这项工作得到了补助R01-CA169354支持&#160;和R01-047822 &#160;来自美国国立卫生研究院和退伍军人事务部（ECB）优异奖。 RKP是由NIH T32 CA009287-30A1，一个ASCO青年研究者奖，加州乳腺癌研究项目奖学金，和美国癌症协会的指导式研究学者格兰特的支持; BAZ是由美国国立卫生研究院资助AI079320支持。 JM是由美国国立卫生研究院T-32培训资助T32-AI07290- 25，T32-AI07290-24和美国癌症协会博士后奖学金PF-12-052-01-CSM奖学金支持。

注:所有的动物实验均按照批准的斯坦福大学，Palo Alto的VA HCS，以及卫生机构AnimalCare和使用委员会的指导方针国家机构进行。 1.准备样品采集和处理（所需时间:〜10-15分钟） <ol><li>如先前所描述的6接种鼠B16F0黑素瘤细胞（0.5-1×10 6）皮下处或附近在雌性C57BL / 6小鼠的腹部正中线。可替代地，接种的肿瘤细胞，无需额外的解剖位点（ 如侧翼，乳腺脂肪垫，等等）。 注意:任何数量的肿瘤（植入或自发）可以使用该协议被使用和评价。例如，其它常用的同基因肿瘤的C57BL / 6包括结肠线MC38，Lewis肺癌（LLC），和前列腺TRAMP-C2线。同时接种到免疫缺陷小鼠的人异种移植物也可在此方式被评估，但应注意的是，在p肿瘤浸润白细胞rofiles必然会改变按照宿主小鼠的免疫缺陷状态。 <ol><li>使用完全培养基（含10％FBS和添加剂如 RPMI）或其他适当的含蛋白的缓冲液（ 例如，HBSS或PBS + 1％FBS或1％BCS）。寒意媒体或缓冲至4℃之前安乐死。 </li></ol></li><li>建立收集管和过滤器切除肿瘤;放置这些冰上。 <ol><li>使用1个50毫升的锥形管每肿瘤切​​除。将在每一个40-70微米的细胞悬液过滤器。另外，使用的15毫升锥形管与金属滤网。使用5ml的注射器柱塞或粗笔在15ml试管的顶部，以创建一个杯形的过滤器。 </li></ol></li><li>准备切除肿瘤的处理手段;用手术镊子和剪刀精切除和处理肿瘤。用70％的乙醇清洗切除/肿瘤样品之间的仪器。对于较大数量Ø˚F样品（ 如 &gt; 10），我们建议由多个样本的研究人员同时处理，以避免肿瘤样本之间的处理时间显著的差异。 </li></ol> 2.肿瘤切除术（所需时间:约5分钟/肿瘤） <ol><li>安乐死单独使用二氧化碳或其他批准的方法的B16肿瘤的小鼠。 </li><li>确保鼠标在手术阶段。用70％乙醇喷雾肿瘤区域和擦拭过量关闭;这有助于防止任何毛发目前蔓延。用于非裸鼠中，根据需要剃肿瘤周围的皮肤区域之前切除。 </li><li>使用镊子和剪刀，切除皮下肿瘤整块切除 ，包括覆及周围皮肤。所采取的"缘"皮肤的量可以变化，但典型地切除2-3毫米肿瘤周围的皮肤。 <ol><li>在这一点上权衡切除肿瘤（+/-周围的皮肤）。将切除的皮肤/肿瘤节内的PL保护过滤器ASTIC锥形管。 </li></ol></li></ol> 3.准备一个单细胞瘤悬架（所需时间:5-7〜最小/肿瘤） <ol><li>使用镊子和剪刀直言不讳肿瘤及覆/周围皮肤。百果使用，直到组织各大部分被加工成1-2毫米的部分剪样品。 注:通常情况下，整个肿瘤进行处理;可替代地，对于较大的肿瘤，肿瘤的代表性部分可被切碎，并进行处理。 </li><li>从一次性5或10毫升注射器使用柱塞，机械地分解对担保滤波器的切碎的肿瘤组织。 </li><li>使用移液器，用5-10毫升冷介质或缓冲洗涤处理肿瘤组织。这将通过过滤器冲洗单细胞。 </li><li>重复上述两个步骤几次，以确保总人数和洗液的体积大致相同，同样大小的肿瘤。 <ol><li>清洗处理肿瘤组织后，观察皮肤和/或其他结缔组织的小片段应在滤波器被留下。 </li><li>另外，对于肿瘤，其中单独的机械分解可能并不足够，消化用胶原酶和/或其它酶可以在切碎过程之前进行。这将允许进行更彻底的单细胞悬浮液。但是，应该指出的是，这样的消化协议可以影响表面抗原表达20，所以应该小心在解释这些结果。 </li></ol></li><li>洗，并通过添加媒体/缓冲液在4℃下500×g离心离心5分钟沉淀细胞。 </li></ol>的单细胞悬液4. FACS染色（所需时间:〜30-60分钟） <ol><li>重悬的细胞在冰冷的FACS缓冲液（PBS + 1％FBS）中。 </li><li>使用计数台盼蓝和血球，或自动细胞计数活细胞。 </li><li>确定每卷总活细胞的产量。这个数据可以用来推断绝对数字与FACS数据相结合肿瘤浸润的白细胞。 </li><li>确定细胞的数目，以进行染色并通过FACS分析。这将通过几个因素（ 例如肿瘤组织学类型，肿瘤的年龄和大小，浸润白细胞的平均百分比等）的影响。 注意:在B16黑色素瘤中，我们发现肿瘤浸润白细胞的低百分比（TIL;通常&lt;5％）相比，肿瘤细胞。因此，至少1×10 6个总肿瘤（肿瘤+白细胞浸润）细胞通过流式细胞仪通常收集用于分析。 <ol><li>使用锥虫染色计数数据，被染色用于FACS的细胞的计算量。例如，如果典型的TIL构成总肿瘤细胞的5％（许多肿瘤细胞会死亡锥虫染色），因子到这一点的肿瘤的单细胞悬浮液的总数被染色。 </li><li>对于不太常见的白细胞亚群，例如 CCR9 + PDC）使用总肿瘤细胞的更大数目（例如2-3×10 6），用于FACS染色，鉴于这些细胞在单细胞悬浮液的相对低的频率。 </li></ol></li><li>染色肿瘤单细胞悬液的FACS。 <ol><li>对活/死判别，使用可固定染色或未染色可固定（ 例如碘化丙啶）都可以使用。如果细胞染色无活/死的歧视，必须小心谨慎考虑到潜在的对抗体的非特异性结合。 </li><li>染色的细胞悬浮液用于使用标准FACS染色协议特定白细胞亚群。块的细胞，以减少非特异性抗体用含1％大鼠血清和Fc嵌段（抗CD16 / 32），以便事先为10分钟至染色与抗原特异性抗体的PBS / FBS的约束力。包括正确的同型对照抗体，以保证染色是具体的。 </li><li>在这一点上，后在FACS染色完成后，固定用4％甲醛或其他类似的固定剂的样品。在4°C的大存储样本RK（以避免荧光团的淬灭），直到收集和分析的时间。 注:存储固定的样品进行长时间可导致改变的荧光团21的信号和强度。 </li><li>收集样品上的流式细胞仪。最佳流式细胞仪的设置将根据仪器和激光/检测器设置的类型不同而不同。 注:此外，可以存在实验之间变化，即使使用相同的机器。因此，进行适当的机器设置（ 例如使用校准珠），以确保前FACS收集和每个实验的分析，适当的补偿设置。 </li></ol></li><li> leukoctye亚群分析<ol><li>使用FACS数据分析软件，分析数据。对于活/死染色，通过门控出死细胞开始。使用抗体对白细胞特异性标记（ 例如抗CD45）的活细胞内识别白细胞。 <ol><li>在此之后，使用各种趣纳克计划，以确定具体的子集，这取决于染色方案。例如，为了找出CD4 + T细胞，然后从CD45 +人口SSC 罗区，并选择CD19-CD3 +细胞;从该CD4 + T细胞可以识别22。 </li></ol></li><li>分析数据并将其显示在许多不同的方式，以确保一致性和识别的模式或趋势（ 即使用总数量，百分比，或比率来量化子集）。 注:例如，我们看住总CD45 +细胞共收集肿瘤细胞（ 图1）的百分比。此外，我们然后计算肿瘤内各白细胞亚群（例如CD4 + T细胞）的百分比，并比较这些作为对照组和试验组（ 图3）之间的比率。 </li></ol></li></ol>

