这项工作得到了 NIH 赠款 CA194013、CA192064 和 U54GM104942 的部分支持。WVCTSI 被确认为 VVK、AB 和 TDE 的启动。作者感谢 m. Gencheva 博士和 k. Steinberger 的帮助与说明性实验。内容完全是作者的责任, 不一定代表 NIH 的官方观点。

所有的动物工作都是按照西弗吉尼亚大学 IACUC 批准的议定书进行的。
1. 探头的合成与标定
<ol><li>微粒pO2敏感的林肯-小波探针 注意:林肯-小波纳米微晶的合成和制备如参考13 中所述。他们是非常稳定的, 可以保持在室温下多年。林肯-小波微粒探针的 EPR 线宽是一个pO2敏感参数。林肯-小波纳米微晶显示了理想线性依赖性的线宽对氧气浓度的范围从缺氧条件高达 760 mmHg的pO 2 分部压力13, 与内在线宽的值在缺席氧和斜坡的氧依赖性 (以 mG/mmHg) 略有变化的不同批次的晶制剂。因此, 每个特定批次都需要校准。<ol>
<li>重60毫克的林肯-小波纳米微晶。</li>
		<li>对于氧敏感性校准, 暂停纳米微晶在3毫升的 Dulbecco 的修改鹰介质 (DMEM) 介质 (在浓度20毫克/毫升) 和油脂实验5分钟的冰与探针 sonicator 20 赫使用7瓦特的力量在5毫升玻璃圆底管。</li>
		<li>将微气泡纳米微晶的1毫升放在 L 波段 (1.2 GHz) EPR 光谱仪的表面线圈谐振器中, 并获得连续波 (CW) epr 谱在37°c 的生理温度和氧浓度 0, 1, 2, 4, 8和20.9%。通过用气体控制器提供的气体混合物冒泡溶液, 保持氧气浓度, 并使用恒温器上的水浴保持温度。使用以下 EPR 光谱仪采集参数: 调制振幅, 100 毫克;调制频率, 100 赫;扫宽, 5 克;扫时间, 六十年代。</li>
		<li>为了达到更好的信噪比 (信噪比), 使用调制幅度值的60% 的线宽 (例如, 使用调制振幅 0.6 g 的线宽为1克)。</li>
		<li>替代简化校准程序: 记录空气冒泡和缺氧解决方案中的 EPR 光谱14。在后一种情况下, 根据参考14, 增加10毫米葡萄糖和 100 U/毫升葡萄糖氧化酶到1毫升探针溶液中, 保持样品中的缺氧。</li>
		<li>用洛伦兹函数拟合 EPR 谱, 求出线宽、长波。 将纳米微晶的灵敏度评估为pO 2 作为在po2 上的依赖的斜率, 即作为值 (空气−LW 缺氧)/p o 2air, 其中,和长波缺氧分别为空气和缺氧条件下的光谱线宽;pO2air = 152 mmHg。</li>
	</ol></li>
	<li>双功能 pH 和氧化还原探针, NR 注意:NR 探针是按照参考11中的描述合成的。它是稳定的室温, 无论是固体和水溶液。合成的 NR 探针保持在4摄氏度。氮超精细分裂, 一个 N 和信号振幅衰减率是对 pH 敏感的 NR 探针的光谱参数 (探针 pK a = 6.6 在37°c, pH 敏感性的范围从5.6 到 7.6) 和探针的减少容量 微环境, 分别。<ol>
<li>从冰箱中取下 NR, 让容器温暖到室温 (10-15 分钟)。重量6.34 毫克的 NR, 溶解在1毫升盐水溶液, 并调整 ph 值为7.2 与小整除数盐酸或氢氧化钠使用 pH 计。使用准备好的 NR 溶液 (10 毫米) 作为库存解决方案。</li>
		<li>执行 NR 探针的 pH 校准, 如下所示 (请参阅参考11)。首先, 添加0.1 毫升的 NR 库存解决方案, 0.9 毫升2毫米钠磷酸盐缓冲, 150 毫米氯化钠。滴定获得的 1 mM NR 溶液与整除数的盐酸或氢氧化钠的 ph 值, 使用 ph 表。使用附加在恒温器上的水浴控制温度。</li>
		<li>用 L 波段 EPR 光谱仪记录样品在1.5 毫升离心管内的 EPR 谱。使用以下 EPR 光谱仪采集参数: 调制振幅, 2.5 克;调制频率, 100 赫;扫宽, 60 克;扫时间, 二十年代。</li>
		<li>测量超精细分裂常数 (N) 作为 EPR 谱的低和高场分量之间距离的一半, 并绘制它与 ph 值之间的间距, 以提供 pH 值的 L 波段 EPR 测量的校准曲线.</li>
	</ol></li>
