Background: The mechanical conditioning (MeCo) score is a multigene expression signature that is acquired by cancer cells in the primary breast tumor and is reflective of their responsiveness to ECM stiffness caused by tumor fibrosis. Chromatin remodeling downstream of mechanotransduction allows cancer cells to retain these acquired aggressive features even in the absence of mechanical stimulation from the primary tumor microenvironment, for instance, after dissemination through systemic circulation during metastasis. Importantly, patients who have high MeCo score tumors are at higher risk of developing metastatic breast cancer, compared to those with low MeCo scores. Moreover, circulating tumor cells (CTCs) are associated with a higher rate of metastatic dissemination, making CTC detection in the circulation of patients with breast cancer a significant prognostic biomarker for breast cancer metastasis. Beyond their enumeration per blood volume units, specific prognostic features of CTCs are not fully explored. We sought to determine whether MeCo scores increase stepwise along the metastatic cascade, from primary tumors to CTCs to distant metastatic colonization, using patient-matched biopsies. Methods: CTCs were isolated from the peripheral blood of two patient cohorts: patients with early-stage breast cancer using immunomagnetic enrichment/FACS methodology; and patients with late-stage breast cancer using the ANGLE Parsortix microfluidics system. Gene expression profiling using RNA-seq was performed on CTCs and matched primary tumors (PTs) in the early-stage cohort, and on CTCs and matched metastases (METs) for the late-stage cohorts. A quantile normalization approach was used to allow comparison across cohorts and MeCo scores were computed for all samples. The Wilcoxon matched-pairs signed rank test was performed for the comparison of MeCo scores from matching samples within each cohort; the Mann–Whitney unpaired test was used to compare MeCo scores of CTCs across cohorts. Results: In 12 pairs of patients with early-stage breast cancer, MeCo scores in CTCs were significantly higher than in their matched PTs (p= 0.026). Additionally, in 26 pairs of metastatic patient CTCs and METs, MeCo scores were significantly higher in METs compared to matched CTCs (p= 0.0004). MeCo scores of CTCs were similar between patients with early- and late-stage breast cancers, despite differing CTC isolation strategies (epitope-dependent and microfluidics size gradient). Notably, 98% of the genes in the MeCo score were present across evaluable CTC, MET, and PT samples. Conclusions: Our results show that the MeCo score is higher in CTCs than in PTs, and higher in METs compared to CTCs, in early- and late-stage breast cancer, respectively (i.e., PT < CTC < MET). Therefore, the MeCo score is progressively higher throughout the metastatic cascade in breast cancer. These findings demonstrate that mechanical conditioning from primary tumors is retained during metastatic progression, after mechanical induction by ECM stiffness is lost, as cancer cells disseminate through systemic circulation. Additionally, these findings support that cancer cells with higher MeCo scores are more competent with—and potentially selected for—metastatic progression. Importantly, these findings provide a novel feature of CTCs, mechanical conditioning (MeCo), which is associated with higher capacity for metastasis. Furthermore, since the CTC MeCo score is elevated even in early-stage breast cancer, it could provide, in addition to CTC enumeration, a potential prognostic indicator to improve metastatic risk assessment in early disease.
背景：机械适应（MeCo）评分是一种多基因表达特征，由原发乳腺癌中的癌细胞获得，反映了它们对肿瘤纤维化引起的细胞外基质（ECM）硬度的反应性。机械传导下游的染色质重塑使得癌细胞即使在缺乏原发肿瘤微环境的机械刺激（例如，在转移过程中通过体循环播散后）的情况下，仍能保留这些获得的侵袭性特征。重要的是，与MeCo评分低的患者相比，MeCo评分高的肿瘤患者发生转移性乳腺癌的风险更高。此外，循环肿瘤细胞（CTCs）与更高的转移播散率相关，使得乳腺癌患者循环中CTCs的检测成为乳腺癌转移的重要预后生物标志物。除了按血容量单位进行计数外，CTCs的具体预后特征尚未得到充分探索。我们试图利用患者匹配的活检样本，确定MeCo评分是否沿着转移级联（从原发肿瘤到CTCs，再到远处转移定植）逐步增加。 方法：从两个患者队列的外周血中分离CTCs：早期乳腺癌患者使用免疫磁珠富集/FACS方法；晚期乳腺癌患者使用ANGLE Parsortix微流控系统。对早期队列的CTCs和匹配的原发肿瘤（PTs），以及晚期队列的CTCs和匹配的转移灶（METs）进行了RNA-seq基因表达谱分析。采用分位数归一化方法以便在不同队列间进行比较，并计算所有样本的MeCo评分。使用Wilcoxon配对符号秩检验比较每个队列内匹配样本的MeCo评分；使用Mann-Whitney非配对检验比较不同队列间CTCs的MeCo评分。 结果：在12对早期乳腺癌患者中，CTCs的MeCo评分显著高于其匹配的PTs（p=0.026）。此外，在26对转移性患者的CTCs和METs中，METs的MeCo评分显著高于匹配的CTCs（p=0.0004）。尽管采用了不同的CTC分离策略（表位依赖性和微流控尺寸梯度），早期和晚期乳腺癌患者的CTCs MeCo评分相似。值得注意的是，MeCo评分中98%的基因在可评估的CTC、MET和PT样本中均存在。 结论：我们的结果表明，在早期和晚期乳腺癌中，CTCs的MeCo评分分别高于PTs，而METs的MeCo评分又高于CTCs（即PT < CTC < MET）。因此，在乳腺癌的整个转移级联过程中，MeCo评分逐渐升高。这些发现表明，在癌细胞通过体循环播散后，即使ECM硬度引起的机械诱导已经消失，原发肿瘤的机械适应在转移进展过程中仍然得以保留。此外，这些发现支持具有更高MeCo评分的癌细胞更具备转移进展的能力，并可能在转移过程中被选择。重要的是，这些发现提供了CTCs的一个新特征——机械适应（MeCo），它与更高的转移能力相关。此外，由于即使在早期乳腺癌中CTC的MeCo评分也已升高，除了CTC计数外，它还可能提供一个潜在的预后指标，以改善早期疾病的转移风险评估。
肿瘤(癌症)患者之家