Philosophiae Doctor - PhD / The current diagnostic, prognostic, predictive and therapeutic monitoring methods used for breast cancer are limited. Thus, research into more specific, sensitive and effective strategies is required. Breast cancer is the most prevalent form of cancer in women worldwide and accounts for the most common cause of death in women every year. Cancer development is characterized by a wide spread of genetic abnormalities of gene sequences that can be used in detecting and monitoring treatment of the disease as a result of altered gene expression patterns which leave a trail of biomarkers. Seven candidate genes (Gene 1-7) were identified from a previous in silico study and their gene products (BRG 1-7) were annotated to be good candidate breast cancer biomarkers. Differential gene expression analysis using quantitative real-time PCR (qRT-PCR) validated the over-expression of Gene 3, Gene 4 and Gene 7 in a breast cancer cell line (MCF7), of which Gene 7, annotated as a Natural killer group 2, member D (NKG2D) ligand, was observed to be the most over-expressed gene. The innate immune system is the first line of the body's physiological defense against diseases and the natural killer (NK) cells, are central to mediating this type of immunity. NK cells are activated when a specific surface receptor such as the NKG2D receptor binds its ligands expressed by tumor cells. To evade being detected by the immune system, cancer cells are reported to shed off the NKG2D ligands and are expected to be present in the bodily fluids of cancer patients. Also, chemotherapeutics have been reported to suppress the natural anti-tumour immune response, thus should be taken into account when designing optimal therapy for cancer patients. The aim of this research was to validate these candidate genes as effective breast cancer biomarkers using several in silico methods as well as molecular techniques and study the effect of Gene 7 on modulating the effect of several pro-apoptotic compounds. The in silico part of the study investigated the functional, protein interaction, pathways, and tissue expression specificity of the candidate biomarkers using computational software such as DAVID, STRING, KEGG, Genecards and GEA. Also an in silico validation of the prognostic/predictive values of the genes was analysed using SurvExpress, KMplot, and GOBO. Protein expression of selected genes was analysed by Western blot, and immunofluorescence analysis. BRG 7 gene was cloned into pcDNA3.1 vector using recombinant DNA technology while commercial shRNA construct was used to 'knock-down' Gene 7 expression. The two constructs were used to transfect MCF-7 and MCF-12A cells. Over-expression and 'knock down' Gene 7 in transfected cells was confirmed using western blot analysis. Stably transfected cells were then treated with three pro-apoptotic compounds (Camptothecin, Doxorubicin and DMSO) for 24 hours. The apoptotic cells were stained with 3, 4, 5, 6-tetrachloro-2', 4', 5', 7' tetraiodofluorescein (TCTF) and then analysed using flow cytometry. Functional analysis linked Gene 1, Gene 2, Gene 4, Gene 6 and Gene 7 to different cancer related processes. The pathway analysis showed Gene 1, Gene 2, Gene 4 and Gene 7 were involved in pathways that can be linked to cancer modulation. The protein-protein interaction analysis showed only BRG 2 was directly linked to two major hallmarks of cancer (Apoptosis and Autophagy). Breast cancer associated Transcription factors were shown to regulate these genes. Gene 1 and Gene 5 as well as the three genes observed to be highly expressed in the qRT-PCR study were validated to differentially express in breast cancer. An additional protein (BRG 8) was identified and postulated to be a good biomarker candidate for breast cancer based on its direct interaction with BRG 7 and estrogen receptor protein (ESR). The prognostic value of the candidate genes were monitored in two datasets (DATA1 and DATA2) in SurvExpress. DATA1 showed that Gene 6 and Gene 8 while DATA2 showed that Gene 3, Gene 6 and Gene 7 were valuable candidate genes in breast cancer prognosis. The survival curves from the two datasets showed the combined genes could predict the outcome of breast cancer patients undergoing treatments. A plot box output from SurvExpress showed most of the genes were differentially expressed comparing two risk groups. The Kaplan Meier plotter confirmed, Gene 1, Gene 3, Gene 4 and Gene 7 have a significant P-value in predicting the survival outcome based on gene differential expression value. GOBO analysis showed the genes may accurately predict the survival outcome of estrogen positive subtype, ERBB2 subtype of estrogen receptor negative and lymph node negative subtype of ER- tumours, but not all subtype of ER- tumours. Western blot analysis showed BRG 7 may be highly expressed in MCF-7 as compared to MCF-12A, BRG 8 was found to be expressed in all cancer cell types analyzed except for MCF-7 and HT29. BRG 2 was found to be expressed in all cancer types analyzed. immunofluorescence analysis showed BRG 3, BRG 4 and BRG 7 are differentially expressed in breast cancer cell line and are more localized on the cell membrane when compared to the breast non-cancer cell line. Over-expression and gene knock down in cells were successfully confirmed with Western blot analysis. Stably transfected MCF-12A cell for over-expression of BRG7 protein, resulted in cell senescent and the cell stopped growing while stably transfected MCF-7 over-expressing BRG7 did not show any morphological changes. Apoptosis was enhanced in cells treated with camptothecin, doxorubicin and DMSO overexpressing BRG7. Apoptosis was reduced in camptothecin and DMSO treated gene 'knock-down' cells but not doxorucin treated. BRG7 gene 'knock down' in transfected cells showed varying response to all three pro-apoptotic compounds. From this study Gene 3, 5, 7 and 8 and their protein levels were confirmed to be differentially expressed in breast cancer cells and could serve as putative biomarkers for breast cancer. However the variance in the effectiveness of individual genes suggests that the set of genes would perform better than individual gene. The modulating role of BRG7 in drug induced apoptosis, suggest it could probably play an important role in personalised medicine and could serve as a biomarker to monitor the prognosis and/or therapeutic outcome of pro-apoptotic drugs in breast cancer patients. These findings will be further investigated in human breast tissues to validate these data.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Suppression of anti-tumour immunity by the tumour microenvironment remains a major barrier to the development of broadly effective immunotherapies to treat solid tumours. Cytotoxic natural killer (NK) cells are vital to anti-cancer immunity and have shown clinical efficacy for treating hematologic malignancies. However, NK cell therapies have failed to be effective against solid tumours as cytotoxic NK cells become dysfunctional in the tumour microenvironment. While tumours hinder cytotoxic NK cells, they stimulate the tumour-promoting functions of regulatory NK cells. The mechanisms that dictate NK cell polarization and their fate in the tumour microenvironment remain poorly defined but harbour key therapeutic potential. Glucose-driven cellular metabolism has emerged as a central regulator of NK cell anti-tumour activity. Notably, tumour cells have deregulated metabolism, causing a metabolically hostile environment that is low in glucose and oxygen and high in metabolic waste. In the work presented, we demonstrate that NK cells expanded from cancer patients or healthy donors exert strong anti-tumour activity and dismantle the immunosuppressive tumour microenvironments of advanced ovarian and lung cancer. As a result, expanded NK cells were capable of sensitising initially non-responsive patient tumours to PD1 checkpoint-blockade therapy. Further, we uncover that the activity of cellular metabolic pathways plays a key role in NK cell functional fate in tumour microenvironment. We show that the tumour microenvironment induces paralysis of cytotoxic NK cell glucose metabolism to cause their dysfunction. However, reprogramming of NK cell metabolism through expansion arms expanded NK cells with enhanced metabolic flexibility which enabled their anti- tumour activity to be paradoxically strengthened by the tumour microenvironment. We further identify that regulatory NK cells have a distinct metabolic program compared to cytotoxic NK cells, including lower glucose-driven metabolism, that is amenable with the tumour microenvironment. Our work provides new mechanistic insight into how NK cell fate is regulated and how the pathological environment of a tumour capitalizes on this. This knowledge provides new therapeutic targets to intervene with the suppression of cytotoxic immunity in tumours. Further, this work identifies that expanded NK cells are a promising therapeutic candidate that exploit the metabolic hostility of the tumour microenvironment and synergize with other immunotherapies. / Thesis / Candidate in Philosophy / Harnessing the body’s natural immune defenses against cancer in the form of immunotherapy has emerged as a powerful treatment modality. Over the past decade, immune cell therapies have revolutionized the treatment of blood cancers like leukemia and lymphoma. Yet despite the potential, immune cell therapies have failed to be broadly effective against solid tumours because the anti-cancer activity of immune cells, such as Natural Killer (NK) cells, becomes severely impaired by the tumour environment. In this work, we identify that NK cells expanded from cancer patients and healthy donors overcome suppression by tumours and eliminate detectable tumour in pre-clinical models of advanced ovarian and lung cancer. These expanded NK cells also enhanced the functions of other immunotherapies. Further, we shed new light on how NK cells become dysfunctional in tumours. We uncover that NK cells undergo a metabolic energy crisis in tumours that causes their dysfunction, but that expanded NK cells have increased metabolic fitness which allows them to overcome this energy crisis and remain highly functional. Finally, we also characterize the metabolism of a subset of NK cells that are tumour-promoting and find that they harbour metabolic advantages to thrive in tumours. Overall, our work provides new insight as to how to overcome immunosuppression by tumours. This work identifies that expanded NK cells are a promising therapeutic candidate that exploit the hostility of tumours and synergize with other immunotherapies.

