The tumour microenvironment (TME) plays a critical role in therapeutic response and clinical outcomes in cancer. Senescent stromal cells have been shown to promote tumour progression; however, the role of senescent vascular endothelial cells (VECs) in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we aimed to explore the effects and potential mechanisms of senescent VECs in OSCC progression.

Cisplatin was used to induce senescence in two endothelial cell lines. Senescence-associated β-galactosidase (SA-β-gal) staining, immunoblotting, cell cycle and proliferation assays, and migration and invasion assays were performed to access senescence development and biological behavior. Additionally, RNA sequencing analysis, multiplex immunohistochemical staining, immunoblotting, and xenograft mouse models were used to investigate the senescence-associated secretory phenotype of senescent VECs during OSCC progression and its potential molecular mechanisms.

Cisplatin-induced senescent VECs exhibited senescence-related changes, including positive SA-β-gal expression and upregulation of p16, p21, and p53, along with attenuated proliferation and migration. Notably, cisplatin-induced VEC senescence promoted OSCC cell proliferation, migration, and invasion by activating complement C3. Increased gene and protein levels of C3 were observed in cisplatin-treated senescent VECs. Inhibition of C3 in vitro and in vivo reduced OSCC cell proliferation and invasion.

Senescent VECs induced by cisplatin promote OSCC proliferation and invasion through complement C3 activation. Targeting complement C3 in senescent VECs may offer a novel therapeutic strategy for OSCC treatment.

Immune checkpoint inhibitors (ICIs) have shown substantial benefit for patients with advanced non-small cell lung cancer (NSCLC). However, resistance to ICIs remains a major clinical challenge. Here, we perform a comprehensive bioinformatic analysis of plasma proteomic profiles to explore the underlying biology of treatment resistance in NSCLC.

The analysis was performed on 388 "resistance-associated proteins" (RAPs) that were previously described as pretreatment plasma proteomic predictors within the PROphet computational model designed to predict ICI clinical benefit in NSCLC. Putative tissue origins of the RAPs were explored using publicly available datasets. Enrichment analyses were performed to investigate RAP-related biological processes. Plasma proteomic data from 50 healthy subjects and 272 patients with NSCLC were compared, where patients were classified as displaying clinical benefit (CB; n=76) or no CB (NCB; n=196). Therapeutic agents targeting RAPs were identified in drug and clinical trial databases.

The RAP set was significantly enriched with proteins associated with lung cancer, liver tissue, cell proliferation, extracellular matrix, invasion, and metastasis. Comparison of RAP expression in healthy subjects and patients with NSCLC revealed five distinct RAP subsets that provide mechanistic insights. The RAP subset displaying a pattern of high expression in the healthy population relative to the NSCLC population included multiple proteins associated with antitumor activities, while the subset displaying a pattern of highest expression in the NCB population included proteins associated with various hallmarks of treatment resistance. Analysis of patient-specific RAP profiles revealed inter-patient diversity of potential resistance mechanisms, suggesting that RAPs may aid in developing personalized therapeutic strategies. Furthermore, examination of drug and clinical trial databases revealed that 17.5% of the RAPs are drug targets, highlighting the RAP set as a valuable resource for drug development.

The study provides insight into the underlying biology of ICI resistance in NSCLC and highlights the potential clinical value of RAP profiles for developing personalized therapies.

PPP1R3G, a regulatory subunit of protein phosphatase 1, plays a critical role in glycogen metabolism and has been implicated in various cancers. This study provides a comprehensive pan-cancer analysis of PPP1R3G, evaluating its expression, diagnostic and prognostic significance, and potential as a therapeutic target.

We performed an extensive pan-cancer analysis of PPP1R3G using several databases to assess its expression and investigate its correlations with clinical outcomes. Our investigation included assessing PPP1R3G's impact on survival, its correlation with immune checkpoints and tumor stemness scores, and its prognostic significance. We also explored its relationship with immunomodulators, genomic profiles, and immunological characteristics, as well as its response to immunotherapy and involvement in various biological pathways.

PPP1R3G expression varied significantly across different cancers and correlated with both diagnostic and prognostic outcomes. Moreover, PPP1R3G was significantly linked to immune checkpoints, immunomodulators, prognosis, immunoregulatory genes, tumor stemness, cellular function, and immune infiltration across numerous cancer types. Further analysis of PPP1R3G-related gene enrichment, mutation profiles, RNA modifications, and genomic heterogeneity revealed that missense mutations were the predominant alteration affecting PPP1R3G.

Overall, the expression of PPP1R3G is closely associated with various cancers and may serve as a potential biomarker for cancer detection.

Tumor growth and spread are sustained by the tumor microenvironment. Inflammatory cells and pathways have a fundamental role in the tumor microenvironment, driving or conditioning the functional activation of other leukocyte subsets and favoring evasion of anti-tumor immunity. One of the inflammatory pathways contributing to cancer-related inflammation is the complement system. Complement has long been considered an immune mechanism associated with immunosurveillance. More recently it emerged as a tumor promoting pathway, due to direct effects on cancer cells or indirect effects via immunosuppression driven by myeloid cells. The role of complement in cancer is complex and ambiguous, and depends on the tumor type and stage, as well as other factors including oncogenic drivers, leukocyte infiltration, interactions with other tumor microenvironment components or tumor cells. Other factors of complexity include the source of complement molecules, its canonical or non-canonical extracellular functions, its potential intracellular activation, and the interaction with other systems, such as the coagulation or the microbiome. Preclinical studies generally demonstrate the involvement of complement activation in smouldering inflammation in cancer and promotion of an immunosuppressive environment. These studies paved the way for clinical trials aimed at enhancing the potential of immunotherapy, in particular by targeting complement-dependent myeloid-sustained immunosuppression. However, the complex role of complement in cancer and the multiplicity of complement players may represent stumbling blocks and account for failures of clinical trials, and suggest that further studies are required to identify patient subsets who may benefit from specific complement molecule targeting in combination with conventional therapies or immunotherapy. Here, we will discuss the anti- or pro-tumor role of complement activation in cancer, focusing on the interactions of complement with immune cells within the tumor microenvironment, in particular the myeloid compartment. Furthermore, we will examine the potential of complement targeting in cancer treatment, particularly in the context of macrophage reprogramming.