	<li>谷胱甘肽敏感 RSSR 探针 注意:RSSR 探针是按照参考15中的描述合成的。将合成的 NR 探针贮存在摄氏4摄氏度。亲脂 RSSR 二硫化双基化合物容易扩散到细胞膜上, 以反应细胞内谷胱甘肽, 并提供了一个可靠的方法, 以确定谷胱甘肽在体内使用 EPR16,17。该方法以 RSSR 探针硫醇谷胱甘肽的主要胞内池的高反应速率为基础。RSSR 双基与谷胱甘肽的反应分裂其二硫化物键 (参见方案 1) 导致两个基片之间的自旋交换被取消, 并在双基谱分量的减少和相应的增加 monoradical 成分。对于双基 RSSR 探针, monoradical 分量振幅的增加率与 gsh 浓度成正比, 是一种方便的谷胱甘肽敏感的 EPR 光谱参数。要评价谷胱甘肽浓度从体内EPR 测量, 前校准率的 RSSR 反应与 GSH 在相应的温度和 pH 值必须执行如下。<ol>
<li>从冰箱中取出 RSSR, 让容器温暖到室温 (10-15 分钟)。在1毫升的亚砜溶液中称量4.05 毫克的 NR, 并将其溶解。使用准备好的 RSSR 解决方案 (10 毫米) 作为库存解决方案。</li>
		<li>确定 RSSR 与 GSH 反应的速率常数 (kobs) 在理想的温度和 pH 值下的数值, 如下所示。</li>
		<li>首先, 添加20µL 的 RSSR 库存溶液 (10 毫米) 到0.98 毫升1毫米的磷酸盐缓冲液, pH 7.2, 150 毫米氯化钠, 获得0.2 毫米 RSSR 探针溶液。</li>
		<li>在 pH 值为7.2 的0.1 米磷酸酯缓冲液中, 制备1、2和5毫米 GSH 浓度的溶液。为了准确地评估靶器官细胞中的 GSH 浓度, 从体内测量中,体外校准必须在 ph 值接近细胞内酸碱度的价值时进行。</li>
		<li>混合等体积的0.2 毫米 RSSR 溶液和一个谷胱甘肽溶液中制备的步骤1.3.4。为探针的最后集中在0.1 毫米和谷胱甘肽在 0.5, 1, 或者2.5 毫米。</li>
		<li>RSSR 和谷胱甘肽溶液混合后立即将样品放入 epr 谐振腔中, 每12秒记录一次 epr 光谱10分钟。然后计算 monoradical 谱振幅增加的动力学。使用以下 EPR 光谱仪采集参数: 调制振幅, 1 克;调制频率, 100 赫;扫宽, 60 克;扫时间, 10-60 秒。</li>
		<li>将所测的 EPR 动力学与 monoexponents 进行拟合, 并计算指数动力学τ的时间常数。线性回归 (1/τ = kobs x [GSH]) 提供了观察到的速率常数值的反应, 谷胱甘肽和 RSSR (例如, 在34摄氏度和 pH 值 7.2, kobs = 2.8 @ 0.2 M-1s-1)11。</li>
	</ol></li>
	<li>pO2、pH 值和 Pi 评估的多功能希望探头 注意:monophosphonated 的三苯甲酯希望探针是按照参考12中的描述合成的, 并保持在4摄氏度。在 ph < < pKa (a 酸型) 和 ph > > pKa (B 基本形式) 中, 希望的 CW EPR 谱表示由双峰, 由于磷超精细分裂, 一个 P.典型的仪器设置如下: 调制振幅, 37.5 毫克;调制频率, 100 赫;扫宽, 0.9 克;扫时间, 20-60 秒。在中间 pH 值 (5 < pH < 8) 中, 当 a 和 B 状态存在时, 希望探针的 EPR 谱为四重唱。希望的单个 EPR 线宽是pO2标记 (精度, ≈ 1 mmHg;pO2范围, 1-100 mmHg)。质子希望 (一种形式) 的分数是 pH 值, 范围从6到 8.0 (精确度, 0.05)。质子交换速率 (用 mG 表示) 与通过光谱模拟提取的 pi 值是一个 pi 标记 (精度, 0.1 毫米, 范围, 0.1-20 毫米)。校准程序是在生理温度 (37 °c), 溶液离子强度 (NaCl, 150 毫米), 和希望探针浓度0.2 毫米, 如前所述引用12,18, 并详述如下。<ol>
<li>从冰箱中取出希望探头, 让容器温暖到室温 (10-15 分钟)。</li>
		<li>称10.7 毫克的希望探针, 溶解在1毫升盐水溶液, 并调整 pH 值为7.4。添加20µL 的希望 (10 毫米) 到0.98 毫升盐水溶液的准备库存解决方案, 以获得 0.2 mM 希望探针解决方案。</li>
		<li>对于探针校准的 pH 值, 滴定0.2 毫米的希望探针溶液中添加了少量的氢氧化钠或 HCl, 最后稀释的样品少于1%。使用国家标准局 (美国) 推荐的参考溶液的 ph 值, 测量 ph 为37°c 的 ph 电极。使用附在循环器上的套接反应烧杯, 在 pH 测量过程中小心地保持参考和滴定溶液的温度。在探针溶液中加入10毫米葡萄糖和 100 U/毫升葡萄糖氧化酶, 保持缺氧状态。</li>
		<li>在缺乏磷酸盐的情况下, 在缺氧条件下, 分别获得 A 和 B 形式的 EPR 谱 (ph ≤5和 ph ≥ 8)。</li>
