease in tumorcharacteristic phenotype (loss of GFAP expression; Fig. 5d) was observed with the fraction of remaining proliferating cells (BrdU-positive; Fig. 5e). Therefore, such conditions enabled to maintain up to 3 subsequent transfers of GB cells in mice, while maintaining their original genotype and phenotype as well as their proliferation capacity. This approach can be basically considered an "in vivo incubator", aimed to propagate primary ...
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... of tumor suppressor mutations. Still, it seems that lack of cell cycle control may facilitate cancer cells transfer and maintenance in vitro. Moreover, we have previously described that there is a higher frequency of homozygous mutations in tumor suppressors as well as mutations resulting in lack of tumor (See figure on previous page.) Fig. 5 Co-injecting glioblastoma tumor cells with basement membrane matrix proteins enables their growth in vivo. a Schematic workflow of sequential in vitro/in vivo culturing of glioblastoma cells. Primary GB tumors from three patients (GB6, GB8 and GB9) were maintained for total ± 30 weeks in vivo vs ± 8 weeks in vitro, before they lost ...
Citations
... For the immunocytochemical analyses of iNSc as well as expression of EGFRvIII cells were seeded on Geltrex (Sigma Aldrich) coated 4-well plates in amount 10, 000 per well. Staining was performed as described previously [9]. Senescence associated (SA)-β-galactosidase assay SA-β-Gal staining was performed using Senescence Cells Histochemical Staining Kit (Sigma Aldrich). ...
It is now accepted that the formation of EGFRvIII, a mutated variant of EGFR, may occur early in the tumorigenesis of glioblastoma. Furthermore, it is speculated that glioblastoma may originate from neural stem cells (NSCs), and EGFRvIII itself is considered a marker of cancer stem cells. Therefore, we decided to test the hypothesis that EGFRvIII alteration can occur as the first one in NSC. We created a model of iNSc showing constitutive and induced expression of EGFRvIII. After a series of analyses, we found that EGFRvIII contributed to the loss of SOX2 and nestin - markers of NSCs in both tested models. Interestingly, however, with constitutive expression of EGFRvIII, a senescence phenomenon was observed, while expression induced by low concentrations of doxycycline increased the rate of cell proliferation. Moreover, we observed senescence in the case of high constitutive EGFRvIII expression. Thus, the results suggest that NSCs may not be the origin of glioblastoma cells, and the other cells simultaneously expressing GFAP and SOX2 should be considered the origin of glioblastoma.
... Although EGFRvIII is found in about 27% of glioblastomas [28], due to difficulties in the long-term in vitro maintenance of cells bearing EGFRvIII [44], we focused on immortalized cell lines. A comparison of various cell lines showed we could distinguish between cells with EGFRwt and those with EGFRvIII. ...
... The establishment of stable GB cell lines was performed as previously described [44]. Briefly, glioma cells were cultured in NSC-like conditions: a 1:1 mixture of Neurobasal medium (Life Technologies, Waltham, MA, USA) and DMEM/F12 (Biowest, Nauille, France), supplemented with N2, B27, antimitotic-antimycotic, Glutamax (all Life Technologies, Waltham, MA, USA), NEAA (Biowest, France), bFGF (40 ng/mL, Peprotech, London, United Kingdom), and EGF (5 ng/mL, Peprotech, London, UK). ...
... Stable AD293 cell lines expressing EGFRvIII mutations (AD293 C16S , AD293 C20S , AD293 C35S , AD293 C38S , and AD293 C42S ) as well as DK-MG vIII-exovIII were obtained by transduction with lentiviruses created using the LENTI Smart Kit (InvivoGen, San Diego, CA, USA) as previously described [44]. The AD293 wt and AD293 vIII cell lines were established by transfecting the AD293 parental line with pLV-puro-EGFRwt and pIRESneo3-EGFRvIII using Fugene HD (Promega, Madison, WI, USA), selecting cells with puromycin or neomycin, respectively (both from InvivoGen, San Diego, CA, USA), and establishing a monoclonal population, as previously described [34]. ...
