Implementation of nanobodies for the design of glioblastoma targeting therapy

Glioblastom multiforme (GBM) je gliom, tumor osrednjega živčnega sistema, najpogostejša oblika možganskega tumorja, za katerim zboli 3-5 bolnikov na 100.000 ljudi. Povprečni čas preživetja bolnikov z GBM je od časa diagnosticiranja 12 do 18 mesecev, ob kombinirani postoperativni terapiji z uporabo temozolomida. Genetska heterogenost GBM je posledica glioblastomskih matičnih celic (GMC). GMC zaradi svoje odpornosti proti kemoterapevtikom in radioterapiji uspešno napadajo zdravo okoljsko tkivo. Zdravljenje je dodatno oteženo zaradi krvno-možganske pregrade (KMP), saj je prehod kemoterapevtikov skozi njo zelo otežen. Za izboljšanje zdravljenja GBM in izid za bolnika je potrebna stalna dostava zdravil v celice glioma, obenem pa je potrebno zmanjšati učinek zdravil na sosednje, zdrave nevrone in celice glije. Novi pristopi zdravljenja GBM so zato zelo zaželeni in potrebni. Za razvoj usmerjenega zdravljenja pa še vedno potrebujemo odkritje bolj specifičnih biomolekularnih označevalcev GMC, kot tudi njihovih tarčnih zdravil, ki bi prehajala KMP. To lahko dosežemo s proteomskim pristopom, ki temelji na nano-protitelesih, ki so eno-domenski antigen-vezavni fragmenti, pridobljeni iz kemelidnih težkoverižnih protiteles, ki zaradi svoje majhnosti lahko prehajajo KMP. V doktorski nalogi smo izdelali knjižnico nano-protiteles in opravili njihovo imunoafinitetno obogatitev, izvedeno na celotnih GMC, in tako pridobili nano-protitelo, specifično za bio-označevalec GMC. Z masno spektrometrijo smo ugotovili, da je nov bio-označevalec mitohondrijski translacijsko-elongacijski dejavnik TUFM. Diferenčno izražanje TUFM smo proučevali na ravni proteinov in mRNA, in sicer v celičnih linijah GBM (U87MG in U251MG), GMC in tkivih GBM, v primerjavi z izražanjem v nevralnih matičnih celicah (NSC) in normalnih možganskih tkivih. S prenosom western smo na ravni proteinov ter qPCR na ravni mRNA potrdili nadizražanje TUFM v GMC. Z imunohistokemijo, na tkivnih rezinah, vklopljenih v parafin, smo potrdili nadizražanje TUFM v tkivih GBM, v normalnem možganskem tkivu pa se TUFM ni izražal. Z imunocitokemijo smo potrdili vstop nano-protitelesa anti-TUFM v GBM celice U87MG, U251MG in GMC ter njegovo vezavo v območje mitohondrijev. Citotoksičnost vezave nano-protitelesa anti-TUFM na antigen smo preverili z metabolnimi testi na celičnih linijah GBM (U87MG in U251MG), GMC ter kontrolnih celičnih linijah – astrocitih, nevralnih matičnih celicah in človeških nesmrtnih keratinocitih. Nano-protitelo anti-TUFM je imelo citotoksičen učinek na vse celične linije GBM, medtem ko toksičnosti anti-TUFM na kontrolnih celičnih linijah nismo opazili. Z vklapljanjem nano-protiteles anti-TUFM v arheosome smo preverili ali obstaja boljši oz. bolj učinkovit dostavni sistem za vnos nano-protitelesa anti-TUFM v celice. Arheosomi niso citotoksični in vivo in imajo edinstvene strukturne lastnosti, ki so osnova za razvoj novih dostavnih sistemov zdravil. Te lastnosti so stabilnost pri visokih temperaturah, nizkem oz. visokem pH, odpornost proti fosolipazam in solem žolčnih kislin ter manjša membranska prepustnost. Nano-protitelesa anti-TUFM smo uspešno vklopili v arheosome in ugotovili, da le-ti vstopajo v celice GBM, U251MG in U87MG. Ugotovili smo, da ima nano-protitelo anti-TUFM, vklopljeno v arheosom, na celice GBM manjši citotoksični učinek kot ga ima samo nano-protitelo anti-TUFM. V doktorski nalogi smo dokazali