Human natural killer (NK) cells are found in virtually all tissues where they act as a first line defense against malignant and virally infected cells. The development of NK cells from CD34+ hematopoietic progenitors is a complex process that involves navigating through different microenvironments and requires contact-dependent interactions with stromal cells. The molecular mediators of NK cell developmental subset trafficking, cell-cell interactions, and maturation have not been fully characterized. This thesis presents 3 studies that aim to uncover contact-dependent interactions that drive human NK cell development. Chapter 2 focuses on defining the adhesome profile of human NK cells from in vitro derived populations, tonsil, and peripheral blood. This study reveals that the tissue origin and developmental stage of NK cells influence the expression of adhesome-associated genes and proteins, as well as the content of cortical actin, which suggests a link between adhesome expression and actin regulation in NK cells. Chapter 3 presents the first comprehensive study on human NK cell development in pediatric tonsil using cyclic immunofluorescence microscopy and imaging mass cytometry. We reveal that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. Chapter 4 demonstrates that neural cell adhesion molecule (NCAM) on stromal cells promotes maintenance of a mesenchymal-like state and subsequently the survival and proliferation of human NK cell precursors. Overall, this thesis provides new insights on previously unknown mediators of NK cell contact-dependent interactions and unveils the first road map of in situ NK cell development.

No description available.

Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.