Phospho-protein detection in solid tumors using phospho-flow cytometry

作者：

David Draper，Ph.D.，科学家，体外运营

DATE:

January 2018

In this article, we will be presenting phospho-protein detection in solid tumors using phospho-flow cytometry and reviewing the analysis of cabozantinib-induced inhibition of PI3K/AKT and JAK-STAT signaling in tumor vs. host immune cells.

这in vivo通过测量药物靶标的途径内的细胞信号传导蛋白的磷酸化状态来评估小分子抑制剂的疗效。当需要对实体瘤的分析时，这可能是挑战。在利用啮齿动物模型的临床前研究中，肿瘤分析最常见的方法是免疫印刷和基于ELISA的技术。然而，这些批量分析方法具有两个主要缺点。它们同时测量多个目标的相对能力是次优。更重要的是，它们无法分析异质肿瘤样本内的多个不同亚群中的靶标。当目标信令蛋白在肿瘤微环境中的不同细胞类型中的相反功能如免疫细胞与肿瘤细胞中时，这可能是重要的，如果药物诱导的效果在一个但不是两个子集中发生的，则甚至临时。

Covance has developed a novelphospho-flow-based platform同时分析多个细胞signaling targets in both the host immune cell and the tumor cell compartments derived from solid tumors. This is accomplished by distinguishing the tumor cells from the infiltrating host immune cells by immunophenotyping for the pan-hematopoietic cell marker CD45, prior to phospho-protein analysis. To demonstrate the utility of this platform, the levels of phosphorylated STAT5 and the downstream target of AKT (S6) were measured in subcutaneous MV-4-11 tumors harvested from mice treated with the FLT3 inhibitor cabozantinib. Figure 1 reveals that cabozantinib reduces the levels of both pS6 and pSTAT5 in the human (h)CD45+ tumor cells. No changes in the levels of these phospho-proteins were observed in the mouse (m)CD45+ host immune cells (not shown). Thus, insight into PI3K/AKT and JAK-STAT signaling activity is gained, which are well documented pathways that regulate both tumor growth and host immunity, and are attractive targets for therapeutic intervention.

MV-4-11 is a model for acute myeloid leukemia (AML), a malignancy driven by genetic mutation in one of several genes. The most common event occurs in FLT3 gene, which results in a constitutively active FLT3 protein that drives cancerous cell proliferation by triggering hyper-activation of PI3K/AKT and JAK-STAT signaling.¹Cabozantib介导的FLT3抑制已经证明抑制MV-4-11肿瘤生长in vivo那which correlated with an inhibition of AKT and STAT signalingin vitro。²Although overactive PI3K/AKT and JAK-STAT5 signaling has been well documented to drive tumor growth in many models, the activity of these signaling proteins are also required for T cell activation and the generation of memory T cell formation.^3.那4.那5.This dichotomy emphasizes the importance of being able to simultaneously analyze phospho-proteins in both solid tumor-derived immune and tumor cells.

Covance的肿瘤磷酸流动具有推导细胞信号场的潜力。在肺和腹膜肿瘤中，2010年描述了固体组织磷流。^6.Since, reports of other successful approaches have been scarce. More recently, an approach termed DISSECT was paired with cytometry and successfully used to measure phospho-proteins in epithelium samples, and later colorectal tumors^。7.那8.It should be noted that these approaches require fixation after tissue harvest, which can alter the epitopes that are bound by the fluorescent antibodies used for both immunophenotyping and signaling analysis. This can limit the amount of data the techniques provide. Solid tumor phospho-flow at Covance does not require fixation, prior to immuno-staining. This facilitates our ability to target distinct populations for analysis. We are working to expand our capabilities. Ongoing efforts are focused on the development of new solid tumor phospho-flow services that will enable the analysis of in vivo treatment induced effects in specific T cell subsets and tumor cells simultaneously.

Contact Covanceto learn more about solid tumor phospho-flow, as well as information on our expanding list of signaling proteins for which we can provide analysis.