Name: experimental melanoma immunotherapy model using tumor vaccination with a hematopoietic cytokine
Uploaded: 2023-02-24T14:30:00
Description: Watch this Scientific Journal Video about Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine at JoVE.com

我们感谢Stephen Schoenberger博士提供B16-Flt3L细胞，并感谢LJI动物和流式细胞术设施的工作人员提供出色的支持。

研究中使用的所有小鼠均在受控温度和湿度的特定无病原体条件下维持并饲养在拉霍亚免疫学研究所（LJI）的动物饲养室中。根据LJI动物护理委员会批准的指南和方案，对8-14周龄的雌性C57BL / 6小鼠进行动物实验。
1.培养的肿瘤细胞植入用的制备
<ol><li>在含有 10% 热灭活 FBS、2 mM 谷氨酰胺、1 mM 丙酮酸钠、1 mM MEM 非必需氨基酸以及青霉素和链霉素各 100 U/mL 的 Ico 改...

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	<li>Zhang, Y., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+and+advances+in+cancer+immunotherapy:+understanding+the+characteristics+of+tumor-infiltrating+immune+cells+and+their+therapeutic+implications.">The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications.</a> Cell &amp; Molecular Immunology. 17 (8), 807-821 (2020).</li><li>Banchereau, J., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+and+the+control+of+immunity.">Dendritic cells and the control of immunity.</a> Nature. 392 (6673), 245-252 (1998).</li><li>Banchereau, 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=Immunobiology+of+dendritic+cells.">Immunobiology of dendritic cells.</a> Annual Review of Immunology. 18, 767-811 (2000).</li><li>Martinez-Lostao, L., Anel, A., Pardo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+cytotoxic+lymphocytes+kill+cancer+cells.">How do cytotoxic lymphocytes kill cancer cells.</a> Clinical Cancer Research. 21 (22), 5047-5056 (2015).</li><li>Maraskovsky, E., 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=Dramatic+increase+in+the+numbers+of+functionally+mature+dendritic+cells+in+Flt3+ligand-treated+mice:+multiple+dendritic+cell+subpopulations+identified.">Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.</a> Journal of Experimental Medicine. 184 (5), 1953-1962 (1996).</li><li>Talmadge, J. E., 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=Intratumoral,+injection+of+adenoviral+Flt3+ligand+has+therapeutic+activity+in+association+with+increased+intratumoral+levels+of+T+cells+but+not+dendritic+cells.">Intratumoral, injection of adenoviral Flt3 ligand has therapeutic activity in association with increased intratumoral levels of T cells but not dendritic cells.</a> Blood. 104 (11), 5280 (2004).</li><li>Curran, M. A., Allison, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vaccines+expressing+flt3+ligand+synergize+with+ctla-4+blockade+to+reject+preimplanted+tumors.">Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors.</a> American Association for Cancer Research. 69 (19), 7747-7755 (2009).</li><li>Simon, S. R., Ershler, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+influences+on+growth+of+B16+murine+melanoma.">Hormonal influences on growth of B16 murine melanoma.</a> Journal of the National Cancer Institute. 74 (5), 1085-1088 (1985).</li><li>Broz, M. L., 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=Dissecting+the+tumor+myeloid+compartment+reveals+rare+activating+antigen-presenting+cells+critical+for+T+cell+immunity.">Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity.</a> Cancer Cell. 26 (6), 938 (2014).</li><li>Salmon, 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=Expansion+and+activation+of+CD103(+)+dendritic+cell+progenitors+at+the+tumor+site+enhances+tumor+responses+to+therapeutic+PD-L1+and+BRAF+inhibition.">Expansion and activation of CD103(+) dendritic cell progenitors at the tumor site enhances tumor responses to therapeutic PD-L1 and BRAF inhibition.</a> Immunity. 44 (4), 924-938 (2016).</li><li>Liu, H. Y., 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=Leveraging+the+Treg-intrinsic+CTLA4-PKCeta+signaling+pathway+for+cancer+immunotherapy.">Leveraging the Treg-intrinsic CTLA4-PKCeta signaling pathway for cancer immunotherapy.</a> Journal for Immunotherapy Cancer. 9 (9), 002792 (2021).</li><li>Kong, K. F., 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=Protein+kinase+C-eta+controls+CTLA-4-mediated+regulatory+T+cell+function.">Protein kinase C-eta controls CTLA-4-mediated regulatory T cell function.</a> Nature Immunology. 15 (5), 465-472 (2014).</li></ol>