Studies have shown that in the pathogenesis of head and neck squamous cell carcinoma, immune circulation obstruction caused by various factors including metabolic abnormalities, gene mutations, and matrix barrier, is a critical factor for the induction of tumor development and progression. Therefore, the immunotherapy strategy of killing head and neck squamous cell carcinoma cells by an enhanced immune circulation mechanism has attracted much attention. In addition, the rapid development of new nucleic acid drug therapy, such as mRNA, oligonucleotide and small guide RNA (sgRNA), has taken immunotherapy of head and neck squamous cell carcinoma (immune checkpoint inhibitors, tumor vaccines, cellular immunotherapy, cytokines and adjuvants, etc.) to a new level. The combination of nucleic acid therapy with immunotherapy developed for its therapeutic properties has brought a new direction for the diagnosis and treatment of head and neck squamous cell carcinoma, and the combination of the two has had considerable curative effect to patients with refractory/recurrent head and neck squamous cell carcinoma. In this review, we summarized the latest progress of nucleic acid therapy applied to conventional immunotherapy for head and neck squamous cell carcinoma, discussed its mechanism of action and efficacy, and looked into the future development trend.

The precise mechanisms by which the complement system contributes to the establishment of an immunosuppressive tumor microenvironment and promotes tumor progression remain unclear. In this study, we investigated the expression and function of complement C5a receptor 1 (C5aR1) in human and mouse cancer-associated dendritic cells (DC). First, we observed an overexpression of C5aR1 in tumor-infiltrating DCs, compared with DCs from the blood or spleen. C5aR1 expression was restricted to type 2 conventional DCs and monocyte-derived DCs, which displayed a tolerogenic phenotype capable of inhibiting T-cell activation and promoting tumor growth. C5aR1 engagement in DCs drove their migration from tumors to tumor-draining lymph nodes, where C5a levels were higher. We used this knowledge to optimize an anticancer therapy aimed at enhancing DC activity. In three syngeneic tumor models, C5aR1 inhibition significantly enhanced the efficacy of poly I:C, a Toll-like receptor 3 agonist, in combination with PD-1/PD-L1 blockade. The contribution of C5aR1 inhibition to the antitumor activity of the combination treatment relied on type 1 conventional DCs and antigen-specific CD8+ T cells, required lymphocyte egress from secondary lymphoid organs, and was associated with an increase in IFNγ signaling. In conclusion, our study highlights the importance of the C5a/C5aR1 axis in the biology of cancer-associated DCs and provides compelling evidence for the therapeutic potential of modulating the complement system to enhance DC-mediated immune responses against tumors.

Neoadjuvant therapy (NAT) is increasingly being used for pancreatic ductal adenocarcinoma (PDAC). This study investigates how NAT differentially impacts PDAC's carcinoma cells and the tumor microenvironment (TME). Spatial transcriptomics was used to compare gene expression profiles in carcinoma cells and the TME of 23 NAT-treated versus 13 NAT-naïve PDACs. Findings were validated by single-nucleus RNA sequencing (snRNA-seq) analysis. NAT induces apoptosis and inhibits proliferation of carcinoma cells and coordinately upregulates multiple complement genes (C1R, C1S, C3, C4B and C7) within the TME. Higher TME complement expression following NAT is associated with increased immunomodulatory and neurotrophic cancer-associated fibroblasts (CAFs); more CD4+ T cells; reduced immune exhaustion gene expression, and improved overall survival. snRNA-seq analysis demonstrates C3 complement is mainly upregulated in CAFs. These findings suggest that local complement dynamics could serve as a novel biomarker for prognosis, evaluating treatment response, and guiding therapeutic strategies in NAT-treated PDAC patients.

The complement system plays an integral role in both innate and adaptive immune responses. Beyond its protective function against infections, complement is also known to influence tumor immunity, where its activation can either promote tumor progression or mediate tumor cell destruction, depending on the context. One such context can be provided by antibodies, with their inherent capacity to activate the classical complement pathway. In recent years, our understanding of the mechanisms governing complement activation by IgG and IgM antibodies has expanded significantly. At the same time, preclinical and clinical studies on antibodies such as rituximab, ofatumumab, and daratumumab have provided evidence for the role of complement in therapeutic success, encouraging strategies to further enhance its activity. In this review we examine the main determinants of antibody-mediated complement activation, highlighting the importance of antibody subclass, affinity, valency, and geometry of antigen engagement. We summarize the evidence for complement involvement in anti-tumor activity and challenges of accurately estimating the extent of its contribution to therapeutic efficacy. Furthermore, we explore several engineering approaches designed to enhance complement activation, including increased Fc oligomerization and C1q affinity, bispecific C1q-recruiting antibodies, IgG subclass chimeras, as well as antibody and paratope combinations. Strategies targeting membrane-bound complement regulatory proteins to overcome tumor-associated complement inhibition are also discussed as a method to boost therapeutic efficacy. Finally, we highlight the potential of complement-dependent cellular cytotoxicity (CDCC) and complement-dependent cellular phagocytosis (CDCP) as effector mechanisms that warrant deeper investigation. By integrating advances in antibody and complement biology with insights from efforts to enhance complement activation in therapeutic antibodies, this review aims to provide a comprehensive framework of antibody design and engineering strategies that optimize complement activity for improved anti-tumor efficacy.

