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京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23843号 / 薬博第850号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 木村 郁夫, 教授 中山 和久, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Glioblastoma multiforme (GBM) is the most deadly and common form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment is known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN comprised of either collagen Type 1 or HyStem-C®, human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBMECs), and LN229 (GBM) cells. A synergistic response between HUVECs and LN229 cells was observed in co-culture, including 10 – 16-fold increased cell proliferation, a decrease in the height of hydrogels of up to 68%, as well as elevated secretion of TGF-β and CXCL12 up to 2.6-fold from Day 8 to 14. These trends correlated with cell colocalization, indicating a chemotactic role for CXCL12 in enabling the migration of LN229 cells towards HUVECs in co-cultures. Von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP) in up to 40% of LN229 cells after 14 days in co-culture in collagen (2.2 mg/mL) and HyStem-C® gels. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® on EC networks, LN229 cell morphology and alignment, cytotoxicity, colocalization, and trans-differentiation. TMZ was observed to primarily affect LN229 cells, with treatment at high concentrations resulting in up to 2.3-fold reduced alignment as well as an increase in cell circularity. Cytotoxicity of up to 94% was also observed up to in LN229 monocultures, and was significantly higher in collagen (1.1 mg/mL) gels. Avastin® treatment resulted in changes to ECs. Network features were significantly reduced and EC cellular proliferation decreased up to 69% with Avastin® treatment. Significant increases in percentages of colocalized and GFAP+/vWF+ cells were also observed when treated with 8 µg/mL Avastin®. This suggests that chemotactic signaling may have been altered. TGF-β secretion was reduced in co-cultures when 150 µM TMZ or 8 µg/mL Avastin® were administered. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadly form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment of particular interest is located near the vasculature, known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN consisting of either collagen type 1 or HyStem-C®, a material made of primarily hyaluronic acid. Human umbilical vein endothelial cells (HUVECs), an immortalized cell line, or primary human brain microvascular endothelial cells (HBMECs) as well as LN229 (GBM) cells were used. A synergistic response was observed between HUVECs and LN229 cells in co-culture, including changes to the extracellular matrix, and signaling factor secretion. Further supporting this data, colocalization between LN229 cells and HUVECs was observed. Colocalization is a phenomenon where two cell types come into physical contact after one moves toward another. This indicated preferential migration, specifically in response to CXCL12. Endothelial cell marker von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP), commonly used to identify GBM cells. This percentage was increased in co-cultures with HBMECs, pointing to differences in the response of primary cells to immortalized cell lines. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® in the PVN model. TMZ was observed to primarily affect LN229 cells, by reducing their alignment as well as causing cell death. Avastin® treatment resulted in changes to ECs. Networks and cell growth were significantly reduced after Avastin® treatment. When either TMZ or Avastin® was administered, the secretion of TGF-β, was reduced.

Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing perfusion-based imaging protocols. Using this method, we examined transport associated with invasion in a murine GL261 cell line. We found that elevated interstitial fluid velocity magnitudes, decreased diffusion coefficients and regions with accumulating flow were significantly associated with invasion. We tested the validity of our invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions indicating location-specific structures may drive both interstitial flow and corresponding invasion heterogeneities. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Over-expression of VEGF-C in the tumor microenvironment neither enhanced drainage nor altered invasion in comparison to our control, indicating other tumor-secreted growth factors, such as VEGF-A, may play a larger role in mediating flow and invasion. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease. / Doctor of Philosophy / Glioblastoma is the most common and deadly primary brain tumor with an average survival of 15 months following diagnosis. Characterized as highly infiltrative with diffuse tumor margins, complete resection and annihilation of tumor cells is impossible following current standard of care therapies. Thus, tumor recurrence is inevitable. Interstitial fluid surrounds all of the cells in the body and has been linked to elevated invasion in glioma, which highlights the importance of this understudied fluid compartment in the brain. The primary objective of this dissertation was to identify specific interstitial fluid transport behaviors associated with elevated invasion surrounding glioma tumors. We first describe our methods to measure interstitial fluid flow in the brain using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI), a clinically used, non-invasive imaging modality. We highlight the versatility of the technique and the possibilities that could arise from widespread adoption into existing imaging projects. Using this method, we examined transport associated with cancer cell invasion in a mouse tumor cell line. We found that interstitial fluid speeds were elevated while diffusion was decreased in regions of invasion. Further, regions that had interstitial fluid flow congregation were significantly associated with invasion. We tested the validity of these invasive trends by extending our analysis to multiple, clinically-relevant tumor locations in the brain. Interestingly, we found invasion did not follow the same trends across brain regions, indicating location-specific structures may drive both interstitial flow and invasion differences. Lastly, we aimed to manipulate flow by engaging with the meningeal lymphatics, an established pathway for interstitial fluid drainage. Following administration of a meningeal lymphatic-relevant protein, we saw no changes in flow or invasion in comparison to our untreated control, indicating other tumor-secreted proteins may play a larger role in these responses. Taken together, by identifying specific transport factors associated with invasion, we may be better equipped to target and treat infiltrative tumor margins, ultimately extending survival in patients diagnosed with this devastating disease.

