Browsing by Subject "cancer"

Chimeric antigen receptor (CAR) T cells are genetically modified usually autologous T cells expressing de novo designed CAR that binds a specific antigen on the surface of the cancer cells, inducing T cell receptor-independent activation and cytotoxic response against the targeted cancer cells. While CAR T cells have been shown to offer effective treatment in acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma, several resistance mechanisms can lead to CAR T cell exhaustion characterized by impaired functions and the expression of inhibitory receptors. The Finnish Red Cross Blood Service has developed novel CARs, differing in structure from the ones currently published. Since the evasion of CAR T cell exhaustion is considered one of the key objectives in the development of CAR T cell therapy, this Master’s thesis project aimed to create a working method to determine the exhaustion of CAR T cells in vitro after long-term repeated stimulation. In order to induce and measure exhaustion, CAR T cells were produced and activated ex vivo in the presence of IL-2 or IL-7/IL-15 cytokines, cultured long-term and repeatedly stimulated by exposure to target cells. CAR T cell cytotoxicity and expansion were determined and the expression of various inhibitory receptors was analyzed. The method enabled the comparison of the designed CAR T cell candidates and the positive control CD19-CD28ζ CAR T cells in long-term cytotoxic potency. In addition, it helped to reveal the surprising difference between IL-2 and IL-7/IL-15 cytokines and their impact on CAR T cell exhaustion. Although CAR T cells produced with IL-2 had poorer expansion during CAR T cell production than CAR T cells produced with IL-7/IL-15, they showed lower expression of exhaustion-related markers supported by better survival, proliferation and cytotoxic activity during long-term repeated stimulation assay.

Wilms tumor is the most common kidney cancer in children. The origin of Wilms tumor is thought to arise from disturbed embryonic development. Wilms tumor is characterized by so called nephrogenic rests consisting of undifferentiated metanephric mesenchyme, that are precursor lesions for tumorigenesis. In addition, the tumors themselves contain tissue types normally found only in the developing kidney and they resemble embryonic kidneys transcriptionally. Since the tumors are heterogenous and contain blastemal, stromal, and epithelial components, all three progenitor populations of the developing kidney, nephron, collecting duct and stromal progenitors, are possible origins of tumors. MAPK/ERK pathway plays a significant role in kidney development for example by affecting nephron progenitor self-renewal and regulating collecting duct progenitor maintenance. Previous studies suggest that MAPK/ERK activity plays a role in Wilms tumor by mediating the intracellular effects of IGF2 overexpression. The aim of this master’s thesis is to compare transcriptional profiles of Wilms tumor to the transcriptional profiles in mouse MAPK/ERK deficient nephron progenitors and ureteric bud epithelium. This is done by utilizing internet-based tool ToppFun and R/Bioconductor tool gage (Generally Applicable Gene-set Enrichment for Pathway Analysis) for Gene Ontology (GO) and KEGG pathway analysis. My analysis revealed several shared GO and KEGG pathways that compose of differentially expressed genes with opposite expression patterns in Wilms tumor and MAPK/ERK deficient renal progenitors. The identified pathways include those previously validated by the host laboratory and some interesting new pathways that will be studied further in the future. The most interesting novel pathways affected in both Wilms tumor and MAPK/ERK deficient kidneys were related to the extracellular matrix (ECM) and chromatin. ECM related GO terms were specifically altered in nephron progenitors and Wilms tumor suggesting that Wilms tumor transformation involves dysregulation of ECM possibly downstream of MAPK/ERK pathway. MAPK/ERK pathway also mediates chromatin level regulation which is also demonstrated by my results. Chromatin related GO terms were upregulated in Wilms tumor and downregulated both in ureteric bud epithelium and nephron progenitors. The second aim of my thesis is to verify the observed gene expression changes by utilizing mouse embryonic kidney cultures and qPCR. The validation was only initial trial and requires further optimization. It did not show significant downregulation in selected validation genes that were chosen for validation. Future goal of my research is to carry out similar analysis in chemotherapy naïve dataset and at different Wilms tumor stages. This will allow avoiding possible chemotherapy induced changes in the outcome and better sorting of tumor progression and its correlation with specific renal progenitor types. In summary, my current results suggest both ECM and chromatin regulation as promising fields for future research.

