Tumor-associated Neutrophils Research Articles

Neutrophils in cancer drug resistance: Roles and therapeutic opportunities.

The tumor microenvironment (TME) is closely associated with the therapeutic response and clinical outcome of cancer drug therapies, which mainly include immunotherapy, chemotherapy and targeted therapy. Neutrophils that infiltrate tumors, also known as tumor-associated neutrophils (TANs), constitute a primary part of the TME. However, the functional importance of TANs in cancer drug therapy has long been overlooked because of their relatively short life span. Recent studies have shown that TANs play crucial protumoral or antitumoral roles in cancer drug treatment, largely because of their diversity and plasticity. This review describes the development, heterogeneity and recruitment of neutrophils in the context of cancer and emphasizes the role and mechanisms of TANs in cancer drug resistance. Additionally, several potential neutrophil-targeted strategies are discussed.

CD8 + T cell proximity analysis of the tumor microenvironment (TME) in patients with pancreatic ductal adenocarcinoma (PDAC) and associated clinical outcomes.

754 Background: Immunosuppressive cells (cancer-associated fibroblasts [CAFs], tumor associated neutrophils [TANs], M2/M1 macrophages) can influence CD8 + T cell infiltration in the PDAC TME. The spatial proximity of these cells to CD8 + T cells and association with clinical outcomes is not well known. We hypothesize the cell distances between CD8 + T cells and these phenotypes: CXC chemokine receptor 4 (CXCR4) + TANs, CAFs, focal adhesion kinase (FAK) + tumor cells, FAK + M2 and M1 macrophages will correlate with survival outcomes. Methods: Twenty-two stage I/II PDAC patients who consented for tissue collection were stratified into two groups: standard responders (n=11) with overall survival (OS) >2 years and poor responders (n=11) with OS ≤ 2 years. Multiplex IHC was performed on specimens using multiple biomarkers (CD8, CXCR4, CD66b, FAK, FAP, CD68, CSF1R, CD163, EPCAM) to identify cell phenotypes. Comprehensive spatial analysis with HALO software was used to measure mean cell distance between CD8 + T cells and other cells. The mean cell-to-cell distance among evaluable specimens was determined by phenotype. Patients were divided into two groups with average cell distance below or above the mean by phenotype. OS of each subgroup was compared using Kaplan-Meier method. Results: Median OS for standard and poor responders was 47.6 months vs 13.1 months (p<.0001), respectively. There was a higher % of CD8 + T cells closer (<50 µm) to CXCR4 + TANs in standard responders (56% vs 28%, p<.0001). There were no significant differences in CD8 + T cell distance to other phenotypes. Longer survival was seen with average cell distance below the mean when comparing CD8 + T cells to FAK + tumor cells (1274 d vs 742 d, p=.306), to CXCR4 + TANs (854 d vs 442 d, p=.947) and to FAP + FAK + CAFs (890.5 d vs 672 d, p=.662). In contrast, longer survival was seen with a distance above the mean comparing CD8 + T cells to M2 (622 d vs 1274 d, p=.732) and to M1 (613 d vs 1397 d, p=.517) macrophages. Conclusions: Our study shows clinical outcomes may be correlated with spatial distance among TME immune cells in PDAC. Longer survival was seen when CD8 + T cells were closer to FAK + tumor cells, CXCR4 + TANs, and CAFs and further away from M2 and M1 macrophages. Larger cohort analyses may better elucidate the prognostic and biological significance of cell distance among TME phenotypes, which may have potential implications for response to immunotherapy.

Neutrophil Engulfment in Cancer: Friend or Foe?

