Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.
The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.
Low toxicity, low immunogenicity, and intrinsic brain tumor tropism of human stem cells are attractive traits for the delivery of therapeutics vehicles1. Novel stem cell-based therapeutics for malignant brain tumors are promising innovations developed in recent years, and the intranasal adaptation of this therapeutic strategy represents a leap towards clinical translation, in that non-invasive and repeated administration might dramatically reduce the barrier for patient applications and may be adaptable for out-patient services without general anesthesia or lengthy in-patient service associated with invasive surgical procedures1,2,3,4.
We and others have pioneered the intranasal route of stem cell delivery to brain tumors and have laid the ground work for some of the basic principles of translational research using mouse xenograft models2,3,4, as well as investigated the migration of stem cells in vivo via magnetic resonance imaging (MRI) reagent carriers2. Through these pilot explorations, we have accumulated substantial experience and gained insight on how to best construct a robust pre-clinical evaluation strategy using well-established patient-derived xenograft (PDX) mouse models of malignant glioma, while maintaining the investigative resolution to examine the nuanced mechanistic details of the sophisticated biological phenomena of the intranasal brain entry of therapeutic stem cells delivered to the nasal cavity. Here, we describe the principles of a standardized operating protocol to demonstrate the current state of experimental investigations using a well-established human neural stem cell line HB1.F3.CD5,6,7,8, which is readily modifiable to adapt to specific tumor models or strategies using human stem cells as therapeutic carriers.
All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC) or equivalent. If there is any uncertainty regarding the specific procedures described herein, do not proceed. Clarify with the institution's IACUC and a designated veterinary staff member.
1. Ensuring Sterility of Cultured Cells and Follow Principles of Aseptic Techniques
2. Preparation of Glioma Cells for In Vivo Experiment2,4
3. Intracranial Implantation to Create Xenograft Mouse Models (Figure 1A)2,4,13
4. In Vivo Bioluminescence Imaging (BLI) of Tumor Growth (Figure 1B)15
NOTE: The patient-derived glioma cells are modified to express firefly luciferase. This allows us to follow tumor progression after intracranial implantation.
5. Whole Brain Irradiation (Figure 1C)2,4,13
NOTE: To enhance the migration of intranasally delivered hNSCs to brain tumors, whole brain irradiation of mice bearing intracranial tumor xenografts can be utilized 4.
6. Genetic Modification of Human Neural Stem Cells (NSCs; Figure 3A)4
7. Hypoxic Preconditioning of Human NSCs (Figure 3A)4
8. Loading of Human NSCs with Oncolytic Virus (Figure 3B-3H)5
9. Loading Stem Cells with Micron-size Paramagnetic Iron Oxides (MPIOs) for In Vivo Tracking via Magnetic Resonance Imaging (MRI; Figure 3B-3H)2
10. Intranasal Delivery of Therapeutic NSCs (Figures 1D and Figure 3D)2,4
11. Survival Analysis (Figure 3E)
12. Tissue Collection and Histology/Immunocytochemistry Verification of Intranasal Stem Cell Brain Entry2,4
Both hypoxic pre-treatment (Figure 4A)4 and CXCR4 overexpression (Figures 4B and 4C)4 significantly upregulate the cell membrane presence of CXCR4 receptors as demonstrated by flow cytometry. The tumor tropism demonstrated by NSCs (blue arrows), is shown in the tumor tissue histology (red circle). The presence of MPIO-labeled (Figure 4D) stem cells in the tumor is confirmed via Prussian blue staining (Figure 4E).4
A tissue analysis indicates that head-only irradiation (Figure 2) induces reduction in tumor volume, as well as the SDF-1 upregulation in tumor and surrounding tissue (Figures 5A and 5B)4, which confirms the hypothesis that CXCR4 receptor upregulated expression in NSCs may facilitate tumor tropism after XRT.
Survival analysis indicate that although irradiation (XRT) alone is beneficial for the survival of tumor-bearing animals (Figure 6A)4, additional benefits can be achieved using HNSCs loaded with OV over NNSCs loaded with OV in the context of XRT (Figure 6B)4. Significant survival benefits can be also seen with genetically engineered CXCR4NSCs in the same contextual therapeutic regimen (Figure 6C)4.