<ol>
	<li>Mantovani, 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=Chemokines+in+the+recruitment+and+shaping+of+the+leukocyte+infiltrate+of+tumors.">Chemokines in the recruitment and shaping of the leukocyte infiltrate of tumors.</a> Semin Cancer Biol. 14 (3), 155-160 (2004).</li><li>Zitvogel, L., Tesniere, A., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+despite+immunosurveillance:+immunoselection+and+immunosubversion.">Cancer despite immunosurveillance: immunoselection and immunosubversion.</a> Nat Rev Immunol. 6 (10), 715-727 (2006).</li><li>Gajewski, T. F., Schreiber, H., Fu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+cells+in+the+tumor+microenvironment.">Innate and adaptive immune cells in the tumor microenvironment.</a> Nat Immunol. 14 (10), 1014-1022 (2013).</li><li>Fridman, W. H., Pages, F., Sautes-Fridman, C., Galon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+contexture+in+human+tumours:+impact+on+clinical+outcome.">The immune contexture in human tumours: impact on clinical outcome.</a> Nat Rev Cancer. 12 (4), 298-306 (2012).</li><li>Curiel, T. 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=Specific+recruitment+of+regulatory+T+cells+in+ovarian+carcinoma+fosters+immune+privilege+and+predicts+reduced+survival.">Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.</a> Nat Med. 10 (9), 942-949 (2004).</li><li>Pachynski, R. K., 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=The+chemoattractant+chemerin+suppresses+melanoma+by+recruiting+natural+killer+cell+antitumor+defenses.">The chemoattractant chemerin suppresses melanoma by recruiting natural killer cell antitumor defenses.</a> J Exp Med. 209 (8), 1427-1435 (2012).</li><li>Hoffman, 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=Transgenic+nude+mice+ubiquitously+expressing+fluorescent+proteins+for+color-coded+imaging+of+the+tumor+microenvironment.">Transgenic nude mice ubiquitously expressing fluorescent proteins for color-coded imaging of the tumor microenvironment.</a> Methods Mol Biol. 1194, 353-365 (2014).</li><li>Mansfield, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+cancer+immunology:phenotyping+immune+cell+subsets+in+situ+in+FFPE+tissue+sections.">Imaging in cancer immunology:phenotyping immune cell subsets in situ in FFPE tissue sections.</a> MLO Med Lab Obs. 46 (6), 12-13 (2014).</li><li>Serres, S., O'Brien, E. R., Sibson, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+angiogenesis,+inflammation,+and+metastasis+in+the+tumor+microenvironment+with+magnetic+resonance+imaging.">Imaging angiogenesis, inflammation, and metastasis in the tumor microenvironment with magnetic resonance imaging.</a> Adv Exp Med Biol. 772, 263-283 (2014).</li><li>Schietinger, 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=Longitudinal+confocal+microscopy+imaging+of+solid+tumor+destruction+following+adoptive+T+cell+transfer.">Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer.</a> Oncoimmunology. 2 (11), e26677 (2013).</li><li>Singh, A. S., Radu, C. G., Ribas, A. P. E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=imaging+of+the+immune+system:+immune+monitoring+at+the+whole+body+level.">imaging of the immune system: immune monitoring at the whole body level.</a> Q J Nucl Med Mol Imaging. 54 (3), 281-290 (2010).</li><li>Kilarski, W. W., 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=Intravital+immunofluorescence+for+visualizing+the+microcirculatory+and+immune+microenvironments+in+the+mouse+ear+dermis.">Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis.</a> PLoS One. 8 (2), e57135 (2013).</li><li>Habibollahi, P., 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=Fluorescent+nanoparticle+imaging+allows+noninvasive+evaluation+of+immune+cell+modulation+in+esophageal+dysplasia.">Fluorescent nanoparticle imaging allows noninvasive evaluation of immune cell modulation in esophageal dysplasia.