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Browsing by study line "Cancer"

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  • Adhikari, Sadiksha (2020)
    Structural variants comprise a large number of variations occurring in the human genome and are detected in many diseases including cancers. To a limited extent, whole exome sequencing (WES) is capable of detecting structural variations (SVs) using algorithms and tools utilizing local assembly, split-reads, discordant read-pairs and read depth methods. However, due to the significantly large size of SVs compared to the reads produced and the presence of repetitive regions in the genome, identification of SVs presents a major challenge. 10X Genomics has developed a technology that requires very low amounts of DNA and uses a linked-reads approach to produce long reads. Recently, linked-read technology has shown promising results in resolving complex SVs. In this thesis, we aimed to assess whether linked-read exome sequencing is able to infer more comprehensive information in SVs compared to WES in multiple myeloma (MM). The disease model was chosen based on the presence of high numbers of SVs in MM patient tumor cells. Here, we report that linked-read sequencing has led to the identification of a potential novel translocation t(1; 14) that significantly impacts the change in expression of genes and could potentially have impact on the prognosis and treatment of multiple myeloma patients. By Long Ranger analysis we detected t(1;14) in six out of eight samples. Further, to study whether the translocation differentially affects the expression levels of any genes, differential gene expression was performed between t(1;14) positive versus t(1;14) wild type samples. The analysis resulted in 107 differentially expressed genes where 4 upregulated and 103 downregulated genes were found in the translocation positive samples. Among the downregulated genes, we found S100A8 and S100A9 genes which are previously shown to be associated with chemoresistance to PAD (bortezomib, doxorubicin and dexamethasone) therapy. The related breakpoints of the event were identified by Manta tool (SV caller) using both linked-read and WES. Therefore, linked-read information does not appear necessary to detect this event. In this study, we found that linked-read sequencing has certain advantages over WES such as low input DNA, increased number and quality of calls and breakpoint information. However, linked-read sequencing technique is limited to the detection of certain SV types in addition to increased cost of sequencing. These two factors must be considered before choosing linked-read sequencing over WES. Somatic mutations and clinically relevant SV were detected equally efficiently by both techniques.
  • Wartiovaara, Linnea (2023)
    Renewal of the intestinal epithelium is driven by the actively dividing and strictly regulated Lgr5-expressing intestinal stem cells (ISCs). As uncontrolled proliferation may lead to colorectal cancer (CRC), ISCs and the regulatory circuit around them could elucidate new targets for colorectal cancer therapy. The regulatory crosstalk between the stem cells and surrounding stroma is under intensive investigation, but little is known about the neuronal control of the intestinal epithelium. Adrenergic signals from sympathetic nervous system regulate hematopoietic and melanocyte stem cells and promote tumorigenesis in e.g., pancreatic, and prostate cancer. Adra2a, one of the nine different adrenergic receptors, is expressed in the ISCs, and adrenergic neurons project neurites to the stem cell niche suggesting a paracrine signaling role. However, whether the adrenergic signaling plays a role in ISC regulation and/or in colorectal cancer remains unknown. The aim of this study was to develop tools to investigate the role of adrenergic signaling in ISC regulation. First, I set up a protocol to delete Adra2a and other genes in intestinal organoids recapitulating the stem cell hierarchy of the intestinal epithelium in vitro, using CRISPR-Cas9 technology. This led to successful deletion of the Ret proto-oncogene while an Adra2a-deficient organoid pool could not be established with these efforts. I differentiated neuroblastoma cells to a catecholaminergic phenotype and cultured them together with intestinal organoids to address the effect of catecholaminergic signaling on the intestinal epithelium in organoid cocultures. The coculture with catecholaminergic cells induced upregulation of the regeneration markers Ly6a and Clu in intestinal organoids, while Reg3b as well as the stem cell markers Lgr5, Olfm4, and Adra2a and the Paneth cell marker Lyz1 were reduced. Third, I assessed the direct effect of a 3-hour norepinephrine (NE) pulse on wild-type organoids with 3’RNA sequencing, however, this did not induce significant changes in the expression levels of the respective regeneration marker genes. Altogether, my work established a CRISPR-Cas9-based method to delete genes of interest in primary intestinal organoids. Further investigation is needed to verify if NE contributes to the regulation of intestinal regeneration and stem cell maintenance.
