A micro-dissection platform for generating uniform-sized patient-derived tumor organoids (PDOs) for personalized cancer therapy

项目摘要 患者来源的肿瘤类器官（PDO），涉及新鲜肿瘤碎片的离体培养， 已经成为预测个性化癌症患者药物反应的有前途的模型 疗法PDO再现了肿瘤微环境（TME），类似于源肿瘤 表型上和基因组上，并且与高通量药物筛选相容。 然而，在PDO中缺乏免疫细胞的保存一直是免疫治疗的主要障碍。 模拟免疫疗法。我们的团队最近展示了一种新型的PDO， 片段作为粘合单元，允许原位保存不同的免疫细胞类型 与肿瘤细胞一起生长这种方法使建模 对免疫检查点抑制剂的患者特异性反应。 生成PDO的第一步之一是解剖患者肿瘤标本 变成小碎片机械解剖，而不是酶消化，是关键， 保持肿瘤细胞与内源性免疫和非免疫之间的体内结合 元素保护内源性免疫细胞的能力，包括肿瘤浸润 淋巴细胞（TIL）的免疫调节对于个性化免疫治疗测试尤其重要。 然而，目前的机械解剖主要依赖于手动切碎肿瘤标本 变成小碎片它产生具有宽尺寸范围的碎片，并且是不精确的， 不可复制太大的碎片遭受营养供应不足， 氧合和活力以及差的药物渗透。太小的碎片不太可能 保留足够的基质细胞以支持PDO生长，和/或内源性免疫细胞， 可能以低浓度存在。 因此，存在对产生可控的肿瘤碎片的更好方法的未满足的需要。 和均匀的大小，并确定最佳的大小，以增加可再生性和产量 PDO可以保留细胞结构和肿瘤结构。该项目旨在 通过开发一种新方法将肿瘤标本机械解剖成 均匀的碎片性能指标包括片段大小均匀性，PDO活力， 免疫细胞的保存和对免疫治疗的肿瘤细胞毒性。其它切割 包括手工切碎在内的方法将被用作基准。

Project Summary Patient-derived tumor organoids (PDO), involving the ex vivo culture of fresh tumor fragments, have emerged as promising models for predicting patient drug response for personalized cancer therapy. PDOs recapitulate the tumor micro-environment (TME), resemble the source tumor phenotypically and genomically, and are compatible with high-throughput drug screening. However, the lack of preservation of immune cells in PDOs has been a major roadblock to modeling immunotherapy. Our team recently demonstrated a new type of PDO that cultures tumor fragments as a cohesive unit, allowing the in situ preservation of diverse immune cell types alongside tumor cells without artificial reconstitution. This approach has enabled the modeling of patient-specific responses to immune checkpoint inhibitors. One of the first steps in the generation of PDOs is the dissection of patient tumor specimen into small fragments. Mechanical dissection, instead of enzymatic digestion, is critical in preserving the in vivo association between tumor cells and endogenous immune and non-immune elements. The ability to preserve endogenous immune cells, including tumor-infiltrating lymphocytes (TIL), is particularly important for personalized immunotherapy testing. However, current mechanical dissection relies primarily on manual mincing of tumor specimen into small fragments. It results in fragments with a broad size range, and is imprecise and irreproducible. Fragments that are too large suffer from inadequate nutrient supply, suboptimal oxygenation and viability, and poor drug penetration. Fragments that are too small are unlikely to preserve sufficient stromal cells to support PDO growth, and/or endogenous immune cells which may be present at low concentrations. As such, there is an unmet need for a better way to generate tumor fragments of controllable and uniform size, and identify optimal size(s) to increase the reproducibility and yield of viable PDOs that can preserve the cellular contexture and tumor architecture. This project aims to address this need by developing a new method to mechanically dissect tumor specimen into uniform fragments. Performance measures include fragment size uniformity, PDO viability, preservation of immune cells, and tumor cytotoxicity in response to immunotherapy. Other cutting methods including manual mincing will be used as benchmarks.