Immunohistochemical detection of EGFRvIII in glioblastoma - Anti-EGFRvIII antibody validation for diagnostic and CAR-T purposes.

The emergence of therapies such as CAR-T has created a need for reliable, validated methods for detecting EGFRvIII in patient tumor cells. Particularly so since previous studies have already suggested that some anti-EGFRvIII antibodies may be non-specific. The present paper evaluates the use of the L8A4 antibody in the immunohistochemical (IHC) and immunocytochemical (ICC) detection of EGFRvIII in 30 glioblastoma specimens, and compares it with other methods such as RT-PCR, MLPA, and FISH. The results indicate that Real-time PCR appears to be a very specific and sensitive method of EGFRvIII detection. ICC analysis with L8A4 also appears specific but requires cell culture. IHC analyses of EGFRvIII returned a number of false positives when using L8A4. Due to the growing need for an effective diagnostic tool before starting immunotherapy methods, such as the CAR-T anti-EGFRvIII or SynNotch CAR-T recognizing EGFRvIII, it is necessary to identify a more reliable and simple method of EGFRvIII detection or improve the specificity of the anti-EGFRvIII antibody, until then, immunocytochemistry may temporarily replace immunohistochemistry.

Increased EGFRvIII Epitope Accessibility after Tyrosine Kinase Inhibitor Treatment of Glioblastoma Cells Creates More Opportunities for Immunotherapy.

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.

The Genetic Diversity of Proteasome Genes in the T1DM Polish Population.

Autoimmune metabolic diseases generate numerous healthy and social problems. The possible association of SNPs in the ubiquitin-proteasome system (UPS) with human pathology is under intensive study. OBJECTIVE: In the present study, the genetic variations in PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) UPS gene cluster was investigated in type 1 diabetes and healthy donors in the Polish population. METHODS: The study comprised 105 patients with type 1 diabetes mellitus (T1DM) and 214 controls. All were genotyped by PCR and restriction digestion analysis or Sanger sequencing. RESULTS: Rs1048990 and rs2348071 were found to be neutral to T1DM (p-value: 0.499 and 0.656, respectively). According to the multiple loci genotype (MLG) analysis, the major homozygote of the tested polymorphisms had a protective effect. The most common MLG in the T1DM group was characterised by simultaneous risk factors at rs11543947, rs2277460, rs2295826 and rs2295827 (pvalue: <0.0001 vs. MGL1). Multiple locus haplotype analysis revealed a similar dependence, with common alleles at all tested loci demonstrating a protective effect, and the rare alleles increasing T1DM risk (p-value: <0.0001 vs. MLH1). CONCLUSION: Our study suggests that the proteasome gene polymorphisms rs11543947, rs2277460, rs2295826, and rs2295827 could be potential markers for T1DM susceptibility in the Polish population.

Proteomic and Transcriptomic Landscapes of Alström and Bardet-Biedl Syndromes.

Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) are rare genetic diseases with a number of common clinical features ranging from early-childhood obesity and retinal degeneration. ALMS and BBS belong to the ciliopathies, which are known to have the expression products of genes, encoding them as cilia-localized proteins in multiple target organs. The aim of this study was to perform transcriptomic and proteomic analysis on cellular models of ALMS and BBS syndromes to identify common and distinct pathological mechanisms present in both syndromes. For this purpose, epithelial cells were isolated from the urine of patients and healthy subjects, which were then cultured and reprogrammed into induced pluripotent stem (iPS) cells. The pathways of genes associated with the metabolism of lipids and glycosaminoglycan and the transport of small molecules were found to be concomitantly downregulated in both diseases, while transcripts related to signal transduction, the immune system, cell cycle control and DNA replication and repair were upregulated. Furthermore, protein pathways associated with autophagy, apoptosis, cilium assembly and Gli1 protein were upregulated in both ciliopathies. These results provide new insights into the common and divergent pathogenic pathways between two similar genetic syndromes, particularly in relation to primary cilium function and abnormalities in cell differentiation.

