Heterogeneity reduces sensitivity of cell death for TNF-Stimuli

Background Apoptosis is a form of programmed cell death essential for the maintenance of homeostasis and the removal of potentially damaged cells in multicellular organisms. By binding its cognate membrane receptor, TNF receptor type 1 (TNF-R1), the proinflammatory cytokine Tumor Necrosis Factor (TNF) activates pro-apoptotic signaling via caspase activation, but at the same time also stimulates nuclear factor kappaB (NF-kappaB)-mediated survival pathways. Differential dose-response relationships of these two major TNF signaling pathways have been described experimentally and using mathematical modeling. However, the quantitative analysis of the complex interplay between pro- and anti-apoptotic signaling pathways is an open question as it is challenging for several reasons: the overall signaling network is complex, various time scales are present, and cells respond quantitatively and qualitatively in a heterogeneous manner. Results This study analyzes the complex interplay of the crosstalk of TNF-R1 induced pro- and anti-apoptotic signaling pathways based on an experimentally validated mathematical model. The mathematical model describes the temporal responses on both the single cell level as well as the level of a heterogeneous cell population, as observed in the respective quantitative experiments using TNF-R1 stimuli of different strengths and durations. Global sensitivity of the heterogeneous population was quantified by measuring the average gradient of time of death versus each population parameter. This global sensitivity analysis uncovers the concentrations of Caspase-8 and Caspase-3, and their respective inhibitors BAR and XIAP, as key elements for deciding the cell's fate. A simulated knockout of the NF-kappaB-mediated anti-apoptotic signaling reveals the importance of this pathway for delaying the time of death, reducing the death rate in the case of pulse stimulation and significantly increasing cell-to-cell variability. Conclusions Cell ensemble modeling of a heterogeneous cell population including a global sensitivity analysis presented here allowed us to illuminate the role of the different elements and parameters on apoptotic signaling. The receptors serve to transmit the external stimulus; procaspases and their inhibitors control the switching from life to death, while NF-kappaB enhances the heterogeneity of the cell population. The global sensitivity analysis of the cell population model further revealed an unexpected impact of heterogeneity, i.e. the reduction of parametric sensitivity

Role of AMP-activated protein kinase in adipose tissue metabolism and inflammation

AMPK (AMP-activated protein kinase) is a key regulator of cellular and whole-body energy balance. AMPK phosphorylates and regulates many proteins concerned with nutrient metabolism, largely acting to suppress anabolic ATP-consuming pathways while stimulating catabolic ATP-generating pathways. This has led to considerable interest in AMPK as a therapeutic target for the metabolic dysfunction observed in obesity and insulin resistance. The role of AMPK in skeletal muscle and the liver has been extensively studied, such that AMPK has been demonstrated to inhibit synthesis of fatty acids, cholesterol and isoprenoids, hepatic gluconeogenesis and translation while increasing fatty acid oxidation, muscle glucose transport, mitochondrial biogenesis and caloric intake. The role of AMPK in the other principal metabolic and insulin-sensitive tissue, adipose, remains poorly characterized in comparison, yet increasing evidence supports an important role for AMPK in adipose tissue function. Obesity is characterized by hypertrophy of adipocytes and the development of a chronic sub-clinical pro-inflammatory environment in adipose tissue, leading to increased infiltration of immune cells. This combination of dysfunctional hypertrophic adipocytes and a pro-inflammatory environment contributes to insulin resistance and the development of Type 2 diabetes. Exciting recent studies indicate that AMPK may not only influence metabolism in adipocytes, but also act to suppress this pro-inflammatory environment, such that targeting AMPK in adipose tissue may be desirable to normalize adipose dysfunction and inflammation. In the present review, we discuss the role of AMPK in adipose tissue, focussing on the regulation of carbohydrate and lipid metabolism, adipogenesis and pro-inflammatory pathways in physiological and pathophysiological conditions

The role of macrophages in obesity-associated islet inflammation and β-cell abnormalities.

