Name: glioblastoma relapse post-resection model for therapeutic hydrogel investigations
Uploaded: 2023-02-24T14:50:00.000+00:00
Duration: 4 min 46 s
Description: Watch this Scientific Journal Video about Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations at JoVE.com

This work was supported by project grants from The National Natural Science Foundation of China (82071767 and 82171781).

All the animal experiments were approved and supervised by the Institutional Review Board and the Animal Ethics Committee of Nanjing Medical University (IACUC-1904004). C57BL/6J female mice, aged 6-8 weeks old, were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Animal preparation
<ol>
	<li>Weigh the mice, and anesthetize them with an intraperitoneal injection of 50 mg/kg pentobarbital sodium (see&#160;Table of Materials). Administer&#160;meloxicam (5mg/kg; i.p.) for perioperative analgesia.&#160;House the mice in a cage under a 12 h/12 h light/dark cycle, an ambient temperature of 24 &#176;C, and a relative humidity of 50%. 
	&#8203;NOTE: For the present study, the initial weight of the mice was about 22 g.</li>
	<li>Remove the hair on the head of a mouse using a laboratory animal shaver, and fix the head and limbs on the stereotaxic apparatus (see Table of Materials). 
	NOTE: Sterilize all equipment before use.</li>
	<li>Disinfect the head of the mouse with at least three alternating rounds of chlorhexidine or povidone-iodine scrub followed by&#160;alcohol, and cover the mouse with a sterile surgical drape. Apply ophthalmic ointment on both eyes to prevent drying.</li>
	<li>Cut the scalp of the mouse about 1 cm along the midline on the top of the right forehead with ophthalmic scissors. 
	NOTE: Confirm&#160;the surgical plane of anesthesia by the absence of pedal reflex&#160;before making the incision.</li>
	<li>Tune the stereotaxic apparatus to ensure the herringbone seam and the front fontanel point are located at the same level.</li>
</ol>
2. Construction of the orthotopic intracranial GBM model
<ol>
	<li>Mark the point 1 mm to the front side of the front fontanel, 1.8 mm to the right side of the front fontanel, and 3 mm down from the front fontanel using a cotton swab dipped with gentian violet2 (see Table of Materials).</li>
	<li>Drill the point using a 1 mm diameter mini cranial drill (see Table of Materials) to create a pore size of about 1 mm diameter and 1 mm depth.</li>
	<li>Remove the exuded cerebrospinal fluid with a sterile cotton swab.</li>
	<li>Aspirate 5 &#181;L of tumor cell suspension (GL261-Luci, 5 &#215; 105 cells suspended in 5 &#181;L of PBS) with a microsyringe. 
	NOTE: The GL261-Luci was obtained from a commercial source (see Table of Materials).</li>
	<li>Align the needle tip of the microsyringe vertically with the skull drilling hole, insert until the needle tip enters the skull plane for 3 mm, and return the needle by 0.5 mm7.</li>
	<li>Open the microsyringe, and inject with a speed of 1 &#181;L/min.</li>
	<li>Retain the needle for 10 min after injection.</li>
	<li>Withdraw the microsyringe slowly, and press the injection point with a sterile dry cotton ball.</li>
	<li>Suture the scalp with a nonabsorbable surgical suture (10-0, see Table of Materials), and disinfect the incision again.</li>
	<li>Monitor the animal&#39;s health, and keep it in warm conditions.</li>
	<li>Move the mouse back to the housing cage after the mouse wakes up.</li>
	<li>Image the mouse with an in vivo bioluminescent imaging system (see Table of Materials) to detect the transplanted tumor on the 10th day after tumor-bearing. 
	NOTE: Luciferase loaded in the GBM cells, GL261, was used for the bioluminescent imaging.&#160;
	<ol>
		<li>Anesthetize the mouse with 1.5% isoflurane with an oxygen flow rate of 0.6L/min. After confirming anesthesia depth via lack of pedal reflex, inject the mouse intraperitoneally with potassium fluorescein (10 mg/mL, see Table of Materials), and 11 s later, perform the in vivo bioluminescent imaging. When the fluorescence value reaches ~5 &#215; 105, the procedure is successful. 
		NOTE:&#160;Anesthetize the animal&#160;before imaging. Maintain anesthesia via nose cone supplied with vaporized isoflurane.</li>
	</ol>
	</li>
	<li>Select the mice with successful tumor-bearing to construct the GBM relapse post-resection model.</li>
</ol>
3. Construction of the GBM relapse post-resection model
<ol>
	<li>When the tumor size in the orthotopic intracranial GBM model mice becomes ~6.5 &#215; 105, select the mice for the postoperative relapse model.</li>
	<li>Weigh the orthotopic intracranial GBM model mice, and anesthetize the mice with an intraperitoneal injection of 50 mg/kg pentobarbital sodium.Administer&#160;meloxicam (5mg/kg; i.p.) for perioperative analgesia.&#160;</li>
	<li>Repeat the process of steps 1.2&#8722;1.5.</li>
	<li>Separate the scalp tissue and skull, and check the drilling hole used to build the orthotopic intracranial GBM model. If the hole has healed, identify the hole using stereotactic apparatus, and follow the procedure mentioned in steps 2.1-2.3.</li>
	<li>Expand the hole diameter to 5 mm with a skull drill, and remove the exuded cerebrospinal fluid with a sterile cotton swab.</li>
	<li>Focus the microscope on the head of the mouse, and adjust the settings to ensure the drilling hole is located in the center of the field of vision.</li>
	<li>Cut the meninges with microscissors, and remove part of the tumor tissue with a micro curette and a micro scalpel under the microscope. 
	NOTE: Regarding the meninges, when the skull is opened, the thin membrane close to the brain tissue is the meninges.</li>
	<li>Stop the bleeding with sterile gauze, and wash the incision with sterile physiological saline.</li>
	<li>Inject the commercially available hydrogel (10 &#181;L, see Table of Materials) into the resection cavity with a 1 mL syringe, suture the scalp with a nonabsorbable surgical suture (10-0), and disinfect the incision again (Figure 1).</li>
	<li>Monitor the animal&#39;s health, and keep the mouse warm.</li>
	<li>Move the mouse back to the housing cage after the mouse wakes up.</li>
	<li>Perform in vivo bioluminescent imaging to detect the transplanted tumor 1 day later to quantify the size of the residual tumor (Figure 2 and Figure 3).</li>
	<li>Weigh the mice every 2 days until the terminal endpoint. 
	NOTE: For the present study, the terminal endpoint was day 30, and the mice were euthanized by an overdose of pentobarbital sodium with an intraperitoneal injection.</li>
</ol>