specifičnost in izrazit zaviralni učinek nano-protitelesa anti-TUFM na rast GMC, kar bi lahko v prihodnje pripomoglo k razvoju specifičnega pristopa za zdravljenje glioblastoma.Glioblastoma multiforme (GBM) is a glioma, a tumor found in central nervous system. It is the most common form of brain tumors, which affects 3-5 patients per 100,000 people. The average survival period of patients with GBM is 12 to 18 months, which includes resection and combination of postoperative therapy with temozolomide. Glioblastoma stem cells (GMC) are responsible for high genetic heterogeneity of GBM, and due to their resistance to chemotherapy and radiotherapy they successfully invade healthy tissue. The treatment is further aggravated, due to difficult transition of chemotherapeutics through blood-brain barrier (BBB). To improve GBM treatment and the outcome of patients, it is necessary to continuously deliver drugs to the glioma cells while reducing the effect of drugs on adjacent, healthy neurons and glial cells. New GBM treatment approaches are therefore much needed. For the development of targeted GBM treatment, we still need the discovery of more specific GMC biomarkers and the corresponding targeting drugs that would pass BBB. This can be achieved by a proteomic approach based on nanobodies, single-domain antigen-binding fragments, derived from camelid heavy chain antibodies that can, due to their small size, pass BBB. In the doctoral thesis, we constructed a nanobody library and biopannings were made on the whole GMCs. We obtained a nanobody specific for a GMC antigen. Mass spectrometry determined that the new biomarker of GMC is the mitochondrial translational-elongation factor TUFM. Differential expression of TUFM was studied at the protein and mRNA levels in the GBM cell lines (U87MG, and U251MG), GMC and GBM tissue, compared to its expression in neural stem cells (NSC) and normal brain tissue. Western blot and qPCR confirmed the TUFM overexpression in GMC. With immunohistochemistry, on paraffin-embedded GBM tissue, we confirmed the TUFM overexpression, whereas the normal brain tissue was negative for TUFM. Immunocytochemistry confirmed the entry of anti-TUFM nanobodies to the U87MG, U251MG and GMC cells and its binding to mitochondria. The cytotoxic effect of anti-TUFM nanobodies on GBM-related cell lines (U87MG, U251MG and GMC), and on control cell lines (astrocytes, NSC and human immortal keratinocytes) was measured through metabolic assays. Anti-TUFM nanobody had cytotoxic effect on all GBM cell lines, while on the other hand no toxicity of anti-TUFM on control cell lines was observed. Anti-TUFM nanobodies were encapsulated to archeosomes and used to verify their delivery in cells. Aerheosomes are not cytotoxic in vivo and have unique structural properties for the development of new drug delivery systems. These properties are stability at high temperatures, extreme pH values, resistance to phospholipases and bile salts and lower membrane permeability. Successfully, anti-TUFM nanobodies were encapsulated in the archeosomes and were found that they could enter the U251MG cells. Therefore, an encapsulated anti-TUFM nanobody exhibited lower cytotoxic effect on GBM cells than the anti-TUFM nanobody itself. In the doctoral thesis, we showed the specificity and pronounced inhibitory effect of anti-TUFM nanobody on GMC growth, which could in the future contribute to the development of a specific approach for the treatment of glioblastoma

