The immune microenvironment in mantle cell lymphoma : Targeted liquid and spatial proteomic analyses

The complex interplay of the tumour and immune cells affects tumour growth, progression, and response to treatment. Restorationof effective immune response forms the basis of onco-immunology, which further enabled the development of immunotherapy. Inthe era of precision medicine, pin-pointing patient biological heterogeneity especially in relation to patient-specific immunemicroenvironment is a necessity for the discovery of novel biomarkers and for development of patient stratification tools for targetedtherapeutics. Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell lymphoma with poor survival and high relapserates. Previous investigations of MCL have largely focused on the tumour itself and explorations of the immune microenvironmenthave been limited. This thesis and the included five papers, investigates multiple aspects of the immune microenvironment withrespect to proteomic analysis performed on tissue and liquid biopsies of diagnostic and relapsed/refractory (R/R) MCL cohorts.Analyses based on liquid biopsies (serum) in particular are relevant for aggressive cases such as in relapse, where invasiveprocedures for extracting tissues is not recommended. Thus, paper I-II probes the possibility of using serum for treatment andoutcome-associated biomarker discovery in R/R MCL, using a targeted affinity-based protein microarray platform quantifyingimmune-regulatory and tumor-secretory proteins in sera. Analysis performed in paper I using pre-treatment samples, identifies 11-plex biomarker signature (RIS – relapsed immune signature) associated with overall survival. Further integration of RIS with mantlecell lymphoma international prognostic index (MIPI) led to the development of MIPIris index for the stratification of R/R MCL intothree risk groups. Moreover, longitudinal analysis can be important in understanding how patient respond to treatment and thiscan further guide therapeutic interventions. Thus, paper II is a follow-up study wherein longitudinal analyses was performed onpaired samples collected at pre-treatment (baseline) and after three months of chemo-immunotherapy (on-treatment). We showhow genetic aberrations can influence systemic profiles and thus integrating genetic information can be crucial for treatmentselection. Furthermore, we observe that the inter-patient heterogeneity associated with absolute values can be circumvented byusing velocity of change to capture general changes over time in groups of patients. Thus, using velocity of change in serumproteins between pre- and on-treatment samples identified response biomarkers associated with minimal residual disease andprogression. While exploratory analysis using high dimensional omics-based data can be important for accelerating discovery,translating such information for clinical utility is a necessity. Thus, in paper III, we show how serum quantification can be usedcomplementary tissue-identified prognostic biomarkers and this can enable faster clinical implementation. Presence of CD163+M2-like macrophages has shown to be associated with poor outcome in MCL tissues. We show that higher expression of sCD163levels in sera quantified using ELISA, is also associated with poor outcome in diagnostic and relapsed MCL. Furthermore, wesuggest a cut-off for sCD163 levels that can be used for clinical utility. Further exploration of the dynamic interplay of tumourimmunemicroenvironment is now possible using spatial resolved omics for tissue-based analysis. Thus, in paper IV and V, weanalyse cell-type specific proteomic data collected from tumour and immune cells using GeoMx™ digital spatial profiler. In paperIV, we show that presence as well as spatial localization of CD163+ macrophage with respect to tumour regions impactsmacrophage phenotypic profiles. Further modulation in the profile of surrounding tumour and T-cells is observed whenmacrophages are present in the vicinity. Based on this analysis, we suggest MAPK pathway as a potential therapeutic target intumours with CD163+ macrophages. Immune composition can be defined not just by the type of cells, but also with respect tofrequency and spatial localization and this is explored in paper V with respect to T-cell subtypes. Thus, in paper V, we optimizeda workflow of multiplexed immunofluorescence image segmentation that allowed us to extract cell metrics for four subtypes ofCD3+ T-cells. Using this data, we show that higher infiltration of T-cells is associated with a positive outcome in MCL. Moreover,by combining image derived metrics to cell specific spatial omics data, we were able to identify immunosuppressivemicroenvironment associated with highly infiltrated tumours and suggests new potential targets of immunotherapy with respect toIDO1, GITR and STING. In conclusion, this thesis explores systemic and tumor-associated immune microenvironment in MCL, fordefining patient heterogeneity, developing methods of patient stratification and for identifying novel and actionable biomarkers

