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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 ...

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1.3K 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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

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 ...

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 ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...