<ol>
	<li>Lim, M., Xia, Y., Bettegowda, C., Weller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+state+of+immunotherapy+for+glioblastoma.">Current state of immunotherapy for glioblastoma.</a> Nature Reviews Clinical Oncology. 15 (7), 422-442 (2018).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+targeting+nanoparticles-hydrogel+hybrid+system+for+combined+chemo-immunotherapy+of+glioma.">In situ targeting nanoparticles-hydrogel hybrid system for combined chemo-immunotherapy of glioma.</a> Journal of Controlled Release. 345, 786-797 (2022).</li><li>Binder, Z. A., O'Rourke, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma:+The+current+state+of+biology+and+therapeutic+strategies.">Glioblastoma: The current state of biology and therapeutic strategies.</a> Cancer Research. 82 (5), 769-772 (2022).</li><li>Kauer, T. M., Figueiredo, J. L., Hingtgen, S., Shah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+therapeutic+stem+cells+implanted+in+the+tumor+resection+cavity+induce+cell+death+in+gliomas.">Encapsulated therapeutic stem cells implanted in the tumor resection cavity induce cell death in gliomas.</a> Nature Neuroscience. 15 (2), 197-204 (2011).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+engineered+TRAIL-overexpressing+adipose-derived+stem+cells+target+and+eradicate+glioblastoma+via+intracranial+delivery.">Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (48), 13857-13862 (2016).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironmental+landscape+of+brain+tumors.">The microenvironmental landscape of brain tumors.</a> Cancer Cell. 31 (3), 326-341 (2017).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunostimulant+hydrogel+for+the+inhibition+of+malignant+glioma+relapse+post-resection.">Immunostimulant hydrogel for the inhibition of malignant glioma relapse post-resection.</a> Nature Nanotechnology. 16 (5), 538-548 (2021).</li><li>Ruan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual-bioresponsive+drug-delivery+depot+for+combination+of+epigenetic+modulation+and+immune+checkpoint+blockade.">A dual-bioresponsive drug-delivery depot for combination of epigenetic modulation and immune checkpoint blockade.</a> Advanced Materials. 31 (17), 1806957 (2019).</li><li>Louveau, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+features+of+central+nervous+system+lymphatic+vessels.">Structural and functional features of central nervous system lymphatic vessels.</a> Nature. 523 (7560), 337-341 (2015).</li><li>Sun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunostimulant+in+situ+hydrogel+improves+synergetic+radioimmunotherapy+of+malignant+glioblastoma+relapse+post-resection.">Immunostimulant in situ hydrogel improves synergetic radioimmunotherapy of malignant glioblastoma relapse post-resection.</a> Advanced Functional Materials. 32 (43), 2205038 (2022).</li><li>Liu, D. K., Sulman, E. P., Wen, P. Y., Kurz, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapies+for+glioblastoma.">Novel therapies for glioblastoma.</a> Current Neurology and Neuroscience Reports. 20 (7), 19 (2020).</li><li>Mellinghoff, I. K., Cloughesy, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+risk+and+efficiency+in+drug+development+for+rare+and+challenging+tumors:+A+new+paradigm+for+glioma.">Balancing risk and efficiency in drug development for rare and challenging tumors: A new paradigm for glioma.</a> Journal of Clinical Oncology. 40 (30), 3510-3519 (2022).</li></ol>

