Cancer research is evolving. Historically concerned with the mechanisms by which malignant cells circumvent cell death signaling and maintain unchecked proliferation, focus has shifted to the complex interactions between the tumor cell and the surrounding microenvironment. Ovarian cancer has one of the highest incidence-to-death ratios of all cancers, and is typically asymptomatic until the later stages, often resulting in metastasis prior to discovery. Naturally occurring phenotypes like lactation and child-bearing (parity) reduce ovarian cancer incidence, but the mechanisms are not understood. As the primary site for ovarian cancer metastasis, and a secondary lymphoid organ capable of mounting potent innate and adaptive immune responses, we believe the omental fat band (OFB) provides a unique opportunity to study complex interactions within the tumor microenvironment. Additionally, we hypothesize that once understood, leukocyte populations within the OFB could be modulated to disrupt the pro-tumorigenic cascade. Using fluorescence-activated cell sorting (FACS) and quantitative realtime PCR (qRT-PCR), we comparatively evaluated the changes in the compositional immune profile of the OFB as a result of parity and cancer. Parous mice were associated with a reduction in macrophages and neutrophils in the OFB, resulting in an inherent "protective state" that was refractory to metastatic cancer cell growth after intraperitoneal implantation. This indicates that the leukocyte populations within the   OFB play an important role in tumor development. Therefore we utilized the potent TH1-type immunomodulatory cytokine IL-12 in a membrane-bound form to circumvent reported side effects, such as hepatic and renal damage, cardiotoxicity and death. Targeted IL-12 delivery to the OFB resulted in delayed disease development, although not protection from subsequent challenge. This was also associated with a reduction tumor-associated macrophages (TAMs) and neutrophils (TANs) within the OFB. Kinetic studies demonstrated that these changes were induced by a significant reduction in neutrophil and macrophage chemoattractants early on in the pro-tumorigenic cascade (7 days post-implantation). This work demonstrates that the OFB is a functionally plastic tissue that can be harnessed and re-mobilized to display an anti-tumorigenic microenvironment. / Ph. D.

The limited transport of conventional chemotherapy within the tumor microenvironment (TME) is due to irregular vascularization, increased tumor interstitial pressure, and a dense extracellular matrix (ECM). The lack of selectivity of anticancer drugs often leads to systemic toxicity and damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is a promising alternative, as tumor-targeting bacteria have been shown to preferentially colonize primary and metastatic tumors and induce anti-tumor effects. In this dissertation, we focus on several aspects of bacteria-nanoparticle conjugates, wherein BBCT is synergistically combined with nanomedicine to augment the efficacy of both treatment modalities. We explore biofabrication of our bacteria-nanoparticle conjugates called NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems) and their interaction with the TME. Specifically, (1) we investigate the effects of two bacteria-NP conjugation chemistry and assembly process parameters of mixing method, volume, and duration, on NP attachment density and repeatability. We evaluate the influence of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS. (2) We investigate the effect of dense stroma and ECM production on the intratumoral penetration of bacteria with a mathematical model of bacterial intratumoral transport and growth. (3) We develop a microfluidic device with multicellular tumor spheroids to study the transport of tumor-targeting bacteria and support real-time imaging and long-term experiments. (4) We develop a new type of bacteria-based bio-hybrid drug delivery system using engineered cell surface display for enhancing the attachment of nanoparticles. / Doctor of Philosophy / Chemotherapy faces challenges in effectively reaching tumors due to factors like irregular blood vessel distribution, increased tumor pressure, and the presence of dense structures such as the extracellular matrix (ECM). This often results in collateral damage to healthy tissues. Bacteria-based cancer therapy (BBCT) offers a promising alternative, utilizing tumor-targeting bacteria to selectively attack tumors. This dissertation focuses on optimizing NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems), which are chemotherapy encapsulating nanoparticle-bacteria assemblies to overcome these challenges and characterizing its behavior in tumors. Firstly, we investigated the optimization of bacteria-nanoparticle attachment, exploring various linkage chemistries and assembly processes to enhance attachment density, viability, and motility. Secondly, we examine how dense stroma and ECM affect bacterial penetration providing insights into intratumoral transport dynamics. Thirdly, we develop a microfluidic device integrated with multicellular tumor spheroids to enable real-time imaging and long-term experimentation on bacteria and drug transport. Lastly, we explore the potential of engineered cell surface display to enhance nanoparticle attachment in NanoBEADS, paving the way for self-propelled and highly targeted drug delivery systems. This dissertation strives to contribute to the transformation of current approaches to cancer treatment by refining drug delivery precision and efficacy while minimizing systemic toxicity.

