The MYC and OCT4 genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal™ 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7⁻EpCAM⁻CD44^+/−Myc^+/−(Oct4, Snai2)^+/−) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7⁺EpCam⁺CD44⁺Snai2⁻). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7⁺EpCam⁺CD44⁻Myc⁺) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7⁺EpCam⁺CD44⁻OCT4⁺). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326⁺CD44⁺CD24⁻CD325⁺) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326⁺CD44⁺CD24⁺Oct4⁺) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis.

该MYC和OCT4基因是与维持多能性有关的已知因素，并且与癌症中更具侵略性的病程，进展和耐药性有关。确定表达Myc和Oct4蛋白的肿瘤细胞亚群将为了解哪些表达MYC和OCT4的肿瘤细胞亚群与转移和耐药相关，以及哪些亚群可以通过抗MYC和抗OCT4治疗靶向提供机会。该研究包括来自新辅助化疗（NACT）后获得的27例管腔B型乳腺癌患者肿瘤的石蜡包埋组织。免疫荧光染色用于鉴定表达Myc，Oct4和Snai2的肿瘤细胞亚群（Opal™7色试剂盒（PerkinElmer，霍普金顿，马萨诸塞州））。用Myc和Oct4蛋白以及Snai2 EMT标记物鉴定出以下肿瘤细胞亚群：具有/不具有Myc，Oct4或Snai2表达的干/祖肿瘤细胞；具有/不具有Myc，Oct4或Snai2表达的分化肿瘤细胞；和其他非肿瘤细胞（CK7⁻炎性浸润内的EpCAM ⁻ CD44 ^+/- Myc ^+/-（Oct4，Snai2）^+/-在肿瘤实质和间质中浸润。通过流式细胞术研究了血液中具有Oct4蛋白表达的循环肿瘤细胞亚群。结果发现，在患者响应于NACT部分消退（PR），肿瘤干细胞的频率为3.6倍增加在非EMT状态（P = 0.038）（CK7 ⁺ EPCAM ⁺ CD44 ⁺ SNAI2 ^- ）。在有转移灶的患者中，具有Myc表达的分化肿瘤细胞（CK7 ⁺ EpCam）的频率增加了统计学上显着的2.5倍⁺ CD44 ^- Myc ⁺），具有Oct4表达的细胞（CK7 ⁺ EpCam ⁺ CD44 ^- OCT4 ⁺）的频率增加了2.7倍。在下一阶段，确定循环肿瘤细胞（CTC）中具有Oct4蛋白表达的亚群的频率和EMT的迹象。在转移患者中，处于EMT状态的肿瘤干细胞的频率（CD326 ⁺ CD44 ⁺ CD24 ^- CD325 ⁺）（p = 0.015）增加了四倍以上，而具有Oct4干蛋白表达的祖细胞的频率（CD326 ⁺ CD44 ⁺ CD24⁺ Oct4 ⁺）（p = 0.016）比没有转移的患者高出近六倍。乳腺癌中存在具有干蛋白Myc和Oct4表达的非干（分化）肿瘤细胞。与没有转移的患者相比，随后发生转移的患者在NACT后残留肿瘤中的含量明显更高。这样的细胞是转移的新的原位标记。