</a> Mol Imaging. 13 (3), 1-11 (2014).</li><li>Balducci, 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=A+novel+probe+for+the+non-invasive+detection+of+tumor-associated+inflammation.">A novel probe for the non-invasive detection of tumor-associated inflammation.</a> Oncoimmunology. 2 (2), e23034 (2013).</li><li>Liu, C., 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=Plasmacytoid+dendritic+cells+induce+NK+cell-dependent,+tumor+antigen-specific+T+cell+cross-priming+and+tumor+regression+in+mice.">Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T cell cross-priming and tumor regression in mice.</a> J Clin Invest. 118 (3), 1165-1175 (2008).</li><li>Hadeiba, 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=CCR9+expression+defines+tolerogenic+plasmacytoid+dendritic+cells+able+to+suppress+acute+graft-versus-host+disease.">CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease.</a> Nat Immunol. 9 (11), 1253-1260 (2008).</li><li>McLean, M., 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+BALB/c+murine+lung+alveolar+carcinoma+used+to+establish+a+surgical+spontaneous+metastasis+model.">A BALB/c murine lung alveolar carcinoma used to establish a surgical spontaneous metastasis model.</a> Clin Exp Metastasis. 21 (4), 363-369 (2004).</li><li>Watkins, S. K., Zhu, Z., Watkins, K. E., Hurwitz, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+immune+cells+from+primary+tumors.">Isolation of immune cells from primary tumors.</a> J Vis Exp. (64), e3952 (2012).</li><li>Shafer-Weaver, K. 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=Cutting+Edge:+Tumor-specific+CD8++T+cells+infiltrating+prostatic+tumors+are+induced+to+become+suppressor+cells.">Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells.</a> J Immunol. 183 (8), 4848-4852 (2009).</li><li>Goodyear, A. W., Kumar, A., Dow, S., Ryan, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+murine+small+intestine+leukocyte+isolation+for+global+immune+phenotype+analysis.">Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis.</a> J Immunol Methods. 405, 97-108 (2014).</li><li>Stewart, J. C., Villasmil, M. L., Frampton, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence+intensity+of+selected+leukocyte+surface+markers+following+fixation.">Changes in fluorescence intensity of selected leukocyte surface markers following fixation.</a> Cytometry A. 71 (6), 379-385 (2007).</li><li>Hackstein, 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=Heterogeneity+of+respiratory+dendritic+cell+subsets+and+lymphocyte+populations+in+inbred+mouse+strains.">Heterogeneity of respiratory dendritic cell subsets and lymphocyte populations in inbred mouse strains.</a> Respir Res. 13, 94 (2012).</li><li>Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid-derived+suppressor+cells:+more+mechanisms+for+inhibiting+antitumor+immunity.">Myeloid-derived suppressor cells: more mechanisms for inhibiting antitumor immunity.</a> Cancer Immunol Immunother. 59 (10), 1593-1600 (2010).</li><li>Curran, M. A., Montalvo, W., Yagita, H., 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=PD-1+and+CTLA-4+combination+blockade+expands+infiltrating+T+cells+and+reduces+regulatory+T+and+myeloid+cells+within+B16+melanoma+tumors.">PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors.</a> Proc Natl Acad Sci U S A. 107 (9), 4275-4280 (2010).</li><li>Schreiber, R. D., Old, L. J., Smyth, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunoediting:+integrating+immunity's+roles+in+cancer+suppression+and+promotion.">Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion.</a> Science. 331 (6024), 1565-1570 (2011).</li></ol>