Tissue analysis confirms that both HNSCs and CXCR4NSCs significantly enhanced the tumor-targeted delivery of OVs, as shown by staining for a viral hexon protein (Figures 7A and 7B)4.
Figure 1: Schematic Flow Chart of the General Animal Procedures Involved. (A)Tumor models are created via xenograft of patient derived glioma cells. (B) Tumor growth is monitored via BLI. (C) Head-only irradiation therapy is delivered as described in Procedure 6. (D) Supine and head-tilt posture is maintained during the therapeutic intranasal stem cell delivery as detailed in Procedure 11. (E) Post therapy survival monitoring, small animal imaging and histological analysis of tissue if necessary are followed up. Please click here to view a larger version of this figure.
Figure 2: The Setup of Animals for Irradiation. Anesthetized mice are positioned between lead shields inside the sample tray allowing for head only irradiation. Please click here to view a larger version of this figure.
Figure 3: A Flow Chart for General Stem Cell Preparation Procedures Involved Prior to Intranasal Delivery. (A-C) Hypoxic pretreatment or genetic modifications of stem cells to overexpress CXCR4. (B-D) Washing, enzymatic dissociation, cell collection, and counting steps are performed for subsequent loading of stem cells with therapeutic or diagnostic cargo. (E) Therapeutic reagents, such as oncolytic viruses, or diagnostic tracers, such as MPIO particles, can be loaded in collected stem cells in appropriate concentrations with periodic shaking and gentle agitation (F). (G-I) The loaded stem cells can then be washed and counted for intranasal delivery into mice. Please click here to view a larger version of this figure.
Figure 4: Analysis of Stem Cell Modifications for CXCR4 Expression and MPIO Loading. (A-C) Flow cytometry can be run to verify the phenotypic modifications of stem cells as a result of hypoxic pre-treatment or genetic engineering. (Details can be found in Dey et al.4) (D-E) The detection of successful MPIO loading of stem cells and in vivo outcomes can be achieved via flow cytometry and Prussian blue staining, respectively. Please click here to view a larger version of this figure.
Figure 5: The Hypothesis that Irradiation Facilitates SDF-1 (Green Signals) Upregulation in Glioma and Surrounding Tissue is Confirmed via Histopathology and Immunocytochemistry of Tissue Sections. Irradiation increases SDF-1 expression in GBM43 xenograft. The analysis of H&E staining and immunocytochemistry of brain tissue sections from control and irradiated mice demonstrates reduced tumor burden but increased SDF-1 levels at the tumor sites after XRT treatment. (A) SDF-1 expression (green) is strongly localized at the tumor site (white boxes) after XRT. (B) Densitometry quantification of the viewing fields demonstrates a four-fold increase in SDF-1 expression in XRT treated animals in comparison to control animals (n = 3 experiments, ***p <0.001, Student's t-test). Details can be found in Dey et al.4 Please click here to view a larger version of this figure.
Figure 6: Survival Benefits of Modified NSCs were Observed (Details can be Found in Dey et al.4). (A) The context of therapeutic benefits of XRT was established (***p <0.001, log rank test). (B) Hypoxic pre-treatment of stem cells loaded with OV enhances animal survival (*p <0.05). C. CXCR4 genetic enhancement further improves survival of animals (**p = 0.0013). Details can be found in Dey et al.4 Please click here to view a larger version of this figure.
Figure 7: Confirmation of OV Delivery by Hypoxia- or CXCR4-enhanced NCSs to Brain Tumor Xenografts is Achieved via Viral Hexon Protein Detection by Immunocytochemistry (Green Dots) of Tissue Sections from Brains of Mice Treated with Either HNSCs (A) or CXCR4NSCs (B), ***p <0.001 t test. Details can be found in Dey et al.4 Please click here to view a larger version of this figure.
