Intra-Cavity Stem Cells Target Post-Surgical Brain Tumor Progression In Novel Resection And Recurrence Mouse Models

Surgical resection is a universal component of therapy against aggressive brain tumors. These cancers include glioblastoma (GBM) and medulloblastoma (MB), which are prevalent in older adults and children, respectively. Due to limited pre-clinical models, little is known about the post-operative microenvironment and how it impacts efficacy of local therapies. Cytotoxic stem cells (SCs) have emerged as a promising approach to target these aggressive post-surgical brain cancers, due in large part to their inherent tumor-homing properties. Our goal is to develop scaffold technology to improve stem cell therapy for post-surgical brain tumors. Though, in order to develop this new stem cell technology, there needs to be animal models in which to test this therapy. Thus, in chapter two, we created the first GBM resection and recurrence model in an immune-competent mice. This model allowed us to observe surgery-mediated events that may potentiate tumor aggressiveness and efficacy of local stem therapies. In chapter three, we tested different scaffold materials that were loaded with cytotoxic stem cells in our GBM resection models. We found that therapeutic stem cells loaded on these materials inhibited post-surgical tumor recurrence. Lastly, in chapter four, we utilize stem cell-scaffold technology in an entirely new model of surgical resection, new disease, and new stem cells for pediatric patients. Using therapeutic stem cells derived from pediatric human patients, this therapy delayed post-surgical tumor recurrence and extended median survival in tumor-bearing mice. These studies demonstrate a novel model of GBM and MB resection and recurrence that provided a platform to develop and advance stem cell technologies in a post-surgical cavity. In addition, the results also suggest that intra-cavity SC therapy is a viable new option to effectively control post-operative tumor growth in GBM and MB patients.Doctor of Philosoph

Reactive astrocytes potentiate tumor aggressiveness in a murine glioma resection and recurrence model

Surgical resection is a universal component of glioma therapy. Little is known about the postoperative microenvironment due to limited preclinical models. Thus, we sought to develop a glioma resection and recurrence model in syngeneic immune-competent mice to understand how surgical resection influences tumor biology and the local microenvironment

Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection

<div><p>Background</p><p>Cytotoxic neural stem cells (NSCs) have emerged as a promising treatment for Medulloblastoma (MB), the most common malignant primary pediatric brain tumor. The lack of accurate pre-clinical models incorporating surgical resection and tumor recurrence limits advancement in post-surgical MB treatments. Using cell lines from two of the 5 distinct MB molecular sub-groups, in this study, we developed an image-guided mouse model of MB surgical resection and investigate intra-cavity NSC therapy for post-operative MB.</p><p>Methods</p><p>Using D283 and Daoy human MB cells engineered to express multi-modality optical reporters, we created the first image-guided resection model of orthotopic MB. Brain-derived NSCs and novel induced NSCs (iNSCs) generated from pediatric skin were engineered to express the pro-drug/enzyme therapy thymidine kinase/ganciclovir, seeded into the post-operative cavity, and used to investigate intra-cavity therapy for post-surgical MB.</p><p>Results</p><p>We found that surgery reduced MB volumes by 92%, and the rate of post-operative MB regrowth increased 3-fold compared to pre-resection growth. Real-time imaging showed NSCs rapidly homed to MB, migrating 1.6-fold faster and 2-fold farther in the presence of tumors, and co-localized with MB present in the contra-lateral hemisphere. Seeding of cytotoxic NSCs into the post-operative surgical cavity decreased MB volumes 15-fold and extended median survival 133%. As an initial step towards novel autologous therapy in human MB patients, we found skin-derived iNSCs homed to MB cells, while intra-cavity iNSC therapy suppressed post-surgical tumor growth and prolonged survival of MB-bearing mice by 123%.</p><p>Conclusions</p><p>We report a novel image-guided model of MB resection/recurrence and provide new evidence of cytotoxic NSCs/iNSCs delivered into the surgical cavity effectively target residual MB foci.</p></div

Daoy human MB cells engineered to express GFP and firefly luciferase form orthotopic xenografts in vivo.

<p>Cultured Daoy human MB cells transduced with lentiviral vectors encoding GFP-FLuc (Daoy-GFPFL) express GFP and luciferase in vitro as determined by white light (A, B) and fluorescence imaging (C, D). Daoy-GFPFL cell number showed linear correlation with Fluc activity (R² = 0.978, P = 0.001, E). Daoy-GFPFL xenografts showed exponential growth with a doubling time of 5.6 days <i>in vivo</i> measured by bioluminescence imaging. Representative bioluminescence images are shown for days 14, 24 and 42 after injection of Daoy-GFPFL cells (F). Representative hematoxylin and eosin staining (G-J) of brain sections show large intra-cerebellar tumors 63 days after injection of Daoy-GFPFL cells. Tumors showed histopathological features of MB, including frequent cell wrapping (white arrowheads) and heightened level of mitosis (black arrowheads). Original magnifications: 15X (G), 40X (A, C), 100X (B, D), 200X (H), 400X (I), 600X (J). Scale bars, 100 μm (A-D) and 200 μm (G-J). n = 5.</p

Therapeutic NSCs delivered within the resection cavity regress and delay re-growth of post-surgical MB.

