Stem/progenitor cell marker expression in clear cell renal cell carcinoma: a potential relationship with the immune microenvironment to be explored
- 作者列表："Ju-Yoon Yoon","Craig Gedye","Joshua Paterson","Laurie Ailles
Abstract Background Clear cell renal cell carcinoma (ccRCC) is a markedly heterogeneous disease in many aspects, including the tumour microenvironment. Our previous study showed the importance of the tumour microenvironment in ccRCC xeno-transplant success rates. In order to better understand the potential relationship between TICs and the immune microenvironment, we employed a multi-modal approach, examining RNA and protein expression (flow cytometry, immunohistochemistry). Methods We first examined the gene expression pattern of 18 stem/progenitor marker genes in the cancer genome atlas (TCGA) ccRCC cohort. Flow cytometry was next employed to examine lineage-specific expression levels of stem/progenitor markers and immune population makeup in six, disaggregated, primary ccRCC specimens. Immunohistochemistry was performed on a commercial ccRCC tissue microarray (TMA). Results The 18 genes differed with respect to their correlation patterns with one another and to their prognostic significance. By flow cytometry, correlating expression frequency of 12 stem/progenitor markers and CD10 resulted in two clusters—one with CD10 (marker of proximal tubular differentiation), and second cluster containing mostly mesenchymal stem cell (MSC) markers, including CD146. In turn, these clusters differed with respect to their correlation with different CD45+ lineage markers and their expression of immune checkpoint pathway proteins. To confirm these findings, four stem/progenitor marker expression patterns were compared with CD4, CD8 and CD20 in a ccRCC TMA which showed a number of similar trends with respect to frequency of the different tumour-infiltrating leukocytes. Conclusion Taken together, we observed heterogeneous but patterned expression levels of different stem/progenitor markers. Our results suggest a non-random relationship between their expression patterns with the immune microenvironment populations in ccRCC.
【摘要】背景肾透明细胞癌 (ccRCC) 是一种多方面显著异质性的疾病，包括肿瘤微环境。我们之前的研究显示了肿瘤微环境在 ccRCC 异种移植成功率中的重要性。为了更好地理解 tic 与免疫微环境之间的潜在关系，我们采用了多模态方法，检测 RNA 和蛋白表达 (流式细胞术，免疫组化)。方法我们首先检测了癌症基因组图谱 (TCGA) ccRCC 队列中 18 个干/祖细胞标记基因的基因表达模式。接下来采用流式细胞术检测 6 例分类的原代 ccRCC 标本中干/祖细胞标志物和免疫群体构成的谱系特异性表达水平。在商业 ccRCC 组织芯片 (TMA) 上进行免疫组织化学。结果 18 个基因在相互关联模式和预后意义方面存在差异。通过流式细胞术，12 个干/祖细胞标志物和 CD10 的相关表达频率导致两个簇-一个与 CD10 (近端肾小管分化的标志物),第二簇主要包含间充质干细胞 (MSC) 标记，包括 cd146。反过来，这些簇在与不同 CD45 + 谱系标记的相关性及其免疫检查点通路蛋白表达方面存在差异。为了证实这些发现，将四种干/祖细胞标记表达模式与 CD4,ccRCC TMA 中的 CD8 和 CD20 显示了许多与不同肿瘤浸润白细胞频率相似的趋势。结论综合起来，我们观察到不同干/祖细胞标志物的异质性但模式化表达水平。我们的结果表明它们的表达模式与 ccRCC 中的免疫微环境群体之间存在非随机关系。
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.