Neutrophils, the most abundant circulating white blood cells, are essential for the initial immune response to infection and injury. Emerging research reveals a dualistic function of neutrophils in cancer, where they can promote or inhibit tumor progression. This dichotomy is influenced by the tumor microenvironment, with neutrophils capable of remodeling the extracellular matrix, promoting angiogenesis, or alternatively inducing cancer cell death and enhancing immune responses. An intriguing yet poorly understood aspect of neutrophil–cancer interactions is the phenomenon of neutrophil engulfment by cancer cells, which has been observed across various cancers. This process, potentially mediated by LC3-associated phagocytosis (LAP), raises questions about whether it serves as a mechanism for immune evasion or contributes to tumor cell death through pathways like ferroptosis. This review examines current knowledge on neutrophil development, their roles in cancer, and the mechanisms of LAP in neutrophil engulfment by tumor cells. We discuss how manipulating LAP impacts cancer progression and may represent a therapeutic strategy. We also explore neutrophils’ potential as delivery vehicles for cancer therapeutic agents. Understanding the complex functions of tumor-associated neutrophils (TANs) and the molecular mechanisms underlying LAP in cancer may open new avenues for effective therapeutic interventions and mitigate potential risks.

Tumor-Infiltrating Lymphocytes and Neutrophils and Their Location in Canine Mammary Neoplasms with a Solid Arrangement: A Prognostic Factor?

In canine mammary neoplasms, greater inflammation is associated with higher histological grade, lymphatic invasion, and metastases. This retrospective study assessed the density of peri- and intratumoral tumor-infiltrating lymphocytes (TILs), tumor-associated neutrophils (TANs), and CD3+ and CD79+ lymphocytes in canine mammary neoplasms with a solid arrangement, and associated such data with histological types, immunophenotype, prognostic factors, cyclooxygenase-2 (Cox-2) expression and overall and cancer-specific survival. Sixty-one neoplasms with a solid arrangement were classified as malignant myoepitheliomas (6/9.8%), solid papillary carcinomas (8/13.1%), carcinomas with a solid pattern (9/14.8%), basaloid carcinomas (BC) (19/31.1%), and malignant adenomyoepitheliomas (19/31.1%). Intra- and peritumoral TILs, TANs, and TCD3+ and BCD79+ lymphocytes were counted, and based on the resulting median, the neoplasms were divided into low or high cell infiltration. BCs had the lowest density of intratumoral TILs (p = 0.02), and luminal B neoplasms showed a significantly higher density of intratumoral TCD3+ than luminal A cases. Neoplasms with a higher density of peritumoral CD3+ and CD79+ had significantly greater proliferative activity. High infiltration of intratumoral BCD79+ lymphocytes was related to nodal metastasis (p = 0.03). Intratumoral TILs and TCD3+ were associated with shorter survival time. Therefore, intratumoral lymphocyte infiltration is possibly an important feature in the progression of cancer and influences the survival in bitches with solid arrangement neoplasms.

Tumor-associated macrophages drive heterogenetic CD10High cancer stem cells to implement tumor-associated neutrophils reprogramming in oral squamous cell carcinoma.

Tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) widely participate in the malignant progression in cancer. Previously, we have demonstrated that M1-like TAMs cascaded a stem-like phenotype of oral squamous cell carcinoma (OSCC). Yet, the underlying mechanisms still need to be demonstrated for the regulation of TAMs on cancer stem cells (CSCs) in OSCC. In this study, we investigated a group of CSCs with increased expression of cluster differentiation 10 (CD10), which acted as a mediator in the interaction network between TAMs and tumor-associated neutrophils (TANs) in OSCC. The results showed a significant association between TAMs infiltrations and increased expression of CD10 among all the CSCs-related molecules in OSCC. Then, we validated that OSCC cells with high CD10 expression possessed increased CSCs characteristics. TAMs could drive the heterogenetic CD10High CSCs by activating the IL6/STAT3/CD10 pathway. Furthermore, CD10High CSCs could recruit and reprogram tumor-associated neutrophils (TANs) in an immunosuppressive state by secreting S100A8/A9 in OSCC. These finding indicated that CD10High CSCs played great roles in signaling crosstalk between TAMs and TANs in OSCC, by which infiltrated TAMs drive CD 10High CSCs to recruit and reprogram TANs in an immunosuppressive state. Herein, we managed to demonstrate that TAMs could directly regulate a heterogenetic cluster of CSCs with high CD10 expression, and CD10High CSCs could recruit and reprogram TANs in OSCC. The novel crosstalk among OSCC-TAMs-CD10High CSCs-TANs might bring new prospects for improving the treatment strategies for OSCC patients.

