Single Cell Analysis of Treatment–Resistant Prostate Cancer: Implications of Cell State Changes for Cell Surface Antigen Targeted Therapies

Targeting cell surface molecules using radioligand and antibody–based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)––a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis (TMA) on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate–resistant PRAD and NEPC than previously anticipated, but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene–regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer (SCLC) subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to novel antigen–directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types. Tumor biopsies were dissociated per previously published protocols (PMID: 35981096). Single cell data for HMP22, HMP23A, HMP23B, HMP24 has not previously been published and is deposited under this GEO ID. Data for HMP04, HMP05, HMP08, HMP11A, HMP11B, HMP13, HMP14, HMP16, HMP17, HMP19, HMP20, HMP25, and HMP26 were re–analyzed, but has previously been published in the context of JAK/STAT signaling in prostate cancer under GSE210358 (PMID 35981096). Data from HP95, HP96, HP97, HP99, HP100, and HP101 are also re–analyzed and has previously been published by our group in the context luminal regeneration under https://duos.broadinstitute.org/ (accession no. DUOS-000115). We have also included processed RDS files for combined samples (N=23) for all cells and tumor cells. Of note, HP and HMP IDs correspond to MSK–HP IDs as presented in this publication in Supplementary Table 3. The list of the re-analyzed sample in GSE210358: SRX16781303 GSM6428952 HMP04 SAMN30111353 SRX16781304 GSM6428953 HMP05 SAMN30111352 SRX16781305 GSM6428954 HMP08 SAMN30111351 SRX16781314 GSM6428955 HMP11_1 SAMN30111350 SRX16781315 GSM6428956 HMP11_2 SAMN30111349 SRX16781316 GSM6428957 HMP13 SAMN30111348 SRX16781317 GSM6428958 HMP14 SAMN30111347 SRX16781319 GSM6428960 HMP16 SAMN30111345 SRX16781318 GSM6428959 HMP15 SAMN30111346 SRX16781320 GSM6428961 HMP17 SAMN30111344 SRX16781321 GSM6428962 HMP19 SAMN30111343 SRX16781322 GSM6428963 HMP20 SAMN30111342 SRX16781323 GSM6428964 HMP25 SAMN30111341 SRX16781325 GSM6428965 HMP26 SAMN30111340

采用放射性配体疗法与基于抗体的疗法靶向细胞表面分子，在多种癌症治疗中已取得显著成效。然而，在谱系可塑性（lineage plasticity）过程中，推定的谱系标志物（尤其是细胞表面分子）的表达变化机制仍不明晰——在此过程中肿瘤细胞会改变自身身份并获得新的致瘤特性。前列腺腺癌（prostate adenocarcinoma, PRAD）向神经内分泌前列腺癌（neuroendocrine prostate cancer, NEPC）的转化是谱系可塑性的典型案例，该转化是一种日益常见的耐药机制，会导致肿瘤对雄激素阻断治疗失去响应，且预示患者预后极差。

为探究前列腺癌谱系可塑性演化过程中谱系标志物的表达变化规律，我们对21例人类前列腺肿瘤活检样本与2株基因工程小鼠模型开展了单细胞分析，并对131例肿瘤样本开展了组织微阵列分析（tissue microarray analysis, TMA）。研究不仅发现去势抵抗性前列腺腺癌与神经内分泌前列腺癌的表型异质性程度远超此前预期，还发现治疗靶向分子——即PSMA、STEAP1、STEAP2、TROP2、CEACAM5与DLL3——的表达在部分基因调控网络（gene-regulatory networks, GRNs）内存在差异。我们同时观察到，神经内分泌前列腺癌与小细胞肺癌（small cell lung cancer, SCLC）亚型共享一套基因调控网络，这提示存在保守的生物学通路，可跨肿瘤类型用于治疗开发。尽管这种极高水平的转录异质性（尤其是细胞表面标志物表达层面）可能会削弱新型抗原靶向疗法的临床响应持久性，但对其进行解析可获得可用于临床试验患者筛选的特征标签，且有望覆盖不同癌症类型。

肿瘤活检样本的解离流程参照已发表方案（PubMed ID: 35981096）。HMP22、HMP23A、HMP23B与HMP24的单细胞数据此前未发表，已上传至基因表达综合数据库（Gene Expression Omnibus, GEO）对应的编号下。HMP04、HMP05、HMP08、HMP11A、HMP11B、HMP13、HMP14、HMP16、HMP17、HMP19、HMP20、HMP25与HMP26的数据为重新分析所得，此前已在前列腺癌JAK/STAT信号通路相关研究中发表，对应GEO数据库编号GSE210358（PubMed ID: 35981096）。HP95、HP96、HP97、HP99、HP100与HP101的数据同样为重新分析所得，此前由本团队在腔面再生相关研究中发表，相关数据可通过https://duos.broadinstitute.org/获取，登录编号为DUOS-000115。我们还提供了合并样本（共23例）的经预处理RDS文件，涵盖所有细胞与肿瘤细胞群体。需注意的是，HP与HMP编号对应本研究补充表3中提及的MSK-HP编号。

本次重新分析的GSE210358数据集样本如下：
SRX16781303 GSM6428952 HMP04 SAMN30111353
SRX16781304 GSM6428953 HMP05 SAMN30111352
SRX16781305 GSM6428954 HMP08 SAMN30111351
SRX16781314 GSM6428955 HMP11_1 SAMN30111350
SRX16781315 GSM6428956 HMP11_2 SAMN30111349
SRX16781316 GSM6428957 HMP13 SAMN30111348
SRX16781317 GSM6428958 HMP14 SAMN30111347
SRX16781319 GSM6428960 HMP16 SAMN30111345
SRX16781318 GSM6428959 HMP15 SAMN30111346
SRX16781320 GSM6428961 HMP17 SAMN30111344
SRX16781321 GSM6428962 HMP19 SAMN30111343
SRX16781322 GSM6428963 HMP20 SAMN30111342
SRX16781323 GSM6428964 HMP25 SAMN30111341
SRX16781325 GSM6428965 HMP26 SAMN30111340