Enhancers are important regulatory elements of DNA, that are bound by transcription factors (TFs) to regulate gene expression. Enhancers control cell type specific gene expression and they can form structures called super-enhancers, that consist of multiple normal enhancers and are bound by high numbers and variety of transcription factors. These super-enhancers are important for defining cell identity and changes in the super-enhancer landscape have been linked to different cancers. In this project, characterization of super-enhancers and their transcription factors composition between primary and cancer cells were studied using genome-wide next-generation sequencing data from multiple assays, such as ChIP-seq, RNA-seq and ATAC-seq. The focus of the project was on the data processing and analysis to identify and characterize the super-enhancers. Analyses included GSEA, heatmap binding analysis, peak and super-enhancer calling and IGV analysis. This project used pancreatic HPDE cell line for primary cells and different cancers with endodermal origin as cancer cell lines. The goal of the thesis was to try show characteristic features of super-enhancers and their features in normal and cancer cells. Data analysis showed that distinct super-enhancers can be identified in cancer cells and defined super-enhancers had typical strong binding for specific transcription factor and histone modification such as histone 3 lysine 27 acetylation (H3K27ac) mark of active enhancers. Super-enhancer regions were located in highly accessible chromatin regions of the genome, and genes that were associated with HPDE super-enhancers could be shown to have association with cell identity. Peak and super-enhancer calling counts varied between cell lines for transcription factors, histone modifications and super-enhancers. Visualization of super-enhancers was successful and could show transcription factor binding and active enhancers that establish the super-enhancer structure. Comprehensive analyses allowed us to characterize typical features of super-enhancers and show differences in the numbers of super-enhancers between primary and cancer cell lines and cancer cell lines of different organ types. Analysis of the transcription factor binding showed unique peaks on some of the super-enhancers, and these peaks might have a role in inducing the super-enhancer structure.

MLH1 is a gene that codes for one of the four mismatch repair (MMR) proteins alongside MSH2, MSH6, and PMS2. The main function of the MMR proteins is to recognize base mismatches and insertion-deletion loops formed during DNA replication and aid in their excision. Inherited heterozygous pathogenic variants in any of the four MMR genes lead to Lynch syndrome, an inherited cancer syndrome that predisposes to multiple different cancer types, most notably colorectal cancer. Loss of the expression of an MMR gene causes MMR-deficiency, which leads to microsatellite instability, the accumulation of mutations in microsatellite regions of the DNA. The higher mutational burden caused by MMR-deficiency is thought to be the main driving force of genomic instability and tumorigenesis in MMR-deficient cells. In addition to MMR, MLH1 and the MMR machinery have roles in other anticarcinogenic cellular processes, such as DNA damage signaling and DNA double-strand break repair. Recently, MLH1 has also been shown to have a significant role in regulating mitochondrial metabolism and oxidative stress responses. The identification of MMR-proficient tumors in Lynch syndrome patients begs the question whether the lower amount of functional MLH1 observed in MLH1 mutation carriers could cause problems with these functions and pose alternative routes to tumorigenesis. In line with this, it has been shown that the role of MLH1 in cell cycle regulation in DNA damage signaling is notably more sensitive to decreased amount of the protein compared to its role in MMR. The main goal of the thesis was to study the effects of decreased MLH1 expression on gene expression, cellular functions, and possible alternative tumorigenic pathways. In order to achieve this, the coding transcriptome of human fibroblast cell lines expressing MLH1 at different levels was sequenced and the resulting data analyzed. The study revealed that decreased MLH1 expression affects cellular functions associated with mitochondrial function and oxidative stress responses in cells with functional MMR. Particularly NRF2-controlled cytoprotective defence systems were observed to be downregulated. Decreased MLH1 expression was also observed to affect several cellular functions associated with reorganization of the cytoskeleton and interactions with the extracellular matrix. These results strengthen the recently made notions that MLH1 has a role in controlling the function of mitochondria and in mitigating oxidative stress, and that these two functions are connected. The study also brings to light new information on the possible role of MLH1 in controlling the organization of the cytoskeleton, which has previously received little attention. Dysfunction of mitochondria, increased oxidative stress, and reorganization of the cytoskeleton, as a result of decreased MLH1 expression, could pose events that facilitate malignant transformation of cells prior to the total loss of MMR function.

Even though cancer treatment modalities have improved during last decades, there is still lack of specific, efficient and curative treatments especially in case of advanced and metastatic cancers. One relatively new approach is to use oncolytic adenoviruses, which selectively infect and kill cancerous cells leaving healthy cells unharmed. These viruses have shown to be effective especially when administered intratumorally and in combination with chemotherapeutics. However this approach has multiple challenges like rapid clearance by antibody neutralization in systemic administration. Another challenge is the cell entry of oncolytic adenovirus, which is mainly mediated by the Coxsackie-Adenovirus receptor and this receptor is downregulated in various cancer cells. Rapid clearance and reduced cell entry thus lead to decreased amount of oncolytic adenovirus in target cells and decreased efficacy. In order to overcome these limitations, this study explored the possibility to use cancer cell derived extracellular vesicles (EVs) as drug delivery system for oncolytic adenovirus. Since oncolytic adenoviruses have shown to be effective especially in combination with chemotherapeutics, the ability of EVs to deliver both oncolytic adenoviruses and chemotherapeutic drug paclitaxel was studied. The aims of this study were to i) study whether oncolytic adenoviruses could be encapsulated inside EVs (EV-virus complex) and load this complex with paclitaxel (EV-virus-PTX complex), ii) discover whether the surface charge or size distribution of EV-virus and EV-virus-PTX complexes differs from the control EVs and iii) study the infectivity/efficacy of EV-virus and EV-virus-PTX complex in comparison to noncapsulated adenovirus in vitro. Since this is a novel approach, the literature review focused on the characteristics, advantages and challenges of oncolytic adenoviruses and EVs. In order to determine whether cancerous cell are able to encapsulate oncolytic adenoviruses inside EVs, A549 lung cancer and PC-3 prostate cancer cells were infected with oncolytic adenovirus and the formed EVs were isolated form conditioned media using differential centrifugation. Paclitaxel was loaded into these EV-virus complexes with incubation. EV-virus complexes were imaged using transmission electron microscopy (TEM) (i). The characteristics of these EV-virus and EV-virus-paclitaxel complexes were studied by determining the surface charge by electrophoretic light scattering and the size distribution by nanoparticle tracking analysis (ii). In order to determine the infectivity/efficacy of these complexes in autologous use, three in vitro level assays were performed (cell viability, immunocytochemistry and transduction assay) (iii). In addition confocal microscopy was used to observe the localization of EV-virus complexes inside the cell. These studies pointed out that both cell lines were able to encapsulate oncolytic adenovirus inside EVs, which was observed by TEM. The size distribution of these EV-virus and EV-virus-PTX complexes may support this observation and the size was in range 50-500 nm. In addition the determined surface charge was shown to be similar in EV-virus and EV-virus-PTX- complexes when compared to control EVs derived from noninfected cells - however more specific assays in order to characterize the surface properties of EV-virus complexes are needed. As a main finding, these EV-virus and EV-virus-PTX complexes were shown to significantly increase the efficacy of oncolytic adenovirus in comparison to free oncolytic adenovirus, paclitaxel and paclitaxel+virus combination in all three in vitro assays. In addition localization of the EV-virus complex was seen with confocal microscopy imaging. These results indicate that EVs may enhance the delivery of oncolytic adenovirus into cancerous cells. Using EVs as a drug delivery system for both oncolytic adenovirus and chemotherapeutic drug paclitaxel was shown to increase the efficacy of oncolytic adenovirus in comparison to free virus. This characteristic could potentially enhance the targeting ability to cancerous cells and thus lead to decreased amount of side-effects of healthy tissues especially in case of chemotherapeutics. These promising results of this novel approach are however preliminary due to relatively low number of repetitions (n~3) and more research is needed especially in order to characterize, purify and concentrate the EV-virus complexes.