The authors declare no conflicts of interest.

Surgery remains the first choice for most GBM patients11. Due to the characteristic of invasive growth of GBM, a small number of tumor cells still remain after micro neurosurgical techniques, resulting in eventual tumor recurrence12. How to inhibit the recurrence of GBM after surgery has become the focus of GBM-related research. However, due to the complex anatomical structure of brain tissue, the construction of a suitable postoperative GBM model has become the primary problem to be solved in this field.
This study developed a GBM relapse post-resection model. In the process of constructing this model, the construction of the orthotopic intracranial GBM model is critical. After this model has been developed successfully, the resection needs to be performed at the right time. The recommended time is when the fluorescence value of the tumor size is about 6.5 &#215; 105. To reduce the mortality of the mice, resection under anesthesia was performed with 40 mg/kg 1% pentobarbital sodium by intraperitoneal injection. However, the resection was difficult to perform, and the mice often moved due to the small dose of the anesthetic. On this basis, the dose of the anesthetic was increased to 50 mg/kg. After increasing the anesthetic dose, the intraoperative responses of the mice disappeared, and the resection was performed successfully. Isoflurane gas can also be used in this protocol.
In this study, GL261-Luci cells were used to develop the model; therefore, more GBM cell lines must be used to validate the protocol in the future. To make the protocol more convincing, various GBM mouse models, such as genetically engineered GBM mouse models, need to be used. In addition, MRI may be the best means to detect the recurrence of tumors after surgery.
In summary, in this work, a GBM relapse post-resection model has been developed. In this model, tumor recurrence is monitored by assessing the residual tumor&#39;s growth after resection. Although this model cannot be considered to completely mimic tumor recurrence, the resection style in this model is similar to the standard of maximally safe surgery in the clinical treatment of GBM patients. This work provides a convenient and feasible method of constructing the GBM relapse post-resection model and represents an advancement in the field of research on GBM relapse post-resection.