Yes / It is now well established that the tumor microenvironment plays an essential role in the survival, growth, invasion, and spread of cancer through the regulation of angiogenesis and localized immune responses. This review examines the role of the HOX genes, which encode a family of homeodomain-containing transcription factors, in the interaction between prostate tumors and their microenvironment. Previous studies have established that HOX genes have an important function in prostate cancer cell survival in vitro and in vivo, but there is also evidence that HOX proteins regulate the expression of genes in the cancer cell that influence the tumor microenvironment, and that cells in the microenvironment likewise express HOX genes that confer a tumor-supportive function. Here we provide an overview of these studies that, taken together, indicate that the HOX genes help mediate cross talk between prostate tumors and their microenvironment.

Yes / The membrane-type matrix metalloproteinases (MT-MMPs), an important subgroup of the wider MMP family, demonstrate widespread expression in multiple tumor types, and play key roles in cancer growth, migration, invasion and metastasis. Despite a large body of published research, relatively little information exists regarding evidence for MT-MMP expression and function in metastatic prostate cancer. This review provides an appraisal of the literature describing gene and protein expression in prostate cancer cells and clinical tissue, summarises the evidence for roles in prostate cancer progression, and examines the data relating to MT-MMP function in the development of bone metastases. Finally, the therapeutic potential of targeting MT-MMPs is considered. While MT-MMP inhibition presents a significant challenge, utilisation of MT-MMP expression and proteolytic capacity in prostate tumors is an attractive drug development opportunity.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25166号 / 医博第5052号 / 新制||医||1071(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 藤田 恭之, 教授 上野 英樹, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Basal-like breast cancers make up an aggressive subtype of breast cancer with diverse intratumor cell heterogeneity and poor clinical outcomes. These cancers are predominantly triple-negative cancers such that they lack expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). Evidence suggests that basal-like cancers arise from luminal mammary epithelial cells that acquire basal-like traits during tumorigenesis, a process known as cell plasticity. These cancers also present with dramatic remodeling of stromal cell populations such as fibroblasts and immune cells, which become rewired to form a pro-tumorigenic niche. An improved understanding of the cellular mechanisms that drive epithelial plasticity and stromal remodeling may therefore help advance treatment avenues for basal-like breast cancer. This dissertation describes important roles for the Hippo signaling pathway, a pathway involved in development and stem cell traits, in basal-like breast cancer pathogenesis. Using genetic mouse models, we demonstrate that deletion of the Hippo pathway kinases large tumor suppressor kinase (LATS)1 and LATS2 (LATS1/2) in mammary luminal epithelial cells leads to luminal-basal plasticity and the development of basal-like carcinomas through activation of the transcriptional regulators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). These carcinomas are accompanied by remodeling of the mammary stroma, including an accumulation of cancer-associated fibroblasts and a distinct deposition of extracellular matrix. We further present analyses suggesting that LATS1/2 inactivation promotes a tumor-associated niche through reciprocal epithelial-stromal signaling. Together, these results implicate the Hippo pathway as a critical mediator of homeostasis in the mammary gland and suggest that Hippo dysregulation drives basal-like breast cancer initiation by promoting mammary epithelial transformation and stromal remodeling. / 2025-10-29T00:00:00Z

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic reaction driven by pancreatic stellate cells (PSCs). There is accumulating evidence that PSCs influence the malignant phenotype of PDAC. The aim of this study was to analyse the tumour response to radiation treatment in the presence of PSCs and to investigate the cytokine network in the coculture of PSCs and pancreatic cancer cells (PCCs). PSCs were used in coculture with different PCC lines. Clonogenic survival assays of several PCC lines cocultured with PSCs showed decreased radiosensitivity. This effect was abrogated by inhibition of the β1-integrin/FAK signalling pathway. Furthermore, tumour regrowth experiments after irradiation showed that coinjected PSCs were radioprotective for PCCs after single-dose and fractionated irradiation in xenografts. In addition, we examined the expression of 50 proteins in the supernatants of PCCs and PSCs in mono- and coculture conditions. The detected cytokine expression profile of PSCs included many proinflammatory factors. Also, we identified IP-10 as the chemokine with the highest differential upregulation in PSCs by paracrine stimuli from five different PCC lines. Human PDAC with a high stroma component had elevated IP-10 mRNA expression. IP-10 did not stimulate tumour cell growth and migration in our conditions even though several PCCs expressed its cognate receptor CXCR3. Nevertheless, we discovered that in human PDAC samples IP-10 and CXCR3 mRNA levels correlated with the presence of CD3ε, CD4, FoxP3, CTLA4 and CD39 used as surrogate markers for T regulatory cells (Tregs), known to exert an immunosuppressive effect. In conclusion, these data demonstrate that PSCs enhance survival of PCCs to radiation by activating β1-integrin/FAK signalling. Furthermore, the interaction between the tumour stroma in pancreatic cancer may support an immunosuppression by chemoattraction of Tregs following upregulation of IP-10. Further characterisation of the paracrine signalling between PCCs, PSCs and immune cells will improve the understanding of pancreatic cancer biology and could lead to the identification of new targets for multimodal therapy.

Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

Le cancer du sein est le cancer le plus fréquent chez la femme et représente un problème de santé publique majeur. L'agressivité des tumeurs mammaires varie notamment en fonction de leurstatut pour le récepteur α des oestrogènes (ERα). Les cancers du sein n'exprimant pas ERα ont unmauvais pronostic, de part leur capacité métastatique plus importante. Cependant, les facteurs sous jacents à cette plus grande agressivité des cancers ERα-négatifs restent mal compris. Il est aujourd'hui admis que la progression tumorale et la dissémination métastatique dépendent, non seulement des propriétés intrinsèques des cellules cancéreuses, mais également des régulations exercées sur ces cellules par le micro environnement tumoral. Les interactions entre les cellules cancéreuses et les cellules présentes au niveau du site tumoral, telles que les cellules leucocytaires,les cellules endothéliales, ainsi que les cellules stromales, sont nécessaires au développement et à l'évolution de la tumeur. Ces interactions sont médiées via la production d'hormones, de cytokines ainsi que de chimiokines. Les cellules stromales mésenchymateuses (MSC) sont de composants essentiels du stroma tumoral. Leur rôle dans la progression des tumeurs reste, pour le moment, très controversé. L'objectif de notre projet a été de comprendre les raisons pour lesquelles les MSC peuvent favoriser ou inhiber le développement tumoral. Nous nous sommes, dans un premier temps,intéressés aux interactions entre les cellules cancéreuses mammaires et les MSC. Nous avons déterminé si le fait que les cellules cancéreuses soit métastatiques ou non modifiait le phénotype des MSC et leur réponse dans les régulations de la croissance tumorale. Nous avons ainsi constaté quel es facteurs sécrétés spécifiquement par les cellules cancéreuses métastatiques ERα-négativesinduisaient la production de certaines chimiokines, dont CXCL5. Ces chimiokines peuvent êtressécrétées par les cellules du microenvironnement mais également par les cellules cancéreuses ellesmêmes.Nous avons donc étudié le rôle de CXCL5 dans l'agressivité des tumeurs mammaires. Nousavons ainsi montré que ces chimiokines induisent, in vitro, une augmentation des propriétésprolifératives, invasives et migratoires des cellules cancéreuses. Cette étude nous à permis demontrer que les chimiokines et les interactions entre les cellules cancéreuses et les MSC pouvaientêtre impliquées dans la progression tumorale ainsi que dans l'agressivité des tumeurs mammaires. / Breast cancer remains in Europe and USA the first cause of death by cancer for women.Breast cancer aggressiveness relies in particular on estrogen receptor α (ERa) status. Breast cancers which do not express ERα are more metastatic and have a poorer prognosis, than ERα-positivetumors. However underlying factors involved in these invasive properties are poorly understood.Today, it is established that tumor progression is regulated by intrinsic cancer cells properties, and byinteractions between cancer cells and surrounding microenvironment. Several evidences suggest thatleukocytes, endothelial cells, fibroblasts and infiltrating cells present in stromal compartment caninteract with tumor cells through the production of hormones, cytokines and chemokines.Mesenchymal stromal cells (MSC) belong also to the stromal compartment. Recent studies havehighlighted their potential role in cancer growth and metastasis. However, the ability of MSC to favor orprevent cancer progression remains controversial. The aim of this work was to understand the roles ofMSC in tumor progression and to explain the differential effects of MSC on cancer cells, depending onthe type of cancer cells involved. First, we were analyzed MSC and cancer cells interactions, anddetermined if metastatic cancer cells could affect MSC phenotypes and its response in terms of tumorgrowth. We observed that metastatic breast cancer cells secreted factors, which could highly enhancethe release by MSC of several chemokines, including CXCL5. CXCL5 can be secreted by stromal cellsbut also by cancer cells themselves. We next showed in vitro that CXCL5 increased proliferative,invasive and migratory properties of breast cancer cells. This study allowed us to demonstrate thatchemokines play a role in the cross-talk between MSC and breast cancer cells, and that they play akey role in tumor proliferation and aggressiveness.