Sarcoma ecosystems : spatial characterization and prognostic significance

Sarcoma is a highly heterogeneous disease with complex biological activities and unique tumor microenvironments (TME) in distinct subtypes. The limited treatment options and inadequate responses to current therapies necessitate a deeper understanding of sarcoma biology and personalized treatment strategies. Our research comprehensively explores the sarcoma TME through advanced spatial analysis and investigates sarcoma's molecular and genetic profiles through transcriptome and genome sequencing. In paper I, we focused on undifferentiated pleomorphic sarcoma (UPS) using multiplex immunofluorescence (mIF) staining for in-depth spatial analysis of B cell populations and lymphocyte aggregates (LAs). LAs in UPS were found to be associated with longer overall survival (OS) and metastasis-free survival (MFS). Moreover, we unveiled distinct maturation profiles among B cell subsets, indicative of different phenotypes that contribute to functional ecosystems in TME. LA-positive tumors displayed a more well-differentiated B cell profile throughout the entire tumor section, not limited in LA regions. We introduced the B-index, an integrated measurement tool combining B cell abundance and maturity, which demonstrated predictive power for both MFS and OS. Using the TissUUmap tool, we identified B cell desert areas characterized by extremely low B cell infiltration. LA-positive tumors displayed smaller and more fragmented B cell desert areas. In paper II, we performed double immunohistochemistry to study CD11c-positive antigen-presenting cells (APCs) and CD8- positive cells in 177 soft tissue sarcoma (STS) patients. We found that CD11c-CD8 interactions in the TME were associated with improved MFS and OS. Transcriptomic analysis in The Cancer Genome Atlas (TCGA) sarcoma cohort supported the prognostic significance of combining CD11c with CD8, irrespective of FOXP3 levels and in the presence of CD274 (PD-L1). In paper III, we conducted transcriptome and targeted DNA sequencing in 91 synovial sarcomas, identifying three distinct Synovial Sarcoma Clusters (SSCs) mirroring histological subtypes. SSC-I was characterized by high cell proliferation and immune evasion with an unfavorable prognosis. SSC-II was dominated by vascularstromal components and correlated with better outcomes. SSC-III displayed biphasic differentiation, genomic complexity, and immune checkpoint-mediated immune suppression, leading to adverse outcomes, even after a good histological response to neoadjuvant treatment. In paper IV, we analyzed Ewing sarcoma (ES) transcriptome signatures in four previously published cohorts and identified 29 prognostic RNA-binding protein (RBP) genes, from which we constructed and validated an RBP-associated prognostic risk model (RPRM). The RPRM demonstrated stable predictive value for prognosis, with NSUN7 emerging as an independent and favorable prognostic marker. In summary, our research integrates spatial analysis of the sarcoma TME to identify unique immune features and prognostic markers. Moreover, we use transcriptomic and genomic analyses to categorize specific sarcoma types for more detailed survival stratification. This work provides a deeper insight into the sarcoma TME and suggests an improved grouping strategy, aiming to shape the development of personalized immunotherapy in the future

Identification of genes and pathways involved in the development and progression of glomerular diseases

