Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America.
PLoS One. 2023 Oct 10;18(10):e0287126. doi: 10.1371/journal.pone.0287126. eCollection 2023.
Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.
雄激素剥夺疗法（ADT）是高危和晚期前列腺癌的标准治疗方法；然而，从雄激素依赖性前列腺癌（ADPC）发展为致命且不可治愈的去势抵抗性前列腺癌（CRPC），以及（在少数情况下）神经内分泌前列腺癌（NEPC）是很常见的。由于对既定治疗方法的获得性耐药以及晚期前列腺癌（PC）的巨大异质性，确定有效的靶向治疗方法具有挑战性。为了简化潜在有效的前列腺癌治疗药物的鉴定，我们开发了一种适应性强的半自动方案，该方案优化了细胞生长，并利用自动化技术提高了稳健性、重现性和通量，同时整合了活细胞成像和终点活力测定，以评估体外药物疗效。在这项研究中，为包括：BPH-1 和 RWPE-1（非肿瘤性）、LNCaP 和 VCaP（ADPC）、C4-2B 和 22Rv1（CRPC）、DU 145 和 PC3（雄激素受体缺失的 CRPC）以及 NCI-H660（NEPC）在内的大型代表性人前列腺细胞系建立了 96 孔板中 72 小时药物筛选的培养条件。在筛选新的或重新利用的药物之前，针对每种细胞系进行了实时成像和终点活力测定的细胞生长和 72 小时汇合优化，以验证方案的有效性。我们通过验证第一个 BET 和 CBP/p300 双重抑制剂 EP-31670 是一种有效化合物，可以减少 ADPC 和 CRPC 细胞生长的既定发现，证明了该方案的有效性和可靠性。此外，我们发现胰岛素样生长因子-1 受体（IGF-1R）抑制剂 linsitinib 是一种针对高度致命和耐药的 NEPC NCI-H660 细胞的潜在药理药物。该方案可用于其他癌症类型，代表了一种适应性强的策略，可以优化特定于测定的细胞生长条件，并同时评估多种细胞系的药物疗效。