		<li>利用相应的光谱获得固有的光谱参数。即, 将谱线作为洛伦兹函数的卷积, 与期望探针的未解析超超精细结构近似的高斯函数进行模拟。计算的 EPR 谱与实验谱的拟合产生P和洛伦兹线宽 (ΔLpp) 的值, 由横向松弛率决定, 1/t2 (其中 1/t2 = (√3/2) Lpp用于连续波 EPR 中射频吸收线的测量导数), 以及高斯分布的宽度, G。 注意:以下是根据步骤1.4.2 中指定的条件测量的光谱所获得的参数:p(a) = 3.63 G, 一个p(B) = 3.37 g;1/吨2(A) = 23.6 毫克;1/吨2(B) = 9 毫克;G (A) = 40 毫克;G (B) = 45 毫克 (参见参考8)。</li>
		<li>获取希望在中间 pH 值 (5 < pH < 8) 中的 EPR 谱 .利用在非耦合或松散耦合系统中的多个站点之间的交换理论, 根据先前描述的 18, 模拟获得的 EPR 谱的高场分量。使用为 A 和 B 状态获取的内部参数 (请参见步骤 1.4.5) 以减少变量数。将计算出的光谱与实验结果相匹配, 以查找分数 A (pa) 的值, 并将 pA值的依赖性绘制为 pH。将 pA的依赖性用于进一步研究中的 ph 值校准曲线。 注意:用标准滴定曲线拟合 pA的 pH 依赖性提供了离解常数的值, pKA (希望) = 6.98。在体内研究中, 获取希望探针的完整 EPR 谱是不切实际的, 因为需要额外的时间来获得 EPR 谱中磷超精细分裂的低和高场分量之间的间隙。因此, 在进一步的实证研究中, 仅对 EPR 谱的高场分量进行了测量和分析。</li>
		<li>对于pO2的探针校准, 获取希望探针在各种氧浓度下的 EPR 谱。</li>
		<li>通过使用气体控制器提供的混合气体冒泡来控制解决方案的pO2值。使用附加在恒温器上的水浴控制溶液温度 (37 °c)。</li>
		<li>模拟 EPR 谱并将其与步骤1.4.6 中描述的实验结果相匹配, 以确定氧诱导松弛率的值。 注意:氧诱导松弛率的值为0.49 毫克/mmHg 的希望探针的 A 和 B 形式, 测量37°c8。</li>
		<li>对于 [Pi] 的探针标定, 在各种磷酸盐浓度下获得希望探针的 EPR 谱。使用在 pka (pka = 6.9 在37°c)18和滴定它与各种磷酸盐浓度附近的 pH 值的希望根治解决方案。按照上述步骤, 保持温度和气体组成。</li>
		<li>模拟 EPR 谱, 并将其与步骤1.4.6 中描述的实验结果相匹配, 以确定 Pi 引起的汇率值。 注意:在进一步研究中, pi 诱导汇率对 pi 的依赖性被用作校准。</li>
	</ol></li>
</ol>2. 乳癌的小鼠模型
<ol><li>MMTV-PyMT 自发性肿瘤模型<ol>
<li>使用4-8 周大的女性朋友病毒 B 型易感性/NIH (FVB/N) 小鼠乳腺肿瘤病毒启动子 (MMTV) 多瘤中 T 抗原 (PyMT +) 小鼠自发形成乳腺肿瘤为体内 EPR 研究. </li>
		<li>为比较正常乳腺和肿瘤的组织微环境, 请使用 PyMT 癌基因 (PyMT−、"野生型") 20 中缺乏年龄匹配的 littermate 女性.</li>
		<li>在异氟醚麻醉 (见下面的探针传递) 的情况下, 每周在四周内对小鼠进行 L 波段 EPR 光谱学。</li>
		<li>麻醉使用空气异氟醚 (3% 异氟烷), 并将鼠标放在一个可调节的桌子上, 右, 侧位与肿瘤 (乳腺) 接近表面线圈谐振器。</li>
		<li>在鼠标放置后, 通过 intratissual (it) 注入进行探针管理, 调整 epr 光谱仪, 获得5-10 分钟的 epr 谱。</li>
		<li>在同一 EPR 会议期间, 测量2-3 乳腺肿瘤 (从 MMTV-PyMT + 小鼠) 或非肿瘤携带乳腺 (从 PyMT−小鼠)。</li>
	</ol>
</li>
	<li>原位 MET-1 肿瘤模型<ol>
<li>生长 FVB/N 背景 Met-1 小鼠乳腺癌细胞在37摄氏度, 5% CO2和95% 相对湿度在 DMEM 含有10% 胎牛血清 (血清), 10 µg/毫升胰岛素, 5 ng/毫升 rhegf 促, 1% PSA (青霉素 G 钠, 硫酸链霉素, 和两性霉素 B), 以80% T175 瓶汇合。</li>
		<li>用10毫升 PBS (1.54 毫米的2PO4, 155 毫米氯化钠, 2.71 毫米 Na2HPO4-7H2O, 没有氯化钙或氯化镁, pH 值 = 7.4), 吸入介质并冲洗附着细胞。</li>
		<li>通过添加5毫升0.25% 胰蛋白酶-EDTA 溶液和摇瓶, 分离细胞。当细胞分离时, 加入10毫升 DMEM, 其中含有10% 的血清, 并收集细胞。</li>
