Tumor initiation and progression is dependent on the acquisition and accumulation of multiple driver mutations that acti­vate and fuel oncogenic pathways and deactivate tumor suppressor networks. This complex continuum of non-stochastic genetic changes in accompaniment with error-prone mitoses largely explains why tumors are a mosaic of different cells. Contrary to the long-held notion that tumors are dominated by genetically-identical cells, tumors often contain many different subsets of cells that are remarkably diverse and distinct. The extent of this intratumor heterogeneity has bewildered cancer biologists’ and clinicians alike, as this partly illuminates why most cancer treatments fail. Unsurprisingly, there is no “wonder” drug yet available which can target all the different sub-populations including rare clones, and conquer the war on cancer. Breast tumors harbor ginormous extent of intratumoral heterogeneity, both within primary and metastatic lesions. This revelation essentially calls into question mega clinical endeavors such as the Human Genome Project that have sequenced a single biopsy from a large tumor mass thus precluding realization of the fact that a single tumor mass comprises of cells that present a variety of flavors in genotypic compositions. It is also becoming recognized that intratumor clonal heterogeneity underlies therapeutic resistance. Thus to comprehend the clinical behavior and therapeutic management of tumors, it is imperative to recognize and understand how intratumor heterogeneity arises. To this end, my research proposes to study two main features/cellular traits of tumors that can be quantitatively evaluated as “surrogates” to represent tumor heterogeneity at various stages of the disease: (a) centrosome amplification and clustering, and (b) mitotic frequency. This study aims at interrogating how a collaborative interplay of these “vehicles” support the tumor’s evolutionary agenda, and how we can glean prognostic and predictive information from an accurate determination of these cellular traits.

No description available.

Like with many cancers, a single ovarian tumor can display remarkable diversity in genetics, epigenetics, expression profiles, microenvironment and cell differentiation and plasticity. This so-called intratumoral heterogeneity (ITH) is thought to greatly increase mortality by enabling tumors to adapt quickly to therapy, metastasize, and recur, thus the study of ITH holds great clinical significance. Clonal evolution and cancer stem cell (CSC) theory are two models for the initiation and propagation of a tumor, which offer differing views on the way that ITH is developed and maintained. In the clonal evolution model, cancer arises from a single cell and, through genetic instability, proliferates into a diverse population of daughter cells, which develop additional mutations and undergo Darwinian selection under the influence of the tumor microenvironment. Each cell of the clonal evolution model may be capable of initiating a tumor independently. In CSC theory, cancer arises from the transformation of a stem cell that has the capacity to self-renew and differentiate into a diverse population of daughter cells. Each cell is NOT capable of tumorigenesis as most are terminally differentiated and do not harbor self-renewing capabilities. According to CSC theory, small, rare subpopulations of CSCs persist throughout chemotherapy and are responsible for repopulating the heterogeneous tumor post-treatment. The hypothesis that CSCs may play a role in ovarian cancer progression is the subject of this thesis. Many studies have detected the presence of stem cell markers and dysregulated stem cell signaling pathways in ovarian cancer, but doubts remain as to the existence of ovarian CSCs; critics have pointed out inherent flaws in experimental designs meant to identify and characterize CSCs. For example, the presence of cancer cells which express the stem cell marker CD133 has been correlated to both positive and negative impacts on prognosis. Further challenging the study of ovarian CSCs is the lack of consensus on the true cell of origin for ovarian cancer - whether it be from the fallopian tube epithelium or ovarian surface epithelium, or elsewhere in the peritoneal cavity - this will have important implications for the identification and characterization of tumorigenic ovarian CSCs. Advocates of clonal evolution theory have put forth incredible effort to reveal the extent of inter and intra-tumoral heterogeneity in ovarian cancer, and from these data there has arisen a general consensus that cancer cell populations do evolve in a step-wise fashion, accumulating additional mutations over time. The involvement of cancer stem cells in this progression and how exactly they fit in (as a cell of origin or arising from genetic mutations), as well as their significance for different cancer types, is a question worth answering. Despite the challenges facing the study of ovarian CSCs, the clinical impact of cells with stem-like properties has been repeatedly demonstrated, especially with regard to metastatic processes and chemoresistance. Moreover, new drugs which target stem cell pathways have proven effective in the treatment of ovarian cancer. The existence of a rare subset of cells that have enhanced tumor-initiating properties is apparent in ovarian cancer, and more work is needed to characterize the unique identifiers and behavior of these cells in vivo. Future experiments involving lineage tracing promise to deepen our understanding of the nature of ovarian CSCs and address whether normal stem cells might serve as the cell of origin.

BACKGROUND: Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50µm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH). METHODS: Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests. RESULTS: Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (p CONCLUSION: Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.

Le cancer colorectal est marqué par une importante mortalité dans les stades avancés du fait du fort taux de récidives tumorales après chimiothérapie. La prédiction de l'efficacité et de la toxicité des cytotoxiques s'impose comme un des enjeux majeurs de ces prochaines années. Parce que la majorité des anticancéreux sont pris en charge par les enzymes et transporteurs dont l'expression est contrôlée par le niveau d'expression et d'activation des xénosenseurs CAR et PXR, il est fort probable que ces xénosenseurs puissent représenter des facteurs prédictifs à prendre en compte dans la prise en charge des cancers. Notre équipe a récemment montré que les récepteurs des xénobiotiques PXR (NR1I2) (Raynal et al, 2010) et CAR (NR1I3) sont exprimés dans des lignées cellulaires et des tissus coliques humains. Leur surexpression dans les lignées coliques LS174T et T84 entraine leur résistance à l'irinotécan et à son métabolite actif le SN38 alors que leur inhibition antagonise cette résistance. Des dosages intra- et extra-cellulaires du SN38 et du SN38-G, ainsi que la quantification des ARNm des l'UGT1As et du transporteur MDR1, montrent que CAR et PXR augmentent le métabolisme détoxifiant et l'efflux du SN38. L'impact de la surexpression de ces xénosenseurs sur la viabilité des cellules LS174T à différentes classes de cytotoxiques (anti-métabolites, intercalants, inhibiteurs de topoisomérases, poisons du fuseau) a ensuite été évaluée. Nous avons observé que l'expression de CAR ou PXR conduit à une forte chimiorésistance au paclitaxel, au docétaxel et au 4-hydroxy-cyclophosphamide alors que PXR entraîne une sensibilisation marquée au cisplatine et au carboplatine en augmentant la quantité d'adduits de platine sur l'ADN. Les études du transcriptome de nos modèles cellulaires nous ont permis d'identifier les gènes cibles impliqués dans ces variations de cytotoxicité. Des études de confirmation par modulation pharmacologique ou ARNs interférents de ces gènes cibles sont en cours et nous permettront de préciser les mécanismes mis en jeu dans les variations de chimiosensibilité. Ces travaux devraient permettent de mieux appréhender le rôle des xénosenseurs CAR et PXR sur le métabolisme intra-tumoral des cytotoxiques et potentiellement sur la réponse à des chimiothérapies variées. / Colorectal cancer is characterized by high mortality in advanced stages due to the high rate of tumor recurrence after chemotherapy. The prediction of the efficacy and toxicity of cytotoxic drugs represents a major challenge in the coming years. Because the majority of cancer drugs are supported by the enzymes and transporters whose expression is controlled by the level of expression and activation of the xenosensors CAR (NR1I3) and PXR (NR1I2), it is likely that they may represent predictive factors in the management of cancer. Our team has recently shown that xenobiotic receptors PXR (Raynal et al, 2010) and CAR are expressed in cell lines and human colon tissues. Their overexpression in colon cancer cell lines LS174T and T84 leads to resistance to irinotecan and to its active metabolite SN38, while their inhibition reverse this resistance. Irinotecan metabolites detection assays of SN38 and SN38G, and the quantification of UGT1As and MDR1 mRNA, show that CAR and PXR increase the detoxifying metabolism and the efflux of SN38. The impact of overexpression of these xenosensors on LS174T cell viability to different classes of cytotoxic agents (anti-metabolites, DNA intercalators, topoisomerase inhibitors, antimitotic agents) was then evaluated. We observed that the expression of CAR or PXR results in a significant drug resistance to paclitaxel, docetaxel and 4-hydroxy-cyclophosphamide whereas PXR leads to a marked sensitization to cisplatin and carboplatin by increasing the amount of platinum adducts the DNA. Microarray studies of our cell models allowed us to identify the target genes potentially involved in these changes in cytotoxicity. Further studies by pharmacological modulation or interfering RNAs of these target genes are in progress and will allow us to clarify the mechanisms involved in the changes in chemosensitivity. This work should help us to understand the impact of CAR and PXR xenosensors on the intratumoral metabolism of cytotoxic drugs and potentially on the response to various chemotherapies.

Glioblastomas are highly vascular brain tumors that are characterized as heterogeneous, comprised of an anatomically and functionally irregular blood brain barrier (BBB) that contributes to variable drug distribution and possibly associated pharmacodynamics (PD) responses. Standard pharmacokinetic (PK) approaches that are based on whole tumor homogenates and accordingly averaged drug concentrations are limited in their ability to depict regional variations in drug concentrations, and could lead to faulty assessments of drug distribution and activity. Given the paucity of quantitative information on intratumoral PK/PD variability of anticancer drugs, the goal of this project was to characterize the regional PK and PD properties of gefitinib, an EGFR inhibitor, in brain tumors, in the context of biological characteristics, and use the information to develop mechanistic PK/PD models that may be valuable to understand why some anticancer drugs are inactive. Towards this end, in vitro cytotoxicity assays and pilot in vivo studies were first conducted and identified U87VIII mutant cell line as a gefitinib sensitive orthotopic brain tumor model with suitable growth characteristics to allow for immunohistochemical (IHC) analysis of tumor biological characteristics for further studies. Subsequently, in vitro PD studies identified phosphorylated-ERK1/2 (pERK) as a PD marker for this cell line. Thereafter, to set up a framework to obtain intratumoral PK/PD information in vivo, a novel tumor sectioning protocol was devised and sensitive and robust PK (LC-MS/MS) and PD (Meso Scale Discovery, MSD, electrochemiluminescence-based assay) methods were developed to enable the use of minimal amounts of tumor samples to assess the intratumoral PK and PD characteristics of gefitinib. Mice bearing orthotopic U87VIII mutant tumors were administered gefitinib at doses of 150 mg/kg and 50 mg/kg orally (p.o.), followed by collection of plasma and tumor samples at various time points, based on a serial sacrifice study design. Serial tumor sections were obtained in four distinct regions according to the aforementioned protocol for PK, PD and IHC measurements. An IHC index called microvessel pericyte index (MPI), a measure of BBB integrity, reflected the variability in gefitinib brain tumor concentrations, and was used to bin the data to generate three intratumoral PK/PD data sets. These data sets were then used to develop a set of hybrid physiologically based PK/PD models accounting for variable BBB permeability within the tumor. Each model consisted of a forcing function describing plasma concentration profile, a tissue compartment to represent the drug disposition within the tumor, and target-response compartments for the PD model. The intratumoral variation in the PKs of gefitinib was accurately described by the MPI classifications and ranged about 2-fold, and was responsible for the associated PD variability. In summary, using a novel tumor sectioning protocol and sensitive analytical methods in an orthotopic glioblastoma (GBM) model, the intratumoral variability of gefitinib PK/PD could be binned according to BBB integrity and enabled the development of a mechanistic hybrid physiologically based PK/PD models, which provides a means to assess the influence of tumor heterogeneity on drug response. / Pharmaceutical Sciences

Le microbiote humain est défini par l’ensemble des microbes habitant un site corporel en particulier. Les différents microbiotes de l’Homme, notamment le microbiote intestinal qui est le plus étudié, peuvent moduler de nombreux mécanismes biologiques dont le métabolisme et la réponse immunitaire. Un débalancement du microbiote au niveau des espèces qui le composent, ou dysbiose, a été associé à plusieurs maladies inflammatoires comme le diabète, l’obésité, mais aussi divers types de cancer. De plus, il a été démontré que les bactéries pouvaient avoir un impact sur la réponse des patients aux thérapies contre le cancer. Le cancer du sein est le cancer le plus mortel chez la femme. Or, l’étiologie de la maladie reste