Chronic, unresolved tissue inflammation is a well-described feature of obesity, type 2 diabetes mellitus (T2DM) and other insulin-resistant states. In this context, adipose tissue and liver inflammation have been particularly well studied; however, abundant evidence demonstrates that inflammatory processes are also activated in pancreatic islets from obese animals and humans with obesity and/or T2DM. In this Review, we focus on the characteristics of immune cell-mediated inflammation in islets and the consequences of this with respect to β-cell function. In contrast to type 1 diabetes mellitus, the dominant immune cell type causing inflammation in obese and T2DM islets is the macrophage. The increased macrophage accumulation in T2DM islets primarily arises through local proliferation of resident macrophages, which then provide signals (such as platelet-derived growth factor) that drive β-cell hyperplasia (a classic feature of obesity). In addition, islet macrophages also impair the insulin secretory capacity of β-cells. Through these mechanisms, islet-resident macrophages underlie the inflammatory response in obesity and mechanistically participate in the β-cell hyperplasia and dysfunction that characterizes this insulin-resistant state. These findings point to the possibility of therapeutics that target islet inflammation to elicit beneficial effects on β-cell function and glycaemia

Resistance to Cell Death and Its Modulation in Cancer Stem Cells

Accumulating evidence has demonstrated that human cancers arise from various tissues of origin that initiate from cancer stem cells (CSCs) or cancer-initiating cells. The extrinsic and intrinsic apoptotic pathways are dysregulated in CSCs, and these cells play crucial roles in tumor initiation, progression, cell death resistance, chemo- and radiotherapy resistance, and tumor recurrence. Understanding CSC-specific signaling proteins and pathways is necessary to identify specific therapeutic targets that may lead to the development of more efficient therapies selectively targeting CSCs. Several signaling pathways-including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/Β-catenin&and expression of the CSC markers CD133, CD24, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain CSC properties. Studying such pathways may help to understand CSC biology and lead to the development of potential therapeutic interventions to render CSCs more sensitive to cell death triggered by chemotherapy and radiation therapy. Moreover, recent demonstrations of dedifferentiation of differentiated cancer cells into CSC-like cells have created significant complexity in the CSCs hypothesis. Therefore, any successful therapeutic agent or combination of drugs for cancer therapy must eliminate not only CSCs but differentiated cancer cells and the entire bulk of tumor cells. This review article expands on the CSC hypothesis and paradigm with respect to major signaling pathways and effectors that regulate CSC apoptosis resistance. Moreover, selective CSC apoptotic modulators and their therapeutic potential for making tumors more responsive to therapy are discussed. The use of novel therapies, including small-molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of CSCs, immunotherapy, and noncoding microRNAs may provide better means of treating CSCs

Identifying noise sources governing cell-to-cell variability

Phenotypic differences often occur even in clonal cell popu- lations. Many potential sources of such variation have been identified, from biophysical rate variance intrinsic to all chem- ical processes to asymmetric division of molecular compo- nents extrinsic to any particular signaling pathway. Identifying the sources of phenotypic variation and quantifying their con- tributions to cell fate variation is not possible without accurate single cell data. By combining such data with mathematical models of potential noise sources it is possible to characterize the impact of varying levels of each noise source and identify which sources of variation best explain the experimental ob- servations. The mathematical framework of information theory provides metrics of the impact of noise on the reliability of a cell to sense its environment. While the presence of noise in a single cellular system reduces the reliability of signal trans- duction its impact on a population of varied single cells remains unclear

