Name: ビデオ: In vitroでの癌性神経浸潤のモデリング:背根神経節と癌細胞を共培養する方法 - 概念
Uploaded: 2023-04-30T13:42:18.000+00:00
Duration: 1 min 31 s
Description: 1.3K ビュー. 出典: Na'ara, S. et al. Developing Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016) このビデオでは、DRG細胞と癌細胞を共培養して神経向性と神経浸潤を研究するin vitroの方法を示しています。

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Isolating the Dorsal Root Ganglia (DRG)

<ol>
	<li>Use a stereomicroscope with 4X magnification.</li>
	<li>Position the spine on a non-adherent, non-absorbing platform (Telfa/nylon). Position the spine face up&#160;at the same craniocaudal orientation.</li>
	<li>Remove any spinal muscles and connective tissue. Use the ribs as a landmark for nerves as they leave the spine. Ribs also protect the nerves from surgical tools. The DRG are located at the cervical, thoracic, and lumbar sites of the vertebrae.</li>
	<li>Next, using scissors, cut the vertebral body in the midline and expose the spinal cord and DRG roots by gentle lateral retraction of the vertebral body.</li>
	<li>In the intercostal space, follow the peripheral nerve medially along the rib lateral to the DRG.</li>
	<li>Identify the DRG. It looks like the yolk of a &#39;fried egg&#39; lying on the nerve.</li>
	<li>Cut the intercostal nerve distal to the DRG, leaving 2&#8211;3 mm of nerve distal to the ganglia to be used for retraction. This &#34;tail&#34; will be removed after harvesting.</li>
	<li>Grasp the nerve using forceps. Pinching the DRG body itself will cause neural cell damage and should be avoided.</li>
	<li>Approach the DRG proximally along its attachment to the spinal cord via its anterior and posterior roots.</li>
	<li>Apply gentle retraction to the DRG by pulling the efferent nerve (intercostal nerve stump) laterally and cut the anterior and posterior roots close to the DRG.</li>
	<li>Keep the DRG in a 35 mm Petri dish filled with fresh, ice-cold DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% sodium pyruvate.</li>
</ol>

2. Implantation in the Petri Dish

NOTE: Perform the next steps on ice using pre-cooled pipette tips to prevent ECM solidification.

<ol>
	<li>Under a stereomicroscope at 2&#8211;4X magnification, place a 35 mm glass-bottom Petri dish on a paper grid. 
	IMPORTANT: Work on ice in order to keep the ECM in liquid state. Create a 1 mm diameter spot (approximately 20 &#181;L) of growth factor-depleted ECM at the center of the grid.</li>
	<li>Under direct visualization, place the DRG at the center of the ECM spot close to the bottom of the dish. 
	NOTE: The cancer cells used in this protocol are murine pancreatic cancer cells (KPC) established from freshly isolated tumor specimens from KPC mice, as described elsewhere.</li>
	<li>Harvest 40,000 cancer cells from confluent cultures, wash them once with PBS, and resuspend them in 40 &#181;L ECM on ice.</li>
	<li>Under direct visualization of the grid using a stereomicroscope, measure 500 &#181;m at each direction from the DRG. At this point, slowly seed 10,000 cells/10 &#181;L ECM. Use a 2&#8211;10 &#181;L pre-cooled tip to inject the cells close to the dish bottom, avoiding their spread in the matrix or detachment of the matrix. (Figure 1).</li>
	<li>Leave the dish within a laminar flow hood for 10&#8211;15 min&#160;to solidify. Avoid ECM drying.</li>
	<li>Slowly add DMEM, prepared as previously mentioned, against the sidewall of the plate. Add enough medium to cover the ECM (about 2 mL).</li>
	<li>Put the dish back in the incubator at 37 &#176;C.</li>
	<li>Replace the medium with a fresh medium on the following day.</li>
	<li>Replace the medium every two days.</li>
</ol>

