下面的协议指的是鼠标 MoDC 的隔离, 但总体原则可能也适用于其他 DC 子集单元。12-16 周大的 C57Bl/6j 小鼠被维持在美国的实验室动物 Care–accredited 动物设施认证协会。所有议定书均经斯坦福大学和特拉维夫大学机构动物护理和使用委员会批准。
1. 肿瘤相关单核细胞源性 DC 的分离
<ol><li>在层流罩中, 从 CO2安乐死小鼠中取出肿瘤, 并将肿瘤放置在没有胎牛血清的 RPMI 培养基中。 注意: 强烈建议在切除肿瘤之前, 用70% 乙醇喷出老鼠, 并将其喷洒, 以尽量减少毛皮的潜在污染物。确定肿瘤不超过 25-40 毫米2 , 因为较大的肿瘤有更少的 DC, 往往是脆弱的, 容易遭受细胞死亡。如果需要更多的 DC, 每只老鼠都可以在多个地点注射肿瘤细胞。</li>
	<li>在无菌层流罩下, 用手术剪刀将肿瘤劈成小块 (大约 1 x 1 毫米2)。<ol>
<li>将所有肿瘤片段从一只老鼠添加到一个30毫升无菌的扁平底管与磁性搅拌棒, 其中包含5毫升的汉克的平衡盐溶液 (HBSS), 2 毫克/毫升胶原酶 IV, 0.01 毫克/毫升 DNase i。 注意: 髓细胞通过糖基化产生能量, 即使在稳定状态下也是如此。因此, 只使用含有葡萄糖的培养基 (如 HBSS、RPMI 和 DMEM), 因为葡萄糖饥饿的量只有 10-15 分钟, 在24小时内会导致细胞死亡和凋亡. 只使用低内毒素胶原酶 IV, 胶原酶是由革兰氏阳性产生的细菌 (histolyticum 梭菌)。</li>
	</ol>
</li>
	<li>在37个oC 孵化器中搅拌约 200-400 rpm, 用磁力搅拌器进行20-30 分钟。</li>
	<li>添加5毫升完整的介质, 并大力重新悬浮。</li>
	<li>过滤细胞通过一个70µm 细胞过滤器。离心机位于 4 oC 400 区域合作框架内, 5-10 分钟颗粒状细胞。 注意: 不要试图在酶消化后直接分离细胞, 因为有些肿瘤是坏死的, 含有相对较多的细胞碎片或细胞外基质。在15% 密度梯度介质上应用细胞, 只获得细胞。</li>
	<li>悬浮9毫升密度梯度介质与1毫升 10x PBS 调整渗透压, 并获得 100% Percoll 的库存解决方案。将1.5 毫升密度梯度介质溶液与8.5 毫升 HBSS 混合, 并与肿瘤衍生细胞颗粒混合。在室温下, 在15% 密度梯度介质的顶部轻轻地层2毫升的 DMEM, 在400个区域合作框架内离心机为20分钟。丢弃上清液, 因为细胞会在管子的底部形成一个小球。<ol>
<li>两次清洗颗粒细胞, 重新悬浮在10毫升 HBSS 含有2% 的血清, 1% 青霉素/链霉素, 10 毫米 EDTA (隔离缓冲), 离心机在4个oC 400 区域合作框架为5-10 分钟颗粒细胞。</li>
		<li>在光镜下用台盼蓝计数 hemocytometer 上的细胞。</li>
	</ol>
</li>
	<li>在 1 mL 隔离缓冲区中重新挂起 1 x 108单元格, 并在4个oC 处孵化它们, 并将 CD11b+共轭磁性珠的 30 ul 作为15分钟。 注意: 避免使用 CD11c-conjugated 珠, 因为这会激活 DC 并诱发细胞死亡。不要试图阻止 FcγRII 和 FcγRIII 与 anti-CD16/32 抗体。阻断抗体结扎 FcγR, 诱导强磷酸化的 MAP 激酶和素数的 DC。<ol>
<li>在 4oC 中, 在400区域合作框架中添加9毫升的隔离缓冲和离心细胞, 以消除多余的未绑定珠子。</li>
	</ol>
</li>