Phenotypical Flexibility of the EGFRvIII-Positive Glioblastoma Cell Line and the Multidirectional Influence of TGFß and EGF on These Cells-EGFRvIII Appears as a Weak Oncogene.

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 TGFß (transforming growth factor ß). 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 TGFß 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 TGFß. CONCLUSIONS: The roles of TGFß 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).

Regeneration difficulties in patients with FQAD can limit the use of iPSc-based cell therapy.

Etiopathogenesis of fluoroquinolone-associated disability (FQAD) syndrome is not fully understood, yet research could progress by utilizing induced pluripotent stem cells (iPSc) from people with this syndrome. Similarly, iPSc, or rather their derivatives, could be used in their therapy, not only for FQAD but also for other disorders in which generated autologous iPSc and their derivatives might be helpful. Urine was collected from ten donors with FQAD, and reprogramming of these cells was conducted with the use of Epi5TM Episomal iPSC Reprogramming Kit. IPSc were generated in one out of ten person's urine cells. While urinary cells are considered the easiest mature cells to be reprogrammed into iPSc, the urinary cells from six consecutive donors quickly became senescent. Stable urine primary cell cultures could not be obtained from the three remaining donors. Repeated attempts to reprogram epithelial cells were not successful. During parallel studies conducted for healthy donors, reprogramming success was achieved in six out of ten cases. These data may suggest serious limitations in the regeneration system of individuals with FQAD. Consequently, it indicates that therapy with autologous iPSc derivatives may face serious difficulties in their case, still, the first iPSc cell line from a person with FQAD was established.

Role of Senescence in Tumorigenesis and Anticancer Therapy.

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.

Gaps and Doubts in Search to Recognize Glioblastoma Cellular Origin and Tumor Initiating Cells.

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.

A way to understand idiopathic senescence and apoptosis in primary glioblastoma cells - possible approaches to circumvent these phenomena.

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 tumor, while its primary cultures are difficult to maintain in vitro. We previously reported that senescence is one of the major mechanisms responsible for primary GB cells stabilization failure, to a lesser extent accompanied by apoptosis and mitotic catastrophe-related cell death. METHODS: We made an attempt to circumvent difficulties with glioblastoma primary cultures by testing 3 different approaches aimed to prolong their in vitro maintenance, on a model of 10 patient-derived tumor specimens. RESULTS: Two out of ten analyzed GB specimens were successfully stabilized, regardless of culture approach applied. Importantly, cells transduced with immortalizing factors or cultured in neural stem cell-like conditions were still undergoing senescence/apoptosis. Sequential in vivo/in vitro cultivation turned out to be the most effective, however, it only enabled to propagate cells with preserved molecular profile up to 3rd mice transfer. Nevertheless, it was the only method that impeded these phenomena long enough to provide sufficient amount of material for in vitro/in vivo targeted analyses. Interestingly, our data additionally demonstrated that some subpopulations of several stabilized GB cell lines undergo idiopathic senescence, however, it is counterbalanced by simultaneous proliferation of other cell subpopulations. CONCLUSIONS: In the majority of primary glioma cultures, there has to be an imbalance towards apoptosis and senescence, following few weeks of rapid proliferation. Our results indicate that it has to be associated with the mechanisms other than maintenance of glioblastoma stem cells or dependence on proteins controlling cell cycle.

[Glioblastoma with BRAFV600E mutation and numerous metastatic foci: a case report].

Glioblastoma, the most malignant astrocytic tumour, is associated with limited survival and thus rare metastases. We analysed a particularly interesting case - a 51-year-old male diagnosed within 2 years with primary and recurrent glioblastoma, isocitrate dehydrogenase (IDH)-wild type, as well as with numerous extra-central nervous system (CNS) metastatic foci. Genetic material obtained from primary and recurrent tumours, as well as from pulmonary metastasis was analysed and compared at a molecular level. Next generation sequencing (NGS) analysis revealed BRAFV600E mutation, detected only in 2-5% of glioblastomas, in both the primary tumour and pulmonary metastases. Importantly, this mutation provides a possible therapeutic option as it constitutes a target for clinically approved inhibitors. This case study not only demonstrates a molecular comparison of primary, recurrent and metastatic glioblastoma, but also emphasizes the need for precise molecular diagnostics, which may facilitate treatment choice, especially in tumours currently lacking efficient treatment.

EGFRvIII: An Oncogene with Ambiguous Role.

Epidermal growth factor receptor variant III (EGFRvIII) seems to constitute the perfect therapeutic target for glioblastoma (GB), as it is specifically present on up to 28-30% of GB cells. In case of other tumor types, expression and possible role of this oncogene still remain controversial. In spite of EGFRvIII mechanism of action being crucial for the design of small active anticancer molecules and immunotherapies, i.e., CAR-T technology, it is yet to be precisely defined. EGFRvIII is known to be resistant to degradation, but it is still unclear whether it heterodimerizes with EGF-activated wild-type EGFR (EGFRWT) or homodimerizes (including covalent homodimerization). Constitutive kinase activity of this mutated receptor is relatively low, and some researchers even claim that a nuclear, but not a membrane function, is crucial for its activity. Based on the analyses of recurrent tumors that are often lacking EGFRvIII expression despite its initial presence in corresponding primary foci, this oncogene is suggested to play a marginal role during later stages of carcinogenesis, while even in primary tumors EGFRvIII expression is detected only in a small percentage of tumor cells, undermining the rationality of EGFRvIII-targeting therapies. On the other hand, EGFRvIII-positive cells are resistant to apoptosis, more invasive, and characterized with enhanced proliferation rate. Moreover, expression of this oncogenic receptor was also postulated to be a marker of cancer stem cells. Opinions regarding the role that EGFRvIII plays in tumorigenesis and for tumor aggressiveness are clearly contradictory and, therefore, it is crucial not only to determine its mechanism of action, but also to unambiguously define its role at early and advanced cancer stages.

Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation.

Despite intensive research no therapies targeted against the oncogenic EGFRvIII are present in the clinic. One of the reasons is the elusive nature of the molecular structure and activity of the truncated receptor. The recent publications indicate the EGF-bound wild-type EGFR to trans-phosphorylate the EGFRvIII initiating aberrant signaling cascade. The elevated stability of the mutant receptor contributes towards oncogenic potential, preventing termination of signaling by receptor degradation. Here, we show that inhibition of phosphatases leads to a marked increase in phosphorylation of wild-type EGFR and EGFRvIII, indicating that both undergo cyclic rounds of phosphorylation and dephosphorylation on all investigated tyrosine residues, including Tyr1045. Still, we observe elevated stability of the mutant receptor, suggesting phosphorylation as insufficient to cause degradation. Hyperphosphorylation of EGFRvIII was hindered only by EGFR tyrosine kinase inhibitors. Co-immunoprecipitation as well as semi-native Western blotting structural analyses together with functional investigation of EGFRvIII's phosphorylation following depletion of wild-type EGFR by shRNA or EGF-mediated degradation indicated homodimerization as the predominant quaternary structure of the mutant receptor. Dimers were observed only under non-reducing conditions, suggesting that homodimerization is mediated by covalent bonds. Previous reports indicated cysteine at position 16 to mediate covalent homodimerization. Upon its substitution to serine, we have observed impaired formation of dimers and lower phosphorylation levels of the mutated oncogene. Based on the obtained results we propose that EGFRvIII is predominantly regulated dynamically by phosphatases that counteract the process of trans-phosphorylation occurring within the homodimers.

Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma.

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene.

Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors.

BACKGROUND: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. METHODS: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. RESULTS: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. CONCLUSIONS: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.

Efficient and simple approach to in vitro culture of primary epithelial cancer cells.

Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice.We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs-5 compared with 8 and PEBCs-6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents.In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses.