The process by which living cells are phagocytosed and digested to death is called cell death by phagocytosis, a term that has just recently been generalized and redefined. It is characterized by the phagocytosis of living cells and the cessation of cell death by phagocytosis. Phagocytosis of dead cells is a widely discussed issue in cancer, cell death by phagocytosis can stimulate phagocytosis and stimulate adaptive immunity in tumors, and at the same time, do not-eat-me signaling is an important site for cancer cells to evade recognition by phagocytes. Therefore, we discuss in this review cell death by phagocytosis occurring in cancer tissues and emphasize the difference between this new concept and the phagocytosis of dead tumor cells. Immediately thereafter, we describe the mechanisms by which cell death by phagocytosis occurs and how tumors escape phagocytosis. Finally, we summarize the potential clinical uses of cell death by phagocytosis in tumor therapy and strive to provide ideas for tumor therapy.

We observed that the tumor microenvironment (TME) in metastatic epithelial ovarian cancer (EOC) and in other solid tumors can reprogram normal neutrophils to acquire a complement-dependent suppressor phenotype characterized by inhibition of stimulated T cell activation. This study aims to evaluate whether serum markers of neutrophil activation and complement at diagnosis of EOC would be associated with clinical outcomes.

We conducted a two-center prospective study of patients with newly diagnosed EOC (N = 188). Blood and ascites fluid were collected at diagnosis for biomarker analysis. Patients were evaluated for progression-free survival (PFS) and overall survival (OS).

The median OS was 47 months (95 % CI: 34-58) and the median PFS was 12 months (95 % CI: 11-15). Pre-treatment serum levels of genomic DNA (gDNA), markers of neutrophil degranulation (myeloperoxidase [MPO]) and neutrophil extracellular traps (NETs) (citrullinated histone H3 [CitH3]), and complement activation (C3b/c) were each associated with worse OS in univariate analysis. In multivariate analyses controlling for age, stage, and optimal debulking, serum gDNA, MPO, and CitH3 remained associated with worse OS, while C3b/c levels were not. In an exploratory analysis, the largest magnitude of difference in 2-year OS occurred in patients with low C3b/c and low CitH3 compared to all other patients (87 % vs 46 % survival, respectively). In ascites fluid, increased factor H, a negative regulator of complement activation, was associated with improved OS in univariate analysis.

These results point to serum gDNA, NETs, and complement activation as potential prognostic biomarkers in patients with newly diagnosed EOC.

Clinically, multimodal therapies are adopted worldwide for the management of cancer, which continues to be a leading cause of death. In recent years, immunotherapy has firmly established itself as a new paradigm in cancer care that activates the body's immune defense to cope with cancer. Immunotherapy has resulted in significant breakthroughs in the treatment of stubborn tumors, dramatically improving the clinical outcome of cancer patients. Multiple forms of cancer immunotherapy, including immune checkpoint inhibitors (ICIs), adoptive cell therapy and cancer vaccines, have become widely available. However, the effectiveness of these immunotherapies is not much satisfying. Many cancer patients do not respond to immunotherapy, and disease recurrence appears to be unavoidable because of the rapidly evolving resistance. Moreover, immunotherapies can give rise to severe off-target immune-related adverse events. Strategies to remove these hindrances mainly focus on the development of combinatorial therapies or the exploitation of novel immunotherapeutic mediations. Nanomaterials carrying anticancer agents to the target site are considered as practical approaches for cancer treatment. Nanomedicine combined with immunotherapies offers the possibility to potentiate systemic antitumor immunity and to facilitate selective cytotoxicity against cancer cells in an effective and safe manner. A myriad of nano-enabled cancer immunotherapies are currently under clinical investigation. Owing to gaps between preclinical and clinical studies, nano-immunotherapy faces multiple challenges, including the biosafety of nanomaterials and clinical trial design. In this review, we provide an overview of cancer immunotherapy and summarize the evidence indicating how nanomedicine-based approaches increase the efficacy of immunotherapies. We also discuss the key challenges that have emerged in the era of nanotechnology-based cancer immunotherapy. Taken together, combination nano-immunotherapy is drawing increasing attention, and it is anticipated that the combined treatment will achieve the desired success in clinical cancer therapy.

Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2) is a nucleocytoplasmic protein initially identified as a regulator of membrane-bound sodium-hydrogen exchanger 3 (NHE3). In the cytoplasm, NHERF2 regulates the activity of G protein-coupled receptors (GPCRs), including beta-2 adrenergic receptor (2β-AR), lysophosphatidic acid receptor 2, and parathyroid hormone type 1 receptor. In the nucleus, NHERF2 acts as a coregulator of transcription factors such as sex-determining region Y protein (SRY), involved in male sex determination, and estrogen receptor alpha (ERα). ERα is a ligand-dependent transcription factor that controls mammary gland growth and differentiation during puberty and pregnancy and plays a major role in the development and progression of breast cancer tumors. Altogether, the regulatory functions of NHERF2 on ion channels, GPCRs, and nuclear transcription factors have a modulatory effect on signal transduction pathways, metabolic homeostasis, cell proliferation and differentiation, neurotransmission, muscle contraction, and renal electrolyte balance. This work highlights NHERF2 functions in the cytoplasm and nucleus and underscores the nuclear mechanisms through which NHERF2 participates in the regulation of gene expression and tumor growth and progression in breast cancer.

Gastric cancer continues to be a leading global health concern, with current therapeutic approaches requiring significant improvement. While the disruption of iron metabolism in the advancement of gastric cancer has been well-documented, the underlying regulatory mechanisms remain largely unexplored. Additionally, the complement C5a-C5aR pathway has been identified as a crucial factor in gastric cancer development. The impact of the complement system on iron metabolism and its role in gastric cancer progression is an area warranting further investigation. Our research demonstrates that the C5a-C5aR pathway promotes gastric cancer progression by enhancing iron acquisition in tumor cells through two mechanisms. First, it drives macrophage polarization toward the M2 phenotype, which has a strong iron-release capability. Second, it increases the expression of LCN2, a high-affinity iron-binding protein critical for iron export from tumor-associated macrophages, by activating endoplasmic reticulum stress in these cells. Both mechanisms facilitate the transfer of iron from macrophages to cancer cells, thereby promoting tumor cell proliferation. This study aims to elucidate the connection between the complement C5a-C5aR pathway and iron metabolism within the tumor microenvironment. Our data suggest a pivotal role of the C5a-C5aR pathway in tumor iron management, indicating that targeting its regulatory mechanisms may pave the way for future iron-targeted therapeutic approaches in cancer treatment.