		<li>离心机的细胞悬浮在 132 x g 10 分钟4摄氏度。使用 hemocytometer 和并用重悬将每个单元格计数为每100µL 最小 DMEM 的 1 x10 6 单元格。</li>
		<li>使用胰岛素注射器 (29G1/2 针), 慢慢地将100µL 肿瘤细胞悬浮液注入8周大的雌性 FVB/N 型野生小鼠的4乳房脂肪垫中。</li>
		<li>通过触诊 (大约2-3 周后出现)、生长 (视觉) 和老鼠健康 (视觉) 来监测肿瘤的起始。</li>
		<li>使用卡尺测量每星期一次肿瘤尺寸, 并使用该方程确定肿瘤体积: <img alt="Figure " src="/files/ftp_upload/56624/56624eq1v2.jpg">
</li>
	</ol>
</li>
</ol>3. 对体内功能测量的探针传递
<ol><li>使用微粒林肯-小波探针 (协议 I) 用于 pO2在原位肿瘤模型中植入肿瘤细胞, 并将其内化的林肯-小波纳米微晶作为先前描述的14, 21, 并详述如下.<ol>
<li>在 MET-1 肿瘤模型的情况下, 为林肯-小波纳米微晶内化成 MET-1 细胞, 在小波中暂停林肯-纳米微晶 DMEM, 浓度为20毫克/毫升, 油脂实验与探针 sonicator 在20赫上使用 7 W 功率在5毫升玻璃圆底管在冰上5分钟。</li>
		<li>添加100µL (2 毫克林肯-小波) 的悬浮到一个 T75 瓶与10毫升培养基含有 MET-1 细胞 (约30% 汇合)。所有的程序都发生在一个安全的内阁和媒体含有青霉素和链霉素, 以尽量减少潜在的感染。</li>
		<li>孵化细胞在37°c 72 小时, 直到他们达到80% 融合。</li>
		<li>吸媒体。用10毫升 PBS 冲洗细胞五次。用5毫升胰蛋白酶-EDTA 分离细胞。收集细胞。离心机如上文所述的步骤2.2.4。用排除染料染色细胞的样本, 以确定细胞的生存能力和数量。</li>
		<li>将单元格以 1 x 106的浓度 (每100µL 最小 DMEM) 暂停。</li>
		<li>使用胰岛素注射器, 慢慢地注入100µL 细胞悬浮液, 其中包含了林肯-小波纳米微晶的4乳房脂肪垫8周大的雌性 FVB/N 野生型小鼠, 如步骤2.2.5 所述。</li>
		<li>监测肿瘤的起始和生长情况, 如步骤2.2.6 和2.2.7 中所述。</li>
	</ol>
</li>
	<li>使用微粒林肯-小波探针 (协议 II) 在自发或原位模型。将林肯-OBu 纳米微晶放在感兴趣的地点,例如, 使用胰岛素注射器注入正常乳腺或乳腺肿瘤。</li>
	<li>可溶性探头<ol>
<li>通过麻醉机吸入空气异氟醚混合物 (1.0 升/分和2-3% 异氟醚) 麻醉小鼠, 并将其置于 EPR 光谱仪的间隙。</li>
		<li>调整仪器, 然后注入 NR (10-30 µL, 10 毫米), 希望探针 (10−30µL, 0.5-2 毫米) 在盐水, pH 7.2, 或 RSSR 探针的亚砜 (10 µL, 10 毫米) 解决方案 (it)。</li>
	</ol>
</li>
</ol>4.体内功能测量
<ol><li>对于 EPR 光谱测量, 麻醉小鼠吸入空气异氟醚混合物使用麻醉机, 如步骤3.3.1 所述。</li>
	<li>使用 L 波段 (1.2 GHz) EPR 光谱仪进行功能测量, 如下所示。<ol>
<li>将表面线圈谐振器放到正常乳腺或乳腺肿瘤上, 调整光谱仪。</li>
		<li>在植入后数周内, 从植入微粒探针中获取5−10的 EPR 谱。在可溶探针的情况下, 探针注入后立即获得 EPR 谱5-10 分钟。</li>
		<li>分析 NR 探针的 EPR 谱, 找出超精细分裂, 一个 N 和信号振幅, I (t).使用在步骤1.2.4 中获得的校准曲线, 将N的值转换为 pH 值。分析了信号振幅 i (t) 的衰减率与初始振幅的相对变化, i (t = 0), 以每秒任意单位 (s-1) 计算。</li>
		<li>拟合谷胱甘肽敏感 RSSR 探针的 EPR 谱 monoradical 分量的增加, 以获得指数动力学的时间常数来计算 gsh 浓度。</li>
		<li>将多功能希望探针的高场分量的 EPR 谱与实验结果 (步骤 1.4.5) 相匹配, 以产生 pH 值, pO2和 Pi。</li>
	</ol>
</li>
</ol>5. 统计分析
<ol><li>进行数据处理和统计分析。使用皮尔逊的 r 相关测试 (对于通常分布的数据集) 和长矛的秩阶相关 (对于数据分布被拒绝正态分布的数据集) 进行相关分析。</li>
</ol>