The authors have nothing to disclose.

在进行肿瘤微环境的详细分析在确定的机制和免疫调节的影响是至关重要的。与免疫治疗的增加存在于人类的临床领域，了解这些药物对肿瘤浸润白细胞的影响变得必要在确定它们的作用机理。在人类中，有经常存在在获得和分析肿瘤组织对白细胞子集分析，并且因此外周免疫应答的分析临床和/或后勤困难常常执行。进行临床前研究的动物模型使研究人员更充分地发掘免疫调节剂，肿瘤本身对免疫系统的影响，从而使更多的洞察可能影响临床疗效的机制。 我们的技术允许肿瘤浸润白细胞的详细分析。因为整个肿瘤微环境可以进行采样，肿瘤内免疫的存在的更全局视图可以被评估。并且，最终，亲肿瘤抑制细胞和抗肿瘤效应细胞的平衡可能对肿瘤的生长和临床结果4显著影响。因此，这种类型的分析是在提供与关于本具体白细胞亚群的详细信息的研究者有价值。该详细的数据可用于与其它功能性数据的组合，以进一步阐明肿瘤内免疫机制。 该协议的相对简单性允许一个评估，以尽量减少在组内的可变性较大肿瘤的队列。与成像较为典型和普遍的方法相比（通过IHC或免疫），我们的技术可以非常具体的白细胞亚群，利用多色流式细胞仪鉴定。样品的能力或者整个肿瘤（包括周围）或一节肿瘤给出采样和分析方面的研究人员额外的灵活性。这还允许对超过可通过多个组织切片分析获得由成像，从而使肿瘤微环境的更精确的图象的白细胞显著更多的数据的汇编。 我们利用上皮下肿瘤，其被广泛应用于给定的相对容易肿瘤​​接种和测量的小鼠模型这种技术。因此，该协议将适用于有可能是大多数肿瘤模型的使用。然而，该技术也可应用到肿瘤从原位或自发肿瘤模型切除，尽管对肿瘤切除的额外步骤。虽然B16黑色素瘤并不需要酶消化，以获得单细胞悬浮液，可能存在该步骤是必需的以获得均匀的其他类型的肿瘤，有代表性的单细胞悬浮液。也有优势肿瘤secti那成像项让该不能与我们的技术评估。例如，在肿瘤微环境内的白细胞定位更具体的数据可以通过IHC /中频而获得的，这可能经常是一个有价值的辅助，以通过FACS分析所提供的数据。 总之，我们的技术在肿瘤微环境内的白细胞亚群的评价允许研究者内使用相对简单的协议的肿瘤进行免疫存在的详细分析。它可以应用到大多​​数的小鼠肿瘤模型中，并且可以帮助阐明有关白细胞类型，数量和比率的肿瘤中存在的有价值的信息。

肿瘤的生长和促进和回归之间的平衡是，部分地依赖于存在于所述微1,2-亲和抗肿瘤浸润白细胞的平衡。为了研究肿瘤微环境（TME），并具体指明浸润白细胞亚群，我们在小鼠肿瘤模型开发用于皮下肿瘤的评估的方法。研究肿瘤微环境的重要性是众所周知的，并且在文献中的支持。大量研究表明，亲和抗肿瘤浸润免疫细胞的平衡可影响肿瘤生长的结果，不仅在小鼠而且人类研究（在3,4综述）。例如，Curiel 等。显示恶化的卵巢癌患者的临床结果均与增加的肿瘤浸润调节性CD4 + T细胞（Treg细胞）5百分比的存在。我们自己的工作也呈无效果对白细胞亚群在小鼠黑素瘤模型6，它也与比VEL白细胞趋化减少肿瘤的生长。因此，在一个肿瘤的白细胞亚群的详细分析，现在更广泛的认可和越来越重要。 有许多种方法来评价肿瘤微环境对白细胞浸润;例如团体已经工程化的转基因小鼠来表达各种荧光蛋白，以图像在TME 7，经典免疫组化和保存部8，包括各种成像模态，例如MRI，PET共聚焦显微镜9-11的免疫荧光-一些与能力监控intravitally 10,12。这些可与各种分子成像剂，如纳米颗粒13或新颖造影剂14，其标记的免疫细胞。我们的方法是一种流式细胞仪为基础的流动的方法和具有若干优点。首先，整个肿瘤微环境进行采样;在分析时，整个皮下肿瘤且环绕周边被手术切除处理。这消除了在一个单一的肿瘤的任何潜在的抽样偏差，并给出一个更"全球性"分析的肿瘤的整体。其次，使用多色流式细胞仪来分析白细胞亚群可以让我们更具体地衡量浸润白细胞的表型。取决于所使用的颜色的数量，很具体的子集可被识别。这是很重要的，因为有在一个特定的细胞类型数白细胞亚群 - 或甚至在一般亚型分类 - 具有不同的功能，即是在确定肿瘤的命运可能显著。例如，浆细胞样树突细胞（PDC）有牵连的抗肿瘤免疫15。然而，中pDCs的CCR9 +子集已被证明是致耐受性16和移位巴喷枪这样的一个子集可以具有对肿瘤生长的影响。 我们的方法是适合于皮下或能被切除整块其他肿瘤。在我们手中，肿瘤均匀地安乐死的时间切除。然而，可以想到，作为已经在一些研究中已经完成，即皮下肿瘤可以完全切除，在一个存活手术17周围皮肤的封闭，从而使动物的其他评估。然后在切除肿瘤进行分析。因此，该结果代表在肿瘤的发展的单个时间点。虽然这允许详细研究进入微环境，它也是什么无疑是一个动态过程，静态图片。然而，分离的白细胞（ 例如，通过磁分离或密度梯度）然后可以从肿瘤上皮细胞和间质单独分析，或在其他，功能测定法中使用，以进一步确定它们的表型，因为一直PREVIously描述18。该方法中，然后，将是有兴趣了解肿瘤微环境内的白细胞的组合物在给定时间点的任何调查是有用的，无论是在自然病程的设置，或之后的特定治疗扰动。虽然不是由我们完成的，该过程的变化可能也可能被用来分析在隔离肿瘤的特定部分。例如，给定的肿瘤的大小，所述周边区（S）可被解剖远离肿瘤的中心，可能坏死核心，得到研究者更空间隔离视图肿瘤微环境的。 在肿瘤免疫的新兴领域，无疑将是一个指数越来越多的研究评估在小鼠肿瘤模型新型免疫调节剂。几份报告都强调肿瘤与周围设有内特定的白细胞功能的差异vironment。例如，谢弗-Weaver 等人。表明在抗原特异性T效应CD8 +细胞，而活跃在外围，被转化到的CD8 + T抑制基因细胞，一旦他们贩卖到肿瘤微环境19的小鼠模型。这是由于TGF-β部分，但其他因素是可能的介入。因此，评估白细胞亚群 - 数字和比率，以及功能状态 - 肿瘤本身内将给出一个特定的免疫调节对肿瘤命运的影响的更准确的表示。 我们的技术允许肿瘤的详细分析，并提供了研究者的机会更密切地确定改变白细胞种群比以前的方法。

<table><tbody><tr><td>RPMI</td><td>Cellgro</td><td>10-040</td><td>http://cellgro.com; keep on ice</td></tr><tr><td>FBS</td><td>Cellgro</td><td>35-011-CV</td><td>http://cellgro.com</td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>14-432-22</td><td>fischersci.com</td></tr><tr><td>40 &amp;#956;m filter</td><td>Falcon</td><td>08-771-1</td><td>fischersci.com</td></tr><tr><td>5 ml syringe</td><td>BD</td><td>14-823-35</td><td>fischersci.com</td></tr><tr><td>surgical scissors/forceps</td><td>Roboz</td><td>RS-5910</td><td>roboz.com</td></tr><tr><td>PBS</td><td>Cellgro</td><td>MT-21-030-CM</td><td>http://cellgro.com; keep on ice</td></tr><tr><td>trypan blue</td><td>Cellgro</td><td>MT-25-900-CI</td><td>fischersci.com</td></tr><tr><td>hemacytometer</td><td>Hausser Scientifice&amp;nbsp;</td><td>02-671-54&amp;nbsp;</td><td>fischersci.com</td></tr><tr><td>Live/Dead stain</td><td>Life Technologies</td><td>L34957</td><td>lieftechnologies.com</td></tr><tr><td>FlowJo software</td><td>TreeStar, Inc</td><td></td><td>flowjo.com</td></tr></tbody></table>

evaluation of tumor-infiltrating leukocyte subsets in a subcutaneous tumor model

专门免疫细胞浸润肿瘤微环境调节肿瘤的生长和存活。恶性细胞必须逃避或颠覆抗肿瘤的免疫反应，为了生存和繁荣。肿瘤采取了一些免疫不同的机制"逃逸"，其中包括的致耐受性DC的招募，免疫调节性T细胞（Treg细胞），和髓源抑制细胞（MDSC）抑制细胞毒性抗肿瘤反应的优势。相反地​​，抗肿瘤效应的免疫细胞可以减缓肿瘤的生长和扩增:免疫刺激性树突细胞，天然杀伤细胞，怀有先天抗肿瘤免疫和细胞毒性T细胞都可以参与肿瘤抑制。亲和抗肿瘤白细胞之间的平衡，最终确定的行为和转化细胞的命运;众多的人体临床研究已经证明了这一点。白细胞亚群内。因此，详细的分析肿瘤微环境已经变得越来越重要。在这里，我们描述了用于分析存在于在小鼠肿瘤模型中的肿瘤微浸润白细胞亚群的方法。小鼠B16黑色素瘤肿瘤细胞皮下接种于C57BL / 6小鼠。在指定的时间，肿瘤和周围皮肤手术切除了整块并加工成单细胞悬浮液，然后将其染色的多色流式细胞术。使用各种白细胞子集标记，我们能够比较浸润控制和凯莫瑞表达肿瘤之间白细胞亚群的相对百分比。研究者可以使用这样的工具来研究免疫存在于肿瘤微环境，当与肿瘤生长的传统的卡钳尺寸测量结合时，将有可能让他们阐明变化对肿瘤生长的影响，在免疫组合物中。这样的技术可以应用于任何肿瘤模型，其中肿瘤及其微环境Ç一个被切除和处理。

