Sepsis trains stressed granulocytes to boost nonspecific response and trigger a new wave of inflammation when facing secondary infection. Here, we present a protocol for a murine model of sepsis with secondary infection. We describe steps for cecal ligation and puncture operation and rechallenging with lipopolysaccharide or Pseudomonas aeruginosa during the recovery phase. We also detail steps to characterize the stressed granulocytes by assessing their functional phenotypes and effect on the mortality of rechallenged mice. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.

Abstract Tumor evolution is one of the major mechanisms responsible for acquiring therapy-resistant and more aggressive cancer clones. Whether the tumor microenvironment through immune-mediated mechanisms might promote the development of more aggressive cancer types is crucial for the identification of additional therapeutical opportunities. Here, we identified a novel subset of tumor-associated neutrophils, defined as tumor-associated neutrophil precursors (PreNeu). These PreNeu are enriched in highly proliferative hormone-dependent breast cancers and impair DNA repair capacity.  Mechanistically, succinate secreted by tumor-associated PreNeu inhibits homologous recombination, promoting error-prone DNA repair through non-homologous end-joining regulated by PARP-1. Consequently, breast cancer cells acquire genomic instability, promoting tumor editing and progression. Selective inhibition of these pathways induces increased tumor cell killing in vitro and in vivo. Tumor-associated PreNeu score correlates with copy number alterations in highly proliferative hormone-dependent tumors from breast cancer patients. Treatment with PARP-1 inhibitors counteract the pro-tumorigenic effect of these neutrophils and synergize with combined immunotherapeutic approaches.

Glioblastoma (GBM) is the most common malignant brain tumor in adults, responsible for approximately 225,000 deaths per year. Despite preclinical successes, most interventions have failed to extend patient survival by more than a few months. Treatment with anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint blockade (ICB) monotherapy has been beneficial for malignant tumors such as melanoma and lung cancers but has yet to be effectively employed in GBM. This study aimed to determine whether supplementing anti-PD-1 ICB with engineered extended half-life IL2, a potent lymphoproliferative cytokine, could improve outcomes. This combination therapy, subsequently referred to as enhanced checkpoint blockade (ECB), delivered intraperitoneally, reliably cures approximately 50% of C57BL/6 mice bearing orthotopic GL261 gliomas and extends median survival of the treated cohort. In the CT2A model, characterized as being resistant to CBI, ECB caused a decrease in CT2A tumor volume in half of measured animals similar to what was observed in GL261-bearing mice, promoting a trending survival increase. ECB generates robust immunologic responses, features of which include secondary lymphoid organ enlargement and increased activation status of both CD4 and CD8 T cells. This immunity is durable, with long-term ECB survivors able to resist GL261 rechallenge. Through employment of depletion strategies, ECB's efficacy was shown to be independent of host MHC class I-restricted antigen presentation but reliant on CD4 T cells. These results demonstrate ECB is efficacious against the GL261 glioma model through an MHC class I-independent mechanism and supporting further investigation into IL2-supplemented ICB therapies for tumors of the central nervous system.
©2023 American Association for Cancer Research.

Extracellular vesicles (EVs) released by cells in the bone marrow (BM) are important for regulating proliferation, differentiation, and other processes in hematopoietic stem cells (HSC). TGF-β signaling is now well known to be involved in HSC's quiescence and maintenance, but the TGF-β pathway related to EVs is still largely unknown in the hematopoietic system. We found that the EV inhibitor Calpeptin, when injected intravenously into mice, particularly affected the in vivo production of EVs carrying phosphorylated Smad2 (p-Smad2) in mouse BM. This was accompanied with an alteration in the quiescence and maintenance of murine HSC in vivo. EVs produced by murine mesenchymal stromal MS-5 cells also showed presence of p-Smad2 as a cargo. We treated MS-5 cells with the TGF-β inhibitor SB431542 in order to produce EVs lacking p-Smad2, and discovered that its presence was required for ex vivo maintenance of HSC. In conclusion, we revealed a new mechanism involving EVs produced in the mouse BM that transport bioactive phosphorylated Smad2 as a cargo to enhance the TGF-β signaling-mediated quiescence and maintenance of HSC.
© 2023. The Author(s).

Extracellular vesicles (EVs) released by cells in the BM are important to regulate proliferation, differentiation and other properties of hematopoietic stem cell (HSC). While the TGF-β signaling is now well known since a long time to be involved in HSC’s quiescence and maintenance, the TGF-β pathway related to EVs is still largely unknown in the hematopoietic system. We discovered that EVs inhibitor Calpeptin, intravenously injected in mice, particularly affected the in vivo production of EVs, carrying phosphorylated Smad2 (p-Smad2) in mouse BM. This was accompanied with an alteration in the quiescence and maintenance of murine HSC in vivo . Murine stromal MS-5 cells also expressed p-Smad2 as a cargo. To prove that the signal transducer p-Smad2 was required for HSC maintenance, we treated murine mesenchymal stromal cells (MSC) with the TGF-β inhibitor SB431542, in order to produce EVs without the expression of p-Smad2 and discovered that this mediator was required for maintenance of HSC ex vivo . In conclusion, we discovered a new mechanism, which involved EVs, produced in the mouse BM that, as a cargo, transport bioactive phosphorylated Smad2 to enhance the TGF-β signaling-mediated quiescence and maintenance of HSC.