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作者没有什么可透露的。

所提出的方法允许对化学药品的关键参数进行无创的体内评估, 即pO2, pH 值, 氧化还原状态, 以及间质 Pi 和胞内谷胱甘肽的浓度。磁共振技术, 如 MRI 和低场强 EPR, 是在这些参数的无创体内分析的选择方法。MRI 能直观地解剖结构, 但缺乏功能敏感性。与 MRI 相比, EPR 技术在与功能旋转探针结合使用时, 对微环境的局部参数提供了功能敏感性。根据我们的知识, 没有其他方法可以同时评估活体中的体内生理上重要的化学参数, 如pO2, pHe, Pi, 氧化还原和 GSH。
所描述的协议是基于低场 L 波段 EPR 和特别设计的顺磁探头。图 1中提供的一组四探针可以被认为是有效的体内评估方法。探头的可用性是描述的体内EPR 测量的一个重要先决条件。探头的合成根据已发布的过程11,12,13,15需要在合成有机化学方面的专业知识, 并可能成为方法实现的关键步骤.
微粒探针的应用使组织pO2的重复测量能够在植入1421之后长达几周。可溶性探针显示了对超出氧浓度25以外的参数的扩展的敏感性, 并为空间解析的测量26提供了一个机会。特别是, 应用多功能希望探针为体内同时测量 pH 值、 pO2和胞外空间 Pi 提供了无与伦比的机会。使用单个探针测量三参数, 可以独立于探针分布和测量的时间8进行相关分析。
探头不能同时使用, 除了注入的林肯-小波微粒探针, 它可以与任何可溶探针结合使用, 如图 6所示。因此, 实验设计应基于单独的测量个别注射特定功能的可溶性探针。亲水性胞外 pH 值和氧化还原 NR 探针可以通过 it 注射11,14,26和系统交付27, 而其他两个可溶探针, RSSR 和希望, 只允许 it 交付8,11,12,16. 请注意, RSSR 和希望探测器的未来结构修改可能会通过开发更亲水和毒性较小的探针类似物来解决系统交付25中的后置探针交付限制。
请注意, 光谱 EPR 模式提供了测量的参数的平均值, 这通常以其高异质性为特征。这部分掩盖了参数之间的潜在差异或降低了相关分析的意义。在成像模式中使用所描述的探测器可以获得空间解析的功能信息26。我们相信, 未来的多功能 EPR 成像依赖于进一步发展的相对新方法, 如快速扫描 EPR 成像和 Overhauser 增强 MRI (暗, 也称为质子-电子双共振成像, PEDRI)。最近在相应的专题文章25中讨论了功能 EPR 探针和高级成像技术发展的新方向。

在癌症进展和治疗中的关键作用越来越被赞赏1。在实体肿瘤的重要生理参数中, 组织缺氧2, 酸中毒3,4, 高还原容量5, 细胞内谷胱甘肽6,7,和间质 Pi8有很好的记录。无创性的体内 pO2, pH 值, Pi, GSH 和氧化还原评估提供了独特的洞察力的生物学过程中, 并帮助先进的工具, 临床前筛查抗癌药物和有针对性的治疗策略。通过磁共振成像 (MRI) 和低场强的 EPR 技术, 在组织中合理的射频穿透深度使它们成为无创评估这些参数的最合适方法。MRI 在很大程度上依赖于成像水质子, 并广泛应用于临床设置, 提供解剖分辨率, 但缺乏功能性分辨率。phosphorus-31 核磁共振 (31p-NMR) 测量胞外 Pi 浓度和 pH 值基于来自内源磷酸盐的信号可能有吸引力的特征, 但通常掩盖了几次更高的胞内 Pi 浓度9,10。与此相反, EPR 测量依赖于特殊设计的顺磁探针的光谱学和成像, 以提供功能分辨率。请注意, 由于 epr 的内在敏感性和内生背景 epr 信号的缺乏, 外源 epr 探针比外源核磁共振探针具有更大的优越性。最近开发的双功能 pH 值和氧化还原基团探针11和多功能的三苯基碳探针12为在体内同时测量数个参数提供了无与伦比的机会及其与探针分布和测量时间无关的相关分析。根据我们的知识, 没有其他方法可以同时评估活体中的体内生理上重要的化学参数, 如pO2, pHe, Pi, 氧化还原和 GSH。
探测体内功能测量:
图 1显示用于访问参数的顺磁探头的化学结构, 其中包括微粒和可溶探针。高功能敏感性, 活组织的稳定性和极小的毒性是一些好处, 使微粒探针优于可溶性探针的体内EPR 氧。例如, 微粒探针增加了组织植入部位的保留时间, 与可溶性探针相比, 允许在数周内对组织pO2进行纵向测量。另一方面, 可溶探针通过提供基于 EPR 的成像技术的空间分辨测量, 以及允许多种功能 (pO2、pH 值、Pi、氧化还原和GSH)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56624/56624fig1.jpg"> 图 1。装配评估试验的顺磁探针的化学结构.这包括微粒pO2探针, 林肯-小波 (R = O (CH2)3CH3) 和可溶性探针: 双重作用 pH 值和氧化还原探针, NR;谷胱甘肽敏感探针, RSSR;和多功能pO2, pH 和 Pi 探针的胞外微环境, 希望探针.在提供的引用11、12中描述了这些探测器的合成。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" width="703">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-band EPR spectrometer</td>
			<td style="width:201px;">Magnettech, Germany</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">L-band (1.2 GHz) electron paramagnetic resonance (EPR) spectrometer for collection in vitro and in vivo spectra of paramagnetic molecules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;Temperature &#38; Gas Controller&#160;</td>
			<td style="width:201px;">Noxygen, Germany</td>
			<td style="width:119px;"></td>
			<td>Temperature &#38; Gas Controller designed to control and adjust the temperature and gas composition&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator</td>
			<td style="width:201px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GSH (L-Glutathione reduced)</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td style="width:119px;">G4251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MMTV-PyMT&#160; mice</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Met-1 murine breast cancer cells</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6 wild type mice&#160;</td>
			<td style="width:201px;">Jackson Laboratory</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Exclusion Dye&#160;</td>
			<td style="width:201px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohmeda Fluotec 3&#160;</td>
			<td style="width:201px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoflurane (IsoFlo)</td>
			<td style="width:201px;">Abbott Laboratories</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:201px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate monobasic</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S07051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrochloric acid</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Hydroxide</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;">S8045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose oxydase</td>
			<td style="width:201px;">sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lauda Circulator E100</td>
			<td style="width:201px;">Lauda-Brikmann</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH meter Orion</td>
			<td style="width:201px;">Thermo Scientific&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiNc-BuO probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The Octa-n-Butoxy-Naphthalocyanine probe was synthesizided according to ref 13</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NR probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The Nitroxide probe was synthesizided according to ref 11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RSSR probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The di-Nitroxide probe was synthesizided according to ref 15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOPE probe</td>
			<td style="width:201px;">In house</td>
			<td style="width:119px;"></td>
			<td>The monophoshonated Triarylmethyl probe was synthesizided according to ref 12</td>
		</tr>
	</tbody>
</table>