	<li>在1毫升隔离缓冲器中吸入上清并重新悬浮细胞。将单元格应用于水洗磁柱上。用3毫升的隔离缓冲器将柱面清洗两次。<ol>
<li>从磁铁中移除该列。将6毫升的隔离缓冲器放到柱上, 通过推柱塞将磁性标记的细胞冲出无菌的收集管中。在 4 oC 上, 400 个区域合作框架内的离心细胞为 5-10 分钟。</li>
		<li>在 100 ul 每 1 x 107单元格中重新暂停单元格, 并使用以下荧光共轭抗体进行染色: 宗族阴性 (TCRb、Siglec F、B220、CD19、FceRI 和 Ly6G)、MHCII、Ly-6C。</li>
	</ol>
</li>
	<li>通过浇小细胞, 使用侧向散射 (SSC) 和前向散射 (FSC) 和完全培养基中的培养基来对细胞进行排序, 并辅以 5 ng/毫升的 GM-脑脊液。孵育细胞1小时在 3 7 oC 为了允许巨噬细胞黏附板材。然后, 将松散的和非黏附的细胞转移到新的培养皿中。 注意:鼠标 MoDC 被定义为 CD11b+/CD11c+/MHCIIhi/Ly6C/int 7, 26, 27.Ly6C 的表达可能在肿瘤模型之间有显著差异, 在某些模型中 (如 LMP) MoDCs 完全缺乏 Ly6C 表达。总的来说, 一个100毫米3 B16F10 肿瘤通常包含 5 x 104 DC, 导致大约 4-5 x 106总细胞。在这些细胞中, 10-15% 是免疫细胞, 8-10% 是 MoDC。增加 DC 数可以通过在多个地点注射肿瘤小鼠来实现。只对小的 SSC/FCS 细胞进行排序, 因为其他髓细胞可以表达诸如 CD11c 和 Ly6C 等标记。 可选: 将肿瘤浸润单核细胞分类为小 SSC/FSC 单元, 表达 Ly6Chi , 对 MHCII 是阴性的。之后, 培养的单核细胞体外与 50 ng/毫升 GM 脑脊液获得 DC。</li>
</ol>2. 从外周血中分离单核细胞源 DC
注意: 由于成熟的 MoDCs 在小鼠血液中比较少见, 下面的协议是指它们从排列的单核细胞中的体外派生。
<ol><li>为了增加血液中单核细胞的水平, 麻醉小鼠与4% 异氟醚, 并注射他们皮下 (南卡罗来纳州) 与1微克的 GM 脑脊液在 50 ul PBS。</li>
	<li>1-2 小时后, 使用 CO2来牺牲鼠标。 注意: 不要因为宫颈脱位而牺牲老鼠, 因为这会导致内出血。</li>
	<li>euthanization 后立即用70% 乙醇喷雾鼠标, 用手术剪刀将覆盖心脏的皮肤取出, 在无菌层流罩下。用乙醇清洁剪刀, 并切断心脏的右心房。慢慢地, 用20毫米 EDTA HBSS 用10毫升注射器和25克针将心脏通过右心室冲洗。<ol>
<li>用无菌注射器从胸腔内收集血液, 将其注入含有硫酸硫酸和 EDTA 的无菌管。继续冲洗心脏, 直到肝脏和肺部变成粉红色或白色。</li>
	</ol>
</li>
	<li>将血液应用到密度梯度 mediumand 离心机在400个区域合作框架在室温下的15分钟低刹车。 注意: 使用无内毒素密度梯度培养基 (通常小于0.12 的欧盟每毫升)。<ol>
<li>将单个核细胞收集到新的试管中。在10毫升 HBSS 中重新悬浮, 其中含有2% 的血清, 1% 青霉素/链霉素和10毫米 EDTA (隔离缓冲器), 然后离心在 4 oC 400 区域合作框架内以5-10 分钟的粒细胞。 警告: 仅使用含有葡萄糖至少1克/升的介质。</li>
	</ol>
</li>
	<li>对于 CD11b 正选择, 请在1毫升隔离缓冲区中重新挂起 1 x 108单元格, 并在 15 oC 上将其孵化为50分钟, CD11b-conjugated 磁性珠子的 4 ul。<ol>
<li>在 4 oc 中, 通过在400区域合作框架中添加9毫升的隔离缓冲和离心来清洗多余的珠子, 使其吸入上清液并在1毫升隔离缓冲区中重新挂起单元格。根据制造商的说明, 在水洗磁柱上应用单元格。</li>
		<li>将该列从磁铁中取出, 将6毫升的隔离缓冲液移到柱上, 然后通过推柱塞将磁性标记的细胞冲出无菌的收集管中。400个区域合作框架内的离心细胞为5-10 分钟, 4 oc. 用3毫升隔离缓冲器清洗该列两次。</li>
		<li>在 1 x 107 cells/100 ul 隔离缓冲区中重新挂起单元格, 并使用以下荧光共轭抗体进行着色: 0.1 微克 CD115、0.25 微克 MHCII 和0.1 微克 Ly-6C/1 x 106细胞。</li>
	</ol>
</li>
	<li>通过在小的侧向散射 (SSC) 和小正散射 (FSC) 单元格上的浇口对单元格进行排序。 注: 鼠炎症单核细胞一般定义为 CD115+/MHCIIlo/阴性/Ly6Chi, 而巡逻单核细胞则定义为 CD115+/MHCIIlo/阴性/Ly6C阴性 45。如果不需要两个子集之间的分离, 则可以排序总的 SSClo/FCSlo/CD115+/MHCII劳单元格。无论是天真的还是含肿瘤的小鼠, 都含有大约 5-8 x 104 MoDC 每1毫升的血液和多达 4-5 x 105 MoDC 注射了 GM-CSF 后。其中, 大约70% 个是炎症单核细胞, 30% 个在单核细胞内巡逻。</li>
	<li>在完全介质中, 将细胞集中在 1 x 106细胞/ml 上, 辅以 20 ng/毫升 GM-CSF。1天后, 将不粘附的和松散黏附的细胞转移到新的板块和文化中, 再增加4-5 天。 注意: 直流介质不应辅以丙酮酸。</li>
</ol>3. 肿瘤 IgG 免疫复合物的制备
<ol><li>培养肿瘤细胞在 75 cm2培养瓶到70% 汇合在完全 DMEM 媒介。<ol>
<li>添加2毫升0.25% 胰蛋白酶/EDTA 分离细胞从培养瓶和监测细胞形态学下的光显微镜, 以避免过度 trypsinization。</li>
		<li>添加8毫升完整培养基 (每2毫升胰蛋白酶), 以抑制胰蛋白酶消化, 离心机在4个oC, 400 区域合作框架, 以5-10 分钟的颗粒细胞。 注意: 请确保使用商业 PCR 试剂盒检查支原体的肿瘤细胞。使用鲎鲎裂解法 (拉尔) 化验28) 检测革兰阴性菌和真菌内毒素的存在。血清必须经过0.22 微米的过滤和测试9联邦法规病毒代码。此外, 通过在 LB 琼脂或肉汤上滴下 100-200 ul, 并在 37 oC 中培养2天, 测试培养基和血清。</li>
		<li>再次将细胞从胰蛋白酶和血清中重新悬浮在10毫升 PBS 和离心机中, 在400个区域合作框架内, 5-10 分钟. 吸出上清, 再重复洗涤2次。</li>
	</ol>
</li>
	<li>在室温下将1.8% 缓冲多聚甲醛中的细胞固定为10分钟。<ol>
<li>用10毫升 PBS 和离心机在4个oC 400 区域合作框架中重新悬浮以 5-10 分钟来清洗细胞。</li>
		<li>吸上清液和重复洗两次。 可选: 对于肿瘤摄取化验, 细胞可以标记为5分钟, 在 37 oC 在 PBS 含有1微米羧基琥珀酰亚胺酯 (CFSE)。CFSE, 然后用完整的介质淬火10分钟冰。在含有2% 血清的 PBS 中, 应广泛冲洗细胞以去除残留染料。</li>
	</ol>
</li>
