Thesis advisor: Thomas N. Seyfried / Metastasis is the process by which cancer cells disseminate from the primary neoplasm and invade surrounding tissue and distant organs, and is the primary cause of morbidity and mortality for cancer patients. Most conventional cancer therapies are ineffective in managing tumor metastasis. This has been due in large part to the absence of in vivo metastatic models that represent the full spectrum of metastatic disease. Here I identify three new spontaneously arising tumors in the inbred VM mouse strain, which has a relatively high incidence of CNS tumors. Two of the tumors (VM-M2 and VM-M3) reliably expressed all of the major biological processes of metastasis to include local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Metastasis was assessed through visual organ inspection, histology, immunohistochemistry, and bioluminescence imaging. The metastatic VM tumor cells also expressed multiple properties of macrophages including morphological appearance, surface adhesion, phagocytosis, gene expression (CD11b, Iba1, F4/80, CD68, CD45, and CXCR4) and total lipid composition (glycosphingolipids and phospholipids). The third tumor (VM-NM1) grew rapidly and expressed properties of neural stem/progenitor cells, but was neither invasive nor metastatic. This thesis research also examined the influence of a genelinked up-regulation of the simple ganglioside GM3 in the metastatic VM-M3 tumor. Ganglioside GM3 has been shown to have anti-invasive effects through its ability to modulate integrins and matrix metalloproteases. Additionally, GM3 was previously shown to be elevated in resting macrophages when compared to activated macrophages. The bioluminescent VM-M3 cells (M3/Fluc) contain mostly GM2, GM1, and GD1a with undetectable levels of GM3. Additionally, the M3/Fluc cells express GalNAc-T, a key enzyme for the synthesis of complex gangliosides from GM3, the precursor used for complex ganglioside biosynthesis. Stable transduction of the M3/Fluc tumor with a lentiviral vector containing a cDNA sequence targeting the GalNAc-T gene (Fluc-TNG), resulted in a knock-down of GalNAc-T expression and an up-regulation of GM3 compared to the control (Fluc-csh) transduced M3/Fluc tumor cells. In vivo, the Fluc-TNG cells were significantly less invasive when implanted in the brain and less metastatic when implanted in the flank when compared to the control Fluc-csh tumors. My data indicate that spontaneous brain tumors can arise from different cell types in VM mice and that the ganglioside GM3 can inhibit invasion and metastasis in metastatic cancer cells with macrophage properties. The new VM tumor model will be useful for defining the biological processes of cancer metastasis and for evaluating potential therapies for tumor management. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The study of the development of anti-cancer drugs and preclinical efficacy tests has until today encountered a major problem identified as lack of reliable in vitro tumour models which are able to reflect in vivo tumour conditions. These models provide a clear basis for understanding tumour development processes, assisting in the selection of agents from various chemicals and testing the efficacy of drugs. There are two important characteristics for an in vitro tumour model, i.e. tumour-like structure of cell aggregates, and the in vivo-like culture microenvironment. To meet these two requirements, an in vitro perfusion based three-dimensional tumour model was developed for the three dimensional culture of cancer cells and related anti-cancer drugs tests. In order to assess this model, DLD1 and NCI/ADR cells were cultured in four different models and compared their proliferation rate, cell viability, micro tumour formation and drug responses. In addition, the comparison of static and perfusion culture were done on monolayer and in 3D also. The cells in perfusion culture showed higher proliferation rates and significantly, higher cell viabilities after a 6-day culture compared to statically cultured cells, especially for the cells in the 3D culture. Microtumours (MTs) were formed from this model, which showed significant tumour-like morphological characteristics, a denser and highly stable structure, a higher cell viability, and varied drug responses compared with spheroids. The inhibition effect of paclitaxel and cisplatin, two common type anti-cancer drugs, were tested and a comparative study was carried out using conventional two-dimensional (2D) static culture, spheroids, and the developed 3D MTs model, as well as real human tumour tissues. The results showed that the cells in 2D culture were most greatly inhibited while human tumours showed the lowest drug responses. The efficacy of anti-cancer drugs, tested in conventional 2D static culture, was greatly amplified. Besides, the response of MTs to agents was much closer to that of human tumours, when the values of spheroids are relatively closer to the cells in 2D culture. It is further supported that MTs have more tumour-like characteristics than spheroids. When compared, the inhibition to proliferation of cells in static and perfusion culture showed significantly different drug responses except for the cells on the monolayer. The shown difference between static and perfusion culture can be due to the different culture environment, and further related to the different action mechanisms of anti-cancer agents. The perfusion culture provides a more homogenous and more physiological microenvironment for the in vitro tumour growth, and in vitro perfused 3D cancer model, developed in this thesis, proved valuable for the study of in vitro cancer and related anti-cancer drug tests.

Vitamin E is a group of naturally occurring fat soluble compounds which consists of eight distinct forms of tocopherols and tocotrienols. Although a well-defined physiological function of vitamin E is as an antioxidant, beneficial effects of individual vitamin E compounds on chronic human diseases such as cancer need to be better understood. Studies in this dissertation investigated potential application of gamma-tocopherol (gamma-T), gamma-tocotrienol (gamma-T3) or synthetic derivatives of tocotrienols as anticancer agents in comparison to alpha-tocopherol (alpha-T), its redox-silent acetic acid derivative (alpha-TEA) or alpha-tocotrienol (alpha-T3). Redox-silent derivatives of alpha- and gamma-T3; namely alpha-T3EA and gamma-T3EA exhibited potent anti-proliferative and proapoptotic activities in a murine mammary cancer cell line as well as in human breast cancer cell lines. Moreover, studies using human vascular endothelial cells in cell culture showed that the tocotrienol derivatives exhibited strong antiangiogenic activities which were markedly improved over those of the parent compounds. An antitumor efficacy study using the 66cl-4-GFP syngeneic mouse mammary tumor model showed that each tocotrienol derivative, when delivered in the diet, significantly suppressed mammary tumor growth; however serum and tissue concentrations of these novel compounds were lower than those of alpha-TEA, suggesting that the next generation of vitamin E derivatives will need to be modified to improve bioavailability. On the other hand, some natural-source vitamin E forms, especially gamma-forms, display anticancer activities without any chemical modification in both in vitro cell culture studies and in vivo animal models. Dietary delivery of gamma-T3 suppressed tumor growth in a syngeneic implantation mouse mammary cancer model by inhibiting cell proliferation and inducing apoptosis. Cell culture studies using human breast cancer cells showed that gamma-T3 triggered apoptosis by inducing endoplasmic reticulum (ER)-stress mediated by acid sphingomyelinase (ASMase) action. Activation of stress-activated mitogen-activated protein kinases (MAPKs), JNK and p38, was associated with gamma-T3-induced ER stress followed by upregulation of extrinsic death receptor-5 (DR5) expression in a CHOP transcription factor dependent manner. Gamma-T also triggered extrinsic apoptosis signaling by increasing DR5 mRNA, protein and cell surface expression levels followed by mitochondria-dependent apoptotic signaling. In agreement with in vitro studies, gamma-T delivered in the diet suppressed the tumor growth of MDA-MB-231-GFP human breast cancer cells in a xenograft model but the antitumor activity of gamma-T was hampered by co-administration of alpha-T. The preferential tissue retention of alpha-T over gamma-T could be overcome by use of sesamin, a dietary source of human cytochrome P450 inhibitor. Based on data presented, gamma-T and gamma-T3 show preclinical potential for cancer treatment either as single agents or in combination with other agents. / text

abstract: Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli. These complexities make it difficult to isolate and assess the effects of specific parameters including matrix stiffness and tumor architecture on disease progression. In this regard, morphologically accurate tumor models are becoming instrumental to perform fundamental studies on cancer cell invasion within well-controlled conditions. In this study, the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique was explored to microengineer a 3D breast tumor model. The microfabrication process presented herein enabled precise localization of the cells and creation of high stiffness constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and normal mammary epithelial cells (MCF10A) were embedded separately within the tumor model and cellular proliferation, migration and cytoskeletal organization were assessed. Proliferation of metastatic MDA-MB-231 cells was significantly higher than tumorigenic MCF7 and normal mammary MCF10A cells. MDA-MB-231 exhibited highly migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that were confined within the micropatterned circular features. F-actin staining revealed unique 3D protrusions in MDA-MB-231 cells as they migrated throughout the surrounding matrix. Alternatively, there were abundance of 3D clusters formed by MCF7 and MCF10A cells. The results revealed that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in creating 3D tumor models with well-defined features and tunable stiffness for detailed studies on cancer cell invasion and drug responsiveness. / Dissertation/Thesis / Supplementary Movie 3 / Supplementary Movie 1 / Supplementary Movie 2 / Supplementary Movie 5 / Supplementary Movie 4 / Masters Thesis Bioengineering 2015

Le glioblastome représente le sous-type de tumeur cérébrale le plus fréquent et le plus agressif. Malgré une meilleure compréhension de la maladie ainsi que l'émergence de nouvelles cibles, voire de nouveaux outils thérapeutiques, son pronostic reste inchangé. En effet, l'échec de l'extrapolation des résultats vers la clinique met en exergue la nature complexe de la maladie et la dimension décisive des modèles animaux adéquats et prédictifs dans l'étude des nouvelles thérapies. Et pour cause, un modèle animal idéal doit pouvoir reproduire les caractéristiques histo-pahologiques, génétiques et diagnostics de la pathologie humaine. Il doit avoir également une survie suffisante pour permettre la mise en place et l'évaluation de nouveaux traitements. Au cours de ce travail, nous avons développé un nouveau modèle de tumeur orthotopique chez la souris à partir de cellules de glioblastome humain cultivées en neurosphères. D'une façon similaire aux protocoles cliniques de neuro-imagerie, les techniques classiques d'IRM et les critères radiographiques ont été utilisés afin d'étudier la croissance tumorale de ce modèle ainsi que l'évaluation de sa réponse au traitement à base de temozolomide. Les observations par imagerie ont été complétées et/ou confirmées par examen histologique ainsi que par l'étude du transcriptome. Comme en clinique, ce nouveau modèle orthotopique présente une tumeur invasive et nécrotique, une résistance au temozolomide ainsi que des signatures moléculaires associées aux observations histologiques. De plus, ce modèle tumoral est caractérisé par une dynamique de signalisation promouvant l'invasion, la migration et la résistance à l'apoptose à l'origine de sa survie post-thérapeutique. Ainsi, ce modèle préclinique mime, au plus près, les caractéristiques de la pathologie humaine avec une médiane de survie des animaux de 82 jours, ce qui le rend pertinent pour l'évaluation préclinique des nouvelles stratégies thérapeutiques. / Glioblastoma is the most common and aggressive subtype of brain tumors. Despite a better understanding of the disease and also the emergence of new therapeutic targets and strategies, the prognosis of patients remains unchanged. The failure to extrapolate preclinical results to the clinics highlights the complex nature of the disease and the importance of appropriate and predictive animal models for the study of new therapies. A pertinent animal model should be able to reproduce the characteristic of the human pathology in terms of disease development pattern, histological and transcriptomic specification, drug failure as well as diagnostic features. In this work, we developed a novel orthotopic mouse model derived from human glioblastoma spheres. Like in clinics, conventional MRI techniques and radiographic criteria were used to characterize tumor growth and treatment response to temozolomide. MRI findings have been completed and/or confirmed by histological examination and transcriptomic studies. Like clinically encountered tumors, this new orthotopic tumor model presents an infiltrating growth pattern, resistance to temozolomide and a molecular signature associated with histological features. In addition, this tumor model is characterized by a dynamic signaling pathway, which promotes cell invasion and migration as well as resistance to apoptosis and consequently to treatment. Thus, this preclinical model mimics clinical features of human glioblastoma and has a median host survival time of 82 days, which would be relevant in the assessment of preclinical therapies.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / OBJETIVO: Estabelecer um modelo de tumor no fÃgado de ratos para estudo do comportamento tumoral e avaliar o uso da quimiembolizaÃÃo transarterial. MÃTODOS: Utilizou-se oitenta e oito ratos Wistar, fÃmeas, adultos, pesando entre 175- 284 g . Realizado incisÃo abdominal de 3 cm e implantou-se o carcinossarcoma de Walker 256 no lÃbulo esquerdo do fÃgado. Dividiu-se em trÃs grupos que receberam respectivamente 100x 103 , 200x 103 e 300x 103 cÃlulas tumorais, avaliado a pega do tumor e a sobrevida. Em outro grupo de experimento com 39 animais inoculados com tumor de Walker foi avaliado a sobrevida dos animais apÃs infusÃo do 5-Flourouracil (5-FU) por via intra-peritoneal e intra-arterial. RESULTADOS: O implante do carcinossarcoma de Walker no fÃgado de ratos apresentou desenvolvimento de 100 %, teve um crescimento rÃpido e desenvolvimento de metÃstases tardiamente, levando os animais ao Ãbito entre o sÃtimo e dÃcimo quinto dia. A quimiembolizaÃÃo transarterial à possÃvel de ser realizada experimentalmente. O uso do 5-FU aumentou a sobrevida em comparaÃÃo ao grupo controle. CONCLUSÃO: O modelo de implante do tumor de Walker no fÃgado de ratos à eficiente, de fÃcil reprodutibilidade, e sobrevida mÃdia de 9,96Â0.3 dias. A quimioterapia transarterial hepÃtica pode ser realizada experimentalmente para avaliar diversas drogas. / PURPOSE: An animal model to study the behaviour of liver tumor in rat and its response after use of transarterial chemoembolization. METHODS: We use 88 Wistar rats, all of them were females, adult, weight 175-284 g. Abdominal incison of three cm and implanted the Walker carcinossarcoma 256 at left lobule of the liver. The animals were divided into three grups, that received 100x103 , 200x103, and 300x103 cells. Followed up the animas to avaliate life standing and tumoral development. In another experiment was used 39 animals which already had Walker 256 tumor and we study the survival of the animals after treatment with 5-FU IP or 5-FU IA. RESULTS: The orevall tumor development rate were 100%. Tumor growth was fast, and devolopment metastases on old fase. The animals dead between 7 and 15 day. Its possible to do chemoembolization experimentaly, after the use of 5-FU the rate survival increased. CONCLUSION: The model with Walker 256 tumor developed here is easy to repoduce, efficient, with high tumor development rate observed, the life standing is about 9,96  0,3 days. The chemoembolization experiment allows to assess several drugs.

En recherche et développement pharmaceutiques un candidat thérapeutique doit passer plusieurs barrières précliniques afin de déterminer certains paramètres pharmacocinétiques et pharmacodynamiques avant toute administration à l’homme. Malgré les efforts investis en R&D ces dernières années, l’industrie du médicament a souffert d’un ralentissement dans le développement de nouvelles molécules innovantes. Car avant sa mise sur le marché, tout candidat doit justifier de la sécurité liée à son utilisation mais aussi de sa balance bénéfice/ risque. Les modèles standards utilisés en développement en oncologie ne sont pas assez prédictifs et bien souvent non-adaptés, avec une niche tumorale inexistante. C’est pourquoi il est aujourd’hui essentiel de travailler sur des systèmes plus sensibles et mimant plus fidèlement la pathologie humaine afin d’obtenir des médicaments plus efficaces et moins toxiques pour une meilleure prise en charge. L’utilisation du modèle spontané de cancers comme approche prédictive en oncologie comparée a été rapportée par plusieurs équipes à travers le monde. En effet, les fortes similitudes au niveau histologique, moléculaire et clinique rapportées entre les tumeurs humaines et canines, font de ce modèle un allié essentiel pour l’optimisation du développement pharmaceutique chez l’homme ayant un bénéfice réciproque à la fois à la médecine humaine et vétérinaire.Chez l’homme, les mélanomes constituent l’une des formes les plus agressives des cancers cutanés. Ils représentent 4 à 11% des néoplasies cutanées et seulement 2% des cancers de l’épiderme. Ce sont des tumeurs très immunogènes et de très mauvais pronostic au stade métastatique contribuant au développement d’une réponse immunitaire anti-tumorale bien souvent responsable d’échappements et de résistances au traitement. Et malgré l’amélioration de 50 à 80% de la survie globale à 5 ans ces vingt dernières années, son incidence ne cesse d’augmenter et environ 7000 cas sont rapportés chaque année en France avec plus de 75% de décès liés à ces tumeurs.Bien que rares, ces tumeurs représentent 7% des cancers diagnostiqués chez le chien et environ 160000 cas sont recensés chaque année au niveau mondial. Sa localisation est buccale dans plus de 50% des cas. C’est aussi l’un des cancers les plus agressifs chez le chien, avec une survie globale post-opératoire de 173 jours associé à des métastases ganglionnaires et pulmonaires le plus souvent.Le but de ce travail a été la validation préclinique et clinique du modèle tumoral spontané canin dans la compréhension des mécanismes de cancérogenèse, de dormance tumorale et de développement thérapeutique. Validation préclinique, dans un premier temps via le développement et la caractérisation de modèles cellulaires et murins de mélanomes canins. Puis, dans un second temps, la validation clinique par le biais d’essais thérapeutiques chez le patient chien.A partir de prélèvements issus de deux profils cliniques distincts, deux lignées cellulaires de mélanome canin ont été développées et caractérisées sur le plan pharmacologique, génomique et fonctionnel. Une liste d’altérations génétiques a été établie sur ces deux profils en accord avec la littérature et présentant des points communs avec la pathologie humaine. De plus, il est bien établi que l’hétérogénéité tumorale est responsable de résistances au traitements conduisant aux rechutes, c’est pourquoi nous nous sommes par la suite intéressés à l’étude des populations souches tumorales au sein de notre modèle et à l’identification de marqueur permettant le ciblage de ces cellules pouvant contribuer ainsi à l’avancement de l’enrichissement de l’arsenal thérapeutique oncologique.En conclusion, le patient chien est doté d’un système immunitaire intact et d’une niche tumorale complète, constituant ainsi un système in-vivo très intéressant pour l’homme, pouvant contribuer à avancer la recherche et améliorer grandement nos connaissances sur cette pathologie. / Pharmaceutical development is a long and fastidious process. In fact, each drug candidate has to meet with a certain safety criteria list, pharmacokinetic and pharmacodynamics profiles need to be determined prior to first use in humans and market approval.For years, the pharmaceutical industry has been suffering from a lack of innovative molecules and thus despite the efforts and cost increases in R&D programs. And most novel drug candidates entering clinical trials fail to reach approval, largely because preclinical models used in development do not provide adequate information about their efficacy or toxicity. That’s why; more predictive models of efficiency in oncology, shaping more precisely the human pathology are needed.The study of novel drug candidates in dogs with naturally occurring tumors allows drug assessment in neoplasms sharing many fundamental features with its human counterparts, and thus provides an opportunity to answer questions guiding the cancer drug development path in ways not possible in more conventional models. Moreover, the strong homologies in clinical presentation, morphology, and overall biology between dogs and their human counterparts make companion animals a good model to investigate tumor process from ætiology to tailored treatments.The aim of this project was to validate the canine spontaneous tumor model, by combining preclinical and clinical approaches, in the comprehension of the underlying mechanisms of cancer from carcinogenesis to drug resistance and tumor dormancy and also the discovery of new tools essential for the prediction, diagnosis clinical follow-up and treatment.Metastatic melanoma is one of the most aggressive forms of cutaneous tumors in humans. It constitutes 4 to 11% of skin malignancies and only 2% of the cancers of the epidermis. These highly immunogenic tumors hold a severe prognosis when metastasized and contribute to an immune anti-tumor reaction which could potentially lead to immune escape and resistance to most standard treatment protocols. And even if the 5-year survival has been improved to 50 – 80% over the past decades, its incidence is still in the rise with 7000 cases and 75% related deaths reported every year in France.In dogs, melanomas are one of the most frequently diagnosed malignancies of the oral cavity. These cancers account for 7% of all malignant tumors in dogs and 160000 reported every year worldwide. It also constitutes one of the most aggressive metastasizing tumors with a median post-surgery survival rate of 173 days.We developed and characterized immunucytochemically, pharmacologically and genomically two canine melanoma cell lines from naturally occurring dog tumors with distinct clinical profiles. A list of genetic alterations of these two profiles has also been established and is in accordance with the published literature, presenting same features as human tumors. And because tumor heterogeneity is responsible of resistance to treatment and relapse, we isolated and investigated cancer stem cell populations in our cell line models in order to identify the linked biomarkers which may constitute future potential targets for the expansion of the oncological therapeutic panel.In conclusion, due to its intact immune system, tumor niche and also because it shares the same environment as we do, the canine patient represent a promising opportunity in the advancement of cancer research, the acceleration of translation process and the setting up of more effective and less toxic molecules with dual benefits for the human and veterinary medicine toward better patient care.

No description available.

The objective of this study was to determine the photothermal and photochemical tissue response to carbon nanotube inclusion in laser therapy using experimental and computational methods. In this study, we specifically considered varying types and concentrations (0.01-1 mg/ml) of carbon nanotubes (CNTs), e.g., multi-walled carbon nanotubes (MWNTs), single-walled carbon nanotubes (SWNTs), and single-walled carbon nanohorns (SWNHs). In order to determine the photothermal effect of CNT inclusion, the thermal conductivity and optical properties of tissue representative phantoms with CNT inclusion were measured. Thermal conductivity of tissue phantoms containing CNTs was measured using the hot wire probe method. For identical CNT concentrations, phantoms containing MWNTs had the highest thermal conductivity. Optical properties (absorption and reduced scattering coefficients) of solutions and tissue phantoms containing carbon nanotubes were measured with spectrophotometry and determined by the inverse adding doubling (IAD) method. Inclusion of CNTs in phantoms increased light absorption with minimal effect on scattering and anisotropy. Light absorption of MWNTs was found to be higher than SWNTs and SWNHs. The photochemical response to laser irradiation (wavelength 1064 nm) of CNTs was measured with spin-trap electron paramagnetic resonance (EPR) spectroscopy. Only SWNHs appeared to produce significant levels of ROS production in response to laser excitation in the presence of NADH. We detected the predominant presence of trapped hydroxyl radical (•OH) with a trace of the trapped super oxide (O2•-) radical. These free radicals are highly reactive and could be utilized to cause targeted toxicity to cancer cells. The distribution of CNTs at the cellular level, in phantoms, and in kidney tumors was measured using transmission electron microscopy (TEM) imaging. Samples were imaged following various time periods (2-48h) of incubation and CNTs were observed inside the cell cytoplasm, nucleus, vacuole, and outside cells for the above mentioned time periods. CNTs in phantoms and tumor tissue were randomly and uniformly distributed in the entire volume. Computational model geometries were developed based on CNTs distribution in cells, tissue phantoms, and kidney tumor tissue. In the computational part of this research the temperature response to laser irradiation alone or with CNT inclusion was determined using Penne's bioheat equation which was solved by finite element methods. Experimentally measured thermal conductivity and absorption and reduced scattering coefficients were used as input parameters in Penne's bioheat equation. The accuracy of the model predicted temperature distribution was determined by comparing it to experimentally measured temperature in tissue phantoms and kidney tumors following CNT inclusion and laser therapy. The model determined temperature distribution was in close correspondence with the experimentally measured temperature. Our computational model can predict the effectiveness of laser cancer therapy by predicting the transient temperature distribution. / Ph. D.

<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>