Development of human pancreatic cancer avatars as a model for dynamic immune landscape profiling and personalised therapy

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, a disease with dismal overall survival. Advances in treatment are hindered by a lack of preclinical models. Here we show how a personalised organotypic ‘avatar’ created from resected tissue, allows spatial and temporal reporting on a complete in situ tumour microenvironment, and mirrors clinical responses. Our perfusion culture method extends tumour slice viability, maintaining stable tumour content, metabolism, stromal composition, and immune cell populations for 12 days. Using multiplexed immunofluorescence and spatial transcriptomics, we identify immune neighbourhoods and potential for immunotherapy. We employed avatars to assess the impact of a pre-clinically validated metabolic therapy and show recovery of stromal and immune phenotypes and tumour re-differentiation. To determine clinical relevance, we monitored avatar response to gemcitabine treatment and identified a patient avatar-predicable ..., Acquisition of tissue and blood samples
The study (REC number 19/A056) was approved for the collection of tumour and healthy pancreatic tissue by the Oxford Radcliffe BioBank. The collection of the specimens was supported by the Oxford Centre for Histopathology Research. All patients recruited to the study provided written consent confirming voluntary participation and permission for tissue donation for research. Biopsy punch samples (5mm diameter) were obtained by the pathologist at the John Radcliffe Hospital following surgery provided that clear surgical margins could be determined.
Sectioning and culture of live tumour slices
Samples were transported on ice in unsupplemented low-glucose DMEM media before suspension in agarose scaffolds. Following a manual wash in LG DMEM media, biopsy punches were suspended in 4% low-gelling-temperature agarose and cooled. The agarose scaffold structure was generated by melting the solution and suspending the slice in a small embedding mould. 250 µm..., , # Development of human pancreatic cancer avatars as a model for dynamic immune landscape profiling and personalised therapy

[https://doi.org/10.5061/dryad.qnk98sfr2](https://doi.org/10.5061/dryad.qnk98sfr2)

## Description of the data and file structure

### Spatial Transcriptomics files

Spatial transcriptomic datasets are Excel spreadsheets of gene counts from RNAseq analysis which have been normalised using Q3 normalisation. Cellular areas are designated tumours, stroma, or immune by staining for pan-cytokeratin, alpha-smooth actin and DC45, as described in the methods. Column names are the sample names and row names are the gene names. 

The file 'control_ascorbicAcid_metformin_sample_ID_timepoint.xlsx' gives a key to interpret the sample names in the file 'control_ascorbicAcid_metformin_GeoMX' where

* SegmentLabel = segment designation (tumour, stroma, immune)
* SegmentDisplayName = the sample name given in 'control_ascorbicAcid_metformin_GeoMX'. 'C' or 'T' at the start of the na...

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）是最常见的胰腺癌类型，属于总体生存率极差的恶性肿瘤。当前治疗进展受限于临床前模型的匮乏。本研究展示了一种源自手术切除组织的个性化器官型“化身”模型，可实现完整原位肿瘤微环境的时空解析，并精准复刻临床治疗响应。我们建立的灌注培养（perfusion culture）方法可延长肿瘤切片存活时长，在12天内维持稳定的肿瘤组分、代谢活性、间质组成及免疫细胞群。通过多重免疫荧光（multiplexed immunofluorescence）与空间转录组学（spatial transcriptomics）分析，我们鉴定出免疫微环境区域，并筛选出潜在的免疫治疗靶点。我们利用该“化身”模型评估了一项经临床前验证的代谢疗法的作用，结果显示其可恢复间质与免疫表型，并促进肿瘤再分化。为明确临床相关性，我们监测了“化身”模型对吉西他滨（gemcitabine）治疗的响应，并鉴定出可通过患者“化身”预测的[原文未完成]。

### 样本与血液样本采集
本研究（研究伦理委员会编号19/A056）已获牛津拉德克利夫生物样本库批准，可收集肿瘤与正常胰腺组织。样本采集工作得到牛津组织病理学研究中心的支持。所有纳入研究的患者均签署书面知情同意书，确认自愿参与研究并同意捐赠组织用于科研。约翰·拉德克利夫医院的病理学家在术后获取直径5mm的活检打孔样本，前提是可确定清晰的手术切缘。

### 活体肿瘤切片的制备与培养
样本置于无添加成分的低葡萄糖DMEM培养基中冰上运输，随后悬浮于琼脂糖支架中。经低葡萄糖DMEM培养基手动洗涤后，活检打孔样本被悬浮于4%低熔点琼脂糖（low-gelling-temperature agarose）中并冷却。通过熔融琼脂糖溶液并将切片悬浮于小型包埋模具中，制备得到琼脂糖支架结构。切片厚度为250 μm[原文未完成]。

# 开发人类胰腺癌“化身”模型以用于动态免疫景观谱型分析与个性化治疗
https://doi.org/10.5061/dryad.qnk98sfr2

## 数据与文件结构说明
### 空间转录组学文件
空间转录组学数据集为经Q3标准化（Q3 normalisation）处理的RNA测序基因计数Excel表格。根据研究方法中描述的泛细胞角蛋白（pan-cytokeratin）、α-平滑肌肌动蛋白（alpha-smooth actin）与DC45染色结果，将细胞区域划分为肿瘤区、间质区或免疫区。表格列名为样本名，行名为基因名。

文件`control_ascorbicAcid_metformin_sample_ID_timepoint.xlsx`为`control_ascorbicAcid_metformin_GeoMX`文件中样本名的解析密钥，其中：
* SegmentLabel = 片段标识（肿瘤、间质、免疫）
* SegmentDisplayName = `control_ascorbicAcid_metformin_GeoMX`文件中对应的样本名，样本名开头为“C”或“T”的部分[原文未完成]