	<li>重新悬浮细胞 (PBS) 缓冲 (公共广播辅助与 2% FCS + 5 毫米 EDTA) 和0.5 微克/毫升的 anti-CD16/32, 以阻止潜在的非特异蛋白蛋白相互作用。在 U 形96井/盘中的板材, 浓度为每 100 ul 1 x 105单元格。<ol>
<li>添加不同稀释的肿瘤结合抗体, 范围从5微克-5 ng/1 x 105细胞不等。把盘子在冰上孵化15-20 分钟。 注: IgG 抗体从天真的20-24 周大的雌性小鼠血清中分离出来的蛋白质 A 列, 如24所述。</li>
	</ol>
</li>
	<li>通过添加 150 ul 的 PBS 和离心 4 oC 400 区域合作框架中的板以 5-10 分钟来洗涤细胞。<ol>
<li>扔掉上清液, 重复洗两次。在含有荧光共轭二次抗体的 ul 100。在冰上孵化20分钟。</li>
		<li>通过增加 200 ul 的离心, 在400个区域合作框架中添加 5-10 分钟, 以洗涤细胞. 丢弃上清液并重复清洗。</li>
		<li>用流式细胞仪分析肿瘤的结合度, 确定细胞所需的最小浓度。 注意: 不应在随后的功能检测中使用不增加染色瘤细胞平均荧光强度 (同种) 的抗体。</li>
	</ol>
</li>
</ol>4. 用肿瘤 IgG 芯片活化 MoDC
<ol><li>涂层肿瘤细胞的 IgG 浓度最低, 如3.1 节所述, 通过3.4。3<ol>
<li>在激活 MoDC 与肿瘤 IC 的前一天, 取代 MoDC 培养基, 其中含有 GM-脑脊液。要做到这一点, 轻轻地吸入介质和洗涤细胞一次与预热完整的文化媒体。 注: 分离成熟的肿瘤相关 MoDC 应培养至少 2-3 小时 (甚至一夜之间) 后, 在完全的媒体排序, 没有 GM 脑脊液, 并在激活他们与 IC。</li>
		<li>对于肿瘤摄取分析, 将 CFSE 标记的肿瘤 IC 添加到 MoDC 中, 比例为 1:5 (IC: MoDC), 并在每 1 x 106 DC 的1毫升完整介质中, 隔夜孵化 12-16 小时。</li>
		<li>对于 MoDC 活化实验的血管化分析, 增加肿瘤 IC 在 1:1 (ic: MoDC) 比和孵化过夜 12-16 小时。 注意: 强烈建议包括一个积极的控制好, 其中 MoDC 刺激与1微克/毫升的 LPS 或其他 TLR 激动剂。</li>
		<li>在夜间激活后, 用隔离缓冲剂轻轻地吸入上清液和洗涤细胞三次, 或10毫米 EDTA HBSS。</li>
		<li>分离 DC 从板, 孵化细胞为 2-3 分钟在1毫升 HBSS 包含10毫米 EDTA, 并且分离细胞由剧烈的吹打。</li>
		<li>400个区域合作框架的离心细胞和 1 x 106 DC 在 90 ul PBS 补充了2% 个 FCS, 5 毫米 EDTA (资产管制署缓冲区) 和0.5 微克的阻断抗体。在冰上孵化 5-10 分钟。</li>
		<li>添加 10 ul 的染色抗体混合物的细胞和孵化在冰上15分钟。</li>
		<li>在4个oC 400 区域合作框架中, 向单元格和离心机中添加2毫升的流式缓冲区, 以 5-10 分钟。</li>
	</ol>
</li>
	<li>重新挂起 200 ul 资产管制器缓冲区中的单元格。<ol>
<li>在运行样品之前, 添加 0.5-1 微克/毫升 DAPI 1-2 分钟, 以排除死细胞的分析。不要过度孵化 DAPI, 因为 MoDC 会在 10-15 分钟内把它拿起来。</li>
	</ol>
</li>
</ol>

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J., van der Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+of+established+(pre)malignant+disease+by+synthetic+long+peptide+vaccines.">Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines.</a> Nat Rev Cancer. 8 (5), 351-360 (2008).</li><li>Palucka, K., Banchereau, 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+immunotherapy+via+dendritic+cells.">Cancer immunotherapy via dendritic cells.</a> Nat Rev Cancer. 12 (4), 265-277 (2012).</li><li>Bol, K. F., Schreibelt, G., Gerritsen, W. R., de Vries, I. J., Figdor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Cell-Based+Immunotherapy:+State+of+the+Art+and+Beyond.">Dendritic Cell-Based Immunotherapy: State of the Art and Beyond.</a> Clin Cancer Res. 22 (8), 1897-1906 (2016).</li><li>Rafiq, K., Bergtold, A., Clynes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+complex-mediated+antigen+presentation+induces+tumor+immunity.">Immune complex-mediated antigen presentation induces tumor immunity.</a> J Clin Invest. 110 (1), 71-79 (2002).</li><li>Schuurhuis, D. 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=Immune+complex-loaded+dendritic+cells+are+superior+to+soluble+immune+complexes+as+antitumor+vaccine.">Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine.</a> J Immunol. 176 (8), 4573-4580 (2006).</li><li>Carmi, 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=Allogeneic+IgG+combined+with+dendritic+cell+stimuli+induce+antitumour+T-cell+immunity.">Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity.</a> Nature. 521 (7550), 99-104 (2015).</li><li>Carmi, 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=Akt+and+SHP-1+are+DC-intrinsic+checkpoints+for+tumor+immunity.">Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity.</a> JCI Insight. 1 (18), e89020 (2016).</li><li>Kenkel, J. 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=An+Immunosuppressive+Dendritic+Cell+Subset+Accumulates+at+Secondary+Sites+and+Promotes+Metastasis+in+Pancreatic+Cancer.">An Immunosuppressive Dendritic Cell Subset Accumulates at Secondary Sites and Promotes Metastasis in Pancreatic Cancer.</a> Cancer Res. 77 (15), 4158-4170 (2017).</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>Roslansky, P. F., Novitsky, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Limulus+amebocyte+lysate+(LAL)+to+LAL-reactive+glucans.">Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans.</a> J Clin Microbiol. 29 (11), 2477-2483 (1991).</li><li>Jahr, H., Pfeiffer, G., Hering, B. J., Federlin, K., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxin-mediated+activation+of+cytokine+production+in+human+PBMCs+by+collagenase+and+Ficoll.">Endotoxin-mediated activation of cytokine production in human PBMCs by collagenase and Ficoll.</a> J Mol Med (Berl). 77 (1), 118-120 (1999).</li><li>Zhang, X., Morrison, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+selective+priming+effects+on+tumor+necrosis+factor+alpha+and+nitric+oxide+production+in+mouse+peritoneal+macrophages.">Lipopolysaccharide-induced selective priming effects on tumor necrosis factor alpha and nitric oxide production in mouse peritoneal macrophages.</a> J Exp Med. 177 (2), 511-516 (1993).</li><li>Hirohashi, N., Morrison, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+lipopolysaccharide+(LPS)+pretreatment+of+mouse+macrophages+modulates+LPS-dependent+interleukin-6+production+in+vitro.">Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 production in vitro.</a> Infect Immun. 64 (3), 1011-1015 (1996).</li><li>Deng, H., Maitra, U., Morris, M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+responsible+for+the+priming+of+macrophage+activation.">Molecular mechanism responsible for the priming of macrophage activation.</a> J Biol Chem. 288 (6), 3897-3906 (2013).</li><li>Cella, 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+novel+inhibitory+receptor+(ILT3)+expressed+on+monocytes,+macrophages,+and+dendritic+cells+involved+in+antigen+processing.">A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing.</a> J Exp Med. 185 (10), 1743-1751 (1997).</li><li>Kramer, P. R., Winger, V., Reuben, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+limits+TNF-alpha+production+in+CD16-activated+monocytes.">PI3K limits TNF-alpha production in CD16-activated monocytes.</a> Eur J Immunol. 39 (2), 561-570 (2009).</li><li>Rose, D. 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=Fc+gamma+receptor+cross-linking+activates+p42,+p38,+and+JNK/SAPK+mitogen-activated+protein+kinases+in+murine+macrophages:+role+for+p42MAPK+in+Fc+gamma+receptor-stimulated+TNF-alpha+synthesis.">Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.</a> J Immunol. 158 (7), 3433-3438 (1997).</li><li>Rezzonico, R., Imbert, V., Chicheportiche, R., Dayer, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+of+CD11b+and+CD11c+beta(2)+integrins+by+antibodies+or+soluble+CD23+induces+macrophage+inflammatory+protein+1alpha+(MIP-1alpha)+and+MIP-1beta+production+in+primary+human+monocytes+through+a+pathway+dependent+on+nuclear+factor-kappaB.">Ligation of CD11b and CD11c beta(2) integrins by antibodies or soluble CD23 induces macrophage inflammatory protein 1alpha (MIP-1alpha) and MIP-1beta production in primary human monocytes through a pathway dependent on nuclear factor-kappaB.</a> Blood. 97 (10), 2932-2940 (2001).</li></ol>

所有的作者都宣称他们没有利益冲突, 也没有什么可透露的。

鉴于接种疫苗的小鼠所需的 dc 数量很大 (大约 2-4 x 106 dc 每一只老鼠), 大多数小鼠的疫苗接种策略都是基于从 BM 和脾脏中分离出的 dc 和他们的前体激活。然而, 试图激活肿瘤 DC在体内, 使用相同的条件, 激活脾脏和 BM DC, 往往是不成功的产生有效的免疫力。在随后的两个出版物中, 卡米et al。发现, 血液和肿瘤 MoDC 有明显的差异, 从脾脏和 BM DC, 鉴于他们承担自然高的内在?...

自发现以来, 树突状细胞 (DC) 一直是广泛研究的焦点, 由于其独特的能力, 扭曲 T 细胞分化1。在过去几十年中, 广泛的研究工作试图定义各种 DC 子集及其在肿瘤进展和免疫中的作用2。DCs 由不同的细胞群组成, 它们在模式识别受体、组织分布和迁移和抗原表示能力方面各不相同,3,4,5。与其他 DC 子集相比, 单核细胞衍生 DC (MoDC) 在肿瘤中的丰富程度更大, 可以很容易地从循环或肿瘤浸润的单核细胞6,7中生成。因此, 许多试图利用其相对患病率的临床试验都是基于体内和体内操作自体 MoDC, 以诱导 T 细胞免疫89。
同样, 以 DC 为基础的实验性肿瘤模型接种需要2-3 系列注射, 5-7 天间隔, 1-2 x 106激活 DC 脉冲与肿瘤抗原。因此, 为了实现这一大数目的 DC, 大多数老鼠的研究主要使用 MoDC 培养的骨髓 (BM) 前体在 GM-CSF 7-9 天 (IL-4 是不需要在鼠标设置)10,11。尽管如此, 鉴于 GM-CSF 挖空小鼠有整体正常 DC 隔间12,13, 并考虑到从该区域性获得的混合种群,14这些 DC 的生理相关性已被质疑。
或者, DC 可能经常被隔离从脾脏细胞。然而, DC 只包含大约 0.3-0.8% 的总脾脏细胞 (导致大约 7 x 105 dc 或脾脏) 和这些细胞, 只有 CD103+ DC 和 MoDC 可以迁移回到淋巴器官。由于 MoDCs 包括大约10-15% 的脾脏 DC 人口15,16, 大多数隔离协议产生大约 1 x 105 MoDC 每脾脏。MoDC 的扩张可以通过注射转染 B16 细胞分泌 GM-脑脊液来实现, 导致脾脏 MoDC17增加100倍。然而, 使用 MoDC 开发 DC 疫苗是有限的, 因为这个程序不能在人类和获得的 MoDC 已经高度激活。
除了获得足够数量的 dc, 开发有效的 dc 疫苗抗自体癌细胞的另一个挑战是缺乏足够的危险信号在肿瘤的设置, 以充分激活 dc。诱导的共同刺激信号通常是通过激活模式识别受体 (PRR), 或 c 型凝集素信号通路18,19,20,21。另外一种激活 DC 的方法利用它们通过与表面 Fcγ受体 (FcγR) 的相互作用来处理抗原的能力。事实上, 一些重要的手稿表明, 注射 MoDC 的 BM 前体激活的肿瘤 IgG 芯片可以防止肿瘤生长的预防性设置, 并可导致根除已建立的肿瘤22,23.
在最近的两篇论文中,卡米等发现, 与 BMDC 和脾 DC 相反, MoDC 从血液和肿瘤不能反应的 IgG IC 没有额外的刺激。发现这是由于存在高胞内水平的酪氨酸磷酸调节 FcγR 信号24,25。通过在 dc 中定义一个关键的检查点, 这项工作提供了对成功的基于 dc 的疫苗接种要求的重要洞察力。需要额外的刺激, 以使 FcγR 信号, 并推测信号从其他凝集素受体使用类似的磷酸化级联, 从而强调了避免在其孤立的 DC 启动的必要性。
因此, 本议定书描述了从血液和肿瘤 MoDC 的分离, 这与 BM 和脾脏 DC 明显不同, 并强调了在这一过程中值得考虑的预防措施。