<table><tbody><tr><td>Operating microscope</td><td>&amp;nbsp;Leica</td><td>&amp;nbsp;M205</td><td></td></tr><tr><td>Forceps</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;F4142</td><td></td></tr><tr><td>Surgical blade</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;Z309036</td><td></td></tr><tr><td>Scissors</td><td>&amp;nbsp;Sigma-Aldrich</td><td>&amp;nbsp;S3271</td><td></td></tr><tr><td>35 mm Petri dishes&amp;nbsp; glass bottom</td><td>&amp;nbsp;de groot</td><td>&amp;nbsp;60-627860</td><td></td></tr><tr><td>70% ethanol</td><td>&amp;nbsp;Sigma</td><td></td><td></td></tr><tr><td>Cold PBS</td><td>&amp;nbsp;Biological industries</td><td>&amp;nbsp;02-023-1A</td><td></td></tr><tr><td>DMEM</td><td>&amp;nbsp;Biological industries</td><td>&amp;nbsp;01-055-1A</td><td></td></tr><tr><td>FCS</td><td>&amp;nbsp;Rhenium</td><td>&amp;nbsp;10108165</td><td></td></tr><tr><td>Penicillin and streptomycin</td><td>&amp;nbsp;Biological industries</td><td>&amp;nbsp;01-031-1B</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>&amp;nbsp;Biological industries</td><td>&amp;nbsp;03-042-1B</td><td></td></tr><tr><td>L-Glutamine</td><td>&amp;nbsp;Biological industries</td><td>&amp;nbsp;03-020-1B</td><td></td></tr><tr><td>Growth factor-depleted matrigel</td><td>&amp;nbsp;Trevigen</td><td>&amp;nbsp;3433-005-01</td><td></td></tr></tbody></table>

modeling cancerous neural invasion in vitro: a method to co-culture dorsal root ganglia and cancer cells

Source: Na&#39;ara, S. et al. <a href="https://www.jove.com/video/52990" target="_blank">In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model.</a> J. Vis. Exp. (2016)
In this video, we demonstrate an in vitro method for co-culturing DRG and cancer cells to study neurotropism and neural invasion.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20490/20490fig1.jpg" /> 
Figure 1: Schematic illustration of the protocol steps.

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

Source: Na&#39;ara, S. et al. In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016)
In this video, we ...

Neural invasion is the mechanism of cancer metastasis wherein neurotrophic cancer cells demonstrate an intrinsic ability to grow in and around nerves. To observe this phenomenon in vitro, first, take an isolated murine dorsal root ganglion or DRG - a cluster of cell bodies of sensory neurons.
Place the DRG in an ice-cold medium to retain its viability. Now, transfer the DRG into a drop of chilled extracellular matrix or ECM, which serves as a&#160; three-dimensional scaffold around the DRG. Next, seed the neurotrophic cancer cells into the ECM at an appropriate distance around the DRG.
Allow the ECM to solidify in order to embed the DRG and cancer cells within. Finally, add a suitable growth medium to the ECM and incubate. The nutritive growth media and the ECM proteins provide an optimum microenvironment for the explants.
During incubation, the reciprocal interaction between the explants promotes DRG to sprout small nerve projections or neurites that outgrow radially towards the cancer cells. Simultaneously, cancer cells migrate unidirectionally away from their colonies and invade the newly formed neurites.

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Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

1.3K ビュー. 出典: Na'ara, S. et al. Developing Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model. J. Vis. Exp. (2016) このビデオでは、DRG細胞と癌細胞を共培養して神経向性と神経浸潤を研究するin vitroの方法を示しています。 

ビデオ: In vitroでの癌性神経浸潤のモデリング:背根神経節と癌細胞を共培養する方法 - 概念

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

頭頸部扁平上皮癌における神経周囲の浸潤のためのモデル

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

JoVE Journal

がん研究

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

神経周囲浸潤のIn Vivoマウスの坐骨神経モデル

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

頭頸部癌のペリオラル侵食を評価する生体内モデルにおけるひよこチョリオアラントIC膜

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells

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Modeling Cancerous Neural Invasion In Vitro: A Method to Co-culture Dorsal Root Ganglia and Cancer Cells