Chronic kidney disease (CKD) affects millions of people worldwide and is characterized by a reduction in glomerular filtration rate and albuminuria resulting in a gradual loss of kidney function. The high prevalence of the disease, along with the limited treatment options available, makes it a major burden to the health care systems around the world. Current treatment strategies are directed towards slowing the progression and delaying the complications. The glomerulus is the filtration unit of the kidney and a major target of injury, making glomerular diseases one of the leading causes of CKD. The kidney is a challenging organ to study due to tissue heterogeneity, complex disease phenotypes and morphologies. The lack of knowledge on the molecular mechanisms of disease pathogenesis limits the development of new diagnostic and treatment tools. The main aim of this thesis was to gain a better understanding into genes and pathways involved in the development and progression of glomerular diseases. In a long run, our aim is to utilize the gained knowledge to develop new means to diagnose and treat CKD. In the first part of the thesis (paper I and II), we used single cell RNA-sequencing (scRNAseq) to get insights into the glomerular environment in health and disease, as well as under drug therapy. Paper I: By profiling the glomerulus, we defined the true transcriptomic signatures of specific cell types, gained better insight into their functionality, and identified molecular profiles of rare cell types. By comparing the expression profiles between mouse and human glomerulus, we revealed significant cross-species differences in the main glomerular cells. Paper II: By profiling the molecular signatures of Angiotensin Converting Enzyme-inhibitor (ACEi) in diseased glomerular tissue, we revealed mesangial cells (MCs) to be the main early responder to the treatment. MCs showed downregulation of genes and pathways related to extracellular matrix (ECM) production. Only few transcriptomic changes were detected in the other glomerular cell types. In the second part of the thesis (paper III and IV), we investigated two candidate genes identified through transcriptomic studies. Retinoic acid receptor responder 1 (Rarres1) and Natriuretic peptide receptor 3 (NPR3) were analysed through various in vitro and in vivo experiments. Paper III: We investigated the role of Rarres1 in the glomerulus using various transgenic mouse lines, molecular profiling of patient material and in vitro models. We identified Rarres1 as a possible therapeutic target and biomarker of injury for glomerular diseases. In diseases, an up-regulation of Rarres1 expression was observed in endothelial cells, in which it aggravated the glomerular injury. This effect was potentially mediated by the activation of NFκB pathway via tyrosine kinase Axl Paper IV: We analysed the role of NPR3 in the glomerulus, and especially explored the possibility of manipulating glomerular natriuretic peptide (NP) system through NPR3. Pharmacological inhibition of NPR3 showed variable reno-protective effects when profiled in two rodent models of glomerular injury, suggesting that the modulation of the glomerular NP system could be a potential therapeutic target for CKD. However, more studies are needed to optimise the treatment strategy and to further understand the role of NPR3 in kidney tissue. To summarise in this thesis, we have demonstrated the power of transcriptomic approach in gaining new knowledge on the molecular biology of the glomerulus. Moreover, our studies with Rarres1 and NPR3 contribute to identification of possible novel therapeutic approaches and biomarkers

Characterising Chinese Hamster Ovary Cell Line Stability in Bioproduction of Therapeutic Proteins

No abstract

The role of intra-tumoural heterogeneity in resistance to neoadjuvant chemotherapy in breast cancer

Breast cancer is a heterogeneous disease and accumulating evidence suggests that treatment failure may be driven by intra-tumour heterogeneity (ITH). Utilising the current protocol for neoadjuvant (pre-surgery) chemotherapy (NAC) provides the opportunity to study molecular genetic changes between pre- and post-therapy by assessing pre-therapy biopsies and post-therapy surgical resections. Whole exome sequencing was performed on matched pre- and post-treatment cancer cells from 6 patients with oestrogen receptor positive breast cancers that showed partial responses to the chemotherapeutic combination epirubicin/cyclophosphamide. Data analysis was performed to determine differences in genetic aberrations between pre- and post-NAC, and in particular to identify evidence of consistent selection by therapy of aberrations that therefore may define chemotherapy resistance or sensitivity. There were extensive differences in the range of genetic aberrations between pre- and post-NAC. 48 genes were identified for further study based on evidence of mutations conferring a selective advantage or disadvantage during chemotherapeutic response. The relevance of these was screened using siRNA knock-down and assessment of response to epirubicin using cell viability assays in vitro. Two genes were taken forward. Potential loss-of-function mutations in MUC17 were selected against during therapy in patients, and in accordance with this MUC17 knock-down was associated with increased sensitivity in vitro. Potential loss-of-function mutations in PCNX1 were selected for during therapy in patients, and in accordance with this PCNX1 knock-down was associated with resistance. Further work was performed to investigate mechanisms by which these genes modify chemotherapy response, by examining drug loading and ABC transporter expression levels. Data indicate that both genes impact on drug loading, potentially through modulating ABC transporter expression. Also, MUC17 or PCNX1 protein levels were tested as prognostic and predictive markers for breast cancer clinical outcomes using tissue taken from cohorts of patients who received adjuvant chemotherapy or neoadjuvant chemotherapy. Kaplan-Meier survival analyses revealed that low MUC17 expression after neoadjuvant chemotherapy was significantly associated with longer disease free survival, which was in agreement with the selection of MUC17 mutations seen after therapy in the initial patient group, and with the in vitro siRNA findings concerning drug sensitivity. I concluded that MUC17 and PCNX1 are potential markers of response to chemotherapy in breast cancer, and that therapeutic modulation of their activities could enhance chemotherapy responses

Pooled extracellular receptor-ligand interaction screening using CRISPR activation.

Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale

“Rethinking High-Grade Serous Carcinoma: Development of new tools for deep tissue profiling”

Background: High-grade serous ovarian cancer (HGSOC) is the most frequently occurring and most fatal epithelial ovarian cancer (EOC) subtype. The reciprocal interplay of the different components encompassed within the tumour microenvironment (TME) are fundamental for tumour growth, advancement, and therapy response. It is therefore important to be able to deeply characterize the complex and diverse TME with multidimensional approaches. Aims: The main aim of this project was to establish novel multiparametric mass cytometry panels and thoroughly characterise the HGSOC TME. Methods: We first developed a novel 35-marker ovarian TME-based Cytometry by time-of-flight (CyTOF) panel (pan-tumour panel) and utilized it to examine the effects of six different tissue dissociation methods on cell surface antigen expression profiles in HGSOC tumour samples (Paper I). We further established an unique immune panel (pan-immune) for the detailed immunophenotyping of chemo-naïve HGSOC patients. The individual tumour immune microenvironments were characterized with tailored computational analysis (Paper II). With the use of an established merging algorithm— CyTOFmerge—the pan-tumour and pan-immune datasets were merged for a more in- depth immune delineation of the ten ovarian chemo-naïve TME profiles in addition to tumour and stromal cell phenotyping (Paper III). Results: We have established a novel ovarian TME-based CyTOF panel for HGSOC that is capable of delineating the immune, tumour, and stromal cells of the TME. Utilizing this panel, we demonstrated that, although the six tissue dissociation methods have a certain level of influence on the TME antigen expression profiles, inter-patient differences between the tumour samples are still clear. In addition, we identified a previously undescribed stem-like cell subset (Paper I). We have developed a unique 34-marker immune panel and have provided a detailed characterization of the ovarian tumour immune microenvironment of chemo-naïve patients. We identified a high degree of interpatient immune cell heterogenicity and discovered an abundance of conventional dendritic cells (DC), natural killer (NK) cells, and unassigned hematopoietic cells. Certain monocyte and dendritic cell (DC) clusters have shown prognostic relevance within the ovarian TME (Paper II). The merged dataset analysis revealed a new level of complexity with a more in-depth immune (myeloid cells) delineation in addition to tumour and stromal (fibroblast subsets) cell phenotypes. We identified an even higher degree of interpatient TME heterogenicity and a novel tumour cell metacluster, CD45-CD56-(EpCAM-FOLR1-CD24-). As a benefit of integrating the datasets, we identified even higher clinical associations (from 12 [pan-tumour dataset] to 20 [merged dataset]). Furthermore, most of these observed associations were majorly between PFS, OS, and infiltrating immune cell subsets (Paper III). Conclusions and consequences: (Paper I) In conclusion, the panel represents a promising profiling tool for the in-depth phenotyping of the HGSOC TME cell subsets. Although the tissue dissociation methods have influence on the TME antigen expression profiles, inter-patient differences are still clear. (Paper II) Our findings revealed a high degree of heterogeneity and identified phenotypic profiles that can be explored for use in HGSOC phenotypic profiling. (Paper III) Together, the merged sketching illustrates that comprehensive individual TME mapping for HGSOC patients can contribute to a better understanding each patient’s unique micromilieu given the need for more personalized treatment approaches.Doktorgradsavhandlin

The function of the endocannabinoid system and glial cells in vivo in patients with first episode psychosis