Glioblastoma (GBM) is the most common malignant tumor among all central nervous system cancers1,2. Surgery is the first-line treatment for patients with GBM, and chemoradiation is the main adjuvant treatment following surgery. However, tumor recurrence often develops within 3-6 months in most GBM patients with various treatments3,4,5. Hence, there is an urgent need to develop more effective treatment strategies to prevent GBM recurrence.
Recent studies on GBM have mainly focused on primary tumors rather than recurrent tumors6. However, the most common problem that needs to be solved in the clinic is how to inhibit the recurrence of GBM after surgery. Therefore, research on the recurrence of GBM after surgery needs more attention. Bioresponsive therapeutic hydrogels are the most common vector used in studies on tumor recurrence after surgery7,8. However, due to the special structure of the central nervous system, it is difficult to develop a suitable GBM relapse post-resection model9, which is critical for the study of GBM recurrence.
This study has generated an improved GBM relapse post-resection model based on the orthotopic intracranial GBM model used in research on primary GBM. In this model, most of the tumors are removed by surgery with microscopy, and the residual tumor is detected by in vivo bioluminescent imaging and hematoxylin and eosin (H&#38;E) staining. This model mimics the resection state of brain tumor patients and can be used in various studies on GBM relapse.

<table><tbody><tr><td>Gentian violet</td><td>Sigma</td><td>C6158</td><td></td></tr><tr><td>GL261-Luci</td><td>Shanghai Zhong Qiao Xin Zhou Biotechnology Co.,Ltd.</td><td>ZQ0932</td><td></td></tr><tr><td>In vivo bioluminescent imaging system</td><td>Tanon</td><td>Tanon ABL X6</td><td></td></tr><tr><td>Laboratory animal shaver</td><td>Beyotime Biotechnology</td><td>FS600</td><td></td></tr><tr><td>Mice</td><td>Beijing Vital River Laboratory Animal Technology Co., Ltd.</td><td></td><td></td></tr><tr><td>Micro curette</td><td>Belevor Medical Co.,Ltd.</td><td></td><td></td></tr><tr><td>Micro scalpel</td><td>Belevor Medical Co.,Ltd.</td><td></td><td></td></tr><tr><td>Microscope</td><td>Shanghai Xiangfan Instrument Co., Ltd</td><td>JSZ5A/B</td><td></td></tr><tr><td>Microsyringe</td><td>Hamilton</td><td>87943</td><td></td></tr><tr><td>Mini cranial drill</td><td>RWD</td><td>78001</td><td></td></tr><tr><td>Nonabsorbable surgical suture</td><td>Shanghai Yuyan Instruments Co.,Ltd.</td><td></td><td></td></tr><tr><td>Pentobarbital sodium</td><td>ChemSrc</td><td>57-33-0</td><td></td></tr><tr><td>PVA-TSPBA hydrogel&amp;nbsp;</td><td>Aladdin</td><td>9002-89-5</td><td></td></tr><tr><td>Stereotaxic apparatus</td><td>RWD</td><td>68043</td><td></td></tr></tbody></table>