Background and objectives: Cancer is one of the leading causes of death worldwide, and resistance to current treatments demands the continuous development of novel cancer therapies. Cancer immunotherapy aims to induce anticancer immune responses that selectively target cancer cells. Viruses can also be harnessed to elicit tumor-specific immune responses and to improve the response rates of other concomitant cancer therapies. The purpose of this study was to develop a novel viral vector-based cancer vaccine for intratumoral immunotherapy. By using the previously developed PeptiENV cancer vaccine platform, the vector viruses were coated with cell-penetrating peptide (CPP) sequence-containing tumor peptides in an attempt to further drive the immune responses elicited by the vector against cancer cells. The efficacy of the PeptiENV complex as a cancer vaccine was assessed by following its effects on tumor growth and the development of local and systemic antitumor immune responses. Methods: The PeptiENV complex formation was assessed by a surface plasmon resonance (SPR) analysis. Dendritic cell (DC) activation and antigen cross-presentation were studied using the murine JAWS II dendritic cell line. The development of cellular immune responses against tumor antigens was first studied by immunizing mice with the PeptiENV complex. The antitumor efficacy and immunity of intratumoral PeptiENV administration were then studied using the murine melanoma models B16.OVA and B16.F10.9/K1. In addition to intratumoral PeptiENV treatment, some of the B16.F10.9/K1-implanted mice were also treated with an anti-PD-1 immune checkpoint inhibitor (ICI) to study the PeptiENV complex as a biological adjuvant for ICIs. Results: The SPR analysis confirmed that CPP-containing peptides can be stably anchored onto the viral envelope of the viral vector. The in vitro results showed that the PeptiENV complex does not hamper the presentation of antigens at the surface of DCs. Additionally, the viral vector was found to activate DCs seen as a change in the cells’ morphology and surface protein expression. Immunizing mice with the PeptiENV complex induced a robust antigen-specific cytotoxic T cell response. Upon intratumoral administration in vivo, the PeptiENV cancer vaccine was not capable of inducing tumor growth control against B16.OVA melanoma, although it did still elicit robust systemic and local antitumor T cell responses. In the treatment of B16.F10.9/K1 melanoma, however, the PeptiENV complex induced efficient tumor growth control, which resulted in a significant survival benefit. Additionally, co-administration of anti-PD-1 resulted in an additive therapeutic effect. Discussion and conclusions: The present study describes a novel, highly immunogenic viral vector-based cancer vaccine that has the potential to be used as an adjuvant treatment for ICI therapy. Subsequent studies could be conducted to gain a deeper understanding of the immunological mechanisms underlying the antitumor efficacy of the cancer vaccine complex. Moreover, this novel PeptiENV complex could also be further developed as an infectious disease vaccine platform against emerging pandemics. However, the effects of pre-existing antiviral immunity on the efficacy of the cancer vaccine should be explored in future studies.

