Ferroptotic therapy in cancer: benefits, side effects, and risks.

Ferroptosis is a type of regulated cell death characterized by iron accumulation and uncontrolled lipid peroxidation, leading to plasma membrane rupture and intracellular content release. Originally investigated as a targeted therapy for cancer cells carrying oncogenic RAS mutations, ferroptosis induction now exhibits potential to complement chemotherapy, immunotherapy, and radiotherapy in various cancer types. However, it can lead to side effects, including immune cell death, bone marrow impairment, liver and kidney damage, cachexia (severe weight loss and muscle wasting), and secondary tumorigenesis. In this review, we discuss the advantages and offer an overview of the diverse range of documented side effects. Furthermore, we examine the underlying mechanisms and explore potential strategies for side effect mitigation.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

A guideline on the molecular ecosystem regulating ferroptosis.

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases.

Epigenetic modulation of antitumor immunity for improved cancer immunotherapy.

Epigenetic mechanisms play vital roles not only in cancer initiation and progression, but also in the activation, differentiation and effector function(s) of immune cells. In this review, we summarize current literature related to epigenomic dynamics in immune cells impacting immune cell fate and functionality, and the immunogenicity of cancer cells. Some important immune-associated genes, such as granzyme B, IFN-γ, IL-2, IL-12, FoxP3 and STING, are regulated via epigenetic mechanisms in immune or/and cancer cells, as are immune checkpoint molecules (PD-1, CTLA-4, TIM-3, LAG-3, TIGIT) expressed by immune cells and tumor-associated stromal cells. Thus, therapeutic strategies implementing epigenetic modulating drugs are expected to significantly impact the tumor microenvironment (TME) by promoting transcriptional and metabolic reprogramming in local immune cell populations, resulting in inhibition of immunosuppressive cells (MDSCs and Treg) and the activation of anti-tumor T effector cells, professional antigen presenting cells (APC), as well as cancer cells which can serve as non-professional APC. In the latter instance, epigenetic modulating agents may coordinately promote tumor immunogenicity by inducing de novo expression of transcriptionally repressed tumor-associated antigens, increasing expression of neoantigens and MHC processing/presentation machinery, and activating tumor immunogenic cell death (ICD). ICD provides a rich source of immunogens for anti-tumor T cell cross-priming and sensitizing cancer cells to interventional immunotherapy. In this way, epigenetic modulators may be envisioned as effective components in combination immunotherapy approaches capable of mediating superior therapeutic efficacy.

Itaconic acid induces ferroptosis by activating ferritinophagy.

Itaconic acid is an unsaturated dicarbonic acid. It has a wide range of applications in the industrial production of resins and is also a mediator of immunometabolism in macrophages. Here, we show a previously unrecognized role of itaconic acid in triggering ferroptosis, a form of iron-dependent cell death driven by lipid peroxidation. We found that supraphysiological itaconic acid dose-dependently induces ferroptosis, rather than apoptosis, in human cancer cell lines. Mechanistically, we determined that itaconic acid activates NOCA4-mediated ferritinophagy, which leads to ferroptosis through ferritin degradation and subsequent iron overload and oxidative damage. In contrast, itaconic acid-induced expression and activation of NFE2L2 serves as a defense mechanism to limit ferroptosis by producing antioxidant genes. Consequently, impaired NCOA4 expression prevented, whereas a disrupted NFE2L2 pathway enhanced, sensitivity to itaconic acid-induced ferroptosis in vitro and in xenograft models. These findings establish a dynamic model of metabolite-induced ferroptotic cancer cell death, which may contribute to the development of new targeted therapies.

PPARG-mediated ferroptosis in dendritic cells limits antitumor immunity.