glioblastoma relapse post-resection model for therapeutic hydrogel investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The process of construction of the GBM relapse post-resection model is shown in Figure 1. The resection cavity after the tumor was partially removed under the microscopy is shown.&#160;The hydrogel was injected into the resection cavity with a syringe to demonstrate the therapeutic effect. The schedule of the experimental design is shown in Figure 2A. After the GBM cells were implanted into the brain of the mice, the tumor growth was tested by in vivo bioluminescent imaging on day 10. The resection was performed on day 11, and the hydrogel was then injected into the resection cavity. The in vivo bioluminescent imaging testing was performed on day 15, day 20, day 25, and day 30 to monitor the residual tumor growth. As shown in Figure 2B,C, the size of the tumors in the GBM relapse post-resection model was significantly smaller than those in the orthotopic intracranial GBM model, as shown by the in vivo bioluminescent imaging testing. On day 25, the tumors recurred significantly post-resection (Figure 2D). The H&#38;E staining confirmed that the GBM relapse post-resection model was constructed successfully and that residual tumors significantly recurred after the resection (Figure 3A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65026/65026fig01.jpg" /> 
Figure 1: Intraoperative view of the tumor resection and injection of the hydrogel. Part of the tumor tissue was removed with a micro curette and micro scalpel under the microscope, and hydrogel was injected into the resection cavity. Scale bars: 50 &#181;m. This figure has been modified from Sun et al.10. <a href="https://www.jove.com/files/ftp_upload/65026/65026fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65026/65026fig02.jpg" /> 
Figure 2: Schedule of the experimental design and the in vivo bioluminescent imaging of mice in the intracranial and post-resection GBM model. (A) The schedule of the experimental design showing that the resection was performed on day 11 and that the in vivo bioluminescent imaging (IV) was performed on day 10, day 15, day 20, day 25, and day 30. (B) The intracranial GBM model mice showed a large tumor size on day 10, (C) the tumor size was significantly reduced after resection on day 11, and (D) the tumor size increased after resection on day 25 in the post-resection GBM model. The control group included the GBM relapse post-resection model mice with no treatment. A total of 42 mice were used in this study. Scale bars: 100 &#181;m. This figure has been modified from Sun et al.10. <a href="https://www.jove.com/files/ftp_upload/65026/65026fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65026/65026fig03.jpg" /> 
Figure 3: H&#38;E staining image of brain tissue from the post-resection GBM model. (A)&#160;An H&#38;E staining image demonstrating the resection cavity, residual tumor, and normal brain tissue. The brain tissue was collected 1 day after the resection. (B) An H&#38;E staining image demonstrating the recurrent tumor and the normal brain tissue. The brain tissue was collected on day 12 after the resection. Scale bars: 100 &#181;m. This figure has been modified from Sun et al.10. <a href="https://www.jove.com/files/ftp_upload/65026/65026fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations at JoVE.com

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective ...

glioblastoma-relapse-post-resection-model-for-therapeutic-hydrogel

Research

JoVE Journal

Cancer Research

1.4K Views.  Nanjing Medical University. The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

Video: Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Medicine

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Bioengineering

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes and are defined as the sudden blockage of a vessel supplying blood to the brain. Within minutes of the blockage, cells begin to die and result in irreparable tissue damage. Current therapeutic treatments focus on clot removal or lysis to allow for the reperfusion and prevent more severe brain damage. Although transient brain plasticity may salvage some of the damaged tissue over time, significant fractions of patients are left with neurological deficits that will never resolve. There is a lack of therapeutic options to treat neurological deficits caused by stroke, emphasizing the need to develop new strategies to treat this growing patient population. Injectable biomaterials are currently being designed to enhance brain plasticity and improve endogenous repair through the delivery of active agents or stem cells. One method to test these approaches is to utilize a rodent stroke model, inject the biomaterial into the stroke core, and assess repair. Knowing the precise location of the stroke core is imperative for the accurate treatment after stroke, therefore, a stroke model that results in a predictable stroke location is preferable to avoid the need for imaging prior to injection. The following protocol will cover how to induce a photothrombotic stroke, how to inject a hydrogel in a controlled and precise manner, and how to extract and cryosection the brain while keeping the biomaterial intact. In addition, we will highlight how these same hydrogel materials can be used for the co-delivery of stem cells. This protocol can be generalized to the use of other injectable biomaterials into the stroke core.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Stroke is the leading cause of disability and the fifth-leading cause of death in the United States. Approximately 87% of all strokes are ischemic strokes ...

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

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Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke