To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Using the proposed immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) approach and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Using ICC-RNAseq, samples captured at a specific spatio-temporal state can be investigated transcriptionally using different biomarkers. This would facilitate our understanding of cell population-specific molecular events driving the disease.

为了研究细胞内异质性，特定细胞群的细胞捕获随后进行转录组分析在新鲜分离的组织中非常有效。然而，这种方法在免疫染色的福尔马林固定石蜡包埋 (FFPE) 切片中颇具挑战性。本研究旨在将标准病理学技术、免疫染色和激光捕获显微切割与全 RNA 测序和生物信息学分析相结合，以表征具有异质性黄体酮受体 (PR) 表达的 FFPE 乳腺癌细胞群。免疫细胞化学分析表明，60% 的 MCF-7 细胞混合高表达 PR。使用提出的基于免疫细胞化学的靶向 RNA-seq (ICC-RNAseq) 方法和计算机功能分析表明，PR 高细胞群与涉及免疫调节和炎症通路（例如 NF-κB 和干扰素信号传导）的转录本的上调有关. 相比之下，PR 低细胞群与参与代谢和线粒体过程以及 EGFR 和 MAPK 信号传导的基因的上调有关。这些发现在 FACS 分选的 PR 高和 PR 低 MCF-7 细胞以及异位过表达 PR 的 MDA-MB-231 细胞中得到了交叉验证和证实。使用 ICC-RNAseq，可以使用不同的生物标志物对在特定时空状态下捕获的样本进行转录研究。