Dendritic cells (DCs) are antigen-presenting cells of the immune system, which play a key role in antitumor immunity by activating cytotoxic T cells. Here, we report that elevated ferroptosis, a lipid peroxidation-mediated cell death, impairs the maturation of DCs and their function in tumor suppression. Ferroptosis is selectively induced in DCs by the GXP4 inhibitor RSL3, but not the SLC7A11 inhibitor erastin. Ferroptotic DCs lose their ability to secrete pro-inflammatory cytokines (TNF and IL6) and express MHC class I in response to the maturation signal of lipopolysaccharide. Moreover, ferroptotic DCs fail to induce CD8+ T cells to produce IFNG/IFNγ. Mechanistically, PPARG/PPARγ, a nuclear receptor involved in the regulation of lipid metabolism, is responsible for RSL3-induced ferroptosis in DCs. Consequently, the genetic depletion of PPARG restores the maturation and function of DCs. Using immunogenic cell death-based DC vaccine models, we further demonstrate that PPARG-mediated ferroptosis of DCs limits antitumor immunity in mice. Together, these findings demonstrate a novel role of ferroptotic DCs in driving an immunosuppressive tumor microenvironment.

Diagnostic and prognostic value of microRNA-2355-3p and contribution to the progression in lung adenocarcinoma.

The aim of this study was to delve into the clinical significance and biological function of miR-2355-3p in LUAD. Tissues and blood samples from 116 LUAD patients and blood samples of 90 healthy volunteers were collected. The relative expression of miR-2355-3p was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The receiver operating curve (ROC) was plotted for diagnostic value estimation. Kaplan-Meier survival curves were constructed, and multivariate survival analyses were performed for prognostic value estimation. A luciferase reporter assay was carried out to confirm the interaction of miR-2355-3p and ZCCHC14. The CCK-8 and transwell assays were conducted to explore the function of miR-2355-3p on LUAD cells. Rescue experiments were performed to verify the interaction. miR-2355-3p showed an upregulated expression in the samples of LUAD patients. For diagnostic value estimation, the AUC was 0.905 with a sensitivity was 84.5% and specificity of 83.3%. For the estimation of prognostic value, the P-value of log-rank test on K-M curves was 0.002 and 0.006 for overall survival and progression survival, respectively. Based on multivariate Cox regression analysis, miR-2355-3p was a powerful prognostic tool with a P-value of 0.027. ZCCHC14 has binding sites with miR-2355-3p, an expression level, and luciferase activity negatively correlated with miR-2355-3p expression. Knockdown of miR-2355-3p inhibited proliferation, migration, and invasion of LUAD cells, but ZCCHC14 can rescue this inhibition. miR-2355-3p has the potential to be a diagnostic marker and prognostic marker for LUAD. Inhibition of miR-2355-3p in LUAD cells can suppress the progression of LUAD at least partly by direct targeting ZCCHC14.

SMG9 drives ferroptosis by directly inhibiting GPX4 degradation.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that plays an integral role in eliminating abnormal mRNA and corresponding proteins. It is unclear whether the NMD pathway is involved in regulating ferroptosis, which is a type of iron-dependent cell death mainly caused by the inhibition of the antioxidant SLC7A11-GPX4 axis. In this study, we conducted a small-scale RNAi screen and proved that SMG9, a component of the NMD machinery, is a selective driver for ferroptosis in human cancer cells. SMG9 positively regulates ferroptosis independent of its activity in NMD. Instead, SMG9 is a direct binding protein of GPX4 to promote the degradation of GPX4 in response to RSL3 (a GPX4 inhibitor), but not erastin (a SLC7A11 inhibitor). The genetic inhibition of SMG9 increases the accumulation of GPX4 in the mitochondria, thereby preventing mitochondrial oxidative damage, and ultimately favoring ferroptosis resistance in vitro or in xenograft mouse models. Overall, these findings establish a new mitochondrial regulation mechanism that can affect ferroptosis-mediated tumor suppression.

Intratumoral expression of interleukin 23 variants using oncolytic vaccinia virus elicit potent antitumor effects on multiple tumor models via tumor microenvironment modulation.

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.

The HMGB1-AGER-STING1 pathway mediates the sterile inflammatory response to alkaliptosis.

Alkaliptosis is a recently discovered form of regulated cell death driven by intracellular alkalization. However, the immune characteristics and mechanisms of alkaliptosis are still poorly understood. Here, we show that HMGB1, a multifunctional alarm protein that drives innate immunity, is necessary for inflammation caused by alkaliptotic damage. During alkaliptosis, HMGB1 translocation and release from the nucleus to the cytoplasm to the extracellular space requires nuclear DNA damage signals, whereas the FANCD2-dependent (but not ATM-mediated) DNA repair pathway inhibits this process. Once released by alkaliptotic cancer cells, extracellular HMGB1 binds to the AGER receptor in macrophages and then activates the STING1 pathway to produce pro-inflammatory cytokines (e.g., TNF and IL6). Consequently, the pharmacological or genetic inhibition of the HMGB1-AGER-STING1 pathway limits cytokine production during alkaliptosis. These findings provide new insight into the sterile inflammatory response to cell death.

