作者承认分子细胞学设施和纪念斯隆凯特林癌症中心的动物设施提供的技术服务。这项工作得到了 NIH 赠款 CA157686 (R.J.) 和 P30 CA008748 (纪念斯隆凯特林癌症中心支助补助金) 的支持。

所有的程序与动物的对象是批准的机构动物护理和使用委员会在纪念斯隆凯特林癌症中心。
1. 肿瘤细胞的制备
<ol><li>收获亚汇合 Panc02-H7 细胞与0.25% 胰蛋白酶为5分钟在37°c。收集15毫升离心管中的细胞。 注: 细胞生长在 T-225 烧瓶中, 它包含大约 12 x 106细胞每瓶在80% 融合和4毫升胰蛋白酶/烧瓶使用。</li>
	<li>离心机在900克的细胞在 4°c 5 分钟, 通过 resuspending 细胞颗粒在1毫升的 PBS (由吹打上下两次), 并转移悬浮到1.5 毫升离心管冰。</li>
	<li>再次离心细胞在900克在 4°c 5 分钟, 并放弃上清, 不干扰颗粒。把颗粒细胞放在冰上直到注射。</li>
</ol>2. 小鼠的制备与手术
注: 本研究使用8周大、雄性和雌性 C57BL/6J 小鼠。手术条件遵循本机构的 IACUC 规则。器械消毒, 手术工作表面消毒, 动物消毒, 外科医生佩戴无菌手套。
<ol><li>在手术前一天, 麻醉老鼠使用2% 异氟醚, 然后用一把薄剃刀或化学脱毛剂将皮毛沿着股骨的长度移除。</li>
	<li>在手术当天, 麻醉在诱导腔内使用 2% isofluorane 的老鼠。通过脚趾捏刺激和缺乏反应来确认麻醉。</li>
	<li>在眼部应用兽医软膏, 防止麻醉时的干燥。</li>
	<li>将麻醉的老鼠放在腹侧, 用低过敏的胶带轻轻地固定每一个肢体, 使其在肢体中产生轻度的张力。麻醉是维持使用异氟醚提供通过一个精确的蒸发器和鼻锥。</li>
	<li>用 Betadine 清洁注射部位, 再用70% 酒精。重复此过程两次。确保没有松动的头发留在手术领域。</li>
	<li>做一个1厘米切口与小剪刀约2毫米以下和平行的股骨。在侧面用镊子缩回皮肤, 露出下面的肌肉。</li>
	<li>坐骨神经向臀大肌和股二头肌肌肉深部延伸。用小剪刀将这两块肌肉沿筋膜平面分开, 露出坐骨神经下面。用钝夹层释放周围肌肉的神经。</li>
	<li>绘制 3 ul 的癌细胞 (约5万细胞) 从颗粒成 10 ul 注射器。 注: 另一种做法是, 将 PBS 的 3 ul 作为控件绘制。</li>
	<li>将一个小金属铲下的神经在注射点的支持。在可视化的解剖显微镜下, 将针插入神经对金属底座, 使针与神经平行, 在插入时尽可能。小心不要刺穿神经的背部。在整个过程中尽量减少对神经的处理。</li>
	<li>在5秒内慢慢注入神经。在注射区形成灯泡表明注射良好。然后把针放在原地3秒, 然后轻轻地取出针。保持针到位为 3 s 最小化细胞回流的神经。 注: 可对远端神经或脊髓进行注射。在一组实验中保持一致是很重要的。如果细胞外泄, 动物不应纳入实验分析。有经验, 这些事件是非常罕见的。</li>
	<li>把神经放回原来的位置。用上覆的肌肉遮住神经。用适当的镇痛治疗小鼠, 然后用5-0 尼龙缝线闭合皮肤。</li>
	<li>在恢复期间, 将鼠标单独放在干净的笼子里观察, 直到它完全从麻醉中苏醒。 注意: 动物恢复全意识需要5-15 分钟。动物没有被留在无人看管, 直到它恢复了足够的意识, 以维持胸骨卧床。在完全恢复之前, 动物不会返回到其他动物的公司。此后, 每24小时至少评估一次恢复72小时。</li>
</ol>3. 坐骨神经提取
<ol><li>在术后女人天 #7, 弄死与CO2 的鼠标。将鼠标放在腹侧, 用别针稳定远端四肢。</li>
	<li>去除注射肢体和躯干背面的皮肤。</li>
	<li>使用钝夹层, 将坐骨神经深深暴露在肌肉上。</li>
	<li>髂骨和骶骨之间的神经路线。要获得脊髓区的神经, 请将两根骨头分开。首先在坐骨神经所在的狭窄区域插入闭合剪刀, 然后在按住鼠标的同时打开剪刀。在解剖过程中保持坐骨神经的完整性和细腻。</li>
	<li>仔细解剖坐骨神经远端到股骨端, 下部到脊髓。</li>
	<li>注意: 侵入神经非常脆弱, 容易在紧张或强力处理下折断。</li>
	<li>先割下它的远端, 才能收获神经。小心抬起神经, 同时从相邻的组织中释放它。切断神经在近端, 尽可能接近其退出从脊柱。</li>
	<li>用游标卡尺记录下入侵的总长度。这种宏观估计只是指示性的。</li>
</ol>4. 神经处理和量化
<ol><li>将解剖神经嵌入 OCT 化合物中。确保将神经纵向和尽可能平坦的模具底部。</li>
	<li>通过标记字母 P 和 D, 在卡带上显示神经的近端和远端。</li>
	<li>如果不立即切片, 将嵌入的神经放在干冰的顶端。样品可以保存在摄氏-80 摄氏度几个星期。</li>
	<li>部分样品使用恒温器切片在5微米厚度和地方部分在玻璃幻灯片。如有可能, 每张幻灯片应符合2神经节。表示神经的近端。</li>
	<li>使用 H & E 染色着色幻灯片12。</li>
	<li>用可提供高分辨率数字数据的滑动扫描仪对染色的神经部分进行数字扫描。</li>
	<li>使用成像软件, 通过单击测量距离按钮、入侵区域或其他所需参数来量化入侵的长度。为了很好地估计入侵的长度, 使用同一神经的多节 (2 到 4)。</li>
</ol>