Carbon nanotubes (CNTs) are allotropes of carbon, composed of carbon atoms forming a tube-like structure. Their high surface area, chemical stability, and rich electronic polyaromatic structure facilitate their drug-carrying capacity. Therefore, CNTs have been intensively explored for several biomedical applications, including as a potential treatment option for cancer. By incorporating smart fabrication strategies, CNTs can be designed to specifically target cancer cells. This targeted drug delivery approach not only maximizes the therapeutic utility of CNTs but also minimizes any potential side effects of free drug molecules. CNTs can also be utilised for photothermal therapy (PTT) which uses photosensitizers to generate reactive oxygen species (ROS) to kill cancer cells, and in immunotherapeutic applications. Regarding the latter, for example, CNT-based formulations can preferentially target intra-tumoural regulatory T-cells. CNTs also act as efficient antigen presenters. With their capabilities for photoacoustic, fluorescent and Raman imaging, CNTs are excellent diagnostic tools as well. Further, metallic nanoparticles, such as gold or silver nanoparticles, are combined with CNTs to create nanobiosensors to measure biological reactions. This review focuses on current knowledge about the theranostic potential of CNT, challenges associated with their large-scale production, their possible side effects and important parameters to consider when exploring their clinical usage.

This review aims to summarize recent research using animal models, cell models, and human data regarding the role of complement in liver disease. Complement is part of the innate immune system and was initially characterized for its role in control of pathogens. However, evidence now indicates that complement also plays an important role in the response to cellular injury that is independent of pathogens. The liver is the main organ responsible for producing circulating complement. In response to liver injury, complement is activated and likely plays a dual role, both contributing to and protecting from injury. In uncontrolled complement activation, cell injury and liver inflammation occur, contributing to progression of liver disease. Complement activation is implicated in the pathogenesis of multiple liver diseases, including alcohol-associated liver disease, metabolic dysfunction-associated steatotic liver disease, fibrosis and cirrhosis, hepatocellular carcinoma, and autoimmune hepatitis. However, the mechanisms by which complement is overactivated in liver diseases are still being unraveled.

Breast cancer is the most diagnosed malignancy and major cause of cancer death among women population in the worldwide. Ferroptosis is a recently discovered iron-dependent regulated cell death involved in tumor progression and therapeutic response. Moreover, increasing studies have implied that ferroptosis is a promising approach to eliminating cancer cells like developing iron nanoparticles as a therapeutic agent. However, resistance to ferroptosis is a vital distinctive hallmark of cancer. Therefore, further investigation of the mechanism of ferroptosis resistance to enhance its tumor sensitivity is essential for ferroptosis-target breast cancer therapy. Our results revealed that the activation of C5a/C5aR pathway can drive resistance to ferroptosis and reshaping breast cancer immune microenvironment. Accordingly, loading PEG-Fe3O4 with C5aRA significantly improved the anti-tumor effect of PEG- Fe3O4 by inhibiting ferroptosis resistance and increasing macrophage polarization toward M1 phenotype. Our findings presented a novel cancer therapy strategy that combined cancer cell metal metabolism regulation and immunotherapy. The study also provided support for further evaluation of PEG- Fe3O4@C5aRA as a novel therapeutic strategy for breast cancer in clinical trials.

Reliable predictive data are crucial for making accurate treatment decisions in glioma patients, but it can be challenging to obtain due to limited information in many cases. Numerous research studies have indicated the involvement of cyclic adenosine monophosphate (cAMP)-response element binding protein (CREBBP) and E1A binding protein p300 (EP300) in tumorigenesis and tumor progression across various types.

The messenger RNA (mRNA) expression levels of CREBBP and EP300 were retrospectively analyzed in 17 grade-3 glioma patients. The SYBR Green real-time polymerase chain reaction (RT-PCR) technique was employed for mRNA expression analysis, with the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) used as a reference gene for data normalization. In addition, the relationship between CREBBP, EP300 expression and patients' clinical information, imaging features, histologic features, immune factors, and overall survival was assessed through univariate analyses.

The analysis of the data unveiled a statistically significant upregulation of CREBBP and EP300 mRNA expression levels in large gliomas as compared with their smaller counterparts (P < .05). Histological examination using hematoxylin and eosin (H&E) staining exhibited marked cellular heterogeneity, with heightened cell density observed specifically within tumors displaying elevated CREBBP expression levels. In contrast, there was a substantial downregulation of complement 3 and complement 4 within larger tumor volumes when compared with smaller ones (P < .05). However, these findings do not serve as clinically relevant prognostic indicators for glioma.

It is suggested that higher expression levels of CREBBP and EP300 are positively associated with increased tumor volume. Inhibition of CREBBP and EP300 enhances local immunogenicity, leading to the recruitment of immune cells and release of cytokines for effective tumor eradication, ultimately resulting in the inhibition of tumor growth.

Tumor-infiltrating lymphocytes (TILs) are significantly implicated in regulating the tumor immune microenvironment (TIME) and immunotherapeutic response. However, little is known about the impact of the resident and exhausted status of TILs in hepatocellular carcinoma (HCC).

Single-cell RNA sequencing data was applied to discover resident and exhausted signatures of TILs. Survival outcomes, biological function, immune infiltration, genomic variation, immunotherapeutic efficacy, and sorafenib response were further explored the clinical significance and molecular association of TILs in HCC. Moreover, a candidate gene with predictive capability for the dismal subtype was identified through univariate Cox regression analysis, survival analysis, and the BEST website.