  • Dool Muuse Abdillahi, Khalid (2023)
    Pään ja kaulan alueen syövät ovat seitsemänneksi yleisin söpäryhmä. Vuosittain siihen sairastuu 660 000 ja menehtyy 325 000 henkilöä maailmanlaajuisesti. Sen merkittävimmät riskitekijät ovat ikä, tupakointi, alkoholin kuluttaminen sekä HPV-infektio. Sen esiintyvyys on merkittävässä nousussa HPV-infektioiden yleistyessä sekä väestön ikääntymisen myötä. Gerastania on olennainen tekijä potilaan hoidon ja ennusteen kannalta. Siitä huolimatta tätä aihealuetta ja sen yhteyttä pään ja kaulan alueen syöpiin on tutkittu niukasti, sen merkittävyydestä huolimatta. Tällä hetkellä potilaan hoidon- ja ennusteenarvio on yksittäisen tai muutaman kliinikon standardoimattoman arvion käsissä. Tässä narratiivisessa kirjallisuuskatsauksessa perehdytään gerasteniaan ja pään ja kaulan alueen syöpien väliseen yhteyteen kokoamalla PubMed-tietokannasta aihealuetta käsitteleviä artikkeleita hyödyntämällä avainsanoja ’Head and Neck Neoplasms’, ’Frailty’ sekä ’Complications’ sekä ’Therapeutics’. Avainsana ’Therapeutics’ viittaa siis hoitoon, joka pään ja kaulan alueen syövissä on pääosin käsittää kirurgiaa sekä kemosädehoitoa. Tutkimuskysymystä käsitteleviä löydettiin 297 kappaletta, joista 66 hyödynnettiin tutkielman kirjoittamiseen. Tutkielma osoittaa, että pään ja kaulan alueen syöpiin ja niiden hoitoon liittyy monenlaisia komplikaatioita, joilla on osoitetusti huono ennustevaikutus erityisesti gerastenian kanssa. Tutkimus myös osoittaa, että on toivottavaa arvioida systemaattisesti potilaiden gerasteniaa kvantitatiivisesti, sillä pään ja kaulan alueen syöpien potilaat ovat usein hauraita potilaita, joiden kohdalla gerastenian huomioimattomuus voi johtaa väärään tai kohtalokkaaseen hoitoon.
  • Koivula, Julia (2023)
    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.
  • Suominen, Peppi Lotta Josefiina (2023)
    Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells in the bone marrow (BM). MM is the second most common hematological malignancy, accounting for about 14% of blood cancers. Despite the improvements in the treatment of MM, the disease remains incurable, and essentially all patients end up relapsing. Deletion of chromosome 16q, the location of tumor suppressor CYLD, occurs in 35% of MM patients. CYLD is a deubiquitinase, most recognized for its function as a negative regulator of the nuclear factor kappa B (NF-κB) pathway. The loss of CYLD is associated with disease progression and worse survival in MM, but its significance in drug response is unknown. As the loss of CYLD is common in MM, determining its effect on drug response is essential. Analysis of data gained from MM patient samples were used to study the effect of CYLD copy number status on drug response. The treatments were selected based on previous research performed by our group. The effect of homozygous deletion was most significant in inducing resistance to BMS-754807, an inhibitor of insulin-like growth factor 1 receptor. To investigate the role of loss of CYLD on drug response in vitro, cell lines with CYLD knockout (KO) were created using the clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR associated protein 9 (Cas9) technology. An established CYLD-KO cell line was treated with carfilzomib, bortezomib, dexamethasone and BMS-754807 to assess the effect of CYLD-KO. The CYLD-KO slightly increased the sensitivity to BMS-754807, but essentially no differences were detected in response to the drugs. The effect of CYLD-KO was additionally explored to NF-κB - and Wnt-pathway activation by Western blot analysis, but due to technical difficulties, the results were inconclusive. The loss of CYLD is a common genetic aberration in MM, giving a survival benefit for the malignant cells. Based on the results from patient data analysis, the loss of CYLD could promote drug resistance to BMS-754807, and the effect should be further studied with more cell lines with CYLD-KO. As the population ages, and as the median age of newly diagnosed patients is 70, the need for efficient MM therapies increases. Studying the mechanisms behind drug resistance and sensitivity is essential in the aim of improving the efficacy of MM therapy and, in the end, the overall survival of the patients.
  • Bouhlal, Jonas Otto Vilhelm (2022)
    Despite of great advancements in the field of cancer therapy in the past decades, the 5-year survival of acute myeloid leukaemia (AML) patients remains low with high mortality especially in elderly patients, in whom the disease is most often observed. Poor prognosis often results from complex heterogenous molecular abnormalities defining the progress of the disease, while making it more difficult to treat due to intensive treatments only being feasible for younger patients. Our increased understanding of cancer immunology and the potential of immunotherapy has, however, led to promising therapeutic innovations, which give hope for discovering long-lasting and effective treatment options. Natural killer (NK) cell-based immunotherapies are amongst the emerging novel therapeutic approaches that aim to target malignant cells with less toxicity and improved applicability. Using high-throughput drug sensitivity and resistance testing combined with single cell RNA (scRNA) sequencing, this study focused on finding drug compounds that could synergise with NK cells to improve their effectiveness in killing leukemic cells. In this study, many drugs showed promising results in being able to potentiate NK cell cytotoxicity, with daporinad and pevonedistat showing the most notable differences when compared to controls. The potentiating effect of Janus kinase (JAK) inhibitors also suggested a method of increasing NK cell activity against leukemic cells through downregulation of major histocompatibility complex (MHC) class I molecules. In conclusion, findings shed light on the synergetic potential of drugs and NK cells, giving hope for clinically relevant findings following further validation and testing.