The number of glioblastoma (GB) cases is increasing every year, and the currently available therapies remain ineffective. A prospective antigen for GB therapy is EGFRvIII, an EGFR deletion mutant containing a unique epitope that is recognized by the L8A4 antibody used in CAR-T (chimeric antigen receptor T cell) therapy. In this study, we observed that the concomitant use of L8A4 with particular tyrosine kinase inhibitors (TKIs) does not impede the interaction between L8A4 and EGFRvIII; moreover, in this case, the stabilization of formed dimers results in increased epitope display. Unlike in wild-type EGFR, a free cysteine at position 16 (C16) is exposed in the extracellular structure of EGFRvIII monomers, leading to covalent dimer formation in the region of L8A4–EGFRvIII mutual interaction. Following in silico analysis of cysteines possibly involved in covalent homodimerization, we prepared constructs containing cysteine–serine substitutions of EGFRvIII in adjacent regions. We found that the extracellular part of EGFRvIII possesses plasticity in the formation of disulfide bridges within EGFRvIII monomers and dimers due to the engagement of cysteines other than C16. Our results suggest that the EGFRvIII-specific L8A4 antibody recognizes both EGFRvIII monomers and covalent dimers, regardless of the cysteine bridging structure. To summarize, immunotherapy based on the L8A4 antibody, including CAR-T combined with TKIs, can potentially increase the chances of success in anti-GB therapy.
... Our results suggest that primary GB cells became senescent in vitro more easily than pericytes. During these experiments, all media contained EGF [27,28]. ...
... The findings regarding the biological role of EGFRvIII appear incoherent. This may be associated with the lack of an appropriate in vitro model, as cell lines endogenously expressing EGFRvIII are extremely rare, probably due to their in vitro prosenescence [27,29]. However, the DK-MG cell line seems to offer unique features and extreme flexibility during in vitro culture. ...
... To determine the copy number of the EGFRvIII (calculated as the difference between EGFRwt and total EGFR) and EGFRwt genes, real-time PCR analyses at the DNA level were performed [27]. The RPP25 gene was used for normalization. ...
Background:
The biological role of EGFRvIII (epidermal growth factor receptor variant three) remains unclear.
Methods:
Three glioblastoma DK-MG sublines were tested with EGF (epidermal growth factor) and TGFβ (transforming growth factor β). Sublines were characterized by an increased percentage of EGFRvIII-positive cells and doubling time (DK-MGlow to DK-MGextra-high), number of amplicons, and EGFRvIII mRNA expression. The influence of the growth factors on primary EGFRvIII positive glioblastomas was assessed.
Results:
The overexpression of exoEGFRvIII in DK-MGhigh did not convert them into DK-MGextra-high, and this overexpression did not change DK-MGlow to DK-MGhigh; however, the overexpression of RASG12V increased the proliferation of DK-MGlow. Moreover, the highest EGFRvIII phosphorylation in DK-MGextra-high did not cause relevant AKT (known as protein kinase B) and ERK (extracellular signal-regulated kinase) activation. Further analyses indicate that TGFβ is able to induce apoptosis of DK-MGhigh cells. This subline was able to convert to DK-MGextra-high, which appeared resistant to this proapoptotic effect. EGF acted as a pro-survival factor and stimulated proliferation; however, simultaneous senescence induction in DK-MGextra-high cells was ambiguous. Primary EGFRvIII positive (and SOX2 (SRY-Box Transcription Factor 2) positive or SOX2 negative) glioblastoma cells differentially responded to EGF and TGFβ.
Conclusions:
The roles of TGFβ and EGF in the EGFRvIII context remain unclear. EGFRvIII appears as a weak oncogene and not a marker of GSC (glioma stem cells). Hence, it may not be a proper target for CAR-T (chimeric antigen receptor T cells).
... e question is whether each senescent cell should be positive for each senescence marker. For example, the lack of SAHF structures in SA-β-Gal-positive tumor cells stands in favor of the possibility to restore their proliferation [23]. SAHF structures are very stable foci of heterochromatin generated as a result of RB protein action. ...
... Secondly, these cells stabilize in vitro but with previous loss of amplicons acting as an adaptive mechanism. Indeed, in 2019 Janik et al. showed that SA-β-Gal-positive cells were in high percentage polynucleated and undergo abnormal mitoses in early passages [23]. Multipolar spindles, as well as polynuclear cells with asymmetric distribution of phosphorylated histone 3, were easily detected in early passages of glioblastoma tumor cells. ...