这里描述的协议是基于Allison小组的研究。他们证明，B16-Flt3L疫苗与CTLA-4阻断的组合对存活率和肿瘤生长显示出协同作用，而在单独接受B16-Flt3L疫苗或抗CTLA-4抗体治疗的小鼠中没有观察到肿瘤生长减少7。最近的研究揭示了一种新型的Treg内在CTLA4-PKCη信号通路，该通路在调节Treg11的接触依赖性抑制活性中起着重要的强制性作用。单独使用B16-Flt3L疫苗治疗或具有Treg?...

癌症免疫疗法因其毒性副作用较小和反应更持久而被认为是一种有前途的治疗策略。已经开发了几种类型的免疫疗法，包括溶瘤病毒疗法、癌症疫苗、细胞因子疗法、单克隆抗体、过继细胞转移（CAR-T 细胞或 CAR-NK）和免疫检查点阻断1。
对于癌症疫苗，有不同形式的治疗性疫苗，例如基于全细胞的疫苗，蛋白质或肽疫苗以及RNA或DNA疫苗。疫苗接种依赖于抗原呈递细胞（APC）处理肿瘤抗原（包括肿瘤特异性抗原）并将其以免疫原性形式呈递给T细胞的能力。树突状细胞（DC）已知是最有效的APC，被认为在抗肿瘤免疫中起重要作用2，3。这些细胞吸收并处理肿瘤抗原，然后迁移到引流淋巴结（dLN），通过T细胞受体（TCR）和共刺激分子的参与来启动和激活肿瘤特异性T效应（Teff）细胞。这导致肿瘤特异性细胞毒性T细胞（CTL）的分化和扩增，CTL浸润肿瘤并杀死肿瘤细胞4。因此，DC的活化和成熟代表了刺激肿瘤抗原免疫的有吸引力的策略。
已知 Flt3L 可促进表达 MHC II 类、CD11c、DEC205 和 CD86 蛋白的功能成熟 DC 的成熟和扩增5。肿瘤内而非静脉内给药含有 Flt3L 基因 （Adv-Flt3L） 的腺病毒载体已被证明可促进针对口位肿瘤的免疫治疗活性6。Flt3L还被用于基于肿瘤细胞的疫苗，该疫苗由稳定表达逆转录病毒转导的Flt3L的辐照B16-F10细胞组成，作为增强DC对肿瘤抗原交叉呈递的一种方式，从而增加抗肿瘤反应。这里描述的B16-Flt3L肿瘤疫苗接种方案基于James Allison博士的第7组发表的一项研究。在本文中，他们报告说，B16-Flt3L疫苗与CTLA-4阻断相结合，协同诱导对已建立的黑色素瘤的排斥反应，从而提高生存率。
该协议的目标是为黑色素瘤提供临床前免疫治疗模型。这里描述了如何制备和植入肿瘤疫苗的详细程序，以及如何分析实体瘤肿瘤内免疫细胞的组成和功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA&#160;</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>10% heat-inactivated FBS</td>
			<td>Omega Scientific</td>
			<td>FB-02&#160;</td>
			<td>Lot# 209018</td>
		</tr>
		<tr>
			<td>30G needle</td>
			<td>BD Biosciences</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-shape-bottom plate</td>
			<td>SARSTEDT</td>
			<td>83.3926.500</td>
			<td></td>
		</tr>
		<tr>
			<td>B16 cell line expressing Fms-like tyrosine kinase 3 ligand (B16-Flt3L)</td>
			<td>Gift of Dr. Stephen Schoenberger, LJI&#160;</td>
			<td></td>
			<td>Flt3L cDNAs were cloned into the pMG-Lyt2 retroviral vector, as in refernce 5, Supplemental Figure 1</td>
		</tr>
		<tr>
			<td>B16-F10 cell lines</td>
			<td>ATCC</td>
			<td>CRL-6475</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix fixation buffer&#160;</td>
			<td>BD Biosciences</td>
			<td>BDB554655</td>
			<td>Cell fixation buffer (4.2% PFA)&#160;</td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm kit&#160;</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Fixation/Permeabilization Solution Kit</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM)&#160;</td>
			<td>Corning</td>
			<td>10013CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic digital caliper</td>
			<td>Fisherbrand</td>
			<td>14-648-17</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>Flow cytometer data analysis</td>
		</tr>
		<tr>
			<td>GolgiStop (protein transport inhibitor)</td>
			<td>BD Biosciences</td>
			<td>554724</td>
			<td>1:1500 dilution</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#8217;s modified Dulbecco&#8217;s medium (IMDM)</td>
			<td>Thermo Fisher</td>
			<td>12440053</td>
			<td></td>
		</tr>
		<tr>
			<td>LSR-II cytometers&#160;</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr>
			<td>MEM nonessential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>GE17-0891-02</td>
			<td>density gradient specific medium</td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer Hybri-Max liquid</td>
			<td>Sigma</td>
			<td>R7757-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Corning</td>