According to the latest estimations, cancer is the second leading cause of death worldwide. Despite the significant advances in the range of drugs and treatment modalities to treat cancer, the number of deaths is estimated to continue rising, posing serious challenges for the patients, their families, and the healthcare systems. Conventional treatments tend to be associated with severe adverse side effects and treatment resistance. Consequently, safer and more efficient therapy options are urgently needed, especially for the treatment of metastatic tumors refractory to conventional treatments. A new and revolutionizing field in oncology is immunotherapy, in which oncolytic viruses are included. Oncolytic viruses have an inherent or acquired selectivity to replicate exclusively in tumor cells, ultimately destroying them. Simultaneously, they also activate the dormant host’s immune system to fight against the tumor. Adenoviruses, particularly, have shown to be safe, inducing only mild adverse side effects in clinical trials, making them a great candidate for further clinical development. Adenoviruses can be genetically modified to increase their infectivity or improve the anti-cancer immune responses induced by the virus, e.g., through the expression of immunostimulatory molecules. The focus of this thesis was to develop and characterize several genetically modified oncolytic adenoviruses expressing either OX40L alone or OX40L and CD40L, two co-stimulatory molecules capable of engaging both the innate and adaptive arms of the immune system to fight the tumor. The insertion of the transgenes into the E3B-14.7k region of the Ad5/3-∆24 adenovector plasmid was performed using Gibson Assembly® cloning approach. After successful cloning, the recombinant viral genomes were transfected into A549 cells for viral amplification, followed by CsCl purification to produce a high titer viral preparation. The expression of the transgenes was studied in vitro by ELISA and functional assays, showing promising expression levels of functional OX40L and CD40L. However, when the infectivity and virus killing potency were analyzed, in vitro by immunocytochemistry and MTS assay; and in vivo using an immunodeficient mouse model, the data showed that the cloned viruses performed sub-optimally when compared to the control unarmed virus (Ad5/3-∆24). These findings suggest that the insertion of the two transgenes in place of the E3-14.7k gene was detrimental to the fitness of the virus.

Cancer is a worldwide health problem; in 2018 9.6 million people died of cancer, meaning that about 1 in 6 deaths was caused by it. The challenge with cancer drug therapy has been the development of cancer drugs that are effective against cancer but are not harmful to the healthy cells. One of the solutions to this has been antibody-drug conjugates (ADCs), where a cytotoxic drug is bound to an antibody. The antibody binds to specific antigen present on the surface of the cancer cell, thus working as a vessel to carry the drug specifically to the cancer cells. Monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) are mitosis preventing cancer drugs. The auristatins are pentapeptides that were developed from dolastatin 10. MMAE consist of monomethyl valine (MeVal), valine (Val), dolaisoleiune (Dil), dolaproine (Dap) and norephedrine (PPA). MMAF has otherwise similar structure, but norephedrine is replaced by phenylalanine (Phe). They prevent cell division and cancer cell proliferation by binding to microtubules and are thus able to kill any kind of cell. By attaching the auristatin to an antibody that targets cancer cells, they can effectively be used in the treatment of cancer. MMAE and MMAF exist as two conformers in solution, namely as cis- and trans-conformers. The trans-conformer resembles the biologically active conformer. It was recently noted that in solution 50-60 % of the MMAE and MMAF-molecules exist in the biologically inactive cis-conformer. The molecule changes from one conformer to the other by the rotation of an amide bond. However, this takes several hours in body temperature. As the amount of the cis-conformer is significant, the efficacy of the drug is decreased, and the possibility of side effects is increased. It is possible that the molecule leaves the cancer cell in its inactive form, migrates to healthy cells and tissue, and transforms to the active form there, damaging the healthy cell. The goal of this study was to modify the structure of the auristatins so that the cis/trans-equilibrium would change to favor the biologically active trans-conformer. The modifications were done virtually, and the relative energies were computed using high-level quantum chemical methods, at density functional theory (DFT), 2nd order perturbation theory (MP2) and coupled cluster levels. Intramolecular interactions were analyzed computationally, employing symmetry-adapted perturbation theory and the non-covalent interactions analysis. The results suggest that simple halogenation of the benzene ring para-position is able to significantly shift the cis/trans-equilibrium to favor the trans-conformer. This is due to changes in intramolecular interactions that favor the trans-conformer after halogenation. For example, the NCI analysis shows that the halogen atom invokes stabilizing intramolecular interactions with the Dil amino acid; there is no such interaction between the para-position hydrogen and Dil in the original molecules. We also performed docking studies that show that the halogenated molecules can bind to microtubules, thus confirming that the modified structures have potential to be developed into new, more efficient and safe cancer drugs. The most promising drug candidates are Cl-MMAF, F-MMAF, and F-MMAE where 94, 90, and 79 % of the molecule is predicted to exist in the biologically active trans-conformer, respectively.

Tutkielman tarkoitus on tuoda tunnettujen riskitekijöiden rinnalle tuore tieteellinen näkemys suuinfektioiden ja syövän yhteydestä. Tavoitteena on myös vahvistaa käsitystä suunterveyden merkityksestä ihmisen yleisterveyteen. Tutkielma on toteutettu kirjallisuuskatsauksena. Tutkielmassa havaittiin tilastollisesti suuinfektioiden lisäävän potilaan riskiä sairastua syöpään. Tutkielmassa tarkastelluista aineistosta voidaan todeta, että tilastollinen yhteys suuinfektioiden ja syövän välillä on havaittu, mutta mahdollisen kausaliteetin osoittaminen vaatii runsaasti lisätutkimuksia. Suuinfektiot ovat hoidettavissa ja ennaltaehkäistävissä, jolloin voidaan mahdollisesti myös alentaa potilaan riskiä sairastua syöpään.

Matriksin metalloproteinaasi 8 on kollageenia hajottava ihmisen elimistön erittämä entsyymi. Se osallistuu normaaliin kudosten muokkaamiseen ja tulehduksellisiin sairauksiin, kuten syöpään, parodontiittiin ja sydän- ja verisuonitauteihin. Tässä tutkimuksessa aineistona ovat Pubmedistä haetut artikkelit ja FINRISK-tutkimuksessa vuonna 1997 kerätty aineisto 8349 henkilöltä. MMP-8-pitoisuus määritettiin tutkimushenkilöiden seerumista IFMA-menetelmällä. FINRISK-tutkimusaineistoa analysoitiin IMB SPSS Statistics-ohjelman avulla. Tulokseksi saatiin, että seerumin matriksin metalloproteinaasi 8:n pitoisuus on terveillä korkeampi kuin sairailla, kun tarkastellaan koko väestöä. Ikäryhmittäin tarkasteltuna ikäryhmien sisällä pitoisuus on kuitenkin sairailla korkeampi kuin terveillä. Matriksin metalloproteinaasi 8:n pitoisuus laskee iän myötä. Erot pitoisuudessa terveiden ja sairaiden välillä pienentyvät iän myötä. Viitearvot määritettiin tässä tutkimuksessa, ja ne ovat 5,33-241,2 ng/ml kaikille, naisille 5,61-250,1 ng/ml ja miehille 4,96-217,9 ng/ml. Terveillä ja sairailla tupakoitsijoilla pitoisuudet ovat käytännössä samat. Tupakoinnin lopettaneilla erot pitoisuudessa ovat terveiden ja sairaiden välillä suhteellisen suuret. Henkilöillä, joilla on metabolinen oireyhtymä, on matala matriksin metalloproteinaasi 8:n pitoisuus. Pitoisuus on tilastollisesti merkitsevästi matalampi myöhemmin puhkeavan diabeteksen suhteen. Tässä tutkimuksessa MMP-8:n pitoisuudet olivat sydän- ja verisuonisairauksia sairastavilla tutkittavilla henkilöillä matalat. Matriksin metalloproteinaasi 8:n pitoisuus korreloi positiivisesti CRP:n ja erityisen voimakkaasti fibrinogeenin kanssa. Vaikuttaa siltä, että kroonisesti sairailla pitoisuudet ovat matalat, akuutissa tilanteessa pitoisuudet nousevat ja ajan kuluessa laskevat takaisin mataliksi. Tämä koskee erityisesti sydän- ja verisuonitauteja. Hypoteesi tarvitsisi lisää tutkimusta, jotta pystyisimme MMP-8:n avulla seuraamaan ja ennustamaan taudinkulkua paremmin. (212 sanaa)