in vivo epr assessment of ph, po2, redox status, and concentrations of phosphate and glutathione in the tumor microenvironment

该协议表明, 低场电子顺磁共振 (EPR) 技术的能力, 结合功能顺磁性探针, 提供定量信息的化学肿瘤微环境, 包括pO2, pH 值, 氧化还原状态, 间质无机磷酸盐 (Pi) 和胞内谷胱甘肽 (GSH) 的浓度。特别是, 最近开发的可溶解多功能三苯甲的应用为 pH的体内并行测量提供了无与伦比的机会, pO2和pi 在Extracellular 空间 (希望探针)。用单个探针测量三参数, 可以独立于探针分布和测量时间来进行相关分析。

组织pO2使用林肯-小波探针进行评估:
使用步骤1.1 中描述的过程, 我们进行了新制备的林肯-小波纳米微晶悬浮液的标定。图 2显示了林肯-小波探针的线宽的典型氧依赖性, 以及其在缓冲悬浮液中测定的 EPR 谱和在注射细胞肿瘤启动后生长的雌性 C57Bl/6 小鼠乳腺肿瘤组织中的表现。与内化粒子 (步骤 3.1)。使用线宽校准可以计算pO2值 (相当于 图 2中显示的频谱 (b) 的 7.5 mmHg)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56624/56624fig2.jpg"> 图 2.小波粒子的探测器对氧气的 EPR 光谱灵敏度.显示了 DMEM 中小波探针悬浮液的 EPR 谱线宽对氧分压的代表性依赖性。EPR 频谱 (插入a) 测量了0% 的pO2, 37 °c, 并表示一个纯洛伦兹线与宽频带ΔHpp = 113 毫克。线宽随pO2线性增加, 斜率为11.7 毫克/mmHg。所示的频谱 (插入b) 测量了麻醉雌性 C57Bl/6 小鼠乳腺肿瘤组织显示ΔHpp = 204 毫克, 这对应于组织pO2 7.5 mmHg。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
用 NR 探针评价组织胞外 pH 和氧化还原:
使用步骤1.2 中描述的过程, 我们进行了 pH 和氧化还原探针的校准, NR。图 3显示了在 pH 值上的 NR 基的观察到的超精细分裂常数依赖性, 一个 N.依赖性由一个标准滴定曲线描述以 pKa = 6.6 在37°c。N(pH 值) 的这种依赖性作为对应的体内测量的校准曲线, 允许 ph 值从5.6 到7.6 不等, 准确度为 0.05 ph 单位。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56624/56624fig3.jpg"> 图 3.可溶性 NR 探针对 pH 值的 EPR 光谱灵敏度.以不同 ph 值37°c 获得 NR 探针溶液的 l-波段 EPR 谱, 并绘制了观察到的超精细分裂常数 (N) 的 pH 依赖性.实线是与标准滴定曲线相吻合的实验数据,, 产生 pKa = 6.6,N(NR H <img alt="Equation 2" src="/files/ftp_upload/56624/56624eq2.jpg"> </img>+) = 14.24 G,N(R) = 15.27 g 插入: 已获得的 1 mM NR 溶液的 EPR 谱 (pH 6.4)使用以下光谱仪设置: 磁场扫, 80 G;调制振幅, 2.5 G, 承购时间, 二十年代。被测量的超精细分裂常数是 14.63 g 从参考23复制了以约翰威利 & 儿子公司的允许 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig3large.jpg" target="_blank">请单击此处查看此图的较大版本</a>。
图 4举例说明了乳腺肿瘤小鼠模型中的 pH 值和氧化还原测量, 如步骤 3.3 (it 探针注入) 和4节 (光谱采集和频谱分析) 所述。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56624/56624fig4.jpg"> 图 4.组织还原容量的 EPR 评估在体内.插入: 在麻醉雌性 FVB/N 小鼠乳腺肿瘤组织 (10 µL, 10 毫米) 后, NR 探针的 L 波段 EPR 谱测量体内。超精细分裂, 一个 N, 被发现等于 14.72 G, 它对应于 pH 值 e = 6.52 假设肿瘤组织温度34°c 和 pK a = 6.6. 根据中心场谱分量的衰减, 对 NR 还原速率进行了评估。以乳腺肿瘤 () 和乳腺 (o) 为例的动力学例子表明, 肿瘤组织与正常乳腺的降低能力更高。对该动力学的初始部分的分析产生的速率, EPR 信号减少, k红色, 在组织的胞外介质中的 2.5 x 10-3/s 在肿瘤 vs 0.5 x 10-3/s 在正常腺体。从参考11中复制, 并获得约翰威利 & 儿子公司的许可<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig4large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
图 5总结了在肿瘤和正常乳腺支持肿瘤酸性细胞外微环境和高还原容量的雌性 FVB/N MMTV-PyMT 小鼠组中的 pH 值e和氧化还原测量。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56624/56624fig5.jpg"> 图 5.体内EPR 评价组织酸度和降低容量.细胞外组织 pH 值 (填充棒) 和还原率 (空棒) 氮氧自由在正常乳腺和女性 FVB/N 小鼠乳腺肿瘤中的测定,由体内 EPR 测量。误差线表示 SE. 从参考11中复制, 并征得约翰威利 & 儿子公司的许可<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig5large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