  • Karttunen, Konsta Eemeli (2023)
    Transposable elements (TEs) are repetitive DNA elements that have an autonomous capability of replicating and inserting themselves into new loci within genomes. TEs have often been thought to be a part of the non-functional “junk DNA”, but advances in next-generation sequencing technology has revealed that TEs have rich biochemical functions: Specific TEs bind a large fraction of transcription factors and harbor marks of active chromatin in human genomes. However, there have been few genome-wide functional enhancer activity studies on the role of TEs in gene regulation, especially in the context of human cancers. In normal cellular homeostasis, TE activity is tightly controlled by epigenetic mechanisms. In contrast, the cell state in cancer genomes is often permissive for TE activation: genome instability and mutations, notably p53 inactivation, and nonmutational epigenetic reprogramming such as DNA hypomethylation are common characteristics of cancers. TE transcription, somatic retrotransposition and activation of cryptic promoter elements occur frequently in tumors. TE activation is highly heterogeneous between cancer types: for example gastrointestinal tract cancers such as colorectal cancer show high somatic insertion activity, whereas retrotransposition is rare in hematolymphoid malignancies. Due to the widespread TE activation in cancers, we posited that this may also be seen in the activity of cryptic TE enhancers, with specific active families of TEs characteristic to different cell types due to lineage-specific TF binding. We asked if TEs contribute to the enhancer landscape of cancers and to which extent, what are the differences between cancers in the activity and transcription factor binding and whether TE enhancers may have a role in tumorigenesis and the regulation of cancer-specific genes. To functionally study TEs, we utilized a high-resolution, unbiased, genome-wide massively parallel reporter assay (MPRA). We utilized colorectal and hepatocellular cancer cell lines to study the differences and similarities between TE activation and combined the MPRA data with orthogonal epigenetic data to study the in vivo signatures of the TEs. We found that both cell lines show common and highly enriched TE subfamilies that were mostly specific for p53, as well as TEs that were highly unique in both cell lines. By using in vitro methylated MPRA libraries, we found that CpG methylation has relatively minor a role in regulating the enhancer activity of some TE subfamilies in the reporter assay. By comparing the epigenetic context of the TEs, we found that especially colorectal cancer has specific highly active TE subfamilies with signatures of canonical active enhancers. We also used an in silico model to predict TE enhancer to gene contacts and found that these subfamilies regulated genes that were frequently overexpressed. Thus, we present the widespread functional activity of TE enhancers in cancers, providing evidence for further functional validation of TEs and their effects on transcriptional programs and especially dysregulation of gene expression in cancer.
  • Mölsä, Riikka (2023)
    Lung cancer is the number one cause of cancer-related deaths in the world every year. Of all non-small cell lung cancers, lung adenocarcinoma is the most prevalent subtype. There is a great need for better treatment options for lung cancer, of which cancer immunotherapy is an attractive option due to the high mutational burden of lung tumors. Patient-derived lung cancer organoids (lung PDTOs) could provide a new testing platform for these studies as the 3D models better represent the original tumor and its microenvironment compared to often used 2D cell lines. One interesting field is immunopeptidomics, which focuses on discovering tumor peptides presented in the HLA-I molecule on the tumor surface that could elicit an immune response. Using lung adenocarcinoma PDTOs, this study aimed to analyze the immunopeptidome of five PDTOs to discover tumor-specific and immunogenic peptides using PeptiCHIP purification and LC-MS analysis. These findings could be used in PeptiCRAd, a novel cancer vaccine comprised of an oncolytic adenovirus coated with tumor peptides. To elucidate the applicability of PDTOs for virotherapy, three oncolytic adenoviruses, D102, Ad5/3Δ24 and Ad5Δ24-RFP, and their ability to infect, kill, and replicate in lung PDTOs was studied. PDTOs were characterized as epithelial, as they presented epithelial cytokeratin and epithelial layer structures, as indicated by cytoskeletal F-actin staining. The three oncolytic adenoviruses were studied by infecting PDTOs and a difference in killing capacity of the three viruses was shown, potentially due to differences in receptor interactions and expressed transgenes. In addition, D102 and Ad5Δ24-RFP were shown to replicate in PDTOs, which is necessary to induce a strong enough immune response against the virus for immunotherapy efficiency. HLA-I expression was high in all tested models, which indicated that antigens could be presented in the tumor cells. Immunopeptidome analysis did not result in a high yield of peptides, likely due to challenges in sample preparation and patient material being scarce. As the HLA-type of each patient was unknown during this study, more data analyses still need to be done to determine the best immunogenic peptides, which could then be further studied in vitro. However, peptides overexpressed in lung cancer and with cancer benefiting properties were found from PDTOs, which already gives promising results. In conclusion, though additional immunopeptidome studies with an increased yield of peptides are needed to select tumor-relevant immunogenic targets for therapeutical use, as well as additional testing on the optimal oncolytic virus for lung cancer targeted PeptiCRAd immunotherapy, this study proved that oncolytic viruses can infect and kill lung PDTOs, and that HLA-I expressed tumor peptides can be identified from them. This is also one step towards finding better and patient-specific research models for testing therapies and discovering and developing personalized cancer treatments.