incertaine. Récemment, il a été montré que des bactéries pouvaient infiltrer les tissus plus profonds comme le tissu mammaire, formant un microbiote local. Considérant l’impact que la dysbiose peut avoir sur la réponse immunitaire antitumorale et la réponse aux traitements, nous avons émis comme hypothèse qu’une présence bactérienne intratumorale similaire, en composition et en quantité, à celle du tissu normal non-cancéreux affecte la progression du cancer du sein ainsi que le devenir clinique des patientes. La présence du microbiote intratumoral du sein a donc été validée par la détection de plusieurs composants bactériens sur des coupes tumorales à l’aide de marquages moléculaires. Puis, nous avons évalué le rôle potentiel de ce microbiote en quantifiant et identifiant les espèces bactériennes présentes dans les tumeurs et les tissus normaux adjacents des patientes de notre cohorte du cancer du sein. Nos résultats montrent une abondance moins élevée de l’ADN bactérien dans les tumeurs du sein comparativement aux tissus normaux adjacents appariés, suggérant qu’une altération du microbiote mammaire est associée au cancer. De plus, les patientes ayant un signal bactérien très faible dans leur tumeur avaient un nombre de récidives plus élevé. Cette influence de la quantité apparente de bactéries sur le devenir clinique a été observée principalement chez les patientes ayant une tumeur avancée, soit un grade ou un stade élevé, et de sous-types moléculaires Luminal HER2+, HER2+ (non-luminal) et Luminal B. Aucune relation n’a été observée entre la composition bactérienne du microbiote intratumoral mammaire et la récidive. Nos travaux suggèrent une implication pronostique et thérapeutique de la charge bactérienne du microbiote associé aux tumeurs mammaires. / The human microbiota is defined by all the microbes inhabiting a specific body site. The different human microbiota, and in particular the intestinal microbiota which is the most studied, can modulate many biological mechanisms, including metabolism and the immune response. An imbalance in the bacterial species that compose the microbiota, or dysbiosis, has been associated with several inflammatory diseases such as diabetes, obesity, but also various types of cancer. Additionally, bacteria have been shown to impact the response of patients to cancer therapy. Breast cancer is the deadliest cancer in women. However, the etiology of the disease remains uncertain. Recently, it has been shown that bacteria can infiltrate deeper tissues like breast tissue, forming a local microbiota. Considering the impact that dysbiosis can have on the anti-tumor immune response and the response to treatments, we hypothesized that an intratumoral bacterial presence similar in composition and quantity to that of normal non-cancerous tissue affects the progression of breast cancer, as well as the clinical outcomes of patients. The presence of the intratumoral breast microbiota was therefore validated by the detection of several bacterial components on whole tumor sections using molecular staining. Then, we evaluated the potential role of this microbiota by quantifying and identifying the bacterial species present in tumors and adjacent normal tissues of patients in our breast cancer cohort. Our results show a lower abundance of bacterial DNA in breast tumors compared to adjacent paired normal tissues, suggesting that an alteration of the mammary microbiota is associated with breast cancer. In addition, patients with a very low bacterial signal in their tumor had a higher number of recurrences. This influence of the apparent quantity of bacteria on the clinical outcomes has been observed mainly in patients with an advanced tumor, either a high grade or a high stage, and of the Luminal HER2+, HER2+ (non-luminal) and Luminal B molecular subtype. No relationship has been observed between the bacterial composition of the breast intratumoral microbiota and the recurrence. Our work suggests a prognostic and therapeutic implication of the bacterial load of the microbiota associated with breast tumors.

