靶向肿瘤血管是对抗癌症的一种有效方法；然而，激活替代通路来重建受破坏的血管导致肿瘤迅速复长。利用宿主免疫细胞激发和维持强效抗肿瘤反应的免疫治疗已成为最有前景的癌症治疗工具之一，但许多治疗由于产生了耐药机制而失败。因此，我们的目标是检查免疫治疗和抗血管治疗的组合是否能够成功治疗免疫原性差、难治性的黑色素瘤和三阴性乳腺肿瘤模型。我们的研究在B16-F10黑色素瘤和4T1乳腺肿瘤小鼠模型上进行。小鼠接受STING通路激动剂（cGAMP）和破坏血管的剂量CA4P的治疗。监测肿瘤生长。采用多重免疫荧光和流式细胞术综合研究肿瘤微环境（TME）。我们还检查了这种设计治疗是否能使研究中的肿瘤模型对免疫检查点抑制剂（抗PD-1）敏感。在4T1肿瘤中，仅使用STING激动剂cGAMP作为单药治疗不能有效抑制肿瘤生长，因为4T1肿瘤中STING蛋白水平较低。然而，当额外与抗血管剂联合治疗时，获得了显著的治疗效果。在该模型中，获得的效果与TME极化和先天免疫反应的刺激，尤其是NK细胞的激活有关。组合治疗无法激活CD8+ T细胞。由于缺乏PD-1的上调，当额外与抗PD-1抑制剂联合使用时，未观察到改善的治疗效果。在富含STING蛋白的B16-F10肿瘤中，cGAMP作为单药治疗足以诱导强效抗肿瘤反应。在该模型中，治疗效果是由TME与活化的NK细胞浸润有关。cGAMP还引起了CD8+PD-1+ T细胞浸润到TME中，因此，观察到了使用PD-1抑制剂的额外好处。该研究为应用疗法的治疗结果提供了TME对其的重大影响的临床前证据，包括免疫细胞贡献和ICI响应性。我们指出，在抗肿瘤治疗之前，需要进行仔细的TME筛查以实现满意的结果。版权所有©2023 Czapla, Drzyzga, Matuszczak, Cichoń, Rusin, Jarosz-Biej, Pilny和Smolarczyk。

Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models.Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1).The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed.The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results.Copyright © 2023 Czapla, Drzyzga, Matuszczak, Cichoń, Rusin, Jarosz-Biej, Pilny and Smolarczyk.