Single-cell analysis revealed that CD8 + T, CD4 + T, and NK cells were strongly associated with resident and exhausted patterns. Specific resident and exhausted signatures for each subpopulation were extracted in HCC. Further multivariate Cox analysis revealed that the ratio of resident to exhausted CD4 + T cells in TIME was an independent prognostic factor. After incorporating tumor purity with the ratio of resident to exhausted CD4 + T cells, we stratified HCC patients into three subtypes and found that (i) CD4 residencyhighexhaustionlow subtype was endowed with favorable prognosis, immune activation, and sensitivity to immunotherapy; (ii) CD4 exhaustionhighresidencylow subtype was characterized by genome instability and sensitivity to sorafenib; (iii) Immune-desert subtype was associated with malignant-related pathways and poor prognosis. Furthermore, spindle assembly abnormal protein 6 homolog (SASS6) was identified as a key gene, which accurately predicted the immune-desert subtype. Prognostic analysis as well as in vitro and in vivo experiments further demonstrated that SASS6 was closely associated with tumor prognosis, proliferation, and migration.

The ratio of resident to exhausted CD4 + T cells shows promise as a potential biomarker for HCC prognosis and immunotherapy response and SASS6 may serve as a biomarker and therapeutic target for prognostic assessment of HCC.

In different types of cancer treatments, cancer-specific T cells are required for effective anticancer immunity, which has a central role in cancer immunotherapy. However, due to the multiple inhibitions of CD8+ T cells by tumor-related immune cells, CD8+ T-cell mediated antitumor immunotherapy has not achieved breakthrough progress in the treatment of solid tumors. Receptors for sialic acid (SA) are highly expressed in tumor-associated immune cells, so SA-modified nanoparticles are a drug delivery nanoplatform using tumor-associated immune cells as vehicles. To relieve the multiple inhibitions of CD8+ T cells by tumor-associated immune cells, we prepared SA-modified doxorubicin liposomes (SL-DOX, Scheme 1A). In our study, free SA decreased the toxicity of SL-DOX to tumor-associated immune cells. Compared with common liposomes, SL-DOX could inhibit tumor growth more effectively. It is worth noting that SL-DOX could not only kill tumor-related neutrophils and monocytes to relieve the multiple inhibitions of CD8+ T cells but also induce immunogenic death of tumor cells to promote the infiltration and differentiation of CD8+ T cells (Scheme 1B). Therefore, SL-DOX has potential value for the clinical therapeutic effect of CD8+ T cells mediating anti-tumor immunotherapy.

Studies have associated the development of pulmonary leukemia with the activation of the complement system. However, the roles and mechanisms of complement system-related genes (CSRGs) in acute myeloid leukemia (AML) have not been investigated extensively. This study used The Cancer Genome Atlas (TCGA)-AML and GSE37642 datasets. Differentially expressed CSRGs (CSRDEGs) were identified by overlapping genes differentially expressed between the high and low CSRG score groups and key module genes identified in a weighted gene co-expression network analysis. Univariate and multivariate Cox analyses identified CSRG-related biomarkers, which were used to build a prognostic model. After gene set enrichment analysis (GSEA), immune-related and drug-sensitivity analyses were performed in the high- and low-risk groups. Four prognosis-related biomarkers were identified and used to develop a prognostic model: MEOX2, IGFBP5, CH25H, and RAB3B. The model's performance was verified in a test cohort (a subset of samples from the TCGA-AML dataset) and a validation cohort (GSE37642). The GSEA revealed that the high-risk group was mainly enriched for Golgi organization and cytokine-cytokine receptor interactions, and the low-risk group was mainly enriched in the hedgehog signaling pathway and spliceosome. Lastly, two immune cells were found to show differential infiltration between risk groups, which correlated with the risk scores. M1 macrophage infiltration was significantly positively correlated with RAB3B expression. Sensitivity to 36 drugs differed significantly between risk groups. This study screened four CSRG-related biomarkers (MEOX2, IGFBP5, CH25H, and RAB3B) to provide a basis for predicting AML prognosis.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels' alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN 177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN 177STYR (H3N4) of IgG4, EEQYN 227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.

Colon cancer (CC) occurrence and progression are considerably influenced by the tumor microenvironment. However, the exact underlying regulatory mechanisms remain unclear.

To investigate immune infiltration-related differentially expressed genes (DEGs) in CC and specifically explored the role and potential molecular mechanisms of complement factor I (CFI).

Immune infiltration-associated DEGs were screened for CC using bioinformatics. Quantitative reverse transcription polymerase chain reaction was used to examine hub DEGs expression in the CC cell lines. Stable CFI-knockdown HT29 and HCT116 cell lines were constructed, and the diverse roles of CFI in vitro were assessed using CCK-8, 5-ethynyl-2'-deoxyuridine, wound healing, and transwell assays. Hematoxylin and eosin staining and immunohistochemistry staining were employed to evaluate the influence of CFI on the tumorigenesis of CC xenograft models constructed using BALB/c male nude mice. Key proteins associated with glycolysis and the Wnt pathway were measured using western blotting.

Six key immune infiltration-related DEGs were screened, among which the expression of CFI, complement factor B, lymphoid enhancer binding factor 1, and SRY-related high-mobility-group box 4 was upregulated, whereas that of fatty acid-binding protein 1, and bone morphogenic protein-2 was downregulated. Furthermore, CFI could be used as a diagnostic biomarker for CC. Functionally, CFI silencing inhibited CC cell proliferation, migration, invasion, and tumor growth. Mechanistically, CFI knockdown downregulated the expression of key glycolysis-related proteins (glucose transporter type 1, hexokinase 2, lactate dehydrogenase A, and pyruvate kinase M2) and the Wnt pathway-related proteins (β-catenin and c-Myc). Further investigation indicated that CFI knockdown inhibited glycolysis in CC by blocking the Wnt/β-catenin/c-Myc pathway.

The findings of the present study demonstrate that CFI plays a crucial role in CC development by influencing glycolysis and the Wnt/β-catenin/c-Myc pathway, indicating that it could serve as a promising target for therapeutic intervention in CC.

Pancreatic cancer (PC) is associated with some of the worst prognoses of all major cancers. Thymoquinone (TQ) has a long history in traditional medical practice and is known for its anti-cancer, anti-inflammatory, anti-fibrosis and antioxidant pharmacological activities. Recent studies on hypoxia-inducible factor-1α (HIF-1α) and PC have shown that HIF-1α affects the occurrence and development of PC in many aspects. In addition, TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α. Therefore, we speculate whether TQ affects HIF-1α expression in PC cells and explore the mechanism.

