Effect of topographic comparison of electroencephalographic microstates on the diagnosis and prognosis prediction of patients with prolonged disorders of consciousness.

AIMS: The electroencephalography (EEG) microstates are indicative of fundamental information processing mechanisms, which are severely damaged in patients with prolonged disorders of consciousness (pDoC). We aimed to improve the topographic analysis of EEG microstates and explore indicators available for diagnosis and prognosis prediction of patients with pDoC, which were still lacking. METHODS: We conducted EEG recordings on 59 patients with pDoC and 32 healthy controls. We refined the microstate method to accurately estimate topographical differences, and then classify and forecast the prognosis of patients with pDoC. An independent dataset was used to validate the conclusion. RESULTS: Through optimized topographic analysis, the global explained variance (GEV) of microstate E increased significantly in groups with reduced levels of consciousness. However, its ability to classify the VS/UWS group was poor. In addition, the optimized GEV of microstate E exhibited a statistically significant decrease in the good prognosis group as opposed to the group with a poor prognosis. Furthermore, the optimized GEV of microstate E strongly predicted a patient's prognosis. CONCLUSION: This technique harmonizes with the existing microstate analysis and exhibits precise and comprehensive differences in microstate topography between groups. Furthermore, this method has significant potential for evaluating the clinical prognosis of pDoC patients.

Protocol for isolating single cells from human pancreatic cancer tissues and analyzing major immune cell populations using flow cytometry.

Here, we present a protocol for collecting, dissociating, isolating, staining, and analyzing immune cells from pancreatic cancer tissues for flow cytometry. The isolated cells can also be used for single-cell RNA sequencing and other related procedures. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2023).1.

Targeting leucine-rich repeat serine/threonine-protein kinase 2 sensitizes pancreatic ductal adenocarcinoma to anti-PD-L1 immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is not sensitive to immune checkpoint blockade therapy, and negative feedback of tumor immune evasion might be partly responsible. We isolated CD8+ T cells and cultured them in vitro. Proteomics analysis was performed to compare changes in Panc02 cell lines cultured with conditioned medium, and leucine-rich repeat kinase 2 (LRRK2) was identified as a differential gene. LRRK2 expression was related to CD8+ T cell spatial distribution in PDAC clinical samples and upregulated by CD8+ T cells via interferon gamma (IFN-γ) simulation in vitro. Knockdown or pharmacological inhibition of LRRK2 activated an anti-pancreatic cancer immune response in mice, which meant that LRRK2 acted as an immunosuppressive gene. Mechanistically, LRRK2 phosphorylated PD-L1 at T210 to inhibit its ubiquitination-mediated proteasomal degradation. LRRK2 inhibition attenuated PD-1/PD-L1 blockade-mediated, T cell-induced upregulation of LRRK2/PD-L1, thus sensitizing the mice to anti-PD-L1 therapy. In addition, adenosylcobalamin, the activated form of vitamin B12, which was found to be a broad-spectrum inhibitor of LRRK2, could inhibit LRRK2 in vivo and sensitize PDAC to immunotherapy as well, which potentially endows LRRK2 inhibition with clinical translational value. Therefore, PD-L1 blockade combined with LRRK2 inhibition could be a novel therapy strategy for PDAC.

Multi-omics analysis reveals the chemoresistance mechanism of proliferating tissue-resident macrophages in PDAC via metabolic adaptation.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically demonstrates resistance to chemotherapy. Tumor-associated macrophages (TAMs) are essential in tumor microenvironment (TME) regulation, including promoting chemoresistance. However, the specific TAM subset and mechanisms behind this promotion remain unclear. We employ multi-omics strategies, including single-cell RNA sequencing (scRNA-seq), transcriptomics, multicolor immunohistochemistry (mIHC), flow cytometry, and metabolomics, to analyze chemotherapy-treated samples from both humans and mice. We identify four major TAM subsets within PDAC, among which proliferating resident macrophages (proliferating rMφs) are strongly associated with poor clinical outcomes. These macrophages are able to survive chemotherapy by producing more deoxycytidine (dC) and fewer dC kinases (dCKs) to decrease the absorption of gemcitabine. Moreover, proliferating rMφs promote fibrosis and immunosuppression in PDAC. Eliminating them in the transgenic mouse model alleviates fibrosis and immunosuppression, thereby re-sensitizing PDAC to chemotherapy. Consequently, targeting proliferating rMφs may become a potential treatment strategy for PDAC to enhance chemotherapy.

Runx3-overexpression cooperates with ex vivo AKT inhibition to generate receptor-engineered T cells with better persistence, tumor-residency, and antitumor ability.