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

树突状细胞 (DC) 是不同的细胞群, 它们的细胞膜标记物, 迁移模式和分布, 并在其抗原表示和 T 细胞活化能力。由于大多数实验性肿瘤模型的接种都需要数以百万计的 DC, 所以它们被广泛地从骨髓或脾脏中分离出来。然而, 这些 dc 明显不同于血液和肿瘤 DC 在他们的反应免疫复合物 (IC), 大概是其他 Syk 耦合凝集素受体。重要的是, 鉴于 dc 对危险相关分子的敏感性, 在隔离步骤之一中交联激活受体的内毒素或抗体的存在可能导致 DC 的启动, 从而影响参数, 或者至少剂量, 需要激活他们。因此, 我们在这里描述一个详细的协议, 以隔离 MoDC 从血液和肿瘤, 同时避免其过早活化。此外, 还提供了一种用于肿瘤 IC 的 MoDC 活化的协议及其后续分析。

我们最初比较了来自天真自体和同种异体小鼠的抗体与肿瘤细胞的结合能力。为此, B16F10 和 LMP 肿瘤细胞系固定在多聚甲醛和广泛洗涤。B16F10 是黑色素瘤细胞系, 最初是从 C57Bl/6 小鼠的肺转移中分离出来的。LMP 是从 KrasG12D/+、LSL-Trp53R172H/+ 和 Pdx-1-Cre 小鼠中分离出来的胰腺肿瘤细胞, 在129F1 小鼠体内生长稳定。为了获得 IC, 肿瘤细胞在冰上孵化20分钟, 每 1 x 105肿瘤细?...

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Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

单核细胞衍生的 DC (MoDC) 可以感觉到少量的危险相关分子, 因此很容易被引。我们提供了一个详细的协议, 以隔离 MoDC 从血液和肿瘤和他们的活化与免疫复合体, 同时强调应考虑的关键预防措施, 以避免其过早激活。

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

11.3K 次观看.  Tel-Aviv University. 单核细胞衍生的 DC (MoDC) 可以感觉到少量的危险相关分子, 因此很容易被引。我们提供了一个详细的协议, 以隔离 MoDC 从血液和肿瘤和他们的活化与免疫复合体, 同时强调应考虑的关键预防措施, 以避免其过早激活。

视频: Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

Ex 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

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. ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

复杂核型细胞周期捕获细胞的生成与分离

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

正常结肠和结直肠癌患者内皮细胞的分离--一种改进的协议

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

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 ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...