Lin Yen-Yu, Lan Hsin-Yi, Teng Hao-Wei, Wang Ya-Pei, Lin Wen-Chun, Hwang Wei-Lun
Department of Pathology, Fu Jen Catholic University Hospital, Fu Jen Catholic University, New Taipei City 24352, Taiwan.
School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City 24205, Taiwan.
Theranostics. 2025 Mar 18;15(10):4308-4324. doi: 10.7150/thno.110379. eCollection 2025.
Advances in cancer therapies have significantly improved patient survival; however, tumors enriched in cancer stem cells (CSCs) have poor treatment responses. CSCs are a key source of tumor heterogeneity, contributing to therapeutic resistance and unfavorable patient outcomes. In the tumor microenvironment (TME), cell-in-cell (CIC) structures, where one cell engulfs another, have been identified as markers of poor prognosis. Despite their clinical relevance, the mechanisms underlying CIC formation across different tumor cell subpopulations remain largely unknown. Elucidating these processes could provide novel insights and therapeutic opportunities to address aggressive, treatment-resistant cancers. Fluorescent mCherry-carrying colorectal cancer stem cells (CRCSCs) were expanded as spheroids in serum-free media and cocultured with either parental cancer cell-expressing Venus fluorescent protein or CFSE dye-stained immune cells (T cells, M1/M2 macrophages, neutrophils, and NK cells) or treated with EGFR- or PD-L1-targeting antibodies to assess the formation of CIC structures. Genes potentially crucial for the formation of CIC structures were knocked down or overexpressed, and their effects on CIC formation were evaluated. The clinical relevance of the findings was confirmed through analysis of formalin-fixed, paraffin-embedded (FFPE) human colorectal cancer (CRC) specimens. CRCSCs have a strong predilection for serving as the outer cell in a CIC structure and forming homotypic CIC structures predominantly with parental CRC cells. The frequency of CIC structure formation increased when the cells were exposed to anti-PD-L1 antibody treatment. Both the outer CRCSC in a CIC structure and CRCSCs released from a homotypic CIC structure showed enhanced resistance to the cytotoxicity of NK-92MI cells. Restoration of Stathmin1 (STMN1) expression but not knockdown in CRCSCs reduced the homotypic CIC frequency, disrupted the outer cell fate in CIC structures, and increased cell susceptibility to NK-92MI cytotoxicity. In CRC patients, CIC structures are associated with poor tumor differentiation, negative STMN1 expression, and poor prognosis. CSCs play a crucial role in informing CIC structures in CRC. CIC structure formation partially depends on low STMN1 expression and confers a survival advantage under NK cytotoxicity. Targeting this pathway may significantly improve immunotherapy's efficacy for CRC patients.
癌症治疗的进展显著提高了患者的生存率；然而，富含癌症干细胞（CSCs）的肿瘤治疗反应较差。CSCs是肿瘤异质性的关键来源，导致治疗耐药性和不良的患者预后。在肿瘤微环境（TME）中，细胞内细胞（CIC）结构，即一个细胞吞噬另一个细胞，已被确定为预后不良的标志物。尽管它们具有临床相关性，但不同肿瘤细胞亚群中CIC形成的潜在机制仍 largely 未知。阐明这些过程可为治疗侵袭性、耐药性癌症提供新的见解和治疗机会。携带荧光mCherry的结直肠癌干细胞（CRCSCs）在无血清培养基中作为球体扩增，并与表达Venus荧光蛋白的亲代癌细胞或CFSE染料染色的免疫细胞（T细胞、M1/M2巨噬细胞、中性粒细胞和NK细胞）共培养，或用靶向EGFR或PD-L1的抗体处理，以评估CIC结构的形成。对CIC结构形成可能至关重要的基因被敲低或过表达，并评估它们对CIC形成的影响。通过对福尔马林固定、石蜡包埋（FFPE）的人类结直肠癌（CRC）标本进行分析，证实了研究结果的临床相关性。CRCSCs强烈倾向于在CIC结构中作为外层细胞，并主要与亲代CRC细胞形成同型CIC结构。当细胞接受抗PD-L1抗体治疗时，CIC结构形成的频率增加。CIC结构中的外层CRCSC和从同型CIC结构中释放的CRCSCs对NK-92MI细胞的细胞毒性均表现出增强的抗性。在CRCSCs中恢复Stathmin1（STMN1）的表达而非敲低可降低同型CIC频率，破坏CIC结构中的外层细胞命运，并增加细胞对NK-92MI细胞毒性的敏感性。在CRC患者中，CIC结构与肿瘤低分化、STMN1表达阴性和预后不良相关。CSCs在CRC中CIC结构的形成中起关键作用。CIC结构的形成部分取决于低水平的STMN1表达，并在NK细胞毒性下赋予生存优势。靶向该途径可能显著提高CRC患者免疫治疗的疗效。