D8.3 - Project collaboration plan

<p>Plan of how MultiXscale will collaborate internally, with the wider EuroHPC ecosystem as well as with associated partners and supporters, with defined common objectives and activities. </p&gt

Non-animal glioblastoma models for personalized treatment

Glioblastoma is an extremely lethal cancer characterized by great heterogeneity at different molecular and cellular levels. As a result, treatment options have moved far from systemic and universal therapies toward targeted treatments and personalized medicine. However, for successful translation from preclinical studies to clinical trials, experiments must be performed on reliable disease models. Numerous experimental models have been developed for glioblastoma, ranging from simple 2D cell cultures to study the nature of the disease to complex 3D models such as neurospheres, organoids, tissue-slice cultures, bioprinted models, and tumor on chip, as perfect prototypes to evaluate the therapeutic potential of different drugs. The presence of multiple research models is consistent with the complexity and molecular diversity of glioblastoma. The advantage of such models is the recapitulation of the tumor environment, and in some cases the preservation of immune system components as well as the creation of simple vessels. There are also two case studies translating in vitro studies on glioblastoma organoids to patients as well as four ongoing clinical trials using glioblastoma models, indicating high clinical potential of glioblastoma models

Algorithmically deduced FREM2 molecular pathway is a potent grade and survival biomarker of human gliomas

Gliomas are the most common malignant brain tumors with high mortality rates. Recently we showed that the FREM2 gene has a role in glioblastoma progression. Here we reconstructed the FREM2 molecular pathway using the human interactome model. We assessed the biomarker capacity of FREM2 expression and its pathway as the overall survival (OS) and progression-free survival (PFS) biomarkers. To this end, we used three literature and one experimental RNA sequencing datasets collectively covering 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG). The activation level of deduced FREM2 pathway showed strong biomarker characteristics and significantly outperformed the FREM2 expression level itself. For all relevant datasets, it could robustly discriminate GBM and LGG (p 0.74). High FREM2 pathway activation level was associated with poor OS in LGG (p < 0.001), and low PFS in LGG (p < 0.001) and GBM (p < 0.05). FREM2 pathway activation level was poor prognosis biomarker for OS (p < 0.05) and PFS (p < 0.05) in LGG with IDH mutation, for PFS in LGG with wild type IDH (p < 0.001) and mutant IDH with 1p/19q codeletion (p < 0.05), in GBM with unmethylated MGMT (p < 0.05), and in GBM with wild type IDH (p < 0.05). Thus, we conclude that the activation level of the FREM2 pathway is a potent new-generation diagnostic and prognostic biomarker for multiple molecular subtypes of GBM and LGG

Cytoskeletal proteins as glioblastoma biomarkers and targets for therapy

Glioblastoma, the most common primary brain malignancy, is an exceptionally fatal cancer. Lack of suitable biomarkers and efficient treatment largely contribute to the therapy failure. Cytoskeletal proteins are crucial proteins in glioblastoma pathogenesis and can potentially serve as biomarkers and therapeutic targets. Among them, GFAP, has gained most attention as potential diagnostic biomarker, while vimentin and microtubules are considered as prospective therapeutic targets. Microtubules represent one of the best anti-cancer targets due to their critical role in cell proliferation. Despite testing in clinical trials, the efficiency of taxanes, epothilones, vinca-domain binding drugs, colchicine-domain binding drugs and γ-tubulin binding drugs remains to be confirmed. Moreover, tumor treating field that disrupts microtubules draw attention because of its high efficiency and is called “the fourth cancer treatment modality”. Thereby, because of the involvement of cytoskeleton in key physiological and pathological processes, its therapeutic potential in glioblastoma is currently extensively investigated

Coding of glioblastoma progression and therapy resistance through long noncoding RNAs