Novel properties of mature adipocytes in obesity and hyperinsulinemia

Adipose tissue expansion and dysfunction, which leads to obesity and related metabolic diseases (e.g., diabetes and hypertension) are currently the most costly challenges for public health, yet the mechanisms underlying adipocyte functional dysregulation have not been fully elucidated . Investigation is largely hindered by the unique technical limitations associated with handling these large, lipid-filled fat cells. New techniques need to be developed in order to better understand the physiology and pathology of adipocytes. In Paper I, by using an adipocyte-specific reporter mouse, we proved that previously reported adipocyte flow cytometry techniques missed the major adipocyte population. Therefore, we defined several crucial cytometer settings required for large cell types that allow us to analyze and sort both white and brown mature adipocytes. This improved strategy is applicable to sort adipocytes based on size without fixation, which greatly facilitates subsequent downstream analyses. In combination with immunostaining, the presented approach can effectively sort UCP1 positive adipocytes from mouse white and brown adipose tissue. Furthermore, we demonstrated a heterogeneous ADRB2 expression pattern in human adipocytes, which confirmed the applicability of our newly developed technique to further explore other aspects of adipocyte identity. In Paper II, we developed a MAAC (membrane mature adipocyte aggregate culture) system as a novel, high-viability model for human mature adipocytes. With a permeable membrane insert sitting on top to facilitate cell aggregation and nutrition access, adipocytes cultured in the MAAC system maintained adipogenic properties, did not dedifferentiate and had reduced hypoxia. This newly developed in vitro system allows us to compare depot-specific adipocyte gene expression, and analyze the crosstalk between adipocytes and macrophages. In particular, we demonstrated that human adipocytes can be transdifferentiated to brown-like adipocytes under the conditions of rosiglitazone stimulation or PGC-1α overexpression. Taken together, we provided a versatile tool for modulating primary adipocytes, opening up numerous downstream applications. In Paper III, we revealed that a large group of mature human adipocytes express an array of cell cycle-specific markers indicative of a cell cycle re-entry profile, and that this is associated with whole-body insulin resistance. We demonstrated that insulin is a critical driver of adipocyte cell cycle re-entry, subsequently making them vulnerable to undergo cellularsenescence. Our data showed that hyperinsulinemia in obese patients is associated with increased p16 and senescence associated β-galactosidase activity in mature adipocytes. Furthermore, we showed that senescent adipocytes are hypertrophic and develop a senescence- associated secretory phenotype (SASP), defined by the secretion of IL-6, IL-8, and MCP1. These findings challenge the dogma that adipocytes permanently exit the cell cycle upon differentiation and reveals cellular senescence as a new mechanism associated with inflammation-related adipocyte pathology. In conclusion, the research within this thesis has provided important techniques for both in vitro adipocyte modulation and high throughput flowcytometric adipocyte analysis, supporting multiple downstream research applications to investigate mechanisms regulating adipocyte physiology and pathology. Furthermore, we demonstrated the phenomenon of cell cycle re- entry and senescence in human mature adipocytes, thereby introducing novel insights into obesity and hyperinsulinemia-induced adipocyte dysfunction, suggesting potential targets for treating obesity-related metabolic diseases

Role of Diacylglycerol kinase alpha (DGKA) as a therapeutic target in Glioblastoma (GB)

Glioblastoma (GB) is the most common high-grade fatal brain tumor. The standard of care treatment is surgery, followed by radiotherapy and chemotherapy. Despite decades of research, the median life expectancy of patients is still between 12 to 15 months. The activation of multiple receptor tyrosine kinases (RTKs) and/or downstream tumour-intrinsic mutations provide oncogenic stimuli to GB progression and accounts for resistance to current therapies. Identifying a target that is capable of simultaneously disabling of multiple, parallel oncogenic signals can represent an effective therapy. Mounting reports indicate DGK\u3b1 relevance as a therapeutic target across multiple cancers, given its role in different aspects of tumour biology. DGK\u3b1 phosphorylates diacylglycerol (DG) resulting in the production of phosphatidic acid (PA). Both DG and PA are membrane bound secondary messengers that regulate signalling molecules involved in cancer. DGKs act simultaneously as both terminators and activators of DG- and PA-mediated signalling. In order to exploit DGK\u3b1 as a therapeutic target we investigated the role of DGK\u3b1 in GB biology and signalling. Our results show that DGK\u3b1 is required for GB stem-like cell long term viability and stemness maintenance and sensitize tumor cells to temozolomide. Inhibition of DGK\u3b1 strongly impairs NF-\u3baB transcriptional activity and analysis of the TNFR signalling showed that DGK\u3b1 is necessary for FAK and AKT activation downstream TNFa stimulation. Taken together, the results of this study strongly suggest that DGK\u3b1 plays a key role in stemness maintenance contributing FAK, Akt and NF-kB activation upon TNF stimulation and for this reason DGKa might represent a targetable oncogene that links in\ufb02ammation and tumor growth and progression