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line.

The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant-EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.

IDH1R132H in Neural Stem Cells: Differentiation Impaired by Increased Apoptosis.

BACKGROUND: The high frequency of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in diffuse gliomas indicates its importance in the process of gliomagenesis. These mutations result in loss of the normal function and acquisition of the neomorphic activity converting α-ketoglutarate to 2-hydroxyglutarate. This potential oncometabolite may induce the epigenetic changes, resulting in the deregulated expression of numerous genes, including those related to the differentiation process or cell survivability. METHODS: Neural stem cells were derived from human induced pluripotent stem cells following embryoid body formation. Neural stem cells transduced with mutant IDH1R132H, empty vector, non-transduced and overexpressing IDH1WT controls were differentiated into astrocytes and neurons in culture. The neuronal and astrocytic differentiation was determined by morphology and expression of lineage specific markers (MAP2, Synapsin I and GFAP) as determined by real-time PCR and immunocytochemical staining. Apoptosis was evaluated by real-time observation of Caspase-3 activation and measurement of PARP cleavage by Western Blot. RESULTS: Compared with control groups, cells expressing IDH1R132H retained an undifferentiated state and lacked morphological changes following stimulated differentiation. The significant inhibitory effect of IDH1R132H on neuronal and astrocytic differentiation was confirmed by immunocytochemical staining for markers of neural stem cells. Additionally, real-time PCR indicated suppressed expression of lineage markers. High percentage of apoptotic cells was detected within IDH1R132H-positive neural stem cells population and their derivatives, if compared to normal neural stem cells and their derivatives. The analysis of PARP and Caspase-3 activity confirmed apoptosis sensitivity in mutant protein-expressing neural cells. CONCLUSIONS: Our study demonstrates that expression of IDH1R132H increases apoptosis susceptibility of neural stem cells and their derivatives. Robust apoptosis causes differentiation deficiency of IDH1R132H-expressing cells.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line.

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research.

SOX2 and SOX2-MYC Reprogramming Process of Fibroblasts to the Neural Stem Cells Compromised by Senescence.

Tumorigenic potential of induced pluripotent stem cells (iPSCs) infiltrating population of induced neural stem cells (iNSCs) generated from iPSCs may limit their medical applications. To overcome such a difficulty, direct reprogramming of adult somatic cells into iNSCs was proposed. The aim of this study was the systematic comparison of induced neural cells (iNc) obtained with different methods-direct reprogramming of human adult fibroblasts with either SOX2 (SiNSc-like) or SOX2 and c-MYC (SMiNSc-like) and induced pluripotent stem cells differentiation to ebiNSc-in terms of gene expression profile, differentiation potential as well as proliferation properties. Immunocytochemistry and real-time PCR analyses were used to evaluate gene expression profile and differentiation potential of various iNc types. Bromodeoxyuridine (BrdU) incorporation and senescence-associated beta-galactosidase (SA-ß-gal) assays were used to estimate proliferation potential. All three types of iNc were capable of neuronal differentiation; however, astrocytic differentiation was possible only in case of ebiNSc. Contrary to ebiNSc generation, the direct reprogramming was rarely a propitious process, despite 100% transduction efficiency. The potency of direct iNSCs-like cells generation was lower as compared to iNSCs obtained by iPSCs differentiation, and only slightly improved when c-MYC was added. Directly reprogrammed iNSCs-like cells were lacking the ability to differentiate into astrocytic cells and characterized by poor efficiency of neuronal cells formation. Such features indicated that these cells could not be fully reprogrammed, as confirmed mainly with senescence detection. Importantly, SiNSc-like and SMiNSc-like cells were unable to achieve the long-term survival and became senescent, which limits their possible therapeutic applicability. Our results suggest that iNSCs-like cells, generated in the direct reprogramming attempts, were either not fully reprogrammed or reprogrammed only into neuronal progenitors, mainly because of the inaccuracies of currently available protocols.