			<td>10-040-CV</td>
		</tr>
		<tr>
			<td>RS2000 X-ray Irradiator</td>
			<td>Rad Source Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer 40 &#956;m</td>
			<td>Fisherbrand</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer 70 &#956;m</td>
			<td>Fisherbrand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>TL Liberase</td>
			<td>Roche</td>
			<td>477530</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua fixable viability kit</td>
			<td>BioLegend</td>
			<td>423101</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mCD45</td>
			<td>BioLegend</td>
			<td>103135</td>
			<td>Clone: 30-F11 
			Fluorophore: BV570 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD3&#949;</td>
			<td>BioLegend</td>
			<td>100327</td>
			<td>Clone: 145-2C11 
			Fluorophore: PerCP-Cy5.5 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD8</td>
			<td>BioLegend</td>
			<td>100730 
			100724</td>
			<td>Clone: 53-6.7 
			Fluorophore: Alexa Fluor 700, Alexa Fluor 647 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD4</td>
			<td>BioLegend</td>
			<td>100414</td>
			<td>Clone: GK1.5 
			Fluorophore: APC-Cy7 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mFoxp3</td>
			<td>Thermo Fisher Scientific</td>
			<td>11577382</td>
			<td>Clone: FJK-16s 
			Fluorophore: FITC 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-m/hGzmB</td>
			<td>BioLegend</td>
			<td>372208</td>
			<td>Clone: QA16A02 
			Fluorophore: PE 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mIFNg</td>
			<td>BioLegend</td>
			<td>505826</td>
			<td>Clone: XMG1.2 
			Fluorophore: PE-Cy7 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mCD19</td>
			<td>BioLegend</td>
			<td>115543</td>
			<td>Clone: 6D5 
			Fluorophore: BV785 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mGr1</td>
			<td>BioLegend</td>
			<td>108423</td>
			<td>Clone: RB6-8C5 
			Fluorophore: APC/Cy7 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD11b</td>
			<td>BioLegend</td>
			<td>101223</td>
			<td>Clone: M1/70 
			Fluorophore: Pacific blue 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mF4/80</td>
			<td>BioLegend</td>
			<td>123114</td>
			<td>Clone: BM8 
			Fluorophore: PECy7 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mCD11c</td>
			<td>BioLegend</td>
			<td>117328</td>
			<td>Clone: N418 
			Fluorophore: PerCP Cy5.5 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Anti-mMHCII</td>
			<td>BioLegend</td>
			<td>107622</td>
			<td>Clone: M5/114.15.2 
			Fluorophore: AF700 
			Dilution: 1:400</td>
		</tr>
		<tr>
			<td>Anti-mCD103</td>
			<td>BioLegend</td>
			<td>121410</td>
			<td>Clone: 2E7 
			Fluorophore: Alexa Fluor 647 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>Anti-mCD86</td>
			<td>BioLegend</td>
			<td>105007</td>
			<td>Clone: GL-1 
			Fluorophore: PE 
			Dilution: 1:200</td>
		</tr>
		<tr>
			<td>FC-blocker (Rat anti-mouse CD16/CD32)</td>
			<td>BD Biosciences</td>
			<td>553141</td>
			<td>Clone: 2.4G2 
			Dilution: 1:200</td>
		</tr>
	</tbody>
</table>

experimental melanoma immunotherapy model using tumor vaccination with a hematopoietic cytokine