To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression.

Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation.

TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation.

The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.

Background: Metastasis and immunosuppression result in unfavorable prognosis in bladder cancer (BLCA). FGL1 and FGL2 are two members of the fibrinogen-related proteins family, but their potential effects on BLCA remain elusive. Methods: The expression profile of FGL1 and FGL2 in BLCA was analyzed in multiple databases. Furthermore, the expression of FGL2 was validated in BLCA tissues. The predictive capability of FGL2 was evaluated by Kaplan-Meier analysis, univariate analysis, and multivariate Cox regression. A nomogram model was constructed based on FGL2 expression and clinicopathological parameters for clinical practice. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analyses (GSEA) were performed to investigate enrichment in the biological processes. In addition, the correlation between FGL2 and immunological characteristics in the BLCA tumor microenvironment (TME), including tumor-infiltrating immune cells (TICs), cancer-immunity cycles, immune checkpoint molecules (ICPs), immunophenoscores (IPS), and response to anti-PD-L1 immunotherapy was further analyzed. Results: FGL2 was found to be downregulated in BLCA due to hypermethylation of the FGL2 promoter region, which was associated with an unfavorable prognosis. Moreover, BLCA patients with high FGL2 expression exhibited better response to immunotherapy. Conclusions: Our research revealed that FGL2 was downregulated in BLCA and was negatively correlated with DNA methylation. High FGL2 expression was confirmed as an independent risk for prognosis. Moreover, FGL2 is a promising indicator for the response to immunotherapy in patients with BLCA.

Neoadjuvant therapy (NAT) is increasingly being used for pancreatic ductal adenocarcinoma (PDAC) treatment. However, its specific effects on carcinoma cells and the tumor microenvironment (TME) are not fully understood. This study aims to investigate how NAT differentially impacts PDAC's carcinoma cells and TME.

Spatial transcriptomics was used to compare gene expression profiles in carcinoma cells and the TME between 23 NAT-treated and 13 NAT-naïve PDAC patients, correlating with their clinicopathologic features. Analysis of an online single-nucleus RNA sequencing (snRNA-seq) dataset was performed for validation of the specific cell types responsible for NAT-induced gene expression alterations.

NAT not only induces apoptosis and inhibits proliferation in carcinoma cells but also significantly remodels the TME. Notably, NAT induces a coordinated upregulation of multiple key complement genes (C3, C1S, C1R, C4B and C7) in the TME, making the complement pathway one of the most significantly affected pathways by NAT. Patients with higher TME complement expression following NAT exhibit improved overall survival. These patients also exhibit increased immunomodulatory and neurotrophic cancer-associated fibroblasts (CAFs); more CD4+ T cells, monocytes, and mast cells; and reduced immune exhaustion gene expression. snRNA-seq analysis demonstrates C3 complement was specifically upregulated in CAFs but not in other stroma cell types.

NAT can enhance complement production and signaling within the TME, which is associated with reduced immunosuppression in PDAC. These findings suggest that local complement dynamics could serve as a novel biomarker for prognosis, evaluating treatment response and resistance, and guiding therapeutic strategies in NAT-treated PDAC patients.

Cancer immunotherapy, a burgeoning modality for cancer treatment, operates by activating the autoimmune system to impede the growth of malignant cells. Although numerous immunotherapy strategies have been employed in clinical cancer therapy, the resistance of cancer cells to immunotherapeutic medications and other apprehensions impede the attainment of sustained advantages for most patients. Recent advancements in nanotechnology for drug delivery hold promise in augmenting the efficacy of immunotherapy. However, the efficacy is currently constrained by the inadequate specificity of delivery, low rate of response, and the intricate immunosuppressive tumor microenvironment. In this context, the investigation of cell membrane coated nanoparticles (CMNPs) has revealed their ability to perform targeted delivery, immune evasion, controlled release, and immunomodulation. By combining the advantageous features of natural cell membranes and nanoparticles, CMNPs have demonstrated their unique potential in the realm of cancer immunotherapy. This review aims to emphasize recent research progress and elucidate the underlying mechanisms of CMNPs as an innovative drug delivery platform for enhancing cancer immunotherapy. Additionally, it provides a comprehensive overview of the current immunotherapeutic strategies involving different cell membrane types of CMNPs, with the intention of further exploration and optimization.

The involvement of the prostaglandin E2 (PGE2) system in cancer progression has long been recognized. PGE2 functions as an autocrine and paracrine signaling molecule with pleiotropic effects in the human body. High levels of intratumoral PGE2 and overexpression of the key metabolic enzymes of PGE2 have been observed and suggested to contribute to tumor progression. This has been claimed for different types of solid tumors, including, but not limited to, lung, breast, and colon cancer. PGE2 has direct effects on tumor cells and angiogenesis that are known to promote tumor development. However, one of the main mechanisms behind PGE2 driving cancerogenesis is currently thought to be anchored in suppressed antitumor immunity, thus providing possible therapeutic targets to be used in cancer immunotherapies. EP2 and EP4, two receptors for PGE2, are emerging as being the most relevant for this purpose. This review aims to summarize the known roles of PGE2 in the immune system and its functions within the tumor microenvironment. SIGNIFICANCE STATEMENT: Prostaglandin E2 (PGE2) has long been known to be a signaling molecule in cancer. Its presence in tumors has been repeatedly associated with disease progression. Elucidation of its effects on immunological components of the tumor microenvironment has highlighted the potential of PGE2 receptor antagonists in cancer treatment, particularly in combination with immune checkpoint inhibitor therapeutics. Adjuvant treatment could increase the response rates and the efficacy of immune-based therapies.

