iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

Abstract Squamous cell carcinoma (SCC) of the mobile tongue is the most common type of cancer of the oral cavity, accounting for 30-40% of oral cancers. It behaves aggressively and almost half of the affected patients still die of the disease despite great advances in its medical and surgical care. Ameloblastomas are the most common clinically significant type of odontogenic tumors, constituting approximately 1% of all cysts and tumors of the jaw. They are benign but locally invasive tumors with a strong tendency to recur after surgery. Ameloblastic carcinoma combines the histological features of ameloblastoma with cytologic atypia irrespective of the presence or absence of metastasis. The effectiveness of tight junction proteins (claudins 1, 4, 5, 7 and occludin) and cancer-associated fibroblasts (CAFs) as prognostic markers in OTSCC and as markers of malignancy in ameloblastomas was studied. Abundance of CAFs and Claudin 7 derangement was found to be associated with poor disease-specific survival in oral (mobile) tongue cancer. Appearance of CAFs within the epithelial islands of ameloblastoma was found to be a marker of malignancy in the tumor. The prognostic predictability of CAF density, Ki-67 (cell proliferation marker), maspin (tumor suppressor marker) and tumor DNA content (tumor ploidy using image cytometry) in tongue cancers was also tested. CAF density was the only marker strongly predictive of prognosis. In ameloblastomas, α-SMA (for CAFs), Ki-67, epithelial membrane antigen (EMA) and DNA content (using image and flow cytometry) were assessed as markers of ameloblastic carcinoma. Only α-SMA was able to predict ameloblastic carcinoma when found in the epithelial islands. In conclusion, staining for α-SMA and claudin 7 seems to be beneficial for prognostication in tongue cancer, while α-SMA staining may be beneficial in differentiating ameloblastoma from ameloblastic carcinoma.

iii Abstract Glycobiology represents a very progressive subject of cell biology. Protein-saccharide interactions play not only supporting and cell organization role, but they also represent medium for information storage and its decoding. Galectins, group of animal lectins (saccharide-binding proteins), which have selective affinity to ß-galactosides, are multifactorial molecules. They participate in cell-cell and cell-matrix interaction, transmembrane signaling, apoptosis, pre- mRNA splicing and are also present in various types of carcinomas. High expression of galectin-1 has been detected in cancer stroma originated from squamous cell epithelium. In the previous study we established that the fibroblasts - myofibroblasts transition, apart from the known TGF- beta, is also induced by galectin-1. We compared relationship between galectin-1 expression, presence of myofibroblasts and gene expression in tissue samples from patients with head and neck squamous cell carcinoma. Cancer stroma with myofibroblasts was rich in galectin-1 expression in comparison with stroma without myofibroblasts. Moreover, we used microarray analysis (ILLUMINA) to compare the whole genome transcriptome from samples with and without presence of galectin-1. High expression of galectin-1 in tissue samples corresponded with expression...

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Indiana University-Purdue University Indianapolis (IUPUI) / Cachexia is a devastating muscle wasting syndrome affecting multiple biochemical pathways and is a comorbidity of many diseases including pancreatic ductal adenocarcinoma (PDAC). PDAC patients with cachexia commonly experience systemic inflammation, progressive loss of lean muscle and adipose tissue, and cardiac dysfunction. The present workflow identifies proteins and their post-translational modifications extracted from both cardiac and skeletal muscle tissue isolated from a murine model of PDAC-associated cachexia. Reported here are differentially occurring post-translational modifications found on the most abundant contractile proteins. Tissue from mouse muscle samples were collected two weeks after either receiving a sham surgery or orthotopically implanted with PDAC tumor cells, with or without a follow-up chemotherapy treatment of the standard of care agent gemcitabine with nab-Paclitaxel. Whole tissue blocks of gastrocnemius or heart were either flash frozen and pulverized or homogenized in denaturing lysis buffer and then sonicated to facilitate protein extraction. After disulfide bond reduction, cysteine alkylation, and trypsin digestion, the resultant peptides were subjected to molecular barcoding using tandem mass tag isobaric labeling reagents to facilitate multiplexing. The first and second dimension of peptide separation in the multiplexed sample is accomplished with an offline, high pH, reverse phase (RP)-LC fractionation followed by an online RP-LC at lower pH. The use of high-field asymmetric-waveform ion mobility spectrometry provided a last dimension of separation before MSn analyses. ​This novel, proteomic workflow enables deep proteome profiling in the progression of cancer-induced cachexia. The use of multi-dimensional chromatographic separation and differential ion mobility technique have allowed us to identify almost 4,500 proteins groups of gastrocnemius muscle tissue and nearly 7,100 protein groups of myocardium taken from the murine PDAC model of pancreatic cancer. A comprehensive analysis of the data collected from this workflow was used to calculate differential post-translational modifications on major contractile proteins isolated from PDAC model muscle tissue, with or without chemotherapy, when compared to sham surgery controls. Differential post-translational modifications and protein expression changes found to contribute to cancer cachexia may elucidate novel molecular mechanisms and cellular signaling that drive cachexia progression. / 2022-08-23