BACKGROUND: Solid tumors pose unique roadblocks to treatment with chimeric antigen receptor (CAR) T cells, including limited T-cell persistence, inefficient tumor infiltration, and an immunosuppressive tumor microenvironment. To date, attempts to overcome these roadblocks have been unsatisfactory. Herein, we reported a strategy of combining Runx3 (encoding RUNX family transcription factor 3)-overexpression with ex vivo protein kinase B (AKT) inhibition to generate CAR-T cells with both central memory and tissue-resident memory characteristics to overcome these roadblocks. METHODS: We generated second-generation murine CAR-T cells expressing a CAR against human carbonic anhydrase 9 together with Runx3-overexpression and expanded them in the presence of AKTi-1/2, a selective and reversible inhibitor of AKT1/AKT2. We explored the influence of AKT inhibition (AKTi), Runx3-overexpression, and their combination on CAR-T cell phenotypes using flow cytometry, transcriptome profiling, and mass cytometry. The persistence, tumor-infiltration, and antitumor efficacy of CAR-T cells were evaluated in subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: AKTi generated a CD62L+central memory-like CAR-T cell population with enhanced persistence, but promotable cytotoxic potential. Runx3-overexpression cooperated with AKTi to generate CAR-T cells with both central memory and tissue-resident memory characteristics. Runx3-overexpression enhanced the potential of CD4+CAR T cells and cooperated with AKTi to inhibit the terminal differentiation of CD8+CAR T cells induced by tonic signaling. While AKTi promoted CAR-T cell central memory phenotype with prominently enhanced expansion ability, Runx3-overexpression promoted the CAR-T cell tissue-resident memory phenotype and further enhanced persistence, effector function, and tumor-residency. These novel AKTi-generated Runx3-overexpressing CAR-T cells exhibited robust antitumor activity and responded well to programmed cell death 1 blockade in subcutaneous PDAC tumor models. CONCLUSIONS: Runx3-overexpression cooperated with ex vivo AKTi to generate CAR-T cells with both tissue-resident and central memory characteristics, which equipped CAR-T cells with better persistence, cytotoxic potential, and tumor-residency ability to overcome roadblocks in the treatment of solid tumors.

Oxygen microcapsules improve immune checkpoint blockade by ameliorating hypoxia condition in pancreatic ductal adenocarcinoma.

Rationale: Hypoxia in tumor microenvironment (TME) represents an obstacle to the efficacy of immunotherapy for pancreatic ductal adenocarcinoma (PDAC) through several aspects such as increasing the expression of immune checkpoints or promoting fibrosis. Reversing hypoxic TME is a potential strategy to improve the validity of immune checkpoint blockade (ICB). Methods: Here, we synthesized polydopamine-nanoparticle-stabilized oxygen microcapsules with excellent stabilization, bioavailability, and biocompatibility for direct oxygen delivery into tumor sites by interfacial polymerization. Results: We observed oxygen microcapsules enhanced the oxygen concentration in the hypoxia environment and maintained the oxygen concentration for a long period both in vitro and in vivo. We found that oxygen microcapsules could significantly improve the efficiency of ICB against PDAC in vivo. Mechanismly, combined treatments using oxygen microcapsules and ICB could reduce the infiltration of tumor-associated macrophages (TAMs) and polarized pro-tumor M2 macrophages into anti-tumor M1 macrophages. In addition, combined treatments could elevate the proportion of T helper subtype 1 cells (Th1 cells) and cytotoxic T lymphocytes cells (CTLs) to mediate anti-tumor immune response in TME. Conclusion: In summary, this pre-clinical study indicated that reversing hypoxia in TME by using oxygen microcapsules was an effective strategy to improve the performances of ICB on PDAC, which holds great potential for treating PDAC in the future.

Recent Advancements of Nanotechnology-Based Strategies for Overcoming Tumor Microenvironment Hypoxia.

Hypoxia is a typical characteristic of most solid malignancies, which has multiple effects on malignant phenotypes and biological behaviors of tumors including epithelial-mesenchymal-transition (EMT), invasion, migration, metastasis, autophagy, stem cell maintenance, pathological angiogenesis, drug resistance, and immunosuppression. Rcentlyumoand reversing the tumor hypoxic environment via nanotechnology has emerged as a novel therapeutic approach for the treatment of malignancies. The main strategies related to nanotechnology to alleviate or ameliorate hypoxic environment are as follows: (1) Bringing external oxygen to tumor hypoxic microenvironment; (2) Generating oxygen based on nanotechnology in situ; (3) Regulating the structure of the tumor microenvironment; (4) Decreasing oxygen consumption in the tumor microenvironment. In this review, we will discuss these nanotechnologies in detail.

Topological analysis of hepatocellular carcinoma tumour microenvironment based on imaging mass cytometry reveals cellular neighbourhood regulated reversely by macrophages with different ontogeny.

OBJECTIVE: Hepatocellular carcinoma (HCC) tumour microenvironment (TME) is highly complex with diverse cellular components organising into various functional units, cellular neighbourhoods (CNs). And we wanted to define CN of HCC while preserving the TME architecture, based on which, potential targets for novel immunotherapy could be identified. DESIGN: A highly multiplexed imaging mass cytometry (IMC) panel was designed to simultaneously quantify 36 biomarkers of tissues from 134 patients with HCC and 7 healthy donors to generate 562 highly multiplexed histology images at single-cell resolution. Different function units were defined by topological analysis of TME. CN relevant to the patients' prognosis was identified as specific target for HCC therapy. Transgenic mouse models were used to validate the novel immunotherapy target for HCC. RESULTS: Three major types of intratumour areas with distinct distribution patterns of tumorous, stromal and immune cells were identified. 22 cellular metaclusters and 16 CN were defined. CN composed of various types of cells formed regional function units and the regional immunity was regulated reversely by resident Kupffer cells and infiltrating macrophages with protumour and antitumour function, respectively. Depletion of Kupffer cells in mouse liver largely enhances the T cell response, reduces liver tumour growth and sensitises the tumour response to antiprogrammed cell death protein-1 treatment. CONCLUSION: Our findings reveal for the first time the various topological function units of HCC TME, which also presents the largest depository of pathological landscape for HCC. This work highlights the potential of Kupffer cell-specific targeting rather than overall myeloid cell blocking as a novel immunotherapy for HCC treatment.