Although the role of senescence in many physiological and pathological processes is becoming more identifiable, many aspects of senescence are still enigmatic. A special attention is paid to the role of this phenomenon in tumor development and therapy. This review mainly deals with a large spectrum of oncological issues, beginning with therapy-induced senescence and ending with oncogene-induced senescence. Moreover, the role of senescence in experimental approaches, such as primary cancer cell culture or reprogramming into stem cells, is also beginning to receive further consideration. Additional focus is made on senescence resulting from mitotic catastrophe processes triggered by events occurring during mitosis and jeopardizing chromosomal stability. It has to be also realized that based on recent findings, the basics of senescent cell property interpretation, such as irreversibility of proliferation blockade, can be undermined. It shows that the definition of senescence probably requires updating. Finally, the role of senescence is lately more understandable in the immune system, especially since senescence can diminish the effectiveness of the chimeric antigen receptor T-cell (CAR-T) therapy. In this review, we summarize the current knowledge regarding all these issues.
... Mitotic catastrophe (MC) is a newly identi ed type of anticancer mechanism in cancer treatment and MDR prevention and has received more attention in recent years [10,11] . Additionally, a number of studies have shown that MC and senescence are closely related and that cancer cell death by MC is often accompanied by a senescence-like phenotype [12][13][14] . In general, cellular senescence is considered a tumor-suppressive mechanism that complements apoptosis [14] . ...
... Additionally, a number of studies have shown that MC and senescence are closely related and that cancer cell death by MC is often accompanied by a senescence-like phenotype [12][13][14] . In general, cellular senescence is considered a tumor-suppressive mechanism that complements apoptosis [14] . However, senescent cancer cells may not be e ciently eliminated by immune cells due to the impaired anticancer immune response resulting from the cancer microenvironment or cancer therapy [15,16] . ...
... Cellular senescence is a permanent state of cell cycle arrest and has been considered a novel anticancer mechanism [14,17] . In our study, BZML treatment induced the cellular senescence in A549/Taxol cells, showing a signi cantly increased proportion of SA-β-gal-positive cells and some other typical features of cellular senescence, such as attened morphology, increased size and granularity and an elevated lysosomal mass in the cells. ...
Background: Mitotic catastrophe (MC) of cancer cells induced by BZML, a novel colchicine-binding site inhibitor, exerts a significant advantage in overcoming multidrug resistance (MDR) in NSCLC. However, the long cellular death process resulting from MC is not beneficial for anticancer treatment. Here, we study the mechanisms underlying MC occurrence and development to promote the development of anticancer therapies based on drug-induced MC.
Methods: Cellular senescence was confirmed by morphological features, SA-β-Gal and C12FDG staining. Cell cycle analysis and Hoechst 33342 staining were used to detect MC. Relevant signal transduction pathways and protein location were detected by qRT-PCR, westren blot and immunofluorescence. The half-life of proteins was evaluated using the protein synthesis inhibitor cycloheximide. Flow cytometry, MTT assay, crystal violet staining, Hoechst 33342 staining and cell division detection were performed to determine the effects of BZML and/or YM155 on cell fate.
Results: We found that BZML induced p53-dependent cellular senescence in A549/Taxol cells, but not in A549, H1299 and MDA-MB-231 cells. Interestingly, BZML-induced senescence was a secondary effect of MC. In addition, the destruction of the protein-degradation system induced by BZML contributed not only to an increase in p53 protein but also to the accumulation of survivin in the nucleus of A549/Taxol cells. However, in A549 cells, the overexpression of survivin had no effect on apoptosis resistance against BZML and failed to promote BZML-induced MC. The inhibition of survivin did not prevent MC occurrence. Unexpectedly, targeting survivin with YM155 accelerated the death of the MC cells by eliminating senescent cells and strengthening the efficiency of BZML in overcoming the MDR of A549/Taxol cells.
Conclusions: Our data suggest that nuclear accumulation of survivin can delay cellular death during MC by promoting the survival of senescent BZML-treated A549/Taxol cells. Further, depending on the dose sequence, combination therapy with YM155 to inhibit survivin might be a new strategy for potentiating BZML-induced MC to overcome MDR during cancer treatment.
... In the majority of GB, there are no senescence-resistant cells, or cells able to establish stable cell line (dividing infinitely). Although the mechanism of senescence is not fully explained, at least part of the senescence phenomenon is a consequence of mitotic catastrophes [82]. Genetic modifications of GB cells leading to the expression of TERT or SV40 do not prevent the senescence [82]. ...
... Although the mechanism of senescence is not fully explained, at least part of the senescence phenomenon is a consequence of mitotic catastrophes [82]. Genetic modifications of GB cells leading to the expression of TERT or SV40 do not prevent the senescence [82]. Stable cell lines (proliferating for years) can therefore be established from minority of GB. ...