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20230号 / 医博第4189号 / 新制||医||1019(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 武田 俊一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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<p dir="ltr">PDAC, the most common type of pancreatic cancer, is a highly metastatic cancer that has a low survival rate. It is histologically characterized by a thick desmoplastic stroma. Counterintuitively, PCCs can still manage to survive in such a restrictive environment and even metastasize to distant organs. Over the years, efforts have been made to find out the mechanisms underlying these perplexing behaviors. However, questions about the role of ECM accumulation and enhanced stiffness in PCC dissemination remained unanswered. In this dissertation, we aim to advance the material design for tumor modeling, and propose an explanation for the malignant cell behavior in the PDAC TME. This is achieved through the use of hydrogel-based tumor models that recapitulate the elevated stiffness of the tumor tissue. Specifically, hydrogel stiffness was tuned to mimic the PDAC TME to understand how PCCs and CAFs respond to various substrate stiffnesses temporally. Next, we employ bio-orthogonal click chemistries to create hydrogels with on-demand stiffening capabilities, as well as hyaluronic acid deposition in the hydrogel, to investigate the effect of dynamic change in matrix stiffness and composition on PDAC cells and CAFs. Lastly, by leveraging thiol-norbornene, aldehyde-hydrazide, and tetrazine-norbornene click chemistries, we created a microporous hydrogel system with a conformation that combines both the advantage of 3D cell culture and the non-restricting nature of 2D cell culture. Additionally, the system allows the application of modularized user-defined factors, including, but not limited to stiffness and HA deposition to the system. Stiff gel in 2D facilitated cell spreading of Pa03C in the presence of CAF. Despite being more restrictive on cell spreading, stiff gelatin gel in 3D induced cytokines that promote matrix remodeling and spreading cell morphology can be restored by stiffening with HA. Overall, this dissertation demonstrated that ECM component (i.e., HA), culture dimensionality, and cell-cell interaction play a huge role in cell behavior, and these factors may interact with each other and result in synergistic effects.</p>

Pancreatic cancer is one of the most lethal cancers and despite all research efforts the last 50 years, there are still no effective therapy for this terrible disease. Until quite recently most research in the field of pancreatic duct adenocarcinoma (PDAC) was focused on the tumor cells and mechanisms essential for their proliferation and survival. However, the tumor does not only consist of tumor cells, rather a combination of tumor cells and numerous stroma cell types, i.e. the tumor microenvironment. The tumor cells have developed the ability to activate the surrounding cells to produce factors important for the progression of the tumor. Cancer associated fibroblasts (CAFs) are the major stroma component and as much as 70% of the total PDAC tumor mass consists of these cells. In this thesis I have investigated the mechanisms involved in the cross-talk between tumor cells and CAFs and distinguished the local and systemic effects of this communication. Tumor derived IL-1α was identified as an important factor creating the inflammatory profile seen in CAFs. In PDAC patients, IL-1α was detected in 90% of the tumors and high expression was associated with poor clinical outcome. Moreover, the PDAC tumors had elevated expression levels of many inflammatory factors that were induced in CAFs by the tumor derived IL-1α in vitro. Consequently, this high expression of inflammatory factors in CAFs will attract immune cells including tumor associated macrophages (TAMs), dendritic cells (DCs), and CD8+ T cells. This indicates an immune suppressive role of CAFs, protecting the tumor cells by acting as decoy targets for immune cells homing into the tumor. The inflammatory factors produced in the PDAC microenvironment did not only affect the infiltrating immune cells, but had also systemic effects that included decreased levels of blood DCs in PDAC patients. Furthermore, these myeloid and plasmacytoid DCs were partly activated and had a semi mature phenotype and impaired immunostimulatory function. Low levels of blood DCs were direct associated with poor patient prognosis and the same was seen for low expression of ICOSL by the DCs. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor  promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. The findings presented in this thesis indicate an essential role for the cross-talk between tumor cells and stroma in the production of tumor promoting factors. Treatment of PDAC patients with drugs that target the IL-1α signaling pathway could prevent the communication between these cells, thus reduce the amount of inflammatory factors both locally and systemically. Altogether, our findings support the idea that neutralization of the IL-1α signaling molecule could be a promising therapy for pancreatic cancer. / Mindre än 5% av patienterna som drabbas av cancer i bukspottkörteln förväntas överleva i mer än fem år. De typiska symtomen kommer sent och sjukdomen framskrider snabbt. Några av de riskfaktorer som identifierats är tobaksrökning, fetma och typ 2 diabetes. Forskningen har hittills siktat in sig på tumörcellerna och de mekanismer de använder för att överleva och föröka sig. Men en tumör innehåller också normala kroppsceller och vid bukspottkörtelcancer kan så mycket som 70 procent bestå av i sig ofarliga bindvävsceller. Miljön i tumören skapas av samspelet mellan dessa celltyper. De cancerceller som är bäst på att utnyttja omgivningen för sin tillväxt fortlever och för sina egenskaper vidare. En sådan egenskap är att kunna manipulera bindvävsceller till att producera signalsubstanser och tillväxtfaktorer som gynnar tumören. Mekanismerna bakom denna kommunikation har studerats och ett viktigt fynd var att tumörcellerna producerar signalämnet interleukin 1-alpha (IL-1a). Detta protein upptäcktes i 90 procent av de undersökta tumörerna, och var kopplat till dålig prognos hos patienterna. Signalen via IL-1a sätter igång tillverkningen av substanser som behövs för nybildning och tillväxt av blodkärl, i sin tur en förutsättning för att tumören ska leva vidare och växa. Proteinet stimulerar också celldelning i tumören, bidrar till att lura kroppens immunförsvar och underlättar spridning av dottertumörer till andra delar av kroppen. När vi slår ut signaleringen kan tumörcellerna inte längre påverka bindvävscellerna lika effektivt, och således minskar förekomsten av flera faktorer som gynnar tumörtillväxten. IL-1a kan därför vara en lovande kandidat att utforska vidare för framtida som ett läkemedel mot bukspottkörtelcancer.