并发组织 pH 值e、氧化还原和pO2结合 NR 和林肯-小波探针进行评估:
微粒林肯-小波是在预定的利益组织中计划pO2值的重复纵向测量时选择的探针。不幸的是, 微粒探针的一个主要限制是, 它只允许检测氧气和没有其他与生理相关的化学参数在该期间, 不允许组织成像。因此, 在 EPR 中使用可溶性探针具有成像和检测多种生理相关参数的优点。在这方面, 使用注入的林肯-小波粒子的pO2测量可以与双功能 pH 值和氧化还原 nr 探针结合, 因为 nr epr 谱的低场和高场强谱分量不与 epr 线重叠。林肯-小波探头
图 6说明了在对雌性 C57Bl/6 小鼠乳腺肿瘤组织进行 it 注射后, NR 探针立即观察到的典型 EPR 谱。所观察到的 NR 探针的三重光谱与林肯-小波纳米微晶的单一 EPR 线叠加在肿瘤内 (如步骤3.1 所述, 肿瘤生长是由内化微粒的细胞注入所引发的。pH 值是通过将N超精细拆分作为低和高场组件之间的距离来计算的, 并使用图3 (等于 6.88) 中显示的用于图6 中的频谱的校准。请注意, pO2评估是根据对小波信号的线宽的测量在 NR 探针注入之前进行的。NR 信号的衰减提供了组织还原容量的值, 如图 4所示。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56624/56624fig6.jpg"> 图 6.结合林肯-小波和 NR 探针进行多功能评估.EPR 光谱测量的在体内的女性 C57Bl/6 小鼠乳房肿瘤组织后立即对 NR 探针的 it 注射。所观察到的 NR 的三重光谱被叠加在一个小波探针的单一 EPR 线上, 嵌入肿瘤内。从低和高场组分的劈裂中计算出的 pH 值为6.88。NR 信号的衰减提供了组织还原能力的价值。pO2评估是根据小波在 NR 探头注射前的线宽测量结果进行的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
体内细胞内谷胱甘肽的评估:
RSSR 探针与谷胱甘肽之间的硫醚交换反应分裂了探针的二硫化物键, 形成两个 monoradicals22 , 取消 monoradical 碎片之间的分子内自旋交换。图 7A说明了 RSSR 探针与 GSH 的反应对 EPR 谱的影响, 导致 "双基" 谱分量的消失以及 monoradical 成分强度的相应增加。谷胱甘肽与 RSSR 反应的观察速率常数为: kobs = 2.8 0.2 M-1s-1 (T = 34 °c, pH 7.2)11。图 7B示范了用 L 波段 EPR 测定乳腺肿瘤 () 和正常乳腺 (o) 中 monoradical 谱峰值强度变化的动力学。固体线是 monoexponent 的初始部分的配合使用 kobs = 2.8 M-1s-1和屈服 [GSH] = 10.7 毫米和3.3 毫米为肿瘤和正常乳腺, 分别。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56624/56624fig7.jpg"> 图 7.体内EPR 评估细胞内组织谷胱甘肽浓度.(A) 100 µM RSSR 的 X 波段 EPR 谱在孵化后的不同时间点测量, 0.1 米-磷酸盐缓冲液中的2.5 毫米谷胱甘肽, pH 7.2 和1毫米 DTPA 在34摄氏度。动力学分析提供的观察速率常数的反应, 谷胱甘肽和 RSSR, kobs (pH 值 7.2, 34 °c) = (2.8) 0.2) M-1 s-1。(B) 在 RSSR 探针的 monoradical 后, FVB/N 小鼠的乳腺肿瘤 () 和正常乳腺 (o) 中的 L 波段 EPR 测量的光谱峰值强度变化的动力学 (见步骤 3.3.2)。固体线是动力学的最初的部分的适应由 monoexponent, 假设 kobs (pH 值 7.2, 34 °c) = 2.8 M-1s-1和屈服 [GSH] = 10.7 毫米和3.3 毫米为肿瘤和正常乳腺, 分别。从参考11中复制, 并获得约翰威利 & 儿子公司的许可<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig7large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
组织胞外 pH 值的多功能评估pO2和 Pi 使用多功能希望探针:
图 8说明了使用1.4 节所述校准程序获得的希望探针的光谱参数的灵敏度。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56624/56624fig8.jpg"> 图 8.利用希望探针对化学微环境进行多功能评估.(a) 探针的两个电离状态之间的 pH 依赖性平衡方案。(b) 希望的 L 波段 EPR 频谱。(c) 希望的 EPR 线宽为pO2标记 (精度, ≈ 1 mmHg;pO2范围, 1-100 mmHg)。(d) 质子希望的分数是介于6到8.0 之间的 pH 值 (精确度, 0.05)。(e) 依赖于质子交换率 (以 mG 表示) 的希望与无机磷酸盐 (Pi) 浓度提取的光谱模拟 (准确度, 0.1 毫米, 范围, 0.1-20 毫米) 12,18。在具有自然发布组权限的情况下从引用8中复制。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图 9说明了在 FVB/N 野生型乳腺和 MMTV-PyMT 转基因小鼠中进行的多功能测量, 自发地发展成乳腺癌并模拟人类肿瘤分期20。pO2 (mmHg 3 mmHg 在正常组织中的平均值) 和 pHe (3 @ 6.99 和0.03 在正常组织中) 的平均值支持在肿瘤中出现缺氧和酸性区域。最剧烈的变化是观察到的间质 pi 浓度 (1.8, 0.2 毫米, 在正常组织0.07 毫米) 表明间质 pi 浓度作为肿瘤进展的标志物的潜在作用。