<p>White light and fluorescent images of NSC expressing TK (NSC^tk) in culture (A, B) or mixed with fibrin matrix (NSC^tk/fibrin) (C, D), illustrate high transduction efficiency and efficient expression of the construct. Depiction of the <i>in vivo</i> therapy schema, in which tumors were grafted within the cerebellum, resected and treated with therapeutic NSC^tk/fibrin (E). Representative white light (F) and fluorescent (G) images confirm NSC^tk/fibrin implanted within the resection cavity. NSC^tk/fibrin persist longer in the brain without systemic administration of GCV (NSC^tk) compared to with systemic administration of GCV (NSC^tk/GCV) (H). Representative BLI images of NSC^tk and NSC^tk/GCVmice at days 3 and 7 post-surgery are shown above. Daoy-GFPFL cells were seeded with varying concentrations of NSC^tk (I). Tumor cell viability was measured by luminescence 24h after GCV administration. Summary graph and representative BLI show a significant reduction of tumor burden in NSC^tk/fibrin+GCV-treated mice compared to control-treated (J; no NSC and NSC^tk). NSC^tk/fibrin with GCV treatment extended median survival two-fold compared to controls (9 vs. 21 days, log rank *P<0.0001, K). Original magnification: 40X (C, D), 100X (A, B). Scale bars = 100 μm. n = 4 per group in the panel H, n = 4 in no NSC and NSC^TK, n = 7 in the NSC^TK/GCV group in J-K.</p

Comparison of NSC and hp-iNSC migration and killing.

<p>Representative images showing the migration of NSCs to D283 (A) and hp-iNSC to DAOY cells (B). (C-D) Summary graphs demonstrating the velocity (C) and Euclidean Distance of both NSC and hp-iNSC migrating to DAOY and D283. (E-F) Summary graphs demonstrating the efficacy of NSCTK or hp-iNSCTK therapy for DAOY (E) or D283 (F). Original magnification: 100X (A,B). Scale bars = 100 μm. n = 3–4 in each group.</p

NSCs migrate towards human MB in vitro and in vivo.

<p>Migration of NSC-mcF cells (red) the presence (+Daoy) or absence (control) of MB cells (Daoy-GFPFL cells, green) was monitored over 24h (A). Time-lapse images were captured every 20 mins and used to construct single cell tracings (B). Quantitative analysis of single cell tracings demonstrated the presence of Daoy-GFPFL increases the migratory velocity of NSCs (1.61-fold; ****P<0.0001, C), and euclidean distance traveled (1.8-fold; P<0.0001, D). Illustration of NSC and Daoy-GFPFL cells implanted in opposite cerebellar hemispheres (E). Fluorescent images (F-H) 21 days after stem cell implantation shows NSCs co-localize with Daoy-GFPFL tumor cells. Original magnifications: 100X (A, B,), 200X (F, G). n = 4 in panel E.</p

Fluorescence-guided microsurgical resection reduces volumes of orthotopic Daoy-GFPFL xenografts that redevelop.

<p>A scalp incision was made 49 days after injection of Daoy-GFPFL cells and craniotomy (A). Fluorescence was used to visualize the underlying GFP⁺ tumors pre resection (B). Fluorescence-guided microsurgery significantly reduced tumor burden as determined by intra-operative imaging (C, D). Post-operative bioluminescence imaging showed a >92% reduction in mean tumor burden (***P = 0.009, E). Representative white light and bioluminescence images pre- and post-resection are shown (E,F). Representative H&E images (G-J) of brain sections taken immediately post-resection did not show apparent residual tumor within the resection cavity. Recurrent Daoy-GFPFL xenografts (K) grew faster than pre-resection counterparts (data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198596#pone.0198596.g001" target="_blank">Fig 1F</a>), with doubling times of 1.5 vs. 5.6 days, respectively (P = 0.0003). Representative BLI images are shown for days 1, 3, and 5 post-surgery. H&E images (L-N) of brain sections shows recurrent tumor near the resection cavity and cancer cells disseminating through the cerebral spinal fluid (CSF). Original magnifications: 15X (G), 200X (L, H), 400X (I), 600X (J, M, N). Scale bars, 200 μm. RC, Resection Cavity. n = 5 in each group.</p

Cytotoxic human pediatric stem cells derived from patient skin suppress and delay regrowth of post-surgical medulloblastoma.

<p>Illustration of the reprogramming scheme which converts human pediatric fibroblasts into cytotoxic human pediatric induced neural stem cells (hp-iNSC^tk, A). hp-iNSC express the thymidine kinase/RFP lentiviral vector as determined by white light (B) and fluorescent (C) images. Schematic depiction of hp-iNSC^tk migration assay in the presence (+D283) or absence (control) of MB cells (D). The cells were seeded 500 μm apart from each other and migration was tracked over the course of 24h by fluorescent and white light imaging (E). Summary graph shows hp-iNSC^tk cells migrate further in the presence D283-GFPFL cells than in the absence of these cells (3.3-fold, P < 0001, F). D283-GFPFL cells were seeded with varying concentrations of hp-iNSC^tk and tumor cell viability was measured by luminescence 24h after GCV administration (G). Representative BLI images (H) and summary graph (I) showing the <i>in vivo</i> therapeutic efficacy of cytotoxic NSC therapy (hp-iNSC^tk/GCV) compared to control (hp-iNSC^tk). hp-iNSC^tk/GCV treatment extended survival of mice compared to the control hp-iNSC^tk (53 vs 43 days) (J). Post-mortem histopathological analysis showed the presence of larger recurrent tumors in control mice (K) compared with treatment (L) groups. RC and T denotes resection cavity and tumor, respectively. Original magnification: 100X (B, C, E, K, L). Scale bars = 100 μm. n = 5–6 in each group.</p