IL-36γ-armed oncolytic virus exerts superior efficacy through induction of potent adaptive antitumor immunity.

In this study, we aimed to apply the cytokine IL-36γ to cancer immunotherapy by constructing new oncolytic vaccinia viruses (OV) expressing interleukin-36γ (IL-36γ-OVs), leveraging unique synergism between OV and IL-36γ's ability to promote antitumor adaptive immunity and modulate tumor microenvironment (TME). IL-36γ-OV had dramatic therapeutic efficacies in multiple murine tumor models, frequently leading to complete cancer eradication in large fractions of mice. Mechanistically, IL-36-γ-armed OV induced infiltration of lymphocytes and dendritic cells, decreased myeloid-derived suppressor cells and M2-like tumor-associated macrophages, and T cell differentiation into effector cells. Further study showed that IL-36γ-OV increased the number of tumor antigen-specific CD4+ and CD8+ T cells and the therapeutic efficacy depended on both CD8+ and CD4+ T cells. These results demonstrate that these IL36γ-armed OVs exert potent therapeutic efficacy mainly though antitumor immunity and they may hold great potential to advance treatment in human cancer patients.

The dark side of ferroptosis in pancreatic cancer.

Drug-induced ferroptosis, an iron-dependent regulatory necrosis, has been proposed for the therapy of pancreatic ductal adenocarcinoma. However, genetically engineered mouse models have revealed that high-iron diets or deletion of pancreatic GPX4 (a key repressor of ferroptosis) accelerate the development of mutant Kras-driven PDAC by activating the STING1/TMEM173-dependent DNA sensor pathway. Abbreviations ADM: acinar-to-ductal metaplasia; CGAS: cyclic GMP-AMP synthase; DAMP: damage-associated molecular pattern; GPX4: glutathione peroxidase 4; GEMM: genetically engineered mouse models; PDAC: pancreatic ductal adenocarcinoma; PanIN: pancreatic intraepithelial neoplasia, SLC7A11: solute carrier family 7 member 11; STING1: cGAMP-stimulator of interferon response cGAMP interactor 1; TME: tumor microenvironment; 8-OHG: 8-hydroxy-2'-deoxyguanosine.

Oncolytic virus promotes tumor-reactive infiltrating lymphocytes for adoptive cell therapy.

Adoptive cell therapy (ACT) using tumor-specific tumor-infiltrating lymphocytes (TILs) has demonstrated success in patients where tumor-antigen specific TILs can be harvested from the tumor, expanded, and re-infused in combination with a preparatory regimen and IL2. One major issue for non-immunogenic tumors has been that the isolated TILs lack tumor specificity and thus possess limited in vivo therapeutic function. An oncolytic virus (OV) mediates an immunogenic cell death for cancer cells, leading to elicitation and dramatic enhancement of tumor-specific TILs. We hypothesized that the tumor-specific TILs elicited and promoted by an OV would be a great source for ACT for solid cancer. In this study, we show that a local injection of oncolytic poxvirus in MC38 tumor with low immunogenicity in C57BL/6 mice, led to elicitation and accumulation of tumor-specific TILs in the tumor tissue. Our analyses indicated that IL2-armed OV-elicited TILs contain lower quantities of exhausted PD-1hiTim-3+ CD8+ T cells and regulatory T cells. The isolated TILs from IL2-expressing OV-treated tumor tissue retained high tumor specificity after expansion ex vivo. These TILs resulted in significant tumor regression and improved survival after adoptive transfer in mice with established MC38 tumor. Our study showcases the feasibility of using an OV to induce tumor-reactive TILs that can be expanded for ACT.

Pirin is a nuclear redox-sensitive modulator of autophagy-dependent ferroptosis.