  • Junquera Mencia, Ada (2023)
    High-grade serous ovarian cancer (HGSC) presents a complex clinical challenge as it is often diagnosed at advanced stages. Neoadjuvant Chemotherapy (NACT) is commonly used to treat advanced stage (III and IV) patients who cannot undergo primary debulking surgery. However, 80% of them experience relapse and develop resistance to platinum-based chemo therapies. While NACT alters the tumor immune microenvironment in a treatment response specific manner, its underlying mechanisms remain unclear. Understanding the effect of NACT on tumor microenvironment (TME) is crucial to identify novel biomarkers and develop effective therapeutic strategies. Emerging spatially resolved methodologies, including highly multiplexed-imaging and spatial gene-expression profiling approaches provide novel information regarding spatial interactions and mechanisms operating at single-cell level. However, neither of these methods can offer a comprehensive view of the tumor immune landscape while also revealing the molecular mechanisms behind it. To overcome this limitation, this thesis proposes a novel method integrating tissue cyclic immunofluorescence (tCycIF) and GeoMx DSP spatial transcriptomics. tCycIF is a high-throughput multiplexed imaging method, while GeoMx DSP offers sequencing information at near to single-cell resolution in well-defined regions of interest (ROIs), enabling the dissection of the underlying molecular mechanisms. Here I set out to explore the effect of NACT on tumor stroma interface (TSI) and the surrounding tumor and immune cells, particularly IBA1+ macrophages, and CD8+ T cells. HGSC patient-derived pre- and post-NACT FFPE blocks were acquired from Helsinki University Biobank. To preserve morphological and spatial features, adjacent tissue sections underwent tCycIF and GeoMx experiments. Potential regions for spatial transcriptomics (Pre ROIs) were successfully delineated based on tCycIF staining patterns, enabling the identification of IBA1+ macrophage and CD8+ T cell rich areas within the TSI. By overlaying a crop of the tCycIF image onto the GeoMx scan, areas with distinct similarity degrees between tCycIF and GeoMx were generated. The GeoMx-ROIs were ultimately selected based on the Pre-ROIs and their similarity degree with tCycIF scanned images. Tumor and stroma segmentation was performed through a custom segmentation method guided by tumor specific marker pan-cytokeratin staining in GeoMx images, defining distinct spatial compartments for sequencing. Finally, the image-derived data from both techniques was integrated in a single file, enabling a combined subsequent analysis. The novel pipeline developed in this study opens promising research possibilities, as tCycIF-guided ROI selection allows for precise targeting of cell neighborhoods and structures after performing a thorough microenvironment exploration. This approach can potentially be adapted for other uses in a wide range of biomedical fields, beyond the focus of HGSC.