Glioblastoma is the most aggressive and lethal primary brain malignancy, with an average patient survival from diagnosis of 14 months. Glioblastoma also usually progresses as a more invasive phenotype after initial treatment. A major step forward in our understanding of the nature of glioblastoma was achieved with large-scale expression analysis. However, due to genomic complexity and heterogeneity, transcriptomics alone is not enough to define the glioblastoma "fingerprint", so epigenetic mechanisms are being examined, including the noncoding genome. On the basis of their tissue specificity, long noncoding RNAs (lncRNAs) are being explored as new diagnostic and therapeutic targets. In addition, growing evidence indicates that lncRNAs have various roles in resistance to glioblastoma therapies (e.g., MALAT1, H19) and in glioblastoma progression (e.g., CRNDE, HOTAIRM1, ASLNC22381, ASLNC20819). Investigations have also focused on the prognostic value of lncRNAs, as well as the definition of the molecular signatures of glioma, to provide more precise tumor classification. This review discusses the potential that lncRNAs hold for the development of novel diagnostic and, hopefully, therapeutic targets that can contribute to prolonged survival and improved quality of life for patients with glioblastoma

Nanomedicine and immunotherapy

Owing to the advancement of technology combined with our deeper knowledge of human nature and diseases, we are able to move towards precision medicine, where patients are treated at the individual level in concordance with their genetic profiles. Lately, the integration of nanoparticles in biotechnology and their applications in medicine has allowed us to diagnose and treat disease better and more precisely. As a model disease, we used a grade IV malignant brain tumor (glioblastoma). Significant improvements in diagnosis were achieved with the application of fluorescent nanoparticles for intraoperative magnetic resonance imaging (MRI), allowing for improved tumor cell visibility and increasing the extent of the surgical resection, leading to better patient response. Fluorescent probes can be engineered to be activated through different molecular pathways, which will open the path to individualized glioblastoma diagnosis, monitoring, and treatment. Nanoparticles are also extensively studied as nanovehicles for targeted delivery and more controlled medication release, and some nanomedicines are already in early phases of clinical trials. Moreover, sampling biological fluids will give new insights into glioblastoma pathogenesis due to the presence of extracellular vesicles, circulating tumor cells, and circulating tumor DNA. As current glioblastoma therapy does not provide good quality of life for patients, other approaches such as immunotherapy are explored. To conclude, we reason that development of personalized therapies based on a patient’s genetic signature combined with pharmacogenomics and immunogenomic information will significantly change the outcome of glioblastoma patients

Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles

Glioblastoma is one of the deadliest cancers, therefore novel efficient therapeutic approaches are urgently required. One of such are nanobodies, prospective nano-sized bio-drugs with advantageous characteristics. Nanobodies can target intracellular proteins, but to increase their efficiency, the delivery system should be applied. Here, we examined small extracellular vesicles as a delivery system for anti-vimentin nanobody Nb79. Nb79 was loaded in small extracellular vesicles either by incubation with glioblastoma cells, by passive loading into isolated small extracellular vesicles or by sonication of isolated small extracellular vesicles. Small extracellular vesicles secreted by glioblastoma cells were isolated by ultracentrifugation on sucrose cushion. The size distribution and average size of sonicated and non-sonicated small extracellular vesicles were determined by nanoparticle tracking analysis method. The loading of Nb79 into small extracellular vesicles by incubation with cells, passive loading or sonication was confirmed by Western blot and electron microscopy. The effect of small extracellular vesicles on cell survival was determined by WST-1 reagent. Loading of small extracellular vesicles by incubation of cells with Nb79 was unsuccessful and resulted in substantial cell death. On the other hand, as confirmed by Western blot and electron microscopy, sonication is a successful method for obtaining Nb79-loaded small extracellular vesicles. Small extracellular vesicles also had an effect on cell viability. Small extracellular vesicles without Nb79 increased survival of U251 and NCH644 cells for 20–25%, while the Nb79-loaded small extracellular vesicles decreased survival of NCH421k by 11%. We demonstrated that sonication is a suitable method to load nanobodies into exosome, and these small extracellular vesicles could in turn reduce cell survival. The method could be translated also to other applications, such as targeted delivery system of other protein-based drugs