Fms样酪氨酸激酶3配体（Flt3L）是一种造血细胞因子，可促进树突状细胞（DC）的存活和分化。它已被用于肿瘤疫苗中，以激活先天免疫并增强抗肿瘤反应。该协议展示了使用基于细胞的肿瘤疫苗的治疗模型，该疫苗由表达Flt3L的B16-F10黑色素瘤细胞以及肿瘤微环境（TME）中免疫细胞的表型和功能分析组成。描述了培养的肿瘤细胞制备、肿瘤植入、细胞照射、肿瘤大小测量、肿瘤内免疫细胞分离和流式细胞术分析的程序。该方案的总体目标是提供一个临床前实体瘤免疫治疗模型，以及一个研究肿瘤细胞与浸润免疫细胞之间关系的研究平台。这里描述的免疫治疗方案可以与其他治疗方式相结合，例如免疫检查点阻断（抗CTLA-4，抗PD-1，抗PD-L1抗体）或化疗，以提高黑色素瘤的癌症治疗效果。

植入的B16-F10细胞的可见黑点通常在肿瘤植入后~3天在皮肤表面观察到。在肿瘤结节达到≥2mm大小后3，6和9天用肿瘤疫苗治疗小鼠。我们观察到接种疫苗的小鼠组在肿瘤植入后~2周的肿瘤生长显着减少（图1）。在实验结束时，我们分离了肿瘤内免疫细胞，并分析了它们的数量和细胞表面标志物表达，以及如上所述的短暂体外刺激后的细胞因子产生。从白细胞层收集的细?...

Watch this Scientific Journal Video about Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine at JoVE.com

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

该协议提出了一种癌症免疫治疗模型，使用表达Flt3L的B16-F10黑色素瘤进行基于细胞的肿瘤疫苗接种。该协议演示了程序，包括培养的肿瘤细胞的制备，肿瘤植入，细胞照射，肿瘤生长的测量，肿瘤内免疫细胞的分离和流式细胞术分析。