  • Oostveen, Mayke (2023)
    Head and neck cancers form the seventh most common type of cancer globally, and 90% of these cancers are classified as head and neck squamous cell carcinomas (HNSCC). The 5-year survival rate for head and neck cancers remains around 50%, indicating a need for new treatment strategies. This thesis aimed to examine the potential of BH3 mimetics A 1155463, A-1331852, and navitoclax combined with irradiation, as a treatment for HNSCC. BH3 mimetics are compounds able to bind to anti-apoptotic proteins, thereby blocking their ability to exhibit anti-apoptotic effects. A high-throughput screening method was used to characterize the synergistic effects of the BH3 mimetics with different irradiation doses on the viability of twelve HNSCC, normal (NOF) and cancer-associated fibroblastic (CAF), and HPV-16 immortalized oral dysplastic keratinocyte (ODA) cell lines. Additionally, the effects of A-1155463 and A-1331852 on apoptosis and proliferation of two HNSCC cell lines (UT SCC 40 and UT-SCC-42A), in combination with 8 Gy irradiation, were measured. Effects on invasion of UT-SCC-42A cells with the irradiation-mimetic combination were tested in a 3D spheroid model. Lastly, the effect of A 1331852 and navitoclax in combination with immunotherapy drug nivolumab on donor-derived immune cell migration and apoptosis of UT-SCC-40 was assessed with a microfluidic chip assay. All three BH3 mimetics in combination with irradiation synergistically reduced viability in six to ten HNSCC cell lines and ODA cells, but not in NOF or CAF. A-1155463 and A-1331852 induced apoptosis and reduced proliferation in both tested cell lines. The addition of irradiation to the compounds significantly increased the apoptotic ratio in both cell lines and compounds. In the spheroids, A-1331852 significantly reduced cancer cell area and length of invasion both with and without irradiation. A-1155463, unlike navitoclax, had a trend in reducing HNSCC invasion. The combination of A-1331852 with nivolumab increased the immune cell migration of one out of the three donors. These results show the synergistic and apoptosis-inducing effects of the BH3 mimetics combined with irradiation, proving them plausible candidates for HNSCC treatment. The reduction of invasion by A 1331852 suggests a role for Bcl-xL in HNSCC invasion. It is proposed to further investigate the properties of A-1331852 in in vitro cocultures and in vivo studies, with and without irradiation.
  • Tohmola, Tiialotta (2022)
    The current 5-year survival rate of OSCC patients is around 50%. There are no diagnostic tests or markers and the diagnosis is based on histopathologic samples only, which postpones the time of detection and worsens the prognosis of the patient greatly. New diagnostic methods and tools are required to improve the survival rate. This study aimed to find possible biomarkers and develop a new diagnostic method for OSCC by comparing the serum proteomic expression of tongue cancer patients and healthy controls with label-free liquid-chromatography mass spectrometry. The results showed small, but statistically significant difference in protein expression between the patients and healthy controls, as well as a clear separation between the groups based on the peptide data. In addition, no specific networks or cellular pathways were highlighted for OSCC compared to other types of cancers. These results didn’t introduce considerable advances into the diagnostics of OSCC but showed a possibility for finding further distinctive differences between the OSCC patients and healthy controls.
  • Ranta, Amanda Katrianna (2020)
    Ex vivo drug sensitivity testing is used widely in studies aiming at personalizing medicine for acute myeloid leukemia (AML) patients. However, different conditions, such as cytokines used in media and cryopreservation of cells, as well as varying readout methods can affect primary cell viability, cell composition and sensitivity results. Such affects have been previously studied in some AML treatments, however, not with flow cytometry or with venetoclax. In this thesis, we studied the responses of AML patients to venetoclax using ex vivo drug sensitivity testing with various settings. We first tested three media and two sensitivity readout methods on 29 fresh primary AML samples to determine the optimal media and method for determining ex vivo drug sensitivity. We then tested these same variables on 16 cryopreserved samples and compared these results to their fresh counterparts. Finally, we applied our platform to clinical use and tested its capability to predict in vivo responses to venetoclax in ten AML patients. Our platform was able to predict venetoclax responses in nine out of ten patients using condition media coupled with a flow cytometry-based method, determined as optimal in the first phase. Sensitivity results as well as cell composition obtained after cryopreservation differed from their fresh counterparts and, therefore, we conclude that cryopreserved samples should not be used in guiding treatment ex vivo. Our results give valuable information about sources of error associated with ex vivo drug sensitivity testing. Consideration of these results when designing preclinical studies will enhance their reliability and relevance. Ex vivo testing could be in the future implemented into clinical practice in guiding treatment, saving society and patients from costs and unnecessary adverse effects.
  • Azam, Shadi (2022)
    Background: Oral contraceptive (OC) use may increase the risk of specific cancers and mortality. The aims of this study were to investigate the association between OC use and its duration with the risk of breast cancer, to examine the overall mortality associated with OC use and its duration, and finally to identify sociodemographic characteristics of OC use. Methods: Data are derived from the Older Finnish Twin cohort consisted of monozygotic and same-sexed dizygotic twin pairs born before 1958. We included N = 9,607 Finnish twin women aged 18 – 49 years old with information on OC use and other covariates. The information on OC use, reproductive, and lifestyle factors was collected using a mailed questionnaire. The information on breast cancer incidence was obtained from the Finnish Cancer Registry and the data on mortality was collected from the national Population Information System and Statistics Finland. We used Cox proportional hazards regression to estimate the association between OC use and its duration with risk of breast cancer and overall mortality while controlling for potential confounders. Also, we used logistic regression to identify sociodemographic characteristics of OC use. All tests of statistical significance were two-sided. Results: A total of 758 women developed breast cancer during median follow-up of 42.6 years. Women who ever used OC had 20% greater risk of developing breast cancer than women who never used (HR =1.20, 95% CI = 1.02 to 1.40, P = 0.02). Women who used OC for more than 5 years had greater risk of developing the disease than those who used OC for less than 2 years (HR = 1.11, 95% CI = 0.85 to 1.46), however, the results did not reach the statistical significance. Mortality did not significantly different between women who had ever used OC with those who had not used OC while controlling for potential confounders. Current smokers and women who consumed alcohol more than 10 gram/day had the highest odds of ever using OC. Conclusion: Our results suggest that OC use slightly increases the risk of breast cancer, however, no evidence from this study indicates that OC use adversely affect long-term risk for mortality.
