The Role of Egfl6 in Tumor Immunity

卵巢癌（OvCa）具有极高的死亡率，这表明对新的治疗方法的明确需求。 接近。一种这样的方法是免疫检查点抑制剂（ICI）疗法。不幸的是，尽管 由于在许多OvCa患者中存在抗肿瘤效应细胞，ICI疗法在OvCa中具有差的响应率。 其中一个原因可能是大量的免疫抑制性骨髓源性抑制细胞（MDSC）和 肿瘤相关巨噬细胞（TAM）存在于OvCa中。MDSC和TAM均负性调节免疫 在癌症和治疗方法中，可以破坏这些细胞的募集/功能， 反应破坏MDSC/TAM的潜在治疗靶标是Egfl 6。EGF 16是一种发育生长因子 已知其调节细胞迁移、增殖和分化，并在OvCa中高度表达。 我们发现，过度表达Egfl 6的小鼠在两种细胞中的粒细胞和单核细胞数量增加， 骨髓（BM）和脾脏。BM骨髓细胞的Egfl 6处理与整合素相关 p-Syk的依赖性增加以及向IL-10+和S100 A9+粒细胞MDSC的分化。鼠肿瘤 其与对照相比，（i）具有MDSC积累和M2-M4的显著增加， 如TAM，（iii）表达与中性粒细胞胞外陷阱（NET）相关的因子CXCL 2和CLEC 5a， 形成，和（iii）对ICI疗法是难治的。因此，我们假设Egf 16通过Syk激活，促进细胞凋亡。 通过增加肿瘤中MDSC/TAM的数量及其NET的产生，免疫抑制性TME。作为 对此的延伸，我们假设抑制Egf 16将增强ICI疗法的功效。来测试我们 假设，我们提出：SA 1。评估Syk在骨髓细胞上的Egfl 6信号传导中的需要。作为Syk 是粒细胞中整联蛋白信号传导所必需的，我们假设靶向破坏Syk将阻止Egfl 6- 依赖性粒细胞聚集。使用特异性Syk抑制剂化合物和Syk-KO 对于粒细胞/单核细胞，我们将剖析Egfl 6-Syk调节轴。SA 2.分析Egfl 6在NET中的作用 形成和对ICI疗法的抗性。我们发现表达Egfl 6的肿瘤具有增加的水平和NETosis。 并且Egf 16诱导与NETosis相关的因子CXCL 2和CLEC 5a。这种情况发生在存在 以及缺乏ICI治疗。我们假设Egf 16通过诱导NET，限制了抗肿瘤免疫， 促进对ICI疗法的抗性。我们将使用CXCL 2和CLEC 5a介导的抑制/KO方法， 定义Egfl 6诱导NETosis的机制以及Egfl 6诱导的NETosis对ICI细胞毒性的影响 免疫力SA 3.评价Egfl 6中和抗体对ICI治疗和抗肿瘤疗效的影响 免疫力我们将使用免疫活性ICI应答性鼠OvCa模型来评估免疫抑制剂的能力。 Egfl 6中和抗体以克服Egfl 6介导的免疫抑制。结合这些研究， 描述OvCa介导的肿瘤免疫抑制的新机制，并验证EGFL 6抗体作为 增强OvCa患者ICI治疗的新治疗方法。

Ovarian cancer (OvCa) has an extremely high mortality rate, indicating a clear need for new therapeutic approaches. One such approach is immune checkpoint inhibitor (ICI) therapy. Unfortunately, despite the presence of anti-tumor effector cells in many OvCa patients, ICI therapies have poor response rates in OvCa. One reason for this may large numbers of immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) present in the OvCa. Both MDSC and TAM negatively regulate immunity in cancer and therapeutic approaches which can disrupt the recruitment/function of these cells can enhance ICI response. A potential therapeutic target to disrupt MDSC/TAM is Egfl6. Egfl6 is a developmental growth factor which is known to regulate cellular migration, proliferation, and differentiation and is highly expressed in OvCa. We found that mice which overexpress Egfl6 have increased numbers of granulocytes and monocytes in both the bone marrow (BM) and spleen. Egfl6 treatment of BM myeloid cells was associated with an integrin dependent increase in p-Syk, and differentiation to IL-10+ and S100A9+ granulocytic MDSC. Murine tumors which overexpress Egfl6, compared to controls, (i) have a significant increase in MDSC accumulation and M2- like TAM, (iii) express CXCL2 and CLEC5a, factors associated with neutrophil extracellular traps (NETs) formation, and (iii) are refractory to ICI therapy. We thus hypothesize that Egfl6, via Syk activation, promotes an immunosuppressive TME by increasing numbers of MDSC/TAM and their production of NETs in tumors. As an extension of this, we hypothesize that inhibition of Egfl6, will enhance the efficacy of ICI therapy. To test our hypotheses, we propose: SA1. To evaluate the requirement for Syk in Egfl6 signaling on myeloid cells. As Syk is required for integrin signaling in granulocytes, we hypothesize that targeted disruption of Syk will prevent Egfl6- dependent granulocyte accumulation. Using specific Syk inhibitor compounds and Syk-KO granulocytes/monocytes, we will dissect the Egfl6-Syk regulatory axis. SA2. To analyze the role of Egfl6 in NETs formation and resistance to ICI therapy. We found Egfl6 expressing tumors have increased levels and NETosis and that Egfl6 induces CXCL2 and CLEC5a, factors associated with NETosis. This occurs both in the presence and absence of ICI therapy. We hypothesize that Egfl6, by induction of NETs, limits the anti-tumor immunity promoting resistance to ICI therapy. We will use CXCL2 and CLEC5a mediated inhibition/KO approaches to define the mechanisms by which Egfl6 induces NETosis and the impact of Egfl6 induced NETosis on ICI cytotoxic immunity. SA 3. Evaluate the impact of Egfl6 neutralizing antibody on the efficacy of ICI therapy and anti-tumor immunity. We will use an immune competent ICI responsive murine OvCa model to evaluate the ability of an Egfl6 neutralizing antibody to overcome Egfl6 mediated immune suppression. Combined these studies will characterize a novel mechanism of OvCa mediated tumor immune suppression and validate EGFL6 antibody as a novel therapeutic approach to enhance ICI therapy in OvCa patients.