使用造血细胞因子接种肿瘤的实验性黑色素瘤免疫治疗模型

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

该方案提供了临床实体瘤免疫治疗模型和研究平台，研究肿瘤细胞与浸润免疫细胞之间的关系。这种基于细胞的肿瘤疫苗方便，使用简单，并且可以与其他治疗方式（如检查点阻断）结合使用，以实现加法或协同效应，从而产生更有效和高的肿瘤免疫力。帮助演示该程序的是我实验室的研究技术员Ann Balancio。首先，在IMDM中培养B16-F10黑色素瘤细胞，含有10%的热非活性FBS，2毫摩尔谷氨酰胺，1毫摩尔丙酮酸钠，1毫摩尔MEM非必需氨基酸和青霉素和链霉素每毫升100单位。用5%二氧化碳将细胞系保持在37摄氏度。按照文本手稿中的说明播种和收获 B16-F10 细胞。取出培养基并用PBS洗涤烧瓶一次。吸出PBS并加入5毫升0.25%胰蛋白酶-EDTA，然后用力敲击培养瓶的边缘。加入15毫升培养基以中和胰蛋白酶-EDTA，并将烧瓶的内容物倒入50毫升离心管中。用10毫升PBS清洗培养皿表面，然后倒入相同的50毫升离心管中。将细胞在 200 RCF 下离心 5 分钟。弃去上清液，用手指敲击管底部破碎细胞沉淀。加入冷的10毫升PBS，轻轻移液细胞悬液。然后，使用血细胞计数器手动计数细胞。注射前将细胞放在冰上。使用30号针在左胁的50微升冷PBS中皮内植入50，000个细胞B16-F10肿瘤细胞。植入的B16-F10肿瘤细胞的剂量可能需要在50，000至500，000个细胞的范围内进行调整，以成功发展肿瘤。实施后，使用电子数字卡尺每周测量肿瘤长度和宽度三次。计算肿瘤体积，当肿瘤达到文本手稿中所述的两毫米大小时，用肿瘤疫苗治疗小鼠。使用设置为160千伏和25毫安的X射线辐照器，以150灰剂量的伽马射线照射细胞。注射前通过台盼蓝染色计数并检查细胞活力。在与原始肿瘤植入相同的侧翼的50微升冷PBS中皮内注射100万个辐照的B16-Flt3L细胞。在初始细胞植入后的第 3、6 和 9 天距离原发肿瘤部位约 1 厘米。用彩色笔标记疫苗注射部位，以将其与原发肿瘤区分开来。如果最初植入50， 000个B16-F10细胞，建议在第8，11和14天进行疫苗治疗。从每只安乐死小鼠中手术切除带有皮肤的肿瘤，并将其放入装有1毫升10%FBS RPMI 1640培养基的24孔板中。在两毫升消化缓冲液中将肿瘤切成小块，并在37摄氏度下孵育25分钟。加入10毫升10%FBS RPMI 1640培养基以停止消化。使用25毫升血清移液管将细胞转移到40微米细胞过滤器中。然后使用一毫升注射器的柱塞研磨组织。在 500 RCF 下将细胞在 4 摄氏度下离心 5 分钟。将沉淀重悬于5毫升40%密度梯度特定培养基中的PBS稀释至1x浓度。将细胞悬液缓慢加入5毫升含有PBS的80%密度梯度特定培养基上。在室温下以低制动设置以 325 RCF 离心至细胞 23 分钟。离心后，小心地收集在40%和80%密度梯度特定培养基之间的界面处的白细胞层，并将其通过40微米的细胞过滤器。在 500 RCF 下将细胞在 4 摄氏度下离心 5 分钟。将沉淀在两毫升红细胞裂解缓冲液中在室温下孵育五分钟。孵育后，加入10毫升10%FBS RPMI 1640培养基以淬灭RBC裂解缓冲液。在 400 RCF 下将细胞在 4 摄氏度下离心 5 分钟。将细胞重悬于0.5毫升10%FBS RPMI 1640培养基中，并在使用前计算细胞总数以进行进一步分析。通过流式细胞术分析收集脾脏或引流淋巴结作为免疫细胞亚群门控策略的对照。遵循文本手稿中描述的细胞隔离方法。植入的B16-F10细胞的可见黑点通常在肿瘤植入后约三天在皮肤表面上观察到。在肿瘤结节达到或超过两毫米大小后三天，六天和九天用肿瘤疫苗治疗小鼠。在肿瘤植入后约两周，在接种疫苗的小鼠组中观察到显着的肿瘤生长减少。从白细胞层收集的细胞含有许多肿瘤细胞，因此很难轻易定义淋巴细胞群。因此，在流式细胞术分析中，脾细胞被并行用于肿瘤内免疫细胞亚群的适当设门。将CD103阳性、CD11C阳性DC、CD8阳性、CD4阳性和Treg的门控策略与补偿矩阵一起绘制。还提供了所获得账户的代表性数据和每个人口的频率。来自接种疫苗的小鼠的肿瘤内CD103阳性，CD11C阳性DC显示共刺激配体CD86的表达显着升高。接种疫苗的小鼠还显示肿瘤浸润CD8阳性和CD4阳性，Foxp3阴性细胞以及CD8阳性颗粒酶B阳性和干扰素γ阳性CTL增加。原发肿瘤和肿瘤疫苗之间保持足够的距离。这种物理分离对于避免两个肿瘤植入物之间的潜在融合至关重要。

Research

JoVE Journal

Cancer Research

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

原发肿瘤发展小鼠实验模型和腹膜转移<em&gt;通过</em&gt;原位接种卵巢癌细胞

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

该<em&gt;果蝇</em&gt;成虫盘瘤模型：基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

小鼠原位膀胱肿瘤模型和肿瘤检测系统

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

A 3D Organotypic Melanoma Spheroid Skin Model

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

体外肿瘤细胞恢复对 chimeric 抗原受体 t 细胞抗肿瘤功能的预测评价

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

A Melanoma Patient-Derived Xenograft Model

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...