Gene expression data from cancer mammospheres and bulk tumors

Tumorigenic breast cancer cells characterized by CD44 expression and low or undetectable CD24 levels (CD44+/CD24-/low) may be resistant to chemotherapy and therefore responsible for cancer relapse. Paired breast cancer core biopsies before and after neoadjuvant chemotherapy or lapatinib were obtained and as single cell suspensions stained using antibodies against CD24, CD44, and lineage markers, and then analyzed by flow cytometry. Mammosphere (MS) formation in culture was compared before and after treatment. Global gene expression differences between cancer cells bearing CD44+/CD24-/low cells and all other sorted cells, and between cancer MS and the primary bulk invasive cancers were analyzed. We report that CD44+/CD24-/low tumorigenic breast cancer cells were intrinsically chemoresistant ─ chemotherapy led to increased CD44+/CD24-/low cells, increased self-renewal capacity on MS assays, and enhanced tumorigeneicity in immunocompromised SCID/Beige mice. Conversely, in patients with HER2 overexpressing tumors, the EGFR/HER2 tyrosine kinase inhibitor, lapatinib decreased CD44+/CD24-/low cells, with the majority of these patients after conventional therapy achieving pathologic complete response, a validated surrogate marker for long-term survival. Gene transcription pathways that underlie chemoresistant, MS-forming CD44+/CD24-/low cells involve genes belonging to stem cell self-renewal, Wnt signaling, and early development pathways. Keywords: two group comparison Cells from human breast tumors were grown as mammospheres (MS). Isolated single cell suspensions from primary breast cancers were plated onto non-adherent (polyhema-coated) plastic, counted with a hematocytometer, and 20,000 cells were then seeded into a 6-well ultra-low attachment plate supplemented with 2mL MEGM, with the addition of 2 mL of freshly unfrozen MEGM every 3-4 days. Gene expression profiles were taken of both MS and primary bulk tumors and compared with each other.

以CD44阳性表达、CD24低表达或检测不到（CD44+/CD24-/low）为表型特征的致瘤性乳腺癌细胞，可能对化疗产生耐药性，进而促成癌症复发。我们获取了新辅助化疗（neoadjuvant chemotherapy）或拉帕替尼（lapatinib）治疗前后的配对乳腺癌核心穿刺活检样本，将其制备为单细胞悬液，使用针对CD24、CD44及谱系标志物的抗体进行染色，随后通过流式细胞术（flow cytometry）进行分析。对比了治疗前后体外培养中的乳腺球（mammosphere, MS）形成能力。分析了携带CD44+/CD24-/low表型的癌细胞与其余分选细胞之间的全局基因表达差异，以及乳腺球与原发浸润性实体瘤之间的全局基因表达差异。
本研究证实，CD44+/CD24-/low致瘤性乳腺癌细胞本身即具有化疗耐药性——化疗可使CD44+/CD24-/low细胞比例升高，乳腺球形成实验中的自我更新能力增强，且在免疫缺陷SCID/Beige小鼠中的致瘤性提升。反之，在HER2过表达肿瘤患者中，EGFR/HER2酪氨酸激酶抑制剂拉帕替尼可降低CD44+/CD24-/low细胞比例，多数接受常规治疗后的此类患者达到了病理完全缓解（pathologic complete response），这是经临床验证的长期生存替代标志物。
介导化疗耐药、可形成乳腺球的CD44+/CD24-/low细胞的基因转录通路，涉及干细胞自我更新、Wnt信号通路（Wnt signaling）及早期发育相关基因。
关键词：两组比较
从人类乳腺肿瘤分离得到的细胞以乳腺球（mammosphere, MS）的形式进行培养。将从原发性乳腺癌中分离的单细胞悬液接种至聚羟乙基甲基丙烯酸酯（polyhema）包被的非黏附性培养塑料表面，使用血细胞计数板进行细胞计数，随后将20000个细胞接种至6孔超低吸附培养板中，加入2mL MEGM培养基，每3-4天添加2mL新鲜解冻的MEGM培养基。对乳腺球与原发实体瘤样本进行基因表达谱分析，并对二者进行对比。