- 作者列表："Zemek, Rachael M.","Fear, Vanessa S.","Forbes, Cath","Jong, Emma","Casey, Thomas H.","Boon, Louis","Lassmann, Timo","Bosco, Anthony","Millward, Michael J.","Nowak, Anna K.","Lake, Richard A.","Lesterhuis, W. Joost
In this protocol, mice are inoculated with two separate tumors derived from the same cell line. One tumor is removed and assessed before treatment; the other is used to assess the effect of treatment. The therapeutic response to immune checkpoint blockade (ICB) is highly variable, not only between different cancers but also between patients with the same cancer type. The biological mechanisms underlying these differences in response are incompletely understood. Identifying correlates in patient tumor samples is challenging because of genetic and environmental variability. Murine studies usually compare different tumor models or treatments, introducing potential confounding variables. This protocol describes bilateral murine tumor models, derived from syngeneic cancer cell lines, that display a symmetrical yet dichotomous response to ICB. These models enable detailed analysis of whole tumors in a highly homogeneous background, combined with knowledge of the therapeutic outcome within a few weeks, and could potentially be used for mechanistic studies using other (immuno-)therapies. We discuss key considerations and describe how to use two cell lines as fully optimized models. We discuss experimental details, including proper inoculation technique to achieve symmetry and one-sided surgical tumor removal, which takes only 5 min per mouse. Furthermore, we outline the preparation of bulk tissue or single-cell suspensions for downstream analyses such as bulk RNA-seq, immunohistochemistry, single-cell RNA-seq and flow cytometry.
在该方案中，小鼠接种来自相同细胞系的两个单独的肿瘤。一个肿瘤在治疗前切除并评估; 另一个用于评估治疗效果。对免疫检查点阻断 (ICB) 的治疗反应是高度可变的，不仅在不同癌症之间，而且在具有相同癌症类型的患者之间也是如此。这些反应差异背后的生物学机制尚不完全清楚。由于遗传和环境的变异性，确定患者肿瘤样本中的相关因素具有挑战性。鼠类研究通常比较不同的肿瘤模型或治疗方法，引入潜在的混杂变量。该方案描述了双侧鼠肿瘤模型，来源于同基因癌细胞系，对 ICB 显示出对称但二分的反应。这些模型能够在高度均匀的背景下对整个肿瘤进行详细分析，结合几周内的治疗结果知识，并可能用于使用其他 (免疫) 的机制研究疗法。我们讨论了关键考虑因素，并描述了如何使用两种细胞系作为完全优化的模型。我们讨论了实验细节，包括适当的接种技术以实现对称和单侧手术切除肿瘤，每只小鼠只需 5 min。此外，我们概述了大宗组织或单细胞悬浮液的制备，用于大宗 RNA-seq 、免疫组化、单细胞 RNA-seq 和流式细胞术等下游分析。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.