In regulated cell death, genetically encoded molecular machinery destroys cells. This process is not only essential for organ development and homeostasis, but also leads to pathological diseases. One form of regulated cell death is ferroptosis, which is an iron-dependent oxidative cell death caused by lipid peroxidation. Although inducing ferroptosis is an emerging anticancer strategy, the molecular mechanism underlying tumor resistance to ferroptotic cell death is still unclear. Here, we show that pirin (PIR), an iron-binding nuclear protein, plays a previously unrecognized role in mediating ferroptosis resistance in human pancreatic cancer cells. The transcription factor NFE2L2 mediates the upregulation of PIR during ferroptosis caused by small-molecule compounds (e.g., erastin or RSL3). PIR is a nuclear redox sensor and regulator, and increasing it limits the oxidative damage of DNA and the subsequent cytoplasmic transport and extracellular release of HMGB1. In contrast, the depletion of PIR initiates HMGB1-dependent autophagy by binding to BECN1, and subsequently promotes ferroptosis by activating ACSL4. Consequently, in cell cultures and xenograft mouse models, blocking PIR signaling enhances ferroptosis-mediated tumor growth suppression. Together, these findings provide new insights into the molecular mechanisms of autophagy-dependent ferroptosis.

Ferroptotic damage promotes pancreatic tumorigenesis through a TMEM173/STING-dependent DNA sensor pathway.

Ferroptosis is a more recently recognized form of cell death that relies on iron-mediated oxidative damage. Here, we evaluate the impact of high-iron diets or depletion of Gpx4, an antioxidant enzyme reported as an important ferroptosis suppressor, in the pancreas of mice with cerulean- or L-arginine-induced pancreatitis, and in an oncogenic Kras murine model of spontaneous pancreatic ductal adenocarcinoma (PDAC). We find that either high-iron diets or Gpx4 depletion promotes 8-OHG release and thus activates the TMEM173/STING-dependent DNA sensor pathway, which results in macrophage infiltration and activation during Kras-driven PDAC in mice. Consequently, the administration of liproxstatin-1 (a ferroptosis inhibitor), clophosome-mediated macrophage depletion, or pharmacological and genetic inhibition of the 8-OHG-TMEM173 pathway suppresses Kras-driven pancreatic tumorigenesis in mice. GPX4 is also a prognostic marker in patients with PDAC. These findings provide pathological and mechanistic insights into ferroptotic damage in PDAC tumorigenesis in mice.

Whole Transcriptome RNA Sequencing Identified circ_022743, circ_052666, and circ_004452 Were Associated with Colon Cancer Development.

The objective of this study was to identify the key circular RNAs (circRNAs) related to the development of colon cancer. High-throughput RNA sequencing on eight early-stage (ES) and eight later stage (LS) colon tumor tissues, and eight normal tissues, was performed. Differentially expressed circRNAs and differentially expressed mRNAs were identified. Functional enrichment analysis and the miRNA-circRNA-mRNA network were performed. In addition, the differential expression levels of key circRNAs were verified using real-time quantitative PCR (qPCR). In total, 408, 472, and 278 differentially expressed circRNAs were identified in ES versus normal control (N), LS versus N, and LS versus ES groups, respectively. Functional enrichment analysis showed that circ_052666 was significantly enriched in "extracellular matrix/receptor interaction"; circ_022743 was remarkably enriched in "neurotrophin signaling pathway"; and circ_004452 was observably enriched in "TGF-ß signaling pathway." Moreover, key miRNA-circRNA-mRNA relationships, such as hsa-miR-29b/c-3p-circ_052666-COL1A1 and hsa-miR-1294-circ_004452-left-right determination factor 1 (LEFTY1), were identified. Furthermore, qPCR showed consistent results with RNA sequencing. Our findings indicate that key circRNAs, such as circ_022743, circ_052666, and circ_004452, may be involved in colon cancer development, and could be used as potential biomarkers for the diagnosis and treatment of this disease.

In Situ Therapeutic Cancer Vaccination with an Oncolytic Virus Expressing Membrane-Tethered IL-2.