Background: The first-line treatment for patients with advanced prostate cancer (PC) is androgen deprivation therapy. This therapy is initially effective, but after some time tumors relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors show androgen receptor (AR) activity despite castrate levels of circulating testosterone. AR activity could be caused by several mechanisms including; intratumoral androgen synthesis, AR amplification, AR mutations and expression of AR splice variants. The mechanisms controlling CRPC growth in the clinically most relevant metastatic site, the bone, are not fully identified. The purpose of this thesis was therefore to explore AR expression and possible mechanisms behind CRPC growth in PC bone metastases in order to find mechanisms that could be targeted for treatment and/or predict response to certain therapies. Materials and Methods: We have examined hormone-naïve and CRPC bone metastases samples obtained from patients at metastasis surgery, non-malignant and malignant prostate samples obtained from patients at radical prostatectomy, and PC cell lines cultured in vitro. Analysis has been performed using RT-PCR, whole-genome expression arrays, immunohistochemistry, western blotting, FISH, copy number assays and gene ontology analysis. Functional studies have been made by protein overexpression and knock-down in PC cells in vitro and effects studied by evaluation of cell viability, migration, and invasion. Results: We found that high nuclear AR immunostaining (presumed to reflect high AR activity) in bone metastases from CRPC patients was associated with a particularly poor prognosis, while no difference in AR staining was observed between hormone-naïve and CRPC metastases. Further, expression of AR splice variants (AR-V7, AR-V567es) was associated with a high nuclear AR immunostaining score and shown to be increased in CRPC compared to hormone-naïve bone metastases. High levels (levels in the upper quartile) of AR splice variants in CRPC bone metastases was related to disturbed cell cycle regulation and short patients survival. No differences in steroidogenic enzyme levels were detected between CRPC and hormone-naïve bone metastases. Higher levels of enzymes involved in late steps of androgen synthesis (adrenal gland steroid conversion) were observed in bone metastases than in non-malignant and/or malignant prostate tissue, while the enzyme levels in earlier steps (de novo steroidogenesis) were lower in bone metastases. A subgroup of metastases expressed very high levels of AKR1C3, indicating that this group may have an induced capacity of converting adrenal-gland derived steroids into more potent androgens. This was not associated to CRPC but merely with the advanced stage of metastasis. High protein levels of AR splice variants were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR variant levels. Furthermore, about half of the CRPC bone metastases showed androgen receptor gene amplification which was associated with co-amplification of YIPF6, and a gene expression pattern that pointed at decreased osteoclast activity, and consequently decreased bone resorption. Conclusions: The majority of CRPC bone metastases show high nuclear AR immunostaining that seems to be associated with a particularly unfavorable outcome after metastasis surgery. Subgroups of CRPC bone metastases could be identified according to presence of AR amplification and expression levels of AKR1C3 or AR splice variants, which might have clinical relevance for treatment of PC patients. / <p>Författaren är även publicerad med efternamnet Hörnberg.</p>

Résumé : Nous avons d’abord souhaité, dans ce travail de thèse, préciser les mécanismes moléculaires conduisant à l'expression ectopique du récepteur du GIP (glucose-dependent insulinotropic polypeptide receptor, GIPR) dans des adénomes somatotropes provenant de patients présentant une acromégalie avec une réponse paradoxale (stimulation) de l’hormone de croissance au glucose par voie orale. Nous avons montré que l’expression ectopique de GIPR se produit par une activation transcriptionnelle hypomorphe du gène GIPR associée à des anomalies de méthylation dans le corps du gène. L’activation de la voie AMP cyclique par le GIP postprandial dans les adénomes exprimant le GIPR peut représenter un mécanisme alternatif de la tumorigenèse somatotrope en l’absence de mutations de l’oncogène GNAS.Nous rapportons d’autre part une analyse cytogénétique approfondie des adénomes somatotropes, qui nous a