Effective antitumor immunotherapy depends on evoking a cascade of cancer-immune cycles with lymph nodes (LNs) as the initial sites for activating antitumor immunity, making drug administration through the lymphatic system highly attractive. Here, we describe a nanomedicine with dual responsiveness to pH and enzyme for a programmed activation of antitumor immune through the lymphatic system. The proposed nanomedicine can release the STING agonist diABZI-C2-NH2 in the LNs' acidic environment to activate dendritic cells (DCs) and T cells. Then, the remaining nanomedicine hitchhikes on the activated T cells (PD-1+ T cells) through binding to PD-1, resulting in an effective delivery into tumor tissues owing to the tumor-homing capacity of PD-1+ T cells. The enzyme matrix metalloproteinase-2 (MMP-2) being enriched in tumor tissue triggers the release of PD-1 antibody (aPD-1) which exerts immune checkpoint blockade (ICB) therapy. Eventually, the nanomedicine delivers a DNA methylation inhibitor GSK-3484862 (GSK) into tumor cells, and then the latter combines with granzyme B (GZMB) to trigger tumor cell pyroptosis. Consequently, the pyroptotic tumor cells induce robust immunogenic cell death (ICD) enhancing the DCs maturation and initiating the cascading antitumor immune response. Study on a 4T1 breast tumor mouse model demonstrates the prominent antitumor therapeutic outcome of this nanomedicine through creating a positive feedback loop of cancer-immunity cycles including immune activation in LNs, T cell-mediated drug delivery, ICB therapy, and tumor cell pyroptosis-featured ICD.

Cancer remains one of the global leading causes of death and various vaccines have been developed over the years against it, including cell-based, nucleic acid-based, and viral-based cancer vaccines. Although many vaccines have been effective in in vivo and clinical studies and some have been FDA-approved, there are major limitations to overcome: (1) developing one universal vaccine for a specific cancer is difficult, as tumors with different antigens are different for different individuals, (2) the tumor antigens may be similar to the body's own antigens, and (3) there is the possibility of cancer recurrence. Therefore, developing personalized cancer vaccines with the ability to distinguish between the tumor and the body's antigens is indispensable. This paper provides a comprehensive review of different types of cancer vaccines and highlights important factors necessary for developing efficient cancer vaccines. Moreover, the application of other technologies in cancer therapy is discussed. Finally, several insights and conclusions are presented, such as the possibility of using cold plasma and cancer stem cells in developing future cancer vaccines, to tackle the major limitations in the cancer vaccine developmental process.

This study aims to assess the prognostic implications of gene signature of the tertiary lymphoid structures (TLSs) in head and neck squamous cell carcinoma (HNSCC) and scrutinize the influence of TLS on immune infiltration.

Patients with HNSCC from the Cancer Genome Atlas were categorized into high/low TLS signature groups based on the predetermined TLS signature threshold. The association of the TLS signature with the immune microenvironment, driver gene mutation status, and tumor mutational load was systematically analyzed. Validation was conducted using independent datasets (GSE41613 and GSE102349).

Patients with a high TLS signature score exhibited better prognosis compared to those with a low TLS signature score. The group with a high TLS signature score had significantly higher immune cell subpopulations compared to the group with a low TLS signature score. Moreover, the major immune cell subpopulations and immune circulation characteristics in the tumor immune microenvironment were positively correlated with the TLS signature. Mutational differences in driver genes were observed between the TLS signature high/low groups, primarily in the cell cycle and NRF2 signaling pathways. Patients with TP53 mutations and high TLS signature scores demonstrated a better prognosis compared to those with TP53 wild-type. In the independent cohort, the relationship between TLS signatures and patient prognosis and immune infiltration was also confirmed. Additionally, immune-related biological processes and signaling pathways were activated with elevated TLS signature.

High TLS signature is a promising independent prognostic factor for HNSCC patients. Immunological analysis indicated a correlation between TLS and immune cell infiltration in HNSCC. These findings provide a theoretical basis for future applications of TLS signature in HNSCC prognosis and immunotherapy.

Cuproptosis and disulfidptosis, recently discovered mechanisms of cell death, have demonstrated that differential expression of key genes and long non-coding RNAs (lncRNAs) profoundly influences tumor development and affects their drug sensitivity. Clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, presently lacks research utilizing cuproptosis and disulfidptosis-related lncRNAs (CDRLRs) as prognostic markers. In this study, we analyzed RNA-seq data, clinical information, and mutation data from The Cancer Genome Atlas (TCGA) on ccRCC and cross-referenced it with known cuproptosis and disulfidptosis-related genes (CDRGs). Using the LASSO machine learning algorithm, we identified four CDRLRs-ACVR2B-AS1, AC095055.1, AL161782.1, and MANEA-DT-that are strongly associated with prognosis and used them to construct a prognostic risk model. To verify the model's reliability and validate these four CDRLRs as significant prognostic factors, we performed dataset grouping validation, followed by RT-qPCR and external database validation for differential expression and prognosis of CDRLRs in ccRCC. Gene function and pathway analysis were conducted using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) for high- and low-risk groups. Additionally, we have analyzed the tumor mutation burden (TMB) and the immune microenvironment (TME), employing the oncoPredict and Immunophenoscore (IPS) algorithms to assess the sensitivity of diverse risk categories to targeted therapeutics and immunosuppressants. Our predominant objective is to refine prognostic predictions for patients with ccRCC and inform treatment decisions by conducting an exhaustive study on cuproptosis and disulfidptosis.

Immune checkpoint inhibitors (ICIs) dramatically changed the prognosis of patients with NSCLC. Unfortunately, a reliable predictive biomarker is still missing. Commonly used biomarkers, such as PD-L1, MSI, or TMB, are not quite accurate in predicting ICI efficacy.

In this prospective observational cohort study, we investigated the predictive role of erythrocytes, thrombocytes, innate and adaptive immune cells, complement proteins (C3, C4), and cytokines from peripheral blood of 224 patients with stage III/IV NSCLC treated with ICI alone (pembrolizumab, nivolumab, and atezolizumab) or in combination (nivolumab + ipilimumab) with chemotherapy. These values were analyzed for associations with the response to the treatment and survival endpoints.

