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Supplementary Table from Anlotinib Induces a T Cell–Inflamed Tumor Microenvironment by Facilitating Vessel Normalization and Enhances the Efficacy of PD-1 Checkpoint Blockade in Neuroblastoma

Supplementary Figure from Anlotinib Induces a T Cell–Inflamed Tumor Microenvironment by Facilitating Vessel Normalization and Enhances the Efficacy of PD-1 Checkpoint Blockade in Neuroblastoma

Spatial Transcriptomics (ST) technology enables systematic depiction of regional milieu of a tissue, like tumor immuno-microenvironment (iTME). However, a powerful algorithmic framework to dissect spatially resolved niches, and to quantitatively evaluate spatial cell interaction intensity will pave the ways to understand the spatial signature associated mechanism. In this study, we provide a promising framework (StereoSiTE), which is based on space nearest neighbor graph and gene expression profile to spatially resolve iTME and to quantitatively define cell-cell communication intensity. We applied StereoSiTE to dissect the iTME of xenograft model receiving immunoagonist treatment, 7 distinct cellular neighborhoods (CN) were identified, and each CN was considered as the functional unit with exclusive cell type (CT) composition. Further deconvolving the joint matrix covering CNs and CTs indicated the importance of neutrophils in CN6, which was confirmed by pathway enrichment analysis. What’s more, analysis of interaction intensity indicated that the recruited neutrophils preserved tumor protection activity through paired IL-1β/IL-1R after immunoagonist treatment exclusively in CN6. This evidence provided a new possible vision of tumor immune evasion orchestrated by neutrophils. StereoSiTE is believed to be a promising framework of mapping iTME niches using spatial transcriptomics, which could be utilized to spatially reveal tumoribiology mechanisms.HighlightA framework based on space nearest neighbor graph and gene expression profile to spatially resolve iTME and to quantitatively define cell-cell communication intensity (StereoSiTE)

Purpose: Anlotinib has achieved good results in clinical trials of a variety of cancers. However, the effects of anlotinib on the tumor microenvironment (TME) and systemic immunity have not been reported. There is an urgent need to identify the underlying mechanism to reveal new opportunities for its application in neuroblastoma (NB) and other cancers. Understanding the mechanism will hopefully achieve the goal of using the same method to treat different cancers. Experimental Design: This study used bioinformatics, NB syngeneic mouse models, flow cytometry, RNA-seq, and immunofluorescence staining to explore the mechanisms of anlotinib on the TME, and further explored anlotinib-containing combination treatment strategies. Results: We proved that anlotinib facilitates tumor vessel normalization at least partially through CD4+ T cells, reprograms the immunosuppressive TME into an immunostimulatory TME, significantly inhibits tumor growth, and effectively prevents systemic immunosuppression. Moreover, the combination of anlotinib with a PD-1 checkpoint inhibitor counteracts the immunosuppression caused by the upregulation of PD-L1 after monotherapy, extends the period of vascular normalization, and finally induces NB regression. Conclusions: To our knowledge, this study is the first to dynamically evaluate the effect of a multitarget antiangiogenic tyrosine kinase inhibitor on the TME. These findings have very important clinical value in guiding the testing of related drugs in NB and other cancers. Based on these findings, we are conducting a phase II clinical study (NCT04842526) on the efficacy and safety of anlotinib, irinotecan, and temozolomide in the treatment of refractory or relapsed NB, and hopefully we will observe patient benefit.