The post-prognosis lifespan for those suffering with Glioblastoma (GBM) is approximately 13 months with current standard of care. Intratumoral heterogeneity is a common characteristic that hinders GBM treatment in the form of therapy resistant cell subsets and influence on cellular phenotypes. One cell subset in particular, glioma stem cells (GSCs), is frequently left behind in the brain parenchyma once the bulk of the tumor has been resected. Previous research has found that patient-derived GSCs displayed varying invasion responses with and without the presence of interstitial flow. Interestingly, GSCs from a single patient are heterogeneous, displaying differences among sub-colonies derived from the same parental line. To study the motility of cells under flow, PDMS microfluidics are commonly used. Unfortunately, this setup often involves active flow generation using pumps, limiting the number of cell lines that can be imaged at a time. To increase the throughput of GSC sub-colonies imaged simultaneously, we developed a bio-compatible, printable device fabricated to allow for passive, gravity-driven flow through a hydrogel that recapitulates the brain microenvironment, eliminating the need for pumps. Stereo lithography 3D printing was chosen as the manufacturing method for the device, and this facilitated design feature modification when prototyping, increased the potential complexity of future iterations, and avoided some of the hurdles associated with fabricating PDMS microfluidics. This printable imaging device allows for higher throughput live-imaging of cell lines to aid in the understanding of the relationships between intratumoral heterogeneity, invasion dynamics, and interstitial flow. / Master of Science / For those suffering with Glioblastoma, a high-grade brain cancer, the life span post treatment is approximately 13 months. The cells in this and many forms of cancer have physical and biological differences that make successfully eliminating the disease difficult. One of the cell types contributing to this are Glioma Stem Cells (GSCs) that are often left in brain tissue once most of the tumor has been surgically removed. Previous research has found that GSCs from different sources had different responses with and without the simulated or actual presence of flow in brain tissue. This was further complicated when different responses were observed in cells obtained when breaking apart one of the cell lines and propagating these into their own sub-colonies. The current standard for studying the movement of cells under flow is by using compact chips made of a clear silicone rubber. The setup with microfluidics typically requires connection to external tubing and pumps to create flow and this limits the amount of cell types that can be imaged at a time. In order to monitor more cells at a time we created a 3D printable device that uses gravity for flow to go through a gel that mimics brain tissue and these cells of interest. Resin 3D printing was used to make these small devices so that they could be easily re-designed for other experimental purposes in the future. Hopefully this device could be used to more rapidly gain an understanding of cell movement in GBM and other disease models.

Fluid flow is a complex and dynamic process in the brain, taking place at the macro- and microscopic level. Interstitial fluid in particular flows throughout the interstitial spaces within the tissue, interacting with cells and the extracellular matrix. We are coming to find that this interstitial fluid flow plays an important role in both homeostatic and pathologic conditions. It helps to transport chemokines and other molecules such as extracellular vesicles within the environment, clear waste from the brain, and provide biophysical cues to cells. When this flow is disrupted however, such as in glioblastoma or Alzheimer's disease, profound events can occur, for example the build-up of plaques or an increase in tumor cell invasion. While there has recently been an up-tick in interstitial fluid flow research, there is surprisingly little known about its exact nature within the interstitial space and its effects on brain pathology such as glioblastoma. In particular, ways to manipulate and measure brain IFF magnitude at the cellular level are lacking. In this dissertation, a set of tools is created and used to explore the role that interstitial fluid flow magnitude plays in the brain through the lens of glioma invasion. We developed and implemented a flow device that is used in conjunction with an established in vitro tissue culture insert assay to manipulate fluid flow rates through a 3D matrix of tumor cells. We showed that this flow device is biocompatible and accurately recreates flow rates that have been measured previously through the use of MRI. We quantified tumor cell invasion from several glioma cell lines using this device to show a nonlinear trend of invasion in response to increasing fluid flow magnitudes. In addition, we developed a computational model to explore one potential mechanism that fluid flow magnitude might be modulating: autologous chemotaxis. Through this model we showed that increased flow magnitudes such as those seen in gliomas cause an increase in the distribution of the chemokine gradient around a cell of interest, that the morphology of the cell is important to this gradient formation, that temporal effects should not be overlooked, and that within the tumor environment, a minimum distance is required for the invading cell to develop this gradient. Finally, we developed a novel in vivo surgical technique that allows for the manipulation and measurement of interstitial fluid flow within the brain through simultaneous multiphoton imaging. We showed that this technique can be used to modulate interstitial fluid flow, as a mechanism by which to label cells of interest, and as a means to implant and monitor glioma progression. Through these means we further characterize interstitial fluid flow in the brain, allowing for its manipulation and measurement, and examine the ability of increased interstitial fluid flow magnitudes to impact glioma invasion. / Doctor of Philosophy / Fluid flows throughout brain tissue and plays an important role in creating normal conditions for proper brain function. This fluid can also play a role in brain cancer, such as glioblastoma, by causing cancer cells to travel further into the brain which is not desirable. This dissertation seeks to understand fluid flow better by studying how its speed contributes to cancer cell movement which is accomplished through the development of several tools. One tool is a new surgical technique that allows for the measurement and manipulation of fluid flow speed within the mouse brain and visualization of cells of interest, one tool is a flow device that changes fluid flow speed through cells in a gel, and the last is a computational model that predicts how a cell might move under different flow and environmental conditions. The tools were created and utilized, showing several interesting results. Using the flow device, different cancer cell lines were seen to react differently to increased fluid flow speed with two main trends: 1) increased cancer cell movement with increased fluid flow speed and 2) a peak effect where the cell movement started to increase with increasing fluid flow speed and then decreased after a certain fluid flow speed was surpassed. The surgical technique was successful at introducing fluid flow and allowed for reproducible measurements of fluid flow speed. It also was used to introduce stains that show specific cells of interest. The computational model showed that there are specific time and spatial contributions that effect cancer cell movement and that with increased fluid flow speed, cells might be able to more easily utilize a specific mechanism to move. Altogether, this work presents novel insight into fluid flow speed that can be used to further inform the field. It is our hope that the findings from this dissertation can go towards a more comprehensive treatment of a specific type of brain cancer, glioblastoma.

Irreversible electroporation (IRE) is a non-thermal ablation technique that allows for eradication of unresectable tumors in a minimally invasive procedure. While IRE will preferentially kill larger cells over smaller ones, it does not discriminate between cells with larger and small nuclei. Given that one of the hallmarks of cancer cell morphology is larger, more abundant nuclei, our team set out to explore the possibility of preferentially targeting this physical and geometrical characteristic. / Master of Science

Glioblastoma is the most common and lethal malignant brain tumor with a survival rate of 14.6 months and a tumor recurrence rate of ninety percent. Two key causes for glioblastomas grim outcome derive from the lack of applicable prognostic markers and effective therapeutic targets. By employing a loss of function RNAi screen in glioblastoma cells we found a list of 20 kinases that can be considered glioblastoma survival kinases. These survival kinases which we term as survival kinase genes, (SKGs) were investigated to find prognostic markers as well as therapeutic targets for glioblastoma. Analyzing these survival kinases in The Cancer Genome Atlas patient database, we found that CDCP1, CDKL5, CSNK1𝜀, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 genes could be used as prognostic markers for glioblastoma with or without temozolomide chemotherapeutic treatment as a covariate. For the first time, we found that patients with increased levels of NEK9 and PIK3CB mRNA expression had a higher probability of recurrent tumors. We also discovered that expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary glioblastoma tumors was associated with tumor recurrence prognosis. To note, of these recurrent prognostic candidates, PIK3CB expression in recurrent tumor tissue had much higher expression compared to primary tissue. Further investigation in the PI3K pathway showed a strong correlation with recurrence rate, days to recurrence and survival emphasizing the role of PIK3CB in tumor recurrence in glioblastoma. In efforts to find effective therapeutic targets for glioblastoma we used SKGs as potential candidates. We chose the serine/threonine kinase, Casein Kinase 1 Epsilon (CSNK1𝜀) as a target for glioblastoma because multiple shRNAs targeted this gene in our loss of function screen and multiple commercially available inhibitors of this gene are available. Casein kinase 1 epsilon protein and mRNA expression were investigated using computational tools. It was revealed that CSNK1𝜀 expression has higher expression in glioblastoma than normal tissue. To further examine this gene we knocked down (KD) or inhibited CSNK1𝜀 in glioblastoma cells lines and noticed a significant increase in cell death without any significant effect on normal cell lines. KD and inhibition of CSNK1𝜀 in cancer stem cells, a culprit of tumor recurrence, also revealed limited self-renewal and proliferation in cancer stem cells and a significant decrease in cell survival without affecting normal stem cells. Further analysis of downstream effects of CSNK1𝜀 knockdown and inhibition indicate a significant increase in the protein expression of β-catenin (CTNNB1). We found that CSNK1𝜀 KD activated β-catenin, which increased GBM cell death, but can be rescued using CTNNB1 shRNA. Our survival kinase screen, computational analyses, patient database analyses and experimental methods contributed to the discovery of novel prognostic markers and therapeutic targets for glioblastoma. / Ph. D.