Browsing by master's degree program "Translationaalisen lääketieteen maisteriohjelma (Translational Medicine)"

Merkel cell carcinoma (MCC) is a rare, neuroendocrine carcinoma of the skin that is known to have poor prognosis. It is associated with the Merkel cell polyomavirus (MCPyV) and majority of cases harbor this infection. Other risk factors include older age, the male sex, Caucasian skin and increased ultraviolet exposure. Increased lymphocyte invasion into the MCC tumor microenvironment has been reported to infer better survival, but better mechanisms understanding why this occurs this is needed. CCL3 is a chemokine that is implicated in a variety of inflammatory conditions like viral infections and exhibits pro-inflammatory activity mainly through its chemoattractant abilities. In cancer specifically, it functions within the tumor microenvironment by encouraging the trafficking of leukocytes to the tumor site. Transcriptomic data of CCL3 was studied in a cohort of 102 Finnish MCC patients to observe its association with survival, and a variety of clinical-pathological features. The presence of CCL3 in cells was later investigated via immunohistochemistry in 30 formalin-fixed paraffin-embedded Finnish MCC primary tumor tissue samples with varying mRNA expression of CCL3. Macrophages and lymphocytes were found to stain positively for CCL3 and were found exclusively in tumor surroundings. CCL3 was also found to exhibit a MCC-specific survival benefit in patients that harbored higher expression (p=0.031), and was found to be associated with MCPyV positivity (p=0.032). These preliminary findings help establish CCL3’s role in the immune response against MCC and support the need for further studies looking at CCL3 both as a prognostic marker and potential adjuvant therapeutic.

Mucosal associated invariant T (MAIT) cells are a fairly recently described population of innate-like T cells. In humans, MAIT cells represent an exceptionally abundant population in the blood, where they account for 1–10% of all T cells. However, compared to conventional T cells, which can display an almost unlimited T cell receptor (TCR) repertoire, the specificities of MAIT cell TCRs are limited. This evolutionarily conserved subset displays a semi-invariant TCR which recognises riboflavin metabolites, produced by a wide range of bacteria and fungi, and presented on the major histocompatibility complex (MHC)-class I related (MR1) molecule. The function and significance of MAIT cells in health and disease have only started to be unravelled, and it is becoming increasingly clear that MAIT cells are also modulated in non-microbial diseases. Interestingly, MAIT cells have been shown to exhibit a relatively high expression of the transcription factor, Helios, compared to most other T cell subsets. The function of this translational activator and repressor, encoded by the gene IKZF2, in the MAIT cell population remains obscure. This study focused on the functional role Helios may play in regulating the activation of MAIT cells. Thus, by using siRNA to silence Helios expression, and flow cytometry to analyse any potential alterations in MAIT cell activation markers, we aimed to be able to characterise the kinetics and functional role of Helios in peripheral MAIT cells of healthy individuals. Here, we clearly established a striking upregulation of Helios in MAIT cells following 24 hours of stimulation. Moreover, we were able to achieve a >50% knockdown of Helios at the protein level, in this subset of T cells. Nevertheless, no significant difference in any of the activation markers we investigated was present between the MAIT cells with reduced Helios expression and their controls. This could, however, of occurred as a result of the toxicity that the transfection had on the functionally of the T cells. From these results, it is difficult to conclude any clear role for Helios in the activation of MAIT cells, and consequently, further research needs to be performed before any clear conclusions can be drawn.

Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.

Recently, several novel post-translational modifications (PTMs) have been identified as important regulators in biology. Succinylation, the reversible addition of a succinyl group from a free succinyl-CoA into a protein lysine, is one such novel PTM. The last decade of research has unveiled succinylation as a powerful regulator of metabolism, prevalent in every organism it has been studied in and with functional effects on target proteins in several key metabolic pathways. A major contribution of this thesis is to catalogue the recent advances in succinylation research into the most comprehensive literary review currently available on succinylation. While the biological role of this PTM is being established, the relevance of succinylation in human disease has remained unclear. Meanwhile, mitochondrial DNA depletion syndrome caused by defective SUCLA2 (SUCLA2 disease) is a progressive hereditary mitochondrial disease with no available treatment. SUCLA2 disease is caused by defective mutations in the ß-subunit SUCLA2 of the TCA cycle enzyme succinyl-CoA synthetase. While the characteristic manifestations, including impairment of respiratory complexes, and the etiological mutations in this disease are well established, the pathogenic model for SUCLA2 disease has remained incomplete. As succinyl-CoA synthetase shares a substrate, succinyl-CoA, with succinylation, this thesis set out to probe SUCLA2 mutants for a potential succinylation phenotype. An extensive hypersuccinylation phenotype was characterized in fibroblasts and tissue samples from SUCLA2 mutant patients by immunochemical methods. The hypersuccinylation target identities in SUCLA2 mutants were revealed with proteomics by mass-spectrometry. Hypersuccinylation in SUCLA2 mutants was shown to be enriched in proteins participating in mitochondrial energy metabolism, including respiratory complex proteins. In addition, several novel metabolic phenotypes were characterized in SUCLA2 mutants with metabolomics by mass-spectrometry, most prominently a significant depletion of aspartate metabolism. While identification of extensive hypersuccinylation in SUCLA2 mutants establishes a novel concept of succinylation relevance in human metabolic disease, the prospect of altered regulation of the respiratory complexes due to hypersuccinylation lays the foundation for a novel pathogenic model for SUCLA2 disease. Meanwhile, the observed novel metabolic phenotypes significantly contribute to the current understanding on SUCLA2 mutant metabolism and inspire a hypothetical model on how the defective succinyl-CoA synthetase could be circumvented in the TCA cycle of SUCLA2 mutants.