Although the intranasal route of drug delivery has been widely explored for small molecules, nanomedicines, and protein compounds alike18, the application of therapeutic stem cells for intranasal brain tumor targeting is very new in the spectrum of brain tumor therapeutics under development2,3,4. There are intrinsic complexities involved regarding the behaviors of stem cells in the nasal cavity, and molecular details still remain unclear. The size of stem cells and their distribution mechanisms along cranial nerves differ dramatically from those of non-cell biologics or small molecules, and we are currently engaged in detailed mechanistic investigations involving the behaviors of therapeutic stem cells in the nasal epithelia. Molecular interactions involving stem cell surface cytokine receptor expression profiles, as well as extracellular matrix (ECM) adhesion molecules such as integrin, are under investigation to shed light on the underpinnings of the intranasal brain entry of stem cells. The critical steps within the current protocol are multifold: 1) Proper establishment of the intracranial glioma xenograft, as variations in tumor location and growth patterns might affect the chemotaxis that NSCs rely on for migration toward tumor; 2) Proper preparation of the NSCs and their health status and phenotypes that impact the chemotaxic migratory behaviors; 3) Adequate preparation of the nasal cavity with hyaluronidase, which has been demonstrated to improve stem cells migration from nasal cavity into the brain17; and 4) Correct supine posture of mice during intranasal stem cell delivery to prevent cell loss outside of the nostrils, and post-NSC delivery care of the animals. MRI2 or radiotracer-based imaging techniques13 in live animals could be applied for the real-time assessment of in vivo stem cell migration after intranasal delivery.
For a preclinical study using animal models, there are limitations to predict the therapeutic outcome in the translation to clinical human patient studies. There are significant differences in the cranial nerve ending distribution patterns between rodents and humans19,20; thus, strategies to direct therapeutic stem cells to distribute along nerve ending networks more relevant to human brain entry through the intranasal route should be the focus for future research. These details are keys to successful clinical translation efforts, and the principles described herein are essential to establish the basis for further diversification of research parameters designed to distinguish molecular cues and the functional and anatomical differences between mouse models and human application specifics.
In this pilot demonstration of the basic principles of using mouse PDX models of human glioma, we show the potential for diversified strategies to enhance the tumor tropism of hNSCs via hypoxic preconditioning and genetic enhancement in chemotropism promoting CXCR4 receptor expression to target tumor-associated cytokines. The significance of this comprehensive description of intranasal therapeutic stem cell delivery technique is evident, as it is the first step-by-step protocol describing an alternative therapeutic stem cell delivery strategy to the central nervous system (CNS) without invasive approaches typically required for CNS disorders. We aim to bring a robust experimental therapeutic platform to the forefront of developmental medicine for malignant brain tumors, such that greater mechanistic details can emerge from broad research explorations in the scientific community. Future expanded applications to treat CNS disorders of broad complexity and mechanistic diversity can be explored by researchers in the respective fields.
The authors have nothing to disclose.
This work was supported by NIH R01NS087990 (MSL, IVB).
Stereotaxic frame | Kopf Instruments | Model 900 | |
Hypoxic Cell Culture Incubator | ThermoFisher Scientific | VIOS 160i | |
Cell culture supplies (Plastics) | ThermoFisher Scientific | Varies | Replaceable with any source |
Legend Micro 21R Refrigerated Microcentrifuge | ThermoFisher Scientific | 75002490 | Replaceable with any source |
Bench centrifuge Sorvall ST16R | ThermoFisher Scientific | 75004240 | Replaceable with any source |
Micro syringe 702N 25µl (22S/2"/2) | Hamilton Company | 80400 | Flat tip |
Sample Tray for Irradiator | Best Theratronics | A13826 | To set up mice protection with lead shield |
Leica DMi8 Microscope | Leica Microsystem | Custom setup | |
Leica CM1860 UV cryostat | Leica Microsystem | Custom setup | |
Exel International Insulin Syringe | ThermoFisher Scientific | 14-841-31 | |
Corning Phosphate Buffer Saline | Corning Cellgro/ThermoFisher | 21-031-CV | |
Dulbecco's Modified Eagle Medium | Corning Cellgro/ThermoFisher | 11965-084 | |
Trypsin 0.05% | Corning Cellgro/ThermoFisher | 25300054 | |
Hyaluronidase from bovine testes | MilliporeSigma | H3506 |