Modeling Luminal breast cancer heterogeneity: Combination therapy to suppress hormone receptor negative cells in Luminal disease. Homo sapiens

Many Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER-) and progesterone (PR-) receptor-negative cells among the ER+PR+ ones. Currently, the Basal-like ER-PR- Luminobasal subpopulation in Luminal disease is not targeted for treatment. To address the relationships between ER+PR+ and ER-PR- cells in Luminal cancers and tightly control their ratios, we have generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We show that pLUM suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and in pLUM:pLB mixed-cell xenografts in mice. High-throughput screening of FDA-approved oncology drugs reveal pLB cells are sensitive to the EGFR inhibitors Gefitinib and Erlotinib. In mixed-cell 3D colonies and mixed-cell solid mouse tumors, combination therapy with the antiestrogen Fulvestrant and the EGFRi Gefitinib constitutes a robust treatment strategy. We propose that response to combination endocrine/EGFRi therapies in heterogeneous Luminal cancers will improve long-term survival in patients whose primary tumors have been preselected for the appropriate biomarkers. Overall design: The study contains 2 different sample groups measured in triplicate, for a total of 6 individual samples (6 arrays). To generate pLUM, cells from an E tumor were propagated in vitro ~2 months in 1nM E. Live cells were FACS-sorted (Moflo XDP 100, BeckmanCoulter) using CLD3 (mouse Anti-human CLD3-FITC, R&D Systems) and CD49f (PE-Cy5 Rat Anti-Human CD49f, BD Pharmingen) to separate Luminal (CLD3+CD49f-) from Luminobasal (CLD3-CD49f+) cells. Cells were re-plated, cultured another ~2 months in E, then resorted twice to generate pure pLUM (CLD3+CD49f-). They are maintained in E-containing medium and remain Luminobasal-free. To generate pLB, cells from an E+P tumor were plated in vitro ~2.5 months under EWD conditions. They were FACS sorted and the CLD3-CD49f+ subpopulation was re-cultured an additional ~2 months under EWD conditions then resorted twice to yield pure pLB (CLD3-CD49f+). They are maintained in EWD media and remain Luminal-free. Thus, the experiment contains two cell lines and analyzed in triplicate (6 microarrays). RNA from triplicate sorts was profiled using Agilent 4x44K chips. Labeling, hybridization and initial analyses were performed at MOgene LC (www.mogene.com).

许多管腔型乳腺癌（Luminal breast cancer）具有显著异质性，其ER+PR+细胞群中富集有大量雌激素受体（ER）阴性与孕激素受体（PR）阴性的细胞。目前，管腔型乳腺癌中存在的基底样ER-PR-管腔基底（Luminobasal）亚群尚未被纳入治疗靶点范畴。为阐明管腔型乳腺癌中ER+PR+与ER-PR-细胞间的相互关系并精准调控二者比例，我们从同一亲本管腔型人乳腺癌细胞系中成功构建了同基因纯管腔型（pLUM）与纯管腔基底型（pLB）细胞系。实验证实，在体外混合细胞三维集落与小鼠体内pLUM:pLB混合细胞异种移植瘤模型中，pLUM细胞可抑制pLB细胞的增殖。对美国食品药品监督管理局（FDA）获批肿瘤药物的高通量筛选结果显示，pLB细胞对表皮生长因子受体（EGFR）抑制剂吉非替尼（Gefitinib）与厄洛替尼（Erlotinib）敏感。在混合细胞三维集落与小鼠混合实体瘤模型中，采用抗雌激素药物氟维司群（Fulvestrant）联合EGFR抑制剂吉非替尼的治疗方案展现出显著的抗肿瘤效果。我们提出，针对异质性管腔型乳腺癌的内分泌/EGFR抑制剂联合治疗方案，若提前根据合适的生物标志物筛选患者，可改善其长期生存结局。

整体实验设计：本研究包含2组不同样本，每组设置3次生物学重复，共计6个独立样本（6张基因芯片）。
构建纯管腔型细胞（pLUM）时，将雌激素（E）处理的肿瘤细胞在含1nM雌激素的培养基中体外培养约2个月。随后采用贝克曼库尔特Moflo XDP 100流式细胞分选仪，通过CLD3（小鼠抗人CLD3-FITC，R&D Systems）与CD49f（PE-Cy5标记大鼠抗人CD49f，BD Pharmingen）对活细胞进行分选，分离管腔型细胞（CLD3+CD49f-）与管腔基底型细胞（CLD3-CD49f+）。将分选得到的细胞重新铺板，在含雌激素的培养基中继续培养约2个月，随后再进行2次分选，最终获得纯pLUM细胞（CLD3+CD49f-）。该细胞系在含雌激素的培养基中维持培养，无管腔基底型细胞污染。
构建纯管腔基底型细胞（pLB）时，将雌激素+孕激素（E+P）处理的肿瘤细胞在雌激素剥夺（EWD）培养条件下体外铺板培养约2.5个月。随后进行流式细胞分选，挑取CLD3-CD49f+亚群，在雌激素剥夺培养条件下继续培养约2个月，再进行2次分选，最终获得纯pLB细胞（CLD3-CD49f+）。该细胞系在雌激素剥夺培养基中维持培养，无管腔型细胞污染。
综上，本实验包含2株细胞系，每组设置3次生物学重复（共计6张基因芯片）。采用安捷伦4x44K基因芯片对3次分选获得的RNA进行转录组表达谱分析。芯片标记、杂交与初步数据分析均由MOgene LC公司（www.mogene.com）完成。