Successful in situ therapeutic vaccination would allow locally delivered oncolytic virus (OV) to exert systemic immunologic effects on metastases and improve survival. We have utilized bilateral flank tumor models to determine the most efficacious regimens of in situ vaccination. Intratumoral injection with membrane-tethered interleukin -2-armed OV (vvDD-mIL2) plus a Toll-like receptor 9 ligand (CpG) yielded systemic immunization and decreased tumor growth in a contralateral, noninjected tumor. Our main aims were to study the tumor immune microenvironment (TME) after vaccination and identify additional immune adjuvants that may improve the systemic tumor-specific immunity. Immunological profiles in the spleen showed an increased CD8+ T cell/regulatory T cell (Treg) ratio and increased CD11c+ cells after dual injection in one flank tumor. Concurrently, there was increased infiltration of tumor necrosis factor alpha (TNF-α)+CD8+ T cells and interferon gamma (IFN-γ)+CD4+ T cells and reduced CTLA-4+PD-1+CD8+ T cells in the contralateral, noninjected tumor. The anti-tumoral activity depended on CD8+ T cells and IFN-γ, but not CD4+ T cells. Based on the negative immune components still existing in the untreated tumors, we investigated additional adjuvants: clodronate liposome-mediated depletion of macrophages plus anti-PD-1 therapy. This regimen dramatically reduced the tumor burden in the noninjected tumor and increased median survival by 87%, suggesting that inhibition/elimination of suppressive components in the tumor microenvironment (TME) can improve therapeutic outcomes. This study emphasizes the importance of immune profiling to design rational, combined immunotherapy regimens ultimately to impact patient survival.

Oncolytic vaccinia virus delivering tethered IL-12 enhances antitumor effects with improved safety.

Immune checkpoint blockade is arguably the most effective current cancer therapy approach; however, its efficacy is limited to patients with "hot" tumors, warranting an effective approach to transform "cold" tumors. Oncolytic viruses (especially properly armed ones) have positive effects on almost every aspect of the cancer-immunity cycle and can change the cancer-immune set point of a tumor. Here, we tested whether oncolytic vaccinia virus delivering tethered interleukin 12 (IL-12) could turn a "cold" tumor into a "hot" tumor while avoiding IL-12's systemic toxicity. Our data demonstrated that tethered IL-12 could be maintained in the tumor without treatment-induced toxic side effects. Moreover, the treatment facilitated tumor infiltration of more activated CD4+ and CD8+ T cells and less Tregs, granulocytic myeloid-derivedsuppressor cells, and exhausted CD8+ T cells, with increased interferon γ and decreased transforming growth factor ß, cyclooxygenase-2, and vascular endothelial growth factor expression, leading to transformed, immunogenic tumors and improved survival. Combined with programmed cell death 1 blockade, vaccinia virus expressing tethered IL-12 cured all mice with late-stage colon cancer, suggesting immediate translatability to the clinic.

Autophagy-dependent ferroptosis drives tumor-associated macrophage polarization via release and uptake of oncogenic KRAS protein.

KRAS is the most frequently mutated oncogene in human neoplasia. Despite a large investment to understand the effects of KRAS mutation in cancer cells, the direct effects of the oncogenetic KRAS activation on immune cells remain elusive. Here, we report that extracellular KRASG12D is essential for pancreatic tumor-associated macrophage polarization. Oxidative stress induces KRASG12D protein release from cancer cells succumbing to autophagy-dependent ferroptosis. Extracellular KRASG12D packaged into exosomes then is taken up by macrophages through an AGER-dependent mechanism. KRASG12D causes macrophages to switch to an M2-like pro-tumor phenotype via STAT3-dependent fatty acid oxidation. Consequently, the disruption of KRASG12D release and uptake can abolish the macrophage-mediated stimulation of pancreatic adenocarcinomas in mouse models. Importantly, the level of KRASG12D expression in macrophages correlates with poor survival in pancreatic cancer patients. These findings not only identify extracellular KRASG12D as a key mediator of cancer cell-macrophage communication, but also provide a novel KRAS-targeted anticancer strategy. Abbreviations: DAMP, damage-associated molecular pattern; PBMCMs, peripheral blood mononuclear cell-derived macrophages; PDAC, pancreatic ductal adenocarcinoma; s.c., subcutaneously; TAMs, tumor-associated macrophages; TME, tumor microenvironment.