Refining in vivo studies of cancer metastasis with next-generation explant-in-chip perfusion models

我们的总体目标是创建和验证一种新型的“芯片内的细胞”灌注装置，以研究转移的早期阶段，特别是癌细胞停滞、外渗和多个次级组织（脑、肺、肝）内的早期定植。我们估计每年至少有12，000只小鼠被用于转移研究，我们的设备有望通过提供一种用户友好的方法来减少这一数字，该方法使用离体组织来研究转移的早期阶段，而不是对每个实验条件进行体内试验。外植体大小为几毫米，这太大而无法单独通过氧气和营养扩散来支持，但是足够大以包含组织微环境和组织特异性微脉管系统结构的重要部分。为了实现有效的氧气和营养输送，将外植体置于特殊设计的微通道内后，用含营养的培养基灌注，该微通道在外植体周围实现自密封。密封是必要的，以便当在整个外植体上施加压降时，迫使流动通过外植体（灌注融合），而不是围绕外植体（灌注融合）。我们的初步数据表明，灌注保存外植体活力至少6天。通过在灌注液中加入标记的癌细胞，细胞进入血管系统并分布在外植体内的整个微血管网络中，以模拟肿瘤细胞在体内传播的血行途径。从3R的角度来看，这种方法很重要，因为可以从单个动物中分离出多个外植体，并在我们的设备中进行研究。这将实验单位从单个动物减少到单个外植体。由于许多外植体可以从一只小鼠中分离出来，并暴露在一系列不同的处理条件下，我们的设备减少了动物数量，并最大限度地提高了用于研究的每只动物的科学价值。由于离体方法可用于筛选和分类许多实验条件，一旦在离体筛选中确定了最佳药物或药物浓度，该方法允许更精细的后续体内研究。我们的装置捕获并保存了对早期转移具有关键作用的天然组织微环境，同时还提供了在其它离体模型中通常不可用的血管输注途径。我们的方法还提供了一种手段来可视化的时空动态的原生组织微环境内的早期转移，这在体内将需要活体显微镜和植入的光学windows.We的目的是验证我们的设备通过比较金标准在体内模型的实验转移。我们还旨在通过使设计用户友好和举办研讨会以促进其他实验室使用我们的设备来传播我们的技术，以最大限度地影响3R。到本项目结束时，我们将开发并验证一种用户友好的设备，该设备将提供转移性传播中涉及的早期时空事件的单细胞分辨率，减轻了对小鼠中具有技术挑战性和侵入性程序的需要。

Our overall goal is to create and validate a novel "explant-in-chip" perfusion device to study the early stages of metastasis, specifically cancer cell arrest, extravasation, and early colonisation within multiple secondary tissues of interest (brain, lung, liver). We estimate that at least 12,000 mice are used annually for metastasis research, and our device promises to reduce this number by providing a user-friendly approach to investigate the early stages of metastasis using ex vivo tissue, rather than performing in vivo trials for each experimental condition.The explants are several millimetres in size, which is too large to be supported by oxygen and nutrient diffusion alone, but large enough to encompass a significant portion of the tissue microenvironment and tissue-specific microvasculature architectures. To achieve effective oxygen and nutrient delivery, explants are perfused with nutrient-containing medium after being placed within a specially designed microchannel that achieves self-sealing around the explant. Sealing is necessary so that when a pressure-drop is applied across the explant, flow is forced to pass through the explant (per-fusion) as opposed to around the explant (peri-fusion). Our preliminary data demonstrate that perfusion preserves explant viability for at least 6 days. By including labelled cancer cells in the perfusate, the cells enter the vasculature and distribute throughout the microvascular network within the explant to mimic the hematogenous route of tumour cell dissemination in vivo.This approach is important from a 3Rs perspective because multiple explants can be isolated from an individual animal and studied within our device. This reduces the experimental unit from the individual animal to the individual explant. Because many explants can be isolated from a single mouse and exposed to a range of different treatment conditions, our device reduces animal numbers and maximises the scientific value of each animal used for research. Because ex vivo approaches can be used to screen and triage many experimental conditions, the approach allows for more refined follow-on in vivo studies once the optimal drugs or drug concentrations have been identified in ex vivo screens.Our device captures and preserves the native tissue microenvironment which has a critical role on early metastasis, while also providing a route for vascular infusion that is typically unavailable in other ex vivo models. Our approach also provides a means to visualise the spatiotemporal dynamics of early metastasis within the native tissue microenvironment, which in vivo would require intravital microscopy and implantation of an optical window.We aim to validate our device by comparing against gold standard in vivo models of experimental metastasis. We also aim to disseminate our technology for maximum impact on the 3Rs by making the design user-friendly and by hosting workshops to facilitate other laboratories using our device.By the end of this project, we will have developed and validated a user-friendly device that will provide single cell resolution of the early spatiotemporal events involved in metastatic dissemination, alleviating the need for technologically challenging and invasive procedures in mice.