个别测量显示, 在平均值附近有显著的变化。多功能探头的一个重要优点是, 所有参数都是使用单个探针来测量的, 因此可以独立于探针分布和测量时间来进行相关分析, 如图 9b- e。在正常乳腺中, pO2与 pHe之间的正相关关系与肿瘤缺乏相关性, 支持肿瘤依赖于独立于氧浓度的糖酵解;在我们看来, 这是一个例子在体内示范的华宝效应。反过来, 在正常和肿瘤组织中, 间质 [pi] 与pO2之间的负相关关系与高 [pi] (和低 ATP/Pi 比值) 的相关性与低氧生物能学状态的变化一致。供应.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56624/56624fig9.jpg"> 图 9.体内组织pO2, pH 值e和 Pi 评估使用希望探针和 EPR.(a) 设置体内L 波段 epr 测量的照片显示了在 epr 光谱仪的磁体之间的麻醉鼠标, 插入右侧显示了环路谐振器的位置和位置, 在测量组织。注意希望探针的间质胞外定位: 由于体积大的电荷结构, 它不会穿透到细胞中, 并且由于希望与血浆白蛋白络合而引起的信号扩大, EPR 没有检测出来自血液的希望信号24.(b-e)FVB/N 野生型小鼠正常乳腺和 MMTV PyMT 基因小鼠乳腺癌的间质 pO2、pHe和 Pi 值之间的相关性 (N = 23)。为了扩大氧的变化范围, 通过对葡萄糖/葡萄糖氧化酶 (红色符号) 的耗氧酶系统的研究, 建立了间质空间的缺氧条件。蓝色线条表示总数据集的线性拟合。(b) pO2与正常组织中的 pHe之间的正相关 (r = 0.5, p = 0.014 用于黑色符号; r = 0.64, p = 1.8 x 10-4用于总计数据集) vs (c) 没有显著的pO2与 pH 值e之间的相关性 (r = 0.01, p = 0.97 为黑色符号; r = 0.23, p = 0.3 为总数据集) 被发现了。(d) pO2与 Pi 在正常组织中的负相关 (r = −0.51, p = 0.013 用于黑色符号; r = −0.7, p = 2.3 x 10-5用于总计数据集) 和 (e) 在中 (b,底部: r = −0.4, p = 0.079 为黑色符号;r = −0.62, 发现了总计数据集的p = 0.001。自引用8改编而成, 具有自然发布组的权限。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56624/56624fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment at JoVE.com

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

利用可溶性基团和三苯三苯探针对低场 (L 波段, 1.2 GHz) 电子顺磁共振进行了分析, 以评估乳腺癌小鼠模型中肿瘤微环境中生理上的重要参数。

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

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

in-vivo-epr-assessment-ph-po2-redox-status-concentrations-phosphate

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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

8.2K 次观看.  West Virginia University. 利用可溶性基团和三苯三苯探针对低场 (L 波段, 1.2 GHz) 电子顺磁共振进行了分析, 以评估乳腺癌小鼠模型中肿瘤微环境中生理上的重要参数。

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

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

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

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

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

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 ...

科研

癌症研究

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

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 ...

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

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with 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 ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

利用卡尔瓦里亚共同培养和教条法测量骨重塑和再造肿瘤-骨微环境

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

固体肿瘤细胞系侧面人口分析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

通过恶性周围神经鞘肿瘤细胞中絮状等位基因的 离体 消融来定义肿瘤发生的基因功能

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...