  • Sorri, Selma (2022)
    Diffuse large B-cell lymphoma (DLBCL) represents the most common diagnostic entity of lymphoid malignancies. As only 60% of the patients are cured with the current standard of care R-CHOP immunochemotherapy, the quest for better biomarkers and targeted therapies continues. Non-synonymous mutations in the WWE1 domain of an uncharacterized E3 ubiquitin ligase Deltex-1 have been associated with poor outcomes in DLBCL patients. Thus, to elucidate molecular features underlying this observation, this Master’s thesis set out to characterize the expression and subcellular localization of Deltex-1 in a panel of DLBCL cell lines, and to investigate the interaction partners of Deltex-1 in the activated B-cell like (ABC) DLBCL cell line context. The study aimed to gain further knowledge to understand the role that Deltex-1 plays in the pathogenesis of DLBCL, which could be used for inspecting its future possibilities as a prognostic marker or a drug target. Western blot analysis of the cell lysates revealed variable levels of Deltex-1 expression, especially between the ABC-DLBCL cell lines in comparison to germinal centre B-cell like (GCB) DLBCL cell lines. Western blots of separate cytoplasmic and nuclear fractions of the cells showed that Deltex-1 was expressed both in the cytoplasmic and the nuclear fractions of the cells, and the expression levels were reflecting the levels of the whole cell lysates of the same cell lines. The more exact localization of Deltex-1 was observed with immunofluorescence staining and microscopy of fixed cells from a few chosen cell lines. A distinct plasma membrane localization was detected in an ABC-DLBCL cell line U2932. The protein-protein interaction partners of Deltex-1 in the U2932 cell line were screened using proximity-dependent biotin labelling and affinity purification mass spectrometry. The experiments revealed novel associations between Deltex-1 and B-cell receptor signalling regulators, such as B- lymphocyte antigen CD20 and tyrosine protein kinase Lck. Though additional research is needed to define the functional mechanisms of these interactions, these findings might lead to the discovery of the connection between Deltex-1 and lymphomagenesis. In conclusion, this study provides novel information on Deltex-1 expression in the DLBCL context and describes previously unidentified associations of Deltex-1 with B-cell receptor signalling. Yet, more functional experiments are required to clarify the nature of these interactions.
  • Colanglo, Kia Kristiina (2021)
    High-Grade Serous Ovarian Cancer (HGSOC) is the most lethal gynecological cancer in developed countries. Due to lack of early detection methods or targeted treatment options the mortality has not reduced significantly in decades. Standard treatment includes surgery and platinum-taxane chemotherapy, the treatment is very seldom curative. More studies are needed to understand the biology and mechanisms defining chemoresistance, and to develop more personalized treatment schemes. Cancer stem cells are known to resists chemotherapy and therefore this study focuses on the expression of putative cancer stem cell biomarkers in HGSOC. Using Immunohistochemistry (IHC) and Immunofluorescence (IF), a Tissue-Micro Array (TMA) containing 95 patients’ samples was generated and tested for four different potential biomarkers: SOX2, BMI1, C-MYC and ALDH1A1. Scanned slides were evaluated, and results were analyzed using Rstudio as well as Excel analytics. We report that chromogenic IHC staining of individual markers revealed no major differences between expression and Platinum-Free Interval (PFI). Instead, some of the co-expressions and especially triple expressions analyzed with IF resulted in major difference in PFI. Beyond that, ALDH1A1 and SOX2 were found together extremely rarely, and it indicates that it is possible that these two biomarkers are normally not expressed in the same tumor cells. Further study options as well as possible implications are discussed along with the clinical value of the findings.