Transferrin receptor uptakes iron from tumor-associated neutrophils to regulate invasion patterns of OSCC

BackgroundTransferrin receptor (TFRC) uptakes iron-loaded transferrin (TF) to acquire iron and regulates tumor development. Nonetheless, the clinical values and the precise functions of TF-TFRC axis in the development of oral squamous cell carcinoma (OSCC) were still undiscovered, especially the impacts of their regional heterogeneous expression.MethodsImmunohistochemistry (IHC) was used to analyze the expression of TFRC in 106 OSCC patients. Then the prognostic value of TFRC was compared between high and low worst pattern of invasion (WPOI) patients. OSCC cells with low or high expression of TFRC were constructed, and functional experiments were performed to elucidate the effects of TFRC on the migration and proliferation of OSCC cells. Multi-immunofluorescence was applied to stain TF and tumor-associated neutrophils (TANs). The stimulating effects of TF were compared between normal and high TFRC cells in vitro and across different OSCC patients’ subgroups in our sample bank and TCGA database.ResultsHigher TFRC was expressed at invasive tumor front (ITF) in OSCC and correlated with WPOI. Only at ITF in patients with WPOI 4–5, TFRC was a prognostic factor. High TFRC promoted migration and proliferation of cancer cells. Additionally, TANs secreted TF outside. Exogenous TF promoted migration and proliferation of cells with high expression of TFRC. Compared to the TANslowTFRClow OSCC patients, TANshighTFRChigh OSCC patients had poorer clinical outcomes.ConclusionsHigher expression of TFRC at ITF and TANs-TF-TFRC axis promoted OSCC invasion at ITF by facilitating cell migration and proliferation, which may result from increased cellular iron uptake through regulating iron metabolism.

The Role of Tumor-Associated Neutrophils in Early Luminal HER2-Negative Breast Cancer Progression.

To study the predictive role of tumor-associated neutrophils in early luminal HER2-negative breast cancer. This is a retrospective study conducted on 60 women cases aged from 31 to 79 years underwent surgery for luminal HER2-negative ductal breast cancer in tertiary care cancer centre. We first estimated basic morphological signs: tumor size, tumor grade (by Nottingham Histologic Score), tumor infiltrating lymphocytes (TILs), Lymphovascular invasion, hormonal receptors status, proliferative index, and regional lymph nodes metastasis. The expression of intratumoral neutrophils was studied by CD15 immunohistochemistry which was performed using tissue microarrays. The total number of intratumoral neutrophils, were counted in 5 high-power fields. According to the Nottingham histologic score system, grade I cases were detected in 10 cases (16%), grade II in 34 cases (57%), and grade III in 16 cases (27%). Lymphovascular invasion was determined in 23 cases (38%), and perineural invasion in 14 cases (23%). Number of TILs varied from 0 to 14 (counted in 5 HPF) and averaged 4.2±0.5. Luminal A tumor phenotype was detected in 35 cases (58%), and luminal B HER2-negative in 25 cases (42%). Nineteen (32%) women had metastases in regional lymph nodes (N+). The number of tumor microenvironment neutrophils in luminal HER2-negative breast carcinomas ranged from 1 to 10 (counted in 5 HPF) with an average value of 2.7±0.4. High tumor-associated neutrophils concentration significantly correlated with tumor size (<5mm and >20mm) with p=0.05, high grade (p=0.01), high proliferative index ((r=0.67; p=0.05), TILs (p=0.05), Lymphovascular space invasion (p=0.01)and positive regional lymph nodes metastasis (p=0.001), but not perineural invasion (p=0.1) and also, did not correlate with the expression of estrogen (r=0.18) and progesterone (r=0.14) receptors. Tumor-associated neutrophils strongly predict a worse prognosis in early luminal HER2-negative breast cancer.

Dual ferroptosis induction in N2-TANs and TNBC cells via FTH1 targeting: A therapeutic strategy for triple-negative breast cancer.