Higher baseline Tregs, MPV, hemoglobin, and lower monocyte levels were associated with favorable PFS and OS. Moreover, increased baseline basophils and lower levels of C3 predicted significantly improved PFS. The levels of the baseline immature granulocytes, C3, and monocytes were significantly associated with the occurrence of partial regression at the first restaging. Multiple studied parameters (n = 9) were related to PFS benefit at the time of first restaging as compared to baseline values. In addition, PFS nonbenefit group showed a decrease in lymphocyte count after three months of therapy. The OS benefit was associated with higher levels of lymphocytes, erythrocytes, hemoglobin, MCV, and MPV, and a lower value of NLR after three months of treatment.

Our work suggests that parameters from peripheral venous blood may be potential biomarkers in NSCLC patients on ICI. The baseline values of Tregs, C3, monocytes, and MPV are especially recommended for further investigation.

Complement has long been considered a key innate immune effector system that mediates host defence and tissue homeostasis. Yet, growing evidence has illuminated a broader involvement of complement in fundamental biological processes extending far beyond its traditional realm in innate immunity. Complement engages in intricate crosstalk with multiple pattern-recognition and signalling pathways both in the extracellular and intracellular space. Besides modulating host-pathogen interactions, this crosstalk guides early developmental processes and distinct cell trajectories, shaping tissue immunometabolic and regenerative programmes in different physiological systems. This Review provides a guide to the system-wide functions of complement. It highlights illustrative paradigm shifts that have reshaped our understanding of complement pathobiology, drawing examples from evolution, development of the central nervous system, tissue regeneration and cancer immunity. Despite its tight spatiotemporal regulation, complement activation can be derailed, fuelling inflammatory tissue pathology. The pervasive contribution of complement to disease pathophysiology has inspired a resurgence of complement therapeutics with major clinical developments, some of which have challenged long-held dogmas. We thus highlight major therapeutic concepts and milestones in clinical complement intervention.

In ER+ breast cancer, usually seen as the low immunogenic type, the main mechanisms favouring the immune response or tumour growth and immune evasion in the tumour microenvironment (TME) have been examined. The principal implications of targeting the oestrogen-mediated pathways were also considered. Recent experimental findings point out that anti-oestrogens contribute to the reversion of the immunosuppressive TME. Moreover, some preliminary clinical data with the hormone-immunotherapy association in a metastatic setting support the notion that the reversion of immune suppression in TME is likely favoured by the G0-G1 state induced by anti-oestrogens. Following immune stimulation, the reverted immune suppression allows the boosting of the effector cells of the innate and adaptive immune response. This suggests that ER+ breast cancer is a molecular subtype where a successful active immune manipulation can be attained. If this is confirmed by a prospective multicentre trial, which is expected in light of the provided evidence, the proposed hormone immunotherapy can also be tested in the adjuvant setting. Furthermore, the different rationale suggests a synergistic activity of our proposed immunotherapy with the currently recommended regimen consisting of antioestrogens combined with cyclin kinase inhibitors. Overall, this lays the foundation for a shift in clinical practice within this most prevalent molecular subtype of breast cancer.

Osteosarcoma is generally considered a cold tumor and is characterized by epigenetic alterations. Although tumor cells are surrounded by many immune cells such as macrophages, T cells may be suppressed, be inactivated, or not be presented due to various mechanisms, which usually results in poor prognosis and insensitivity to immunotherapy. Immunotherapy is considered a promising anti-cancer therapy in osteosarcoma but requires more research, but osteosarcoma does not currently respond well to this therapy. The cancer immunity cycle (CIC) is essential for anti-tumor immunity, and is epigenetically regulated. Therefore, it is possible to modulate the immune microenvironment of osteosarcoma by targeting epigenetic factors. In this study, we explored the correlation between epigenetic modulation and CIC in osteosarcoma through bioinformatic methods. Based on the RNA data from TARGET and GSE21257 cohorts, we identified epigenetic related subtypes by NMF clustering and constructed a clinical prognostic model by the LASSO algorithm. ESTIMATE, Cibersort, and xCell algorithms were applied to analyze the tumor microenvironment. Based on eight epigenetic biomarkers (SFMBT2, SP140, CBX5, HMGN2, SMARCA4, PSIP1, ACTR6, and CHD2), two subtypes were identified, and they are mainly distinguished by immune response and cell cycle regulation. After excluding ACTR6 by LASSO regression, the prognostic model was established and it exhibited good predictive efficacy. The risk score showed a strong correlation with the tumor microenvironment, drug sensitivity and many immune checkpoints. In summary, our study sheds a new light on the CIC-related epigenetic modulation mechanism of osteosarcoma and helps search for potential drugs for osteosarcoma treatment.

A cardinal principle of oncoimmunology is that cancer cells can be eliminated by tumor-infiltrating cytotoxic CD8 T lymphocytes. This has been widely demonstrated during the last 20 years and also recently harnessed for therapy. However, emerging evidence indicates that even neoplasms showing striking initial responses to conventional and targeted (immuno)therapies often acquire resistance, resulting in tumor relapse, increased aggressiveness, and metastatization. Indeed, tumors are complex ecosystems whose malignant and nonmalignant cells, constituting the tumor microenvironment, constantly interact and evolve in space and time. Together with patient's own genetic factors, such environmental interplays may curtail antitumor immune responses leading to cancer immune evasion and natural/acquired (immuno)therapy resistance. In this context, cancer stem cells (CSCs) are thought to be the roots of therapy failure. Flow cytometry is a powerful technology that finds extensive applications in cancer biology. It offers several unique advantages as it allows the rapid, quantitative, and multiparametric analysis of cell populations or functions at the single-cell level. In this chapter, we discuss a two-color flow cytometric protocol to evaluate cancer cell immunogenicity by analyzing the proliferative and tumor-killing potential of ovalbumin (OVA)-specific CD8 OT-1 T cells exposed to OVA-expressing MCA205 sarcoma cells and their CSC counterparts.