암 미세환경에 의해 조절되는 난소암세포의 항암제 저항성과 전이능 연구

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부(바이오모듈레이션전공), 2021. 2. 송용상.Ovarian cancer is the most lethal type of gynecologic malignancy with high rates of mortality. About 70% of patients diagnosed with the cancer show recurrence of cancer despite the cytoreductive surgery and platinum-based chemotherapy. This is mainly due to the highly metastatic capacity of ovarian cancer and extrinsic mechanisms of chemoresistance exerted by extrinsic factors known as the tumor microenvironment. In this series of studies on the ovarian cancer tumor microenvironment, the effects of the tumor microenvironmental factors (hypoxia, inflammation and malignant ascites) affecting chemoresistance and metastatic potential were investigated. Mitochondria are often referred to as the power plants of a cell. They are vital organelles within a eukaryotic cell, constantly undergoing fission and fusion processes in response to the external stimuli in the surrounding cues. Dysregulation of mitochondrial fission and fusion processes has been shown to be linked to onsets and the progression of many serious diseases such as neurodegenerative diseases, cardiovascular diseases and cancer. Hypoxia is a critical feature of the tumor microenvironment affecting cancer progression through modulation of cancer hallmarks. Hypoxia stimulates the production of reactive oxygen species (ROS), inducing changes in mitochondrial function, metabolism and structures. In this study, we sought to investigate the effect of hypoxia on mitochondrial dynamics of ovarian cancer cells. We investigated alterations in mitochondrial networks under hypoxic conditions (< 1% O2) and the subsequent effect of the mitochondrial alteration on drug response in ovarian cancer cells. Hypoxia-induced ROS caused an increase in mitochondrial fission, a response abolished by free radical scavenging with N-acetylcysteine (NAC) and Trolox. Also, the treatment of hydrogen peroxide (H2O2) decreased inhibitory p-Drp1 (Ser637) content and increased mitochondrial fission. Suppression of mitochondrial fission enhanced the CDDP sensitivity of hypoxic ovarian cancer cells. Lastly, in tumor spheroids from malignant ascites or tissues of patients with advanced-stage ovarian cancer, pre-treatment with Mdivi-1 increased the CDDP sensitivity. Taken together, our results implicate that hypoxia-induced ROS triggers mitochondrial fission and CDDP resistance in ovarian cancer cells. Mitochondrial dynamics of cancer cells adapting to the hypoxic tumor microenvironment could be a promising target for ovarian cancer treatment. Next, a growing body of evidence suggests that inflammation is closely associated with the development and progression of ovarian cancer. Herein, we postulated that TNF-mediated activation of the alternative NFκB pathway promotes invasiveness and resistance to cell death induced by TNF. Among the inflammation-related cytokines, we found that TNF is highly expressed in ovarian cancer tissues, compared to that in the other types of solid tumors. Ovarian cancer cells exposed to TNF for 48 hours showed increased colony-forming capacity and cell cycle progression. Additionally, the gene expression levels of TNF and it receptors (TNFRSF1A and TNFRSF1B) were elevated in the metastatic ovarian cancer tissues than the primary tissues from the same patient. Also, the results from the Matrigel-coated transwell insert assay showed that TNF promotes invasiveness of ovarian cancer cells. To investigate the mechanisms involved