Anti-vimentin, anti-TUFM, anti-NAP1L1 and anti-DPYSL2 nanobodies display cytotoxic effect and reduce glioblastoma cell migration

Background: Glioblastoma is a particularly common and very aggressive primary brain tumour. One of the main causes of therapy failure is the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy, and that have the potential to form new tumours. This study focuses on validation of eight novel antigens, TRIM28, nucleolin, vimentin, nucleosome assembly protein 1-like 1 (NAP1L1), mitochondrial translation elongation factor (EF-TU) (TUFM), dihydropyrimidinase-related protein 2 (DPYSL2), collapsin response mediator protein 1 (CRMP1) and Aly/REF export factor (ALYREF), as putative glioblastoma targets, using nanobodies. Methods: Expression of these eight antigens was analysed at the cellular level by qPCR, ELISA and immunocytochemistry, and in tissues by immunohistochemistry. The cytotoxic effects of the nanobodies were determined using AlamarBlue and water-soluble tetrazolium tests. Annexin V/propidium iodide tests were used to determine apoptotsis/necrosis of the cells in the presence of the nanobodies. Cell migration assays were performed to determine the effects of the nanobodies on cell migration. Results: NAP1L1 and CRMP1 were significantly overexpressed in glioblastoma stem cells in comparison with astrocytes and glioblastoma cell lines at the mRNA and protein levels. Vimentin, DPYSL2 and ALYREF were overexpressed in glioblastoma cell lines only at the protein level. The functional part of the study examined the cytotoxic effects of the nanobodies on glioblastoma cell lines. Four of the nanobodies were selected in terms of their specificity towards glioblastoma cells and protein overexpression: anti-vimentin (Nb79), anti-NAP1L1 (Nb179), anti-TUFM (Nb225) and anti-DPYSL2 (Nb314). In further experiments to optimise the nanobody treatment schemes, to increase their effects, and to determine their impact on migration of glioblastoma cells, the anti-TUFM nanobody showed large cytotoxic effects on glioblastoma stem cells, while the anti-vimentin, anti-NAP1L1 and anti-DPYSL2 nanobodies were indicated as agents to target mature glioblastoma cells. The anti-vimentin nanobody also had significant effects on migration of mature glioblastoma cells. Conclusion: Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) nanobodies are indicated for further examination for cell targeting. The anti-TUFM nanobody, Nb225, is particularly potent for inhibition of cell growth after long-term exposure of glioblastoma stem cells, with minor effects seen for astrocytes. The anti-vimentin nanobody represents an agent for inhibition of cell migration

OligoPrime

With the increasing number of molecular biology techniques, large numbers of oligonucleotides are frequently involved in individual research projects. Thus, a dedicated electronic oligonucleotide management system is expected to provide several benefits such as increased oligonucleotide traceability, facilitated sharing of oligonucleotides between laboratories, and simplified (bulk) ordering of oligonucleotides. Herein, we describe OligoPrime, an information system for oligonucleotide management, which presents a computational support for all steps in an oligonucleotide lifecycle, namely, from its ordering and storage to its application, and disposal. OligoPrime is easy to use since it is accessible via a web browser and does not require any installation from the end user’s perspective. It allows filtering and search of oligonucleotides by various parameters, which include the exact location of an oligonucleotide, its sequence, and availability. The oligonucleotide database behind the system is shared among the researchers working in the same laboratory or research group. Users might have different roles which define the access permissions and range from students to researchers and primary investigators. Furthermore, OligoPrime is easy to manage and install and is based on open-source software solutions. Its code is freely available at https://github.com/OligoPrime. Moreover, an implementation of OligoPrime, which can be used for testing is available at http://oligoprime.xyz/. To our knowledge, OligoPrime is the only software solution dedicated specifically to oligonucleotide management. We strongly believe that it has a large potential to enhance the transparency of use and to simplify the management of oligonucleotides in academic laboratories and research groups