<ol>
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作者没有什么可透露的。

在本协议中, 我们描述了一个在体内小鼠神经入侵模型, 它允许量化的坐骨神经侵袭的胰腺癌细胞。该模型能够研究神经入侵的分子机制。成功的实验使用这项技术需要谨慎的方法, 在三关键步骤的过程中: 1) 注射癌细胞 (步骤 2.7, 2.8), 2) 提取的侵入神经 (步骤 3.4) 和 3) 处理收获的神经 (步骤 4.1)。
注射应该非常小心, 以避免刺穿神经后壁, 导致癌细胞的损失。一旦针到位, ...

神经形成特定的肿瘤微环境, 刺激癌症的生长和迁移1,2,3。神经入侵 (PNI) 是癌细胞在神经和周围侵袭的过程。它可能被认为是一种独特的转移途径, 因为癌症入侵从起源的地方沿神经。PNI 有多种癌症类型, 包括胰腺、前列腺、头 & 颈部、唾液腺、宫颈癌和结直肠癌, 发病率从22% 到 100%1,2不等。PNI 与疼痛相关, 预后较差, 存活率更差1,2。
神经入侵的发展模式对于阐明这一过程的细胞和分子机制, 以及检测候选治疗剂以减少 PNI 是至关重要的。体外方法研究癌症和神经之间的相互作用, 包括与神经外植体的癌细胞共培养4, 背根脊神经5,6,7, 或特定细胞从神经微环境例如雪旺细胞7。然而,体内方法在生理上是相关的, 包括使用癌症小鼠模型, 在这种模式中, 癌症已经被诱导或移植, 并具有占整个神经微环境的优势。在胰腺或前列腺癌的原位模型中, PNI 报告了8,9,10 , PNI 的发病率可能会被记录下来, 但是由于这些器官神经的体积小, 很难看到整个神经, 因此量化 PNI 的程度。我们在这里描述的模型是一个在体内模型中的 PNI, 其中癌细胞通过一个简单的手术程序11注入到小鼠的坐骨神经中。异位移植在神经内侵袭脊髓。从注射部位到脊髓的神经侵入的长度可以测量, 以及神经内的肿瘤体积。重要的是, 侵入神经也可以收集各种化验, 包括显微, 分子分析。可以对多种癌细胞进行检测, 并使用特定化合物进行基因修饰或治疗的宿主老鼠。这一强有力的化验允许肿瘤细胞和宿主微环境被修改, 以调查的机制, PNI。

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

癌细胞通过称为神经侵袭 (PNI) 的过程侵入神经, 癌细胞在神经微环境中增殖和迁移。这种类型的入侵是由多种类型的癌症所表现出来的, 而且在胰腺癌中也经常出现。小鼠胰腺内神经纤维的显微大小使 PNI 在原位鼠模型中的研究更加困难。在这里, 我们描述一个异位的在体内模型的 PNI, 我们注射自体胰腺癌细胞系 Panc02-H7 到小鼠坐骨神经。在该模型中, 麻醉小鼠的坐骨神经被暴露并注入癌细胞。癌细胞从注射点到脊髓的神经向下部侵袭。侵入的坐骨神经, 然后提取和处理与 OCT 的冰冻切片。H & E 和免疫荧光染色这些部分允许量化的入侵程度和变化的蛋白质表达。该模型可应用于 PNI 的多种研究, 考虑了其通用性。使用不同基因修饰的小鼠和/或不同类型的癌细胞, 可以调查 PNI 和不同癌症类型的细胞和分子机制。此外, 治疗药物对神经侵袭的影响可以通过对这些小鼠的治疗来研究。

该方法描述了胰腺癌细胞移植到小鼠坐骨神经中的外科植入, 以建立一个可量化的神经入侵的体内模型。图 1说明了坐骨神经和注射部位的解剖位置。图 2显示了裸鼠的两个坐骨神经。PBS 注入神经 (左) 可与 MiaPaCa-2 癌细胞 (右) 注入的神经进行比较。癌细胞注入的神经被肿瘤浸润, 比用 PBS 注入的神经更厚、更暗。
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An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

神经入侵的体内小鼠坐骨神经模型

我们描述了一个体内小鼠神经入侵的模型, 注射自体胰腺癌细胞进入坐骨神经。该模型允许定量的神经入侵程度, 并支持调查的细胞和分子机制的神经入侵。

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

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Research

JoVE Journal

Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

11.9K 次观看.  Memorial Sloan Kettering Cancer Center. 我们描述了一个体内小鼠神经入侵的模型, 注射自体胰腺癌细胞进入坐骨神经。该模型允许定量的神经入侵程度, 并支持调查的细胞和分子机制的神经入侵。

视频: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

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.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

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

科研

癌症研究

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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.

在头颈部鳞状细胞癌的神经浸润模型

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

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

矫形子宫内膜癌模型，具有从 Vivo 传播和培养的 VX2 细胞中制成的再生淋巴病

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

突变黑色素细胞干细胞引发小鼠黑色素瘤的时空控制

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...