Tumor-associated neutrophils (TANs) play a critical role in the progression and prognosis of triple-negative breast cancer (TNBC), with N2-type TANs known for their pro-tumor characteristics. This study introduces CT-1, a derivative of cryptotanshinone that effectively suppresses TNBC growth while selectively reducing the proportion of N2-type TANs within tumor tissue. Notably, CT-1 induces simultaneous ferroptosis in both N2-type TANs and TNBC cells, a dual mechanism that enhances its therapeutic efficacy. The study identifies ferritin heavy chain 1 (FTH1), a key protein in iron metabolism, as the direct target of CT-1. By targeting FTH1, CT-1 facilitates the interaction between NCOA4 and ferritin, triggering ferritinophagy-mediated ferroptosis. These findings position CT-1 as a promising therapeutic agent, offering a strategy to combat TNBC by inducing ferroptosis in both N2-type TANs and cancer cells. This approach underscores the potential of FTH1 as a therapeutic target for treating TNBC.

The Roles of Myeloid Cells in Breast Cancer Progression.

This chapter reviews tumor-associated myeloid cells, including macrophages, neutrophils, and other innate immune cells, and their multifaceted roles in supporting breast cancer progression and metastasis. In primary tumors, myeloid cells play key roles in promoting tumor epithelial-mesenchymal transition (EMT) and invasion. They can facilitate intravasation (entry into the bloodstream) and colonization, disrupting the endothelial cell layer and reshaping the extracellular matrix. They can also stimulate angiogenesis, suppress immune cell responses, and enhance cancer cell adaptability. In the bloodstream, circulating myeloid cells enable the survival of disseminated tumor cells via immunosuppressive effects and physical shielding. At the metastatic sites, they prime the premetastatic niche, facilitate tumor cell extravasation, and support successful colonization and outgrowth. Mechanistically, myeloid cells enhance cancer cell survival, dormancy escape, proliferation, and mesenchymal-epithelial transition (MET). Nonetheless, substantial gaps in our understanding persist regarding the functional and spatiotemporal diversity, as well as the evolutionary patterns, of myeloid cells during metastatic progression. Myeloid cell plasticity and differential responses to therapies present key barriers to successful treatments. Identifying specific pro-tumoral myeloid cell subpopulations and disrupting their interactions with cancer cells represent promising therapeutic opportunities. Emerging evidence suggests combining immunomodulators or stromal normalizers with conventional therapies could help overcome therapy-induced immunosuppression and improve patient outcomes. Overall, further elucidating myeloid cell heterogeneity and function throughout the process of breast cancer progression and metastasis will enable more effective therapeutic targeting of these critical stromal cells.

Targeting Deltex E3 Ubiquitin Ligase 2 Inhibits Tumor-associated Neutrophils and Sensitizes Hepatocellular Carcinoma Cells to Immunotherapy.

Several E3 ligases have been found to affect the immune microenvironment of hepatocellular carcinoma (HCC) and lead to the resistance of immunotherapy. In this study, genes of E3 ligases are screened based on The Cancer Genome Atlas (TCGA) dataset. Through cytometry by time of flight (CyTOF), flow cytometry, and further experiments, Deltex E3 ubiquitin ligase 2 (DTX2) in HCC cells is identified to promote the infiltration and polarization of tumor-associated neutrophils (TANs) with a protumor phenotype, thus attenuating the infiltration and cytotoxicity of CD8+ T cells partially through C-X-C motif chemokine 2 (CXCL2) and C-X-C motif chemokine 6 (CXCL6). Mechanistically, DTX2 can interact with histone H2B and promote its monoubiquitination at lysine120 (H2BK120ub1), thereby increasing CXCL2 and CXCL6 transcription through histone epigenetic regulation. Different tumor models in vivo demonstrated that DTX2 inhibitor treatment inhibited tumor growth and sensitized HCC cells to the therapeutic effects of programmed cell death protein 1 (PD-1) antibody. In summary, this study identifies DTX2 as a potential target for HCC immunotherapy.

Neutrophil and Colorectal Cancer.

Colorectal cancer (CRC) is often associated with metastasis and recurrence and is the leading cause of cancer-related mortality. In the progression of CRC, recent studies have highlighted the critical role of neutrophils, particularly tumor-associated neutrophils (TANs). TANs have both tumor-promoting and tumor-suppressing activities, contributing to metastasis, immunosuppression, angiogenesis, and epithelial-to-mesenchymal transition. Tumor-promoting TANs promote tumor growth by releasing proteases, reactive oxygen species, and cytokines, whereas tumor-suppressing TANs enhance immune responses by activating T cells and natural killer cells. Understanding the mechanisms underlying TAN mobilization, plasticity, and their role in the tumor microenvironment has revealed potential therapeutic targets. This review provides a comprehensive overview of TAN biology in CRC and discusses both the tumor-promoting and tumor-suppressing functions of neutrophils. Novel therapeutic approaches targeting TANs, such as chemokine receptor antagonists, aim to modulate neutrophil reprogramming and offer promising avenues for improving treatment outcomes of CRC.

CMTM6 promotes hepatocellular carcinoma invasion and metastasis and tumor-associated neutrophil immunoinfiltration through the Wnt/β-catenin pathway

BackgroundCMTM6 has been closely associated with the onset and progression of various tumor types. However, the precise mechanism by which CMTM6 operates in hepatocellular carcinoma remains elusive, necessitating further investigation.MethodsExpression levels of CMTM6 in hepatocellular carcinoma tissues and cells were analyzed using immunohistochemistry and quantitative real-time PCR. The correlation between CMTM6 expression in hepatocellular carcinoma tissues and clinical pathological characteristics, as well as patient prognosis, was investigated. Proliferation and apoptosis of hepatocellular carcinoma cells with silenced or overexpressed CMTM6 were assessed, alongside measurements of β-catenin and Wnt1 protein expression levels. In vivo research was conducted utilizing a murine subcutaneous transplantation model. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed to elucidate the regulatory mechanism of CMTM6. Additionally, CD66b expression levels in tumor tissue were examined using immunohistochemistry, and the immune infiltration of CMTM6 and tumor-associated neutrophils (TANs) was analyzed.ResultsElevated expression levels of CMTM6 in hepatocellular carcinoma tissues and cells were found to be associated with poor patient prognosis. Overexpression of CMTM6 in hepatocellular carcinoma cells was demonstrated to promote cellular proliferation and inhibit apoptosis. Mechanistically, CMTM6 expression levels in hepatocellular carcinoma tissues were observed to positively correlate with β-catenin expression. GSEA and KEGG analysis revealed significant enrichment of CMTM6 in the Wnt/β-catenin pathway, indicating its involvement in pathway regulation. Furthermore, CMTM6 was found to be associated with immune infiltration of TANs in hepatocellular carcinoma tissues.ConclusionCMTM6 plays a pivotal role in the development and progression of hepatocellular carcinoma through regulation of the Wnt/β-catenin pathway via β-catenin. Moreover, CMTM6 demonstrates the capacity to promote immune infiltration of TANs in hepatocellular carcinoma tissues. Consequently, CMTM6 exhibits potential as both an early diagnostic marker and a novel therapeutic target for patients with hepatocellular carcinoma.

Tumor microenvironment in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a highly aggressive and malignant tumor of oral cavity with a poor prognosis and high mortality due to the limitations of existing therapies. The significant role of tumor microenvironment (TME) in the initiation, development, and progression of OSCC has been widely recognized. Various cells in TME, including tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), T lymphocytes, tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs), form a complicated and important cellular network to modulate OSCC proliferation, invasion, migration, and angiogenesis by secreting RNAs, proteins, cytokines, and metabolites. Understanding the interactions among cells in TME provides the foundation for advanced clinical diagnosis and therapies. This review summarizes the current literature that describes the role of various cellular components and other TME factors in the progression of OSCC, hoping to provide new ideas for the novel OSCC treatment strategies targeting the complicated cellular network and factors that mediate the interactive loops among cells in TME.

IGF2BP2 promotes lung adenocarcinoma progression by regulating LOX1 and tumor-associated neutrophils.

Lung adenocarcinoma (LUAD) is the common form of lung cancer and is prone to distant metastasis. IGF2BP2, an m6A modification regulator, is upregulated in lung cancer tissue, but its specific role within the LUAD tumor microenvironment (TME) is unknown. We explored the role of IGF2BP2 in the progression of LUAD. IGF2BP2 expression in LUAD patient specimens and controls was evaluated through bioinformatics, Western blot, and immunohistochemistry. LUAD subcutaneous and orthotopic xenograft tumor models were established, alongside a co-culture system of tumor-associated neutrophils (TANs) and A549 cells. Functional assays assessed IGF2BP2's role under treatment with JX5, an IGF2BP2 inhibitor. Mechanistic assays explored the interaction between IGF2BP2 and LOX1 in 293T cells. IGF2BP2 was significantly upregulated in LUAD tissues, with higher expression levels predicting worse prognosis for patients (p < 0.001). In subcutaneous and orthotopic xenograft models, treatment with JX5, an IGF2BP2 inhibitor, reduced tumor size, volume, and weight (p < 0.001). JX5 also significantly reduced the concentrations of pro-inflammatory cytokines in peripheral blood (p < 0.01 and p < 0.001). Flow cytometry analysis indicated JX5 reduced CD11b+Ly6G+/CD45+ cells (TANs) in the TME (p < 0.001). Mechanistically, JX5 downregulated LOX1 expression in vivo, and co-culture experiments further demonstrated that IGF2BP2 promotes LUAD progression through LOX1-mediated regulation of TAN activity (p < 0.01 and p < 0.001). Overexpression of LOX1 reversed the inhibitory effects of JX5 on TAN infiltration, tumor cell viability, and apoptosis (p < 0.01 and p < 0.001). Additionally, RNA immunoprecipitation revealed that IGF2BP2 binds to LOX1 mRNA at its m6A modification site, stabilizing LOX1 and enhancing its function in the TME. Knockdown of IGF2BP2 accelerated LOX1 mRNA degradation, confirming its role in maintaining LOX1 stability (p < 0.01 and p < 0.001). IGF2BP2 recognizes and stabilizes LOX1 through m6A modification, contributing to TAN-mediated LUAD progression. Overall, these findings offer new insights into LUAD progression and demonstrate that IGF2BP2 is a key regulator that promotes tumor advancement, highlighting the IGF2BP2-LOX1 axis as a potential therapeutic target for LUAD.

Tumor-associated neutrophils attenuate the immunosensitivity of hepatocellular carcinoma.

Tumor-associated neutrophils (TANs) are heterogeneous; thus, their roles in tumor development could vary depending on the cancer type. Here, we showed that TANs affect metabolic dysfunction-associated steatohepatitis hepatocellular carcinoma (MASH-related HCC) more than viral-associated HCC. We attributed this difference to the predominance of SiglecFhi TANs in MASH-related HCC tumors. Linoleic acid and GM-CSF, which are commonly elevated in the MASH-related HCC microenvironment, fostered the development of this c-Myc-driven TAN subset. Through TGFβ secretion, SiglecFhi TANs promoted HCC stemness, proliferation, and migration. Importantly, SiglecFhi TANs supported immune evasion by directly suppressing the antigen presentation machinery of tumor cells. SiglecFhi TAN removal increased the immunogenicity of a MASH-related HCC model and sensitized it to immunotherapy. Likewise, a high SiglecFhi TAN signature was associated with poor prognosis and immunotherapy resistance in HCC patients. Overall, our study highlights the importance of understanding TAN heterogeneity in cancer to improve therapeutic development.

X-ray-activated nanoscintillators integrated with tumor-associated neutrophils polarization for improved radiotherapy in metastatic colorectal cancer.

Radiotherapy, employing high-energy rays to precisely target and eradicate tumor cells, plays a pivotal role in the treatment of various malignancies. Despite its therapeutic potential, the effectiveness of radiotherapy is hindered by the tumor's inherent low radiosensitivity and the immunosuppressive microenvironment. Here we present an innovative approach that integrates peroxynitrite (ONOO-)-mediated radiosensitization with the tumor-associated neutrophils (TANs) polarization for the reversal of immunosuppressive tumor microenvironment (TME), greatly amplifying the potency of radiotherapy. Our design employs X-ray-activated lanthanide-doped scintillators (LNS) in tandem with photosensitive NO precursor to achieve in-situ ONOO- generation. Concurrently, the co-loaded TGF-β inhibitor SB525334, released from the LNS-RS nanoplatform in response to the overexpressed GSH in tumor site, promotes the reprogramming of TANs from N2 phenotype toward N1 phenotype, effectively transforming the tumor-promoting microenvironment into a tumor-inhibiting state. This 'one-two punch' therapy efficiently trigger a robust anti-tumor immune response and exert potent therapeutic effects in orthotopic colorectal cancer and melanoma mouse model. Meanwhile, it also significantly prevents liver metastasis and recurrence in metastatic colorectal cancer. The development of X-ray-controlled platforms capable of activating multiple therapeutic modalities may accelerate the clinical application of radiotherapy-based collaborative therapy.

Machine Learning Integration with Single-Cell Transcriptome Sequencing Datasets Reveals the Impact of Tumor-Associated Neutrophils on the Immune Microenvironment and Immunotherapy Outcomes in Gastric Cancer.

The characteristics of neutrophils play a crucial role in defining the tumor inflammatory environment. However, the function of tumor-associated neutrophils (TANs) in tumor immunity and their response to immune checkpoint inhibitors (ICIs) remains incompletely understood. By analyzing single-cell RNA sequencing data from over 600,000 cells in gastric cancer (GSE163558 and GSE183904), colorectal cancer (GSE205506), and lung cancer (GSE207422), we identified neutrophil subsets in primary gastric cancer that are associated with the treatment response to ICIs. Specifically, we focused on neutrophils with high expression of CD44 (CD44_NEU), which are abundant during tumor progression and exert significant influence on the gastric cancer immune microenvironment. Machine learning analysis revealed 22 core genes associated with CD44_NEU, impacting inflammation, proliferation, migration, and oxidative stress. In addition, multiple immunofluorescence staining and gastric cancer spatial transcriptome data (GSE203612) showed a correlation between CD44_NEU and T-cell infiltration in gastric cancer tissues. A risk score model derived from seven essential genes (AQP9, BASP1, BCL2A1, PLEK, PDE4B, PROK2, and ACSL1) showed better predictive capability for patient survival compared to clinical features alone, and integrating these scores with clinical variables resulted in a prognostic nomogram. Overall, this study highlights the heterogeneity of TANs, particularly the CD44_NEU critical influence on immunotherapy outcomes, paving the way for personalized treatment strategies.

Rapid, Non-Invasive, Accurate Diagnosis and Efficient Clearance of Metastatic Lymph Nodes.

Sentinel lymph node (SLN) biopsy is currently the standard procedure for clinical cancer diagnosis and treatment, but still faces the risks of false negatives and tumor metastasis, as well as time-consuming pathological evaluation procedure. Herein, we proposed a near-infrared-II (NIR-II, 1000-1700 nm) theranostic nanosystem (FLAGC) for rapid, non-invasive, accurate diagnosis and efficient clearance of metastatic lymph nodes in breast cancer. Initialized by chlorin e6 (Ce6), a pH-sensitive amphiphilic amino acid fluorenylmethoxycarbonyl-L-histidine (Fmoc-His) was assembled with Gd3+, luminol, and AgAuSe quantum dots (AAS QDs) to form FLAGC. In FLAGC, luminol and AAS QDs form a NIR-II chemical resonance energy transfer (CRET) system (Luminol-AAS); Ce6 initiates the assembly and also serves as a photosensitizer. Upon subcutaneous injection, FLAGC is easily drained into SLNs, achieving their precise localization. Subsequently, the acidity of tumor microenvironment triggers the rapid disassembly of FLAGC, exposing Luminol-AAS. myeloperoxidase (MPO) secreted by tumor-associated macrophages and neutrophils in SLNs mediates the oxidation of luminol, lighting up AAS QDs through the CRET process for precise diagnosis of metastatic lymph nodes. Moreover, highly efficient clearance of positive lymph nodes is achieved through Ce6-mediated photodynamic therapy. Our strategy provides a new paradigm for identifying and eliminating clinically metastatic lymph nodes.

Tbp and Hprt1 Are Appropriate Reference Genes for Splenic Neutrophils Isolated from Healthy or Tumor-Bearing Mice.

Background/Objectives: Neutrophils have recently gained significant attention due to their heterogeneity in tumor settings. The gene expression profiles of neutrophils from different tumor types are of great interest. Murine splenic neutrophils reflect the immune status of the organism and could be a source of tumor-associated neutrophils in tumor-bearing mice. However, information about appropriate reference genes for RT-qPCR analysis of murine neutrophils in the literature is lacking. The aim of this study was to identify stably expressed reference genes in murine splenic neutrophils. Methods: Bone marrow- and spleen-derived neutrophils were isolated from healthy C57Bl/6 and CBA/LacSto mice. Spleen-derived neutrophils were isolated from mice with Lewis lung carcinoma (LLC) and drug-resistant lymphosarcoma (RLS40). RNA was isolated and used for RT-qPCR analysis of 10 selected reference genes. Analysis of reference gene stability was performed using four different algorithms (BestKeeper, NormFinder, geNorm, ΔCt method), and comprehensive ranking was constructed using RefFinder. Results: The Ct values for the reference genes were in the range of 16.73-30.83 with the highest expression levels observed for B2m and the lowest for Sdha. Differences in the stability ranking performed by different algorithms were observed; however, the overall ranking of the studied reference genes was as follows, from most to least stably expressed: Tbp, Hprt1, Ywhaz, B2m, Gapdh, Actb, Sdha, Eef2, Rack1, and Rpl13a. Using Tbp or Rpl13a for RT-qPCR data normalization significantly affected the interpretation of target gene expression. Conclusions: Tbp and Hprt1 are recommended reference genes for murine splenic neutrophils regardless of their activation status.

The ketogenic diet modulates tumor-associated neutrophil polarization via the AMOT-YAP/TAZ axis to inhibit colorectal cancer progression

Despite significant advances in the diagnosis and treatment of colorectal cancer (CRC), the prognosis for late-stage patients remains poor, highlighting the urgent need for new preventive and therapeutic strategies. Recent studies have focused on the ketogenic diet (KD) and its metabolite, β-hydroxybutyrate (BHB), for their tumor-suppressive effects and modulation of inflammatory responses. Using the azoxymethane (AOM) / dextran sulfate sodium (DSS)-induced mouse CRC model, we found that the ketogenic diet and BHB inhibit pro-tumor N2-type tumor-associated neutrophils (TANs) while promoting the polarization of TANs towards the anti-tumor N1 type. This shift in TANs polarization affects tumor growth and metastasis. The underlying mechanism involves BHB acting on the intracellular receptor histone deacetylases 3 (HDAC3), which modulates the activation of the AMOT-YAP/TAZ axis, leading to the inhibition of pro-carcinogenic factor transcription and release. Moreover, clinical cohort data corroborate these findings, showing that CRC patients with elevated BHB levels have significantly lower rates of lymph node involvement, which is associated with a higher infiltration ratio of anti-carcinogenic N1-type TANs in the tumor microenvironment (TME). These results suggest that BHB levels could serve as a prognostic biomarker for CRC. In conclusion, our findings indicate that BHB derived from KD regulates TANs polarization in CRC via the HDAC3-AMOT-YAP/TAZ axis, effectively inhibiting tumor growth and metastasis. These insights establish a novel theoretical basis for employing the KD in the treatment of CRC and for developing cancer adjuvant immunotherapy strategy based on the polarization of neutrophils.