Multiple Myeloma (MM) is the second most common hematologic malignancy. Despite the advancements in treatment approaches in the last decade, the prevalence of refractory disease leading to relapsed cases has been a major challenge. A wide range of intricate genetic heterogeneity demonstrated by myeloma patients is a credible explanation for the diverse treatment responses observed in patients sharing the same treatment regimens. Pertaining to this, the study aims to identify predictive gene expression biomarkers that forecast response to BCL2 inhibitor venetoclax and treatment outcome to proteasome inhibitor bortezomib. In this study, samples from MM patients were characterized into sensitive and resistant, (1) based on ex vivo response to venetoclax treatment (Resistant n=21; Sensitive n=21), and (2) based on their bortezomib treatment outcome in clinical profiles (Resistant n=12; Sensitive n=15). Associations between the different gene expressions and drug responses were studied using statistical and bioinformatic tools. As a result, we identified that significant (p-value <0.05) overexpression of 36 genes and downregulation of 38 genes appeared to confer resistance to venetoclax drug response in MM patients. Additionally, the functional association of these genes with pathways was determined using a pathway enrichment tool. Furthermore, the study provided evidence that cytogenetic alterations t(11;14) and t(4;14) are significantly (p-value <0.05) associated with differing venetoclax response in MM patients. These findings demonstrated that gene expression biomarkers and chromosomal translocations play a significant role in regulating venetoclax drug response in MM, which can be further utilized to personalize treatments for patients. The knowledge obtained from this work best applies in personalized medicine; whereby fitting treatments to an individual patient’s genomic landscape will enhance patient outcome.

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.

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.

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.

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.

Multiple sclerosis (MS) is a demyelinating autoimmune disease in which peripheral immune cells infiltrate the CNS and damage the insulating myelin sheaths surrounding neurons, creating demyelinated lesions in the spinal cord and the brain. MS is an incurable, life-long disease which causes a range of symptoms resulting from CNS degeneration. Current treatments mostly focus on preventing autoimmune attacks and the formation of lesions, but do not reduce the damage caused by the attacks, or impact the gradual degeneration of the axons of MS patients. This study aimed to establish the potential of MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) as treatments for MS. MANF and CDNF are endoplasmic reticulum (ER) located proteins with unique structure and mode of action. UPR is a cellular stress response that, when triggered by inflammation in MS, can cause the apoptosis of myelinating oligodendrocytes and neurodegeneration. MANF and CDNF are also capable of modulating immune responses and improving regenerative processes in damaged tissues. The capability of these two molecules to protect CNS tissue was tested on mice induced with experimental autoimmune encephalomyelitis (EAE), a disease model for MS. Intravenous injections of MANF or CDNF in two doses were performed every 2nd day for 28 days after disease induction. Behavioral testing (rotarod and open field tests) indicated that both proteins improved motor function before the onset of paralysis. Daily clinical scoring showed a brief therapeutic window after the onset of paralysis, during which MANF and CDNF were able to halt disease progression. Flow cytometry analysis of mice spleens and brains showed no effect on immune cell populations at the end of the 28-day testing period. Immunohistological staining at the end of the experiment showed no differences in levels of neuroinflammation between treatment groups and control mice but showed that treatment with MANF and CDNF clearly reduced the formation of demyelinated lesions over the duration of the disease. These findings suggest the improved motor performances and protection from paralysis provided by treatment by MANF and CDNF may be due to their ability to protect CNS tissue from UPR caused by autoimmune demyelinating attacks. Further research is required to elucidate the mechanics behind this neuroprotective ability, and lead to more effective use of MANF and CDNF.