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

我们的总体目标是创造和验证一种新型的“芯片外植体”灌流设备来研究转移的早期阶段，特别是癌细胞的停滞、外渗和在多个感兴趣的次级组织(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，我们的设备承诺通过提供一种用户友好的方法来使用体外组织来研究转移的早期阶段，而不是针对每个实验条件进行体内试验。外植体大小为几毫米，太大了，仅靠氧气和营养物质的扩散无法支持，但大到足以涵盖组织微环境和组织特有的微血管结构的很大一部分。为了实现有效的氧气和营养输送，外植体被放置在一个特殊设计的微通道中后，被注入含有营养的介质，该微通道实现了外植体周围的自我封闭。密封是必要的，这样当在外植体上施加压降时，流体被迫通过外植体(灌流)，而不是绕着外植体(灌流)。我们的初步数据表明，灌流可以保存外植体至少6天的活力。通过在灌流液中加入标记的癌细胞，这些细胞进入血管系统并分布在外植体内的微血管网络中，以模拟肿瘤细胞在活体中扩散的血源性路径。从3Rs的角度来看，这种方法很重要，因为可以从单个动物身上分离出多个外植体，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身上分离出来，并暴露在一系列不同的治疗条件下，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一种动物的科学价值。由于体外方法可用于筛选和分类许多实验条件，一旦在体外筛选中确定了最佳药物或药物浓度，该方法允许进行更精细的体内后续研究。我们的设备捕获并保存在早期转移中起关键作用的自然组织微环境，同时还提供了其他体外模型中通常无法获得的血管输注途径。我们的方法还提供了一种方法来可视化自然组织微环境中早期转移的时空动力学，这在体内需要活体显微镜和植入光学窗口。我们的目标是通过与实验转移的金标准体内模型进行比较来验证我们的设备。我们的目标也是传播我们的技术，通过使设计变得用户友好并通过举办研讨会来促进其他实验室使用我们的设备，从而最大限度地影响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，该设备将提供涉及转移扩散的早期时空事件的单细胞分辨率，从而减少对小鼠进行具有技术挑战性的侵入性程序的需求。

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.