in this process, we examined the protein expression of classical (p50:p65) and alternative (RelB:P52) NFκB pathway regulators. higher expression of the alternative NFKB transcription factor, RelB was associated with poor survival rates. Using 307 ovarian tumor samples from The Cancer Genome Atlas (TCGA), we found a significant positive correlation between the alternative NFKB pathway genes (RelB and P52) and cancer invasion-related protease (MMP9, PLAU). Protease array revealed that PLAU was upregulated by TNF but this response was absent in the RelB knockdown ovarian cancer cell. Collectively, these findings highlight the involvement of the alternative NFκB pathway-mediated by RelB expression in metastatic processes in ovarian cancer cells. Lastly, to understand cellular interactions, present within the metastatic niche of the ovarian cancer tumor microenvironment, malignant ascites, we utilized single-cell RNA sequencing data from 5 patients with ovarian cancer. We identified 7 distinct cell types based on the expression patterns of cell-type-specific signatures. Macrophages were the most heterogeneous cell type with eleven sub-clusters in the ascites tumor microenvironment. Annotation of macrophage subpopulations was done using MacSpectrum. Also, intra- and inter-tumoral heterogeneities of OC cells in the ascites were assessed. The communication between immune and OC cells was predicted through ligand-receptor interaction analysis with NicheNet. We uncovered that CCL5 is enriched in T cells and NK cells, modulating OC cell survival in the ascites presumably through SDC4. Average SDC4 expression positively correlates with OC cell proportion in each sample. Elevated SDC4 expression predicted poor overall survival of OC patients. Altogether, our study highlights the potential tumor-promoting role of T cells and NK cells in long-term survival outcomes, suggesting that SDC4 is a vital molecule for OC cell survival and a prognostic marker in OC patients.난소암은 사망률이 높은 가장 치명적인 유형의 부인과 악성 종양이다. 암 진단을받은 환자의 약 70%는 종양 감축 수술과 백금 기반 화학 요법에도 불구하고 암의 재발을 보인다. 이것은 주로 난소 암의 고도로 전이되는 능력과 종양 미세 환경으로 알려진 외인성 요인에 의해 발휘되는 화학 내성의 외인성 메커니즘 때문이다. 난소암 종양 미세 환경에 대한 일련의 연구에서, 종양 미세 환경 요인 (저산소증, 염증 및 악성 복수)이 화학 요법과 전이 가능성에 미치는 영향을 조사했다. 미토콘드리아는 종종 세포의 발전소라고 불린다. 미토콘드리아는 진핵 세포 내에서 중요한 소기관이며, 주변 환경의 외부 자극에 반응하여 분열과 융합 과정을 반복한다. 미토콘드리아의 형태적 변화는 퇴행성 신경병, 심혈관 질환 및 암을 포함한 여러 심각한 질병의 발병 및 진행과 관련이 있는 것으로 나타났다. 저산소증은 암 특징(cancer hallmark)의 조절을 통해 암 진행에 영향을 미치는 종양 미세환경의 중요한 특징이다. 저산소증은 미토콘드리아의 기능, 대사 및 구조의 변화를 유도하여 활성 산소 종(ROS)의 생성을 자극한다. 본 연구에서 저산소환경의 난소암 세포에서 미토콘드리아 역학을 조사하였다. 또한 저산소 조건(<1% O2)에서 미토콘드리아 네트워크가 암세포에서 약물 반응에 미치는 영향을 확인하였다. 저산소환경에서의 활성 산소 종은 미토콘드리아 분열을 증가시켰고, 이러한 현상은 항산화제인 N-아세틸 시스테인(NAC) 및 트롤록스(Trolox) 처리 시 완화되었다. 또한, 과산화수소(H2O2) 처리 시 pDrp1(Ser637)의 발현의 감소와 미토콘드리아의 분열이 증가하는 것을 확인하였다. 미토콘드리아 분열 저해 시, 저산소성 난소암 세포의 시스플라틴 감수성을 증가시켰다. 마지막으로, 진행성 난소암 환자의 악성 복수 또는 조직으로부터 분리 한 일차 종양 스페로이드(Speroids)에 Drp1 억제제인 Mdivi-1 전처리시, 시스플라틴 감수성이 증가되었다. 따라서, 난소암세포는 저산소 환경에서 항암제 시스플라틴의 저항성을 일으키고, 이것은 저산소 환경에서 증가한 활성산소를 통한 미토콘드리아 분열 때문이라는 것을 확인했다. 다음으로, 염증이 난소 암의 발생 및 진행과 밀접한 관련이 있다는 증거가 늘어나고 있습니다. 여기에서, 우리는 대체 NFκB 경로의 TNF 매개 활성화가 TNF에 의해 유도 된 세포 사멸에 대한 침입 성과 저항성을 촉진한다고 가설을 세웠다. 염증 관련 사이토 카인 중 TNF는 다른 유형의 고형 종양에 비해 난소 암 조직에서 고도로 발현된다는 것을 발견했습니다. 48 시간 동안 TNF에 노출 된 난소 암 세포는 증가 된 콜로니 형성 능력과 세포주기 진행을 보여 주었다. 또한 TNF 및 수용체(TNFRSF1A 및 TNFRSF1B)의 유전자 발현 수준은 전이성 난소 암 조직에서 동일한 환자의 1 차 조직보다 높게 나타났다. 또한 Matrigel 코팅 된 transwell inserts 분석 결과는 TNF가 난소 암 세포의 침습성을 촉진하는 것으로 나타났다. 이 과정에 관련된 메커니즘을 조사하기 위해 우리는 고전적 (p50 : p65) 및 대안적 (RELB : P52) NFκB 경로 조절제의 단백질 발현을 조사했습니다. 대체 NFKB 경로의 전사 인자 인 RELB의 더 높은 발현은 낮은 생존율과 관련이 있었다. The Cancer Genome Atlas (TCGA)의 307 개의 난소 종양 샘플을 사용하여 대체 NFKB 경로 유전자 (RELB 및 P52)와 암 침습 관련 프로테아제 (MMP9, PLAU)간에 유의 한 양의 상관 관계를 발견했다. 프로테아제 어레이는 PLAU가 TNF에 의해 상향 조절되는 것으로 밝혀졌지만이 반응은 RELB 녹다운 난소 암 세포에는 없었다. 종합적으로, 이러한 발견은 난소 암 세포의 전이 과정에서 RelB 발현에 의해 매개되는 대체 NFκB 경로의 관여를 시사한다. 마지막으로, 난소 암 종양 미세환경인 악성 복수에 존재하는 세포 상호 작용을 이해하기 위해 난소 암 환자 5 명의 단일 세포 RNA 시퀀싱 데이터를 활용했다. 우리는 세포 유형별 시그니처의 발현 패턴을 기반으로 7 개의 별개의 세포 유형을 식별했다. 대식세포는 복수 종양 미세 환경에서 11 개의 하위 클러스터를 가진 가장 이질적인 세포 유형이었다. 대식세포 부분 집단의 해석은 MacSpectrum을 사용하여 수행되었다. 또한 복수에서 OC 세포의 종양 내 및 종양 간 이질성을 평가했다. NicheNet을 통한 리간드-수용체 상호 작용 분석을 통해 면역 세포와 OC 세포 간의 상호작용을 예측했다. 우리는 CCL5가 T 세포와 NK 세포에서 발현하여 아마도 SDC4를 통해 복수에서 OC 세포 생존을 조절한다는 것을 발견했다. 평균 SDC4 발현은 각 샘플에서 OC 세포 비율과 양의 상관 관계가 있었다. 증가 된 SDC4 발현은 OC 환자의 전체 생존율이 낮음을 예측했다. 전체적으로, 우리의 연구는 장기 생존 결과에서 T 세포와 NK 세포의 잠재적인 종양 촉진 역할을 강조하여 SDC4가 OC 세포 생존을위한 필수 분자이자 OC 환자의 예후 마커임을 시사한다.Table of contents Abstract 2 Table of contents 5 List of figures and table 9 List of abbreviations 12 1. Chapter I: Literature review and study summary 14 1.1 Introduction 15 1.2 Molecular regulators of mitochondrial dynamics 16 1.2.1 Mitochondrial fission 20 1.2.2 Mitochondrial fusion 23 1.2.3 Motility and distribution 24 1.3 Tumor microenvironment and mitochondrial dynamics 25 1.3.1 ROS and mitochondrial dynamics 28 1.3.2 Inflammatory microenvironment and mitochondrial dynamics 30 1.3.3 Reduced blood supply and mitochondrial dynamics 31 1.3.4 Intracellular communication and mitochondrial dynamics 32 1.4 Drug resistance and mitochondrial dynamics in cancer 34 1.4.1 Mitochondrial fusion and chemoresistance 40 1.4.2 Mitochondrial fission and chemoresistance 41 1.4.3 Mitochondrial trafficking and chemoresistance 42 1.5 Study summary 43 2. Chapter II: Mitochondrial fission causes cisplatin resistance under hypoxic conditions via ROS in ovarian cancer cells 45 2.1 Introduction 46 2.2 Materials and Methods 48 2.2.1 Cell lines 48 2.2.2 Chemical reagents 48 2.2.3 Bioinformatics 48 2.2.4 Immunoblotting 49 2.2.5 MTT assay 49 2.2.6 Apoptosis analysis 50 2.2.7 Analysis of relative mitochondrial activity and mass 50 2.2.8 Immunocytochemistry and confocal microscopy 50 2.2.9 Detection of relative ROS levels 51 2.2.10 TUNEL assay 51 2.2.11 siRNA transfection 51 2.2.12 Isolation and culture of primary cells and patient-derived spheroids 52 2.2.13 Statistical analysis 53 2.3 Results 54 2.3.1 Hypoxia makes ovarian cancer cells resistant to CDDP irrespective of p53 status 54 2.3.2 Hypoxia increases mitochondrial fission in ovarian cancer cells 62 2.3.3 Hypoxia changes expression and activation status of mitochondrial dynamics protein 72 2.3.4 ROS exerted by hypoxia promotes mitochondrial fission 72 2.3.5 Expression of mRNAs in cancer tissue samples from GEO dataset shows the relevance of hypoxia-induced mitochondrial fission and chemoresistance 81 2.3.6 Inhibition of mitochondrial fission increases the sensitivity of ovarian cancer cells to CDDP in hypoxic conditions 85 2.3.7 Mdivi-1 pretreatment increases CDDP sensitivity of patient-derived spheroids (PDS) from tumor tissues and malignant ascites 104 2.4 Discussion 111 3. Chapter III: RELB stimulated by TNFα promotes invasive capacity of ovarian cancer cells through secretion of urokinase (PLAU/uPa) 116 3.1 Introduction 117 3.2 Materials and Methods 119 3.2.1 Cell culture 119 3.2.2 Invasion assay 119 3.2.3 Cell cycle analysis 119 3.2.4 Reagents and antibodies 120 3.2.5 Cell viability assay 120 3.2.6 Transfection of siRNA 120 3.2.7 Western blotting 120 3.2.8 Bioinformatics analysis 120 3.2.9 Proteome profiler array 121 3.2.10 Statistical analysis 121 3.3 Results 122 3.3.1 TNF is highly expressed in ovarian malignancy compared with other types of solid malignancies 122 3.3.2 TNF promotes ovarian cancer cell growth through cell cycle progression 122 3.3.3 TNF enhances invasion of ovarian cancer cells 125 3.3.4 TNF increases expression of RelB, an alternative NFκB gene, predicting poor prognosis in ovarian cancer patients 125 3.3.5 Knockdown of RelB prevents TNF induced invasion of ovarian cancer cells 125 3.3.6 TNF induces secretion of prometastatic proteases through RelB signaling 132 3.4 Discussion 135 4. Chapter IV: Communication between immune and cancer cells in ovarian cancer ascites through CCL5-SDC4 interaction 138 4.1 Introduction 139 4.2 Materials and Methods 141 4.2.1 Patient samples and sample processing 141 4.2.2 scRNA-seq library preparation and data pre-processing 141 4.2.3 scRNA-seq Data analysis 141 4.2.4 MacSpectrum analysis 142 4.2.5 Pathway enrichment analysis 142 4.2.6 NicheNet analysis 143 4.2.7 TCGA, GEO and GTEx dataset analysis 143 4.2.8 Statistical analysis 144 4.3 Results 145 4.3.1 Clustering of single-cell transcriptomic data identifies cellular heterogeneity of malignant ascites 145 4.3.2 Profiling of macrophage polarization states in ascites TME of ovarian cancer patients with MacSpectrum analysis 149 4.3.3 Comparison of molecular pathways enriched in ovarian cancer cell clusters in malignant ascites reveals intra- and inter-patient heterogeneity 156 4.3.4 Elevated expression of ligands related to the anoikis-resistant phenotype may provide survival advantages to cancer cells in the malignant ascites 164 4.3.5 Effector and exhausted CD8+ T cells and NK cells highly express CCL5 in the malignant ascites 171 4.3.6 SDC4 expression correlates with poor prognosis in ovarian cancer patients 175 4.4 Discussion 180 5. References 184 6. Abstract (Korean version) 206Docto

Crosstalk between Macrophages and Pancreatic β-Cells in Islet Development, Homeostasis and Disease.

Macrophages are highly heterogeneous and plastic immune cells with peculiar characteristics dependent on their origin and microenvironment. Following pathogen infection or damage, circulating monocytes can be recruited in different tissues where they differentiate into macrophages. Stimuli present in the surrounding milieu induce the polarisation of macrophages towards a pro-inflammatory or anti-inflammatory profile, mediating inflammatory or homeostatic responses, respectively. However, macrophages can also derive from embryonic hematopoietic precursors and reside in specific tissues, actively participating in the development and the homeostasis in physiological conditions. Pancreatic islet resident macrophages are present from the prenatal stages onwards and show specific surface markers and functions. They localise in close proximity to β-cells, being exquisite sensors of their secretory ability and viability. Over the years, the crucial role of macrophages in β-cell differentiation and homeostasis has been highlighted. In addition, macrophages are emerging as central players in the initiation of autoimmune insulitis in type 1 diabetes and in the low-grade chronic inflammation characteristic of obesity and type 2 diabetes pathogenesis. The present work reviews the current knowledge in the field, with a particular focus on the mechanisms of communication between β-cells and macrophages that have been described so far