  • Wall, Johanna Tiina Aurora (2023)
    Immunotherapies have exponentially gained interest recently as they are less invasive than traditional cancer treatment. Chimeric Antigen Receptor T cell (CAR T) treatment is among the latest breakthroughs and there are currently five FDA and EMA approved CAR T treatments on the market. Despite their potential, CAR T treatment may have serious adverse effects, they are costly, and the suitable patient population is small. In addition, CAR T treatment works best on haematological cancers, with further challenges in treating solid tumours. Treatment safety is a main concern because CAR Ts may start damaging healthy tissue expressing a target antigen, which can be fatal for some patients. The ongoing research on CAR T treatment for solid tumours is prevalent, but none have been approved by FDA or EMA. This study investigated organs from anti-SSEA-4 CAR T treated mice post an in vivo dose-escalating experiment on immunocompromised mice with human ovarian adenocarcinoma (OVCAR4) xenografts. The study included nine treatment groups in total, four dose-escalating treatment groups, three correlating non-tumour treatment groups and both a tumour control and a no-treatment, no-tumour control group. Differences in the tissues regarding the target antigen SSEA-4, non-transduced T cells and SSEA-4 targeted CAR Ts were analysed with H&E and immunostainings. SSEA-4 expression was found in the kidneys, ovarian follicles and in the gastrointestinal muscular layer. In spite of the SSEA-4 expression on healthy tissues, signs of on-target, off-tumour effects were limited in these organs. T cell infiltrates were found mostly in the intestine, stomach, fallopian tubes, and lungs, of which CAR Ts infiltrated specifically the intestine, stomach, and fallopian tubes. Nonetheless, no clear correlation between endemic SSEA-4 expression and CAR T infiltrates was found. Anti-SSEA-4 CAR Ts had an anti-tumour effect on all studied doses. However, some of the high dose mice showed signs of health deterioration. Despite the weak antigen expression on tumours, many immunologically ‘hot’ tumours with lymphocytes were found which proves a successful tumour infiltration. CAR T dose-limiting, and combinatorial target antigens need to be further investigated to improve treatment safety and before advancing into clinical trials.  
  • Punger, Tatjana (2017)
    Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It arises from skeletal muscle stem cells, which fail to differentiate. Multimodal treatment approach has improved the outcome of RMS during the recent years. However, in case of relapsed or metastatic disease, the prognosis is still very poor. This indicates a high demand for novel targeted therapy approach for RMS. Prox1 transcription factor has been shown to regulate myoblast differentiation into skeletal muscle fibers. Our unpublished observations indicate that Prox1 is highly expressed in RMS tumors and that it is essential for RMS cell growth. The aim of this study was to find novel drug candidates for RMS treatment depending on Prox1 and/or its downstream signaling. High-throughput drug screening with 528 oncology compounds was tested on wild-type and Prox1 silenced rhabdomyosarcoma cells (RD cell line). Gene silencing was performed via lentiviral vectors encoding shRNA for Prox1. In the screening results, we focused on the drugs, which were more potent in Prox1 silenced cells with nano- or micromolar concentrations compared to the wild-type cells. The results revealed 7 potential groups of inhibitors, which had superior suppressive effect on RMS cell viability specifically when Prox1 was silenced. In vitro validation of high-throughput screening results by MTT and luciferase assays confirmed the results. Based on the magnitude of their inhibitory effect and information available on these compounds, three drugs were chosen for further investigation. Two of these compounds also potently inhibited the growth of patient-derived primary RMS cells, which we obtained from the Helsinki University Hospital and named KLHEL1. These drugs were also less toxic to healthy myoblasts. In addition, these two compounds significantly decreased Prox1 mRNA and protein levels in wild-type cells, and completely inhibited the ability of both RD and KLHEL1 cells to form colonies. Combinational exposure to these inhibitors further enhanced the effect compared to a single agent treatment. The present findings demonstrate a potential for repurposing of these drugs for targeted treatment in rhabdomyosarcoma expressing high Prox1 levels.
  • Bütün, Felicia (2021)
    New treatment methods are urgently needed for glioblastoma (GBM), the most common malignant primary brain tumor in adults, that currently lacks any curative treatment. Targeted therapeutic approaches have shown promising results already, but common drug delivery vehicles come with efficacy issues and are restricted by their safety and toxicity profiles. Exosomes, cell-produced nanosized vesicles, have emerged as a new potential carrier for gene therapies in cancer treatment due to their natural material transport properties, biocompatibility, and specificity in transporting cargo to the target cells. These extracellular vesicles have the additional advantage of being able to cross the blood-brain-barrier (BBB), which makes them especially valuable for brain malignancies, such as glioblastomas. So far, gene therapy approaches in exosomes have focused on RNA in cancer treatment, but research findings are limited with plasmid-based gene therapies using exosomes. The main concern has been whether the increased plasmid size would decrease the transfection efficiency of the plasmid into the exosomes. This study aimed at setting-up exosomes as plasmid-based gene therapy nanocarriers. To achieve this, different plasmid-based gene therapies were tested, including the targeting of common aberrations of GBM cells to impair proliferation and the use of cytotoxins to induce apoptosis in the target cells. The plasmids were transfected into exosomes and subsequently inoculated into patient-derived glioblastoma cells with the aim of decreasing the number of glioblastoma cells. The findings of this study demonstrate a successful set-up of an exosome-based gene therapy in patient-derived glioblastoma cells by using engineered HEK293FT cell derived exosomes consisting of a plasmid-based combination gene therapy encoding the cytotoxins Granzyme B and Diphtheria toxin fragment A.
  • Liljeström, Emmi (2021)
    Hepatoblastoma (HB) is the most common malignant pediatric liver tumor. Although developed treatments have increased the survival rate of HB patients, 20-30 % of the patients show lack of response to the currently used treatment. Due to rapid growth and insufficient blood flow, solid tumors, like HB, develop areas with low oxygen levels. This condition is called tumor hypoxia. Tumor hypoxia correlates with poor prognosis, higher metastasis rate and resistance to cancer treatments. In response to hypoxia, cancer cells start to express carbonic anhydrase 9 (CA9) via HIF1. CA9 contributes to the maintenance of alkaline intracellular pH. That promotes tumor development, while the increasingly acidic extracellular space promotes tumor cell invasiveness. CA9 has historically been related to carcinogenic processes in a variety of cancers, and it has been hypothesized that it may be a possible target for cancer therapy. Substances for the inhibition of CA9 already exist, and one of them, SLC-0111, has given promising results in phase I clinical trials as well as several pre-clinical studies. The aim of this study is to describe the expression of CA9 in HB and study their relationship to pathological features in cellular level, especially the viability and migration of cancer cells. Another purpose of the study is to investigate the effect of SLC-0111 on HB cells and to consider its significance as a potential treatment. CA9 is expressed in two different HB cell lines, HUH6 and HB-303-LEF when exposed to hypoxic conditions. Cells show more aggressive behavior under hypoxic conditions. HB-303-LEF migrates more abruptly in hypoxia compared to normoxic cells. Cells from both cell lines in spheroid modeling, in which CA9 was inhibited by SLC-0111, showed lower viability. HB-303-LEF also showed slower migration in hypoxia where it had received the SLC-0111 inhibitor compared to hypoxic cells. HUH6 results were parallel but not statistically significant. Cells behave more aggressively in hypoxia. The use of SLC-0111 contributes to the reduction of viability and migration. It can be considered an interesting discovery for future treatments against HB.
  • Viitala, Emma Wilhelmiina (2022)
    The gastrointestinal (GI) epithelium is composed of a single layer of cells with a turnover time of only a few days. Due to its location at the barrier between GI tract contents and the underlaying mucosa, the epithelium is constantly exposed to stress such as toxic agents and a variety of pathogens and susceptible to injury. Accordingly, the homeostatic growth as well as repair of injury in epithelium must be efficient and strictly regulated. Misregulated repair of the injured epithelium can lead to pathologies such as chronic inflammation or cancer. Underlying stromal cells such as fibroblasts provide growth factors and other signaling molecules regulating the epithelial cell stemness, differentiation and repair, but the stromal regulatory pathways during regeneration are poorly understood. The aim of this study was to establish a consensus view on the heterogeneity of GI fibroblasts, as well as to map potential epithelium derived signals affecting fibroblast function in homeostatic and injury situations using literature review, in silico approaches, and murine primary intestinal fibroblast culture. Seurat and CellChat R packages were used to perform integration and interaction analyses of six previously published mouse and three human single- cell RNA-sequencing datasets of colonic epithelial and mesenchymal cells isolated in homeostatic and/or inflammatory conditions. Murine primary intestinal fibroblasts were treated with identified potential signaling factors ex vivo and 3’RNAseq was performed to identify transcriptional responses. Both mesenchymal and epithelial cell clusters were identified in the scRNAseq data. Interestingly, similar fibroblast populations could be found in the murine and human data. I identified several epithelium-derived signaling molecules potentially targeting GI fibroblasts and focused on Gas6-Axl pathway and lactate. I confirmed high and specific expression of the Gas6 receptor Axl in intestinal fibroblasts, but recombinant Gas6 failed to induce significant changes in cultured primary fibroblasts. Lactate-treated primary intestinal fibroblasts reprogrammed their transcriptome with main alterations in metabolic pathways and induction of neutrophil-attracting chemokines. In this work I suggest a consensus model for GI fibroblast subpopulations and suggest epithelium derived lactate as a powerful means to reprogram fibroblasts.