High resolution mapping of the tumor microenvironment using integrated single-cell, spatial and in situ analysis [Xenium]

Single-cell and spatial technologies that profile gene expression across a whole tissue are revolutionizing the resolution of molecular states in clinical tissue samples. Current commercially available technologies provide whole transcriptome single-cell, whole transcriptome spatial context, or high resolution in situ targeted gene expression analysis. Here, we combine these technologies to explore tissue heterogeneity in large, FFPE human breast cancer sections. In one sample, we identify three molecularly distinct tumor subtypes, enabling us to define cell neighborhoods and biomarkers in the progression towards invasive carcinoma. In another sample, we identify rare boundary cells that sit at the critical myoepithelial border confining the spread of malignant cells. We demonstrate that each technology alone provides information about molecular signatures relevant to understanding cancer heterogeneity. However, it is the integration of these technologies that leads to even deeper insights, ushering in discoveries that will progress oncology research and the development of diagnostics and therapeutics. Two formalin-fixed & paraffin-embedded (FFPE) breast cancer tissue blocks were obtained from Discovery Life Sciences. Sample #1 was annotated by a pathologist to be T2N1M0, Stage II-B, ER+/HER2+/PR−. Sample #2 was characterized as stage pT2 pN1a pMX, ER−/HER2+/PR−. Corresponding dissociated tumor cells for Sample #1, fresh frozen (FF) in liquid nitrogen, were also sampled from the same 2.5 cm biopsy. For the Chromium Flex workflow, two 25 μm curls were pooled as a single replicate. 5 μm sections from Sample #1 were taken from the FFPE tissue using a microtome. Two replicate 5 μm sections were taken each for Visium CytAssist and Xenium. A 5 μm section was also taken from Sample #2 for Xenium.

可对全组织开展基因表达谱分析的单细胞技术与空间组学技术，正颠覆性地提升临床组织样本分子状态的解析分辨率。当前商业化可用的技术可提供全转录组单细胞分析、全转录组空间背景分析，或高分辨率原位靶向基因表达分析。本研究整合上述技术，对大体积福尔马林固定石蜡包埋（Formalin-Fixed Paraffin-Embedded, FFPE）人乳腺癌组织切片的异质性展开探究。
在其中一例样本中，我们鉴定出三种分子特征截然不同的肿瘤亚型，借此明确了向浸润性癌进展过程中的细胞邻域与生物标志物。在另一例样本中，我们发现了位于限制恶性细胞扩散的关键肌上皮边界处的稀有边界细胞。
本研究证实，单一技术仅能提供与解析肿瘤异质性相关的分子特征信息；唯有整合这些技术，方能获得更为深入的认知，进而催生可推动肿瘤学研究及诊断与治疗药物开发的新发现。
本研究从Discovery Life Sciences获取了两例福尔马林固定石蜡包埋（FFPE）乳腺癌组织块。经病理学家注释，样本1的肿瘤分期为T2N1M0、II-B期，免疫表型为雌激素受体（Estrogen Receptor, ER）阳性、人表皮生长因子受体2（Human Epidermal Growth Factor Receptor 2, HER2）阳性、孕激素受体（Progesterone Receptor, PR）阴性。样本2的肿瘤分期为pT2 pN1a pMX，免疫表型为ER阴性、HER2阳性、PR阴性。
从同一份2.5cm活检样本中，我们还采集了对应样本1的解离肿瘤细胞，经液氮速冻后保存（新鲜冷冻, FF）。针对Chromium Flex实验流程，我们将两张25微米厚度的组织卷曲样本混合作为一个生物学重复。使用切片机从样本1的FFPE组织中切取5微米厚度的组织切片。分别为Visium CytAssist与Xenium平台切取两份5微米厚度的重复切片。同时从样本2中切取一份5微米厚度的组织切片用于Xenium平台分析。