Name: in vivo epr assessment of ph, po2, redox status, and concentrations of phosphate and glutathione in the tumor microenvironment
Uploaded: 2018-03-16T13:00:00.000+00:00
Duration: 10 min 46 s
Description: 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

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?...

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/files/ftp_upload/56624/56624fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

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

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

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

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

医学

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

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

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

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 ...

<meta charset="utf8"></head><body>使用 EPRI 和超声对肿瘤氧水平进行临床前成像

Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels.
Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.

<meta charset="utf8"></head><body>肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

Quantitative 3D oxygen maps of murine tumors were imaged noninvasively using Pulse Electron Paramagnetic Resonance. Ultrasound B-mode and Power Doppler were used for anatomy and vascular structure. Images from both modalities were superimposed enabling multi-parametric tumor analysis.

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像 

The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 ...

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

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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

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

在髓系细胞动态实时成像

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像

肿瘤缺氧评估：通过电子顺磁共振进行体内 3D 氧成像