Psychoses are relatively common and often severely debilitating mental disorders with a multifactorial etiological background involving both psychosocial and biological factors. Previously reported associations between the endocannabinoid and immune systems, and psychotic disorders, suggest that they are involved in the etiology of psychosis. Healthy individuals were studied with the selective type 1 endocannabinoid receptor (CB1R) radiotracer [18F]FMPEP-d2, and positron emission tomography (PET), for possible demographic confounders. Radiotracer synthesis and the compound’s behaviour in blood and brain tissues, were in line with reports from previous validation studies. Females had lower availabilities of CB1R than males in 17 discrete brain regions. Separate samples of male patients with first-episode psychosis (FEP) were then studied concurrently in Turku and London, using the CB1R radiotracers [18F]FMPEP-d2 and [11C]MEPPEP respectively. Lower CB1R availability was seen in FEP as compared to healthy controls. The availability of CB1R was also inversely associated with the symptomatology of the psychoses. Translocator protein (TSPO) expression has been postulated to represent glial cell and mitochondrial functions, both of which are influenced by endocannabinoid signalling. Another sample of male and female patients with first episode psychoses was studied using PET with the selective TSPO radiotracer [11C]PBR28. Male and female FEP subjects showed globally lower availability of brain TSPO in comparison to healthy controls. Two concurrent samples of FEP individuals showed persistent elevations of the chemokine CCL22 when compared to population controls. A subgroup of patients with the highest levels of CCL22 also had aberrant levels of other cyto- and chemokines. These results indicate that the immune and brain endocannabinoid systems have become dysregulated in early psychosis. Aberrant glial cell function and/or disturbances in cell metabolism are indicated by the lower availability of TSPO.Endokannabinoidijärjestelmän ja gliasolujen toiminta ensipsykooseissa Psykoosit ovat verrattain yleisiä, vakavia mielenterveyshäiriöitä, joiden syntyyn vaikuttaa sekä psykososiaaliset että biologiset tekijät. Endokannabinoidi- ja immuunijärjestelmien yhteydet psykooseihin, sekä dopamiinijärjestelmän toimintaan, viittaavat näiden järjestelmien toimivan osana psykoosien etiologiaa. Terveiden koehenkilöiden aivojen endokannabinoidijärjestelmän toimintaa tutkittiin tyypin 1 endokannabinoidireseptorin (CB1R) merkkiaineella [18F]FMPEPd2, ja positroniemissiotomografialla (PET), mahdollisten sekoittavien tekijöiden tunnistamiseksi. Merkkiaineen tuotannon laatua kuvaavat tunnusluvut, sekä merkkiaineen käyttäytyminen veressä ja aivokudoksessa, vastasivat aiempien validointitutkimusten tuloksia. Naiskoehenkilöillä oli alhaisemmat [18F]FMPEP-d2:n jakautumistilavuudet 17 aivoalueella verrattuna miehiin. Miespuolisten ensipsykoosipotilaiden aivojen endokannabinoidijärjestelmän toimintaa tutkittiin erikseen Turussa ja Lontoossa PET:lla vastaavasti CB1R merkkiaineilla [18F]FMPEP-d2 ja [11C]MEPPEP. Molempien otosten ensipsykoosipotilailla oli alhaisemmat merkkiaineiden jakautumistilavuudet verrattuna terveisiin koehenkilöihin. Merkkiaineen sitoutumiselle vapaat CB1R:t olivat lisäksi käänteisesti yhteydessä psykoosioireiden vaikeusasteeseen. Aivojen tukisolujen ja näiden mitokondrioiden toimintaan vaikuttavat sekä endokannabinoidiviestintä, että translokaattoriproteiinin (TSPO) toiminta. Ensipsykoosipotilailla oli kauttaaltaan alhaisemmat TSPO PET merkkiaineen [11C]PBR28 jakautumistilavuudet verrattuna terveisiin verrokkihenkilöihin. Ensipsykoosipotilaiden kemokiini CCL22:n pitoisuudet olivat verrokkien pitoisuuksia korkeammat. Korkeimpia CCL22:n pitoisuuksia omaavien potilaiden immuuniviestintä poikkesi muista verrokki- ja potilastutkittavista laaja-alaisesti. Nämä tulokset osoittavat, että immuuni- ja endokannabinoidijärjestelmät toimivat poikkeavasti ensipsykooseissa. TSPO:n poikkeava toiminta viittaa siihen, että aivojen tukisolut ja/tai solujen aineenvaihdunta häiriintyvät psykooseissa

Special considerations for studies of extracellular vesicles from parasitic helminths: A community-led roadmap to increase rigour and reproducibility

Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved