The hope, hype and obstacles surrounding cell therapy.

Cell therapy offers hope, but it also presents challenges, most particularly the limited ability of human organs and tissues to regenerate. Since many diseases are associated with irreversible pathophysiological or traumatic changes, stem cells and their derivatives are unable to secure healing. Although regenerative medicine offers chances for improvements in many diseases, such as type one diabetes and Parkinson's disease, it cannot eliminate the primary cause of many of them. While successes can be expected for diseases such as sickle cell disease, this is not the case for hereditary diseases with varied mutation types or for ciliopathies, which start in embryogenesis. In this complicated medical environment, synthetic biology offers some solutions, but their implementation will take many years. Still, positive examples such as CAR-T therapy offer hope.

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.

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).

Multiomic analysis on human cell model of wolfram syndrome reveals changes in mitochondrial morphology and function.

BACKGROUND: Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. METHODS: We performed transcriptomic and proteomic analysis on WFS human cell model-skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. RESULTS: Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. CONCLUSIONS: Our results show the functional and morphological secondary mitochondrial damage in patients with WFS. Video Abstract.

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.

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.

Defining Glycemic Variability in Very Low-Birthweight Infants: Data from a Continuous Glucose Monitoring System.

BACKGROUND: Glucose variability (GV) is a matter of interest for researches in recent years. It is connected with oxidative stress, which is crucial in the development of multiple complication of prematurity. However, glycemic variability in preterm infants was poorly investigated. This study aims to investigate glycemic variability obtained from a continuous glucose monitoring (CGM) system in a cohort of very low-birthweight (VLBW) infants. METHODS: A prospective, single-center, open cohort study enrolled 74 VLBW infants with a mean birthweight of 1066 g and median gestational age of 28 weeks. A CGM system (Guardian Real-Time CGM®, Medtronic, Northridge, CA) was used to measure interstitial glucose concentration. The glycemic variability was calculated using EasyGV. RESULTS: Most glycemic variability indices in VLBW infants showed log-normal distribution and for these, geometric mean ÷/ × geometric standard deviation (GSD) was calculated: M-value 2.28 (÷/ × 1.82), mean amplitude of glycemic excursions (MAGE) 1.89 (÷/ × 1.34), average daily risk ratio (ADRR) 2.22 (÷/ × 2.56), lability index 0.46 (÷/ × 1.71), J-index 0.46 (÷/ × 1.71), low blood glucose index 2.05 (÷/ × 1.66), high blood glucose index 1.11 (÷/ × 2.44), continuous overlapping net glycemic action (CONGA) 5.54 (÷/ × 1.16), mean of daily differences (MODD) 1.23 (÷/ × 1.38), and coefficient of variation 1.15 (÷/ × 1.31). Only SD of glucose concentration showed a normal distribution: arithmetic mean 1.24 (+/-0.37). ADRR, J-index, MODD, CONGA, and MAGE are moderately to strongly correlated with SD. CONCLUSIONS: In our cohort of VLBW infants, almost all glycemic variability indices showed skewed positive distribution. The natural central tendency measure for the log-normally distributed data is the geometric mean and for statistical variation is the GSD.