我们的研究结果表明，被迫过表达凯莫瑞小鼠的B16肿瘤的增强肿瘤浸润白细胞（的TIL）的百分比。此外，更改为与凯莫瑞表达相关的肿瘤微环境内表示白细胞亚群的相对比率被确定。与许可Pachynski 等 6重新打印。 图1示出，有该表达凯莫瑞与对照组相比，如通过切除天17肿瘤的FACS分析测定中，在肿瘤中显著增加总CD45 +细胞（肿瘤浸润淋巴细胞）。使用染色，冰冻切片B16肿瘤，增加的由肿瘤免疫浸润的CD45 +细胞被证实（ 图2）。 的FACS数据进一步的分析显示了一个相对增加NK细胞，T细胞的平均值，和传统的直流（林B220-的CD11c +）的肿瘤，其中chemeRIN是过表达比对照肿瘤（ 图3）。有白细胞子集是有一定作用的抑制或可以抑制免疫反应，特别是骨髓来源抑制细胞（林的CD11b + GR1 +）和PDC（Lin-的B220 + CD11c的INT PDCA1 +）16,23的比例相对减少。 越来越多的研究表明白细胞亚群的比例在肿瘤微环境内是一个重要的因素，无论是在肿瘤生长方面和临床结果4,24,25。我们的FACS数据进一步分析，以揭示T细胞（总CD3 +）和每个肿瘤细胞NK细胞的数目;这些细胞类型被越来越多地在凯莫瑞表达肿瘤表示与对照相比（ 图4）。 效应（ 即 NK细胞和T细胞），并抑制细胞的直接比较（ 即 MDSC和PDC）肿瘤microenviron内种群<html>精神疾病为肿瘤组然后执行。 图5示出显著增加效应的比率，以抑制细胞群相比，控制凯莫瑞表达肿瘤内。 总体而言，有代表性的结果显示在白血球子群体的肿瘤微环境内凯莫瑞表达的影响，以及这些人群和比率与可见临床益处相一致的方式的有利倾斜。 <img alt="图1" src="/files/ftp_upload/52657/52657fig1.jpg" /> 图1:从凯莫瑞表达VS第17天切除控制在凯莫瑞表达肿瘤增加肿瘤浸润白细胞肿瘤，用于通过流动的CD45 +白细胞浸润（活细胞的百分比）术分析; * P &lt;0.05，通过双尾学生t检验表示平均±SEM具有4个或更多的小鼠每组。 <html> 2"SRC ="/文件/ ftp_upload / 52657 / 52657fig2.jpg"/&gt; 。图2:B16黑色素瘤的TIL成像免疫荧光图像示出了CD45 +细胞浸润（白色箭头）的控制和凯莫瑞表达黑色素瘤切除在第9天;条为25μm。 <img alt="图3" src="/files/ftp_upload/52657/52657fig3.jpg" /> 图3:白细胞亚群凯莫瑞表达肿瘤涂改代表性 FACS分析的数据是在chemerin-通过日志2比TIL子频率的转换表达与对照的肿瘤（PDC浆DC; Lin-的CD11c的INT B220 + PDCA1 +），疾病预防控制中心（传统的DC;林的CD11c 喜 B220-），CD4（CD3 + CD4 +）T细胞，CD8（CD3 + CD8 +）T细胞，总T细胞（CD3 + CD4 +和CD3 + CD8 +），NK细胞（CD3-NK1。 1+），CD11b的（林的CD11b +）单核/巨噬细胞，MDSC（髓源性抑制细胞;林的CD11b + GR1 +），CD19和+ B细胞（CD3-CD19 +）。 古尔4"SRC ="/文件/ ftp_upload / 52657 / 52657fig4.jpg"/&gt; 图4:改变的效应的比率，以抑制白细胞中的B16肿瘤节17 B16黑色素瘤的肿瘤通过FACS所描述进行分析。个别白细胞亚群被确定通过门控。然后计算NK细胞或总T细胞，以在每个肿瘤类型的MDSC或PDC的比率。 <img alt="图5" src="/files/ftp_upload/52657/52657fig5.jpg" /> 图5:增加效应细胞中凯莫瑞表达的B16肿瘤控制或凯莫瑞表达肿瘤通过FACS进行分析。使用FACS数据计算每10,000总肿瘤细胞T细胞和NK细胞的绝对数量。结果来自一个代表性实验。

Watch this Scientific Journal Video about Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model at JoVE.com

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

evaluation-tumor-infiltrating-leukocyte-subsets-subcutaneous-tumor

Nanyang Technological University

Research

JoVE Journal

Medicine

20.0K 次观看.  Washington University School of Medicine. This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. 

视频: Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

科研

癌症研究

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

免疫与感染

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

从原发肿瘤的免疫细胞的分离

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

生物学

Predictive Immune Modeling of Solid Tumors

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

The use of an RNA-based approach to determine quantitative immune profiles of solid tumor tissues and leverage clinical cohorts for immune-oncology biomarker discovery is described through a molecular and informatics protocols.

固体肿瘤的预测免疫建模

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment ...

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

摘要

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此视频中的章节

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

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

从原发肿瘤的免疫细胞的分离

固体肿瘤的预测免疫建模

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

摘要

探索更多视频

此视频中的章节

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

聚酐IL-1α纳米颗粒 体内抗肿瘤 活性评价

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

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

从原发肿瘤的免疫细胞的分离

固体肿瘤的预测免疫建模

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

聚酐IL-1α纳米颗粒体内抗肿瘤活性评价