Cellular origin of glioblastoma (GB) is constantly discussed and remains a controversial subject. Unfortunately, neurobiologists are not consistent in defining neural stem cells (NSC) complicating this issue even further. Nevertheless, some suggestions referring to GB origin can be proposed based on comparing GB to central nervous system (CNS) cells. Firstly, GB cells show in vitro differentiation pattern similar to GFAP positive neural cells, rather than classical (GFAP negative) NSC. GB cells in primary cultures become senescent in vitro, similar to GFAP positive neural progenitors, whereas classical NSC proliferate in vitro infinitely. Classical NSC apoptosis triggered by introduction of IDH1R132H undermines hypothesis stating that IDH-mutant (secondary) GB origins from these NSC. Analysis of biological role of typical IDH-wildtype (primary) GB oncogene such as EGFRvIII also favors GFAP positive cells rather than classical NSC as source of GB. Single-cell NGS and single-cell transcriptomics also suggest that GFAP positive cells are GB origin. Considering the above-mentioned and other discussed in articles data, we suggest that GFAP positive cells (astrocytes, radial glia, or GFAP positive neural progenitors) are more likely to be source of GB than classical GFAP negative NSC, and further in vitro assays should be focused on these cells. It is highly possible that several populations of tumor initiating cells (TIC) exist within GB, adjusting their phenotype and even genotype to various environmental conditions including applied therapy and periodically going through different TIC states as well as non-TIC state. This adjustment is driven by changes in number and types of amplicons. The existence of various populations of TIC would enable creating neoplastic foci in different environments and increase tumor aggressiveness.
... 73 The high heterogeneity of the cell population, the presence of cancer stem cells, and their strong infiltration potential entail enormous difficulties in the development of effective therapy and result in severely limited survival by GBM patients. [74][75][76] A study of tumor samples obtained from 67 GBM patients revealed that the observed decrease in WWOX expression might be caused by a combination of loss of heterozygosity and promoter methylation. What is more, WWOX expression level has been found to positively correlate with Bcl2 and Ki67 level and to have a significant influence on the ErbB4 signaling pathway. ...
Shortly after its discovery in 2000, WWOX was hailed as a tumor suppressor gene. In subsequent years of research, this function was confirmed indisputably. Majority of tumors show high rate of loss of heterozygosity and decreased expression of WWOX. Nevertheless, over the years, the range of its known functions, at the cellular, organ and system levels, has expanded to include metabolism and endocrine system control and CNS differentiation and functioning. Despite of its function as a tumor suppressor gene, WWOX genetic alternations were found in a number of metabolic and neural diseases. A lack of WWOX protein as a consequence of germline mutations results in brain development disturbances and malfunctions.
Impact statement
WW domain-containing oxidoreductase encoded by the WWOX gene is a transcription regulator and a key player in a number of cellular and biological processes such as tumor suppression, cell proliferation, apoptosis induction, steroid metabolism, and central nervous system development. This review provides a comprehensive summary of currently known roles and discusses the importance of WWOX gene for CNS development and functioning.
Mitotic catastrophe (MC) is a newly identified type of anticancer mechanism for multidrug resistance (MDR) prevention. However, the long cellular death process resulting from MC is not beneficial for anticancer treatment. BZML is a novel colchicine-binding site inhibitor which can overcome MDR by inducing MC; however, BZML-induced MC cells underwent a long cellular death process. Thus, to improve anticancer therapies based on drug-induced MC, BZML-induced MC was served as a model to further study the underlying molecular mechanisms in the process of MC. Here, BZML could induce p53-dependent senescence in A549/Taxol cells, a MDR cell line. This senescence was a secondary effect of MC in overcoming MDR. During MC, BZML-induced destruction of protein-degradation system contributed not only to an increase of p53 protein but also to the accumulation of survivin in nucleus of A549/Taxol cells. Importantly, the nuclear accumulation of survivin was not the inducer but the result of BZML-induced MC, and it promoted the survival of senescent cells. Moreover, it provided additional vulnerability and critical opportunities for sequentially applied therapies. Further, targeting survivin with YM155 accelerated the death of MC cells by timely eliminating therapy-induced senescent cells and strengthening the efficiency of BZML in overcoming MDR in A549/Taxol cells. Collectively, nuclear accumulation of survivin delayed cellular death during MC by promoting the survival of BZML-induced senescent A549/Taxol cells. Moreover, “one-two punch” approach to cancer treatment based on combination therapy with YM155 for survivin suppression might be a new strategy for potentiating MC to overcome MDR.
Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.