permis de définir deux groupes d'adénomes, un groupe à faible altération du nombre de copies et un groupe à forte altération du nombre de copies. Deux tumeurs présentaient des réarrangements chromosomiques complexes avec une signature typique de chromothripsis, et une architecture sous-clonale incluant jusqu’à six populations cellulaires différentes, témoignant d’une hétérogénéité intratumorale importante.Dans une collection d'adénomes hypophysaires invasifs comportant la portion intrasellaire et la portion envahissante le sinus caverneux, nous avons montré par RNA-seq des profils d'expression génique divergents, apportant des arguments supplémentaires en faveur de l'hétérogénéité intratumorale dans ces tumeurs bénignes. Les échantillons tumoraux provenant de portions invasives ont montré une surexpression de la voie de transition épithélio-mésenchymateuse et des marqueurs de cellules souches cancéreuses soulignant leur rôle potentiel dans l’acquisition du phénotype invasif des cellules adénomateuses hypophysaires. / AbstractIn this work, we explored the molecular mechanisms of ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in somatotroph adenomas from patients with acromegaly displaying a paradoxical GH increase to oral glucose. We showed that ectopic GIPR expression occurs through hypomorphic transcriptional activation of GIPR gene likely driven by DNA methylation changes. Activation of the cAMP pathway by postprandial GIP may represent an alternative tumorigenic mechanism in GIPR expressing somatotroph adenomas without driver mutations in GNAS oncogene. Cytogenetic profiling defined two groups of adenomas, a low-copy-number alteration (CNA) group and a high-CNA group.Two tumor samples displayed complex chromosomal rearrangements compatible with chromothripsis and showed subclonal architecture with up to six distinct cell population in each tumor, demonstrating an important intratumor heterogeneity.In a collection of invasive pituitary adenomas including the non-invasive intrasellar portions and the portions invading the cavernous sinuses, we showed by RNA-seq different gene expression profiles, providing supplemental evidence for the intratumoral heterogeneity in these benign tumors. Tumor samples from invasive portions showed up-regulation of the epithelial-mesenchymal transition pathway and increased expression of cancer stem-cell markers highlighting their potential role in pituitary tumor cell invasive behavior.

<p>Pancreatic ductal adenocarcinoma is an especially deadly disease having the lowest 5-year survival rate of any major cancer at just 11%. As in many cancers, systemically-delivered chemotherapy forms the backbone of clinical treatment. However, limitations of systemic delivery exacerbated by the unique desmoplastic and avascular microenvironment surrounding the pancreatic tumor cells result in the failed efficacy of current treatments. The high stromal content in the microenvironment, which is especially overabundant in hyaluronic acid, is thought to physically impede drug perfusion into the tissue. Thus, there is clearly a <strong>critical need</strong> to develop novel treatments for pancreatic ductal adenocarcinoma that can overcome these drug delivery barriers. Long-acting injectable implants offer an attractive drug delivery method that can provide <em><strong>sustained</strong></em> drug release directly at the <em><strong>local</strong></em> targeted site, rather than transient, systemic release. Here we use in situ forming implants (ISFIs), which are a low-viscosity solution outside of the body but transition into a solid drug-eluting depot after injection into an aqueous environment. Our <strong>objective</strong> is to develop and characterize an ISFI that can provide sustained release of bioactive hyaluronidase for use as an intratumoral injection to degrade hyaluronic acid in pancreatic tumors. This work was accomplished in four aims. First, a method was developed using diffusion-weighted MRI for noninvasive characterization of the implants. Second, because hyaluronidase is a protein drug, we studied factors affecting protein release from ISFIs, focusing on external factors of the injection site. Third, we showed that basic salt additives can be used to neutralize the acidic environment created by the implants which may improve protein stability. Finally, we formulated an implant to provide sustained release of hyaluronidase and demonstrated retention of its bioactivity both <em>in vitro</em> and <em>ex vivo</em>.</p>