Evolution of Molecularly Imprinted Enzyme Inhibitors: From Simple Activity Inhibition to Pathological Cell Regulation.

Molecular imprinting represents one of the most promising strategies to design artificial enzyme inhibitors. However, the study of molecularly imprinted enzyme inhibitors (MIEIs) remains at a primary stage. Advanced applications of MIEIs for cell regulation have rarely been explored. Using a solid-phase oriented imprinting strategy so as to leave the active site of the enzymes accessible, we synthesized two MIEIs that exhibit high specificity and potent inhibitory effects (inhibition constant at low nM range) towards trypsin and angiogenin. The trypsin MIEI inhibits trypsin activity, tryptic digestion-induced extracellular matrix lysis and cell membrane destruction, indicating its utility in the treatment of active trypsin-dependent cell injury. The angiogenin MIEI blocks cancer cell proliferation by suppressing the ribonuclease activity of angiogenin and decreasing the angiogenin level inside and outside HeLa cells. Our work demonstrates the versatility of MIEIs for both enzyme inhibition and cell fate manipulation, showing their great potential as therapeutic drugs in biomedicine.

Enhancement of angiogenin inhibition by polyphenol-capped gold nanoparticles.

Angiogenin (Ang), is a ribonucleolytic protein that is associated with angiogenesis, the formation of blood vessels. The involvement of Ang in vascularisation makes it a potential target for the identification of compounds that have the potential to inhibit the process. The compounds may be assessed for their ability to inhibit the ribonucleolytic activity of the protein and subsequently blood vessel formation, a crucial requirement for tumor formation. We report an inhibition of the ribonucleolytic activity of Ang with the gallate containing green tea polyphenols, ECG and EGCG that exhibits an increased efficacy upon forming polyphenol-capped gold nanoparticles (ECG-AuNPs and EGCG-AuNPs). The extent of inhibition was confirmed using an agarose gel-based assay followed by fluorescence titration studies that indicated a hundred fold stronger binding of polyphenol-capped gold nanoparticles (GTP-AuNPs) compared to the bare polyphenols. Interestingly, we found a change in the mode of inhibition from a noncompetitive type to a competitive mode of inhibition in case of the GTP-AuNPs, which is in agreement with the 'n' values obtained from the fluorescence quenching studies. The effect on angiogenesis has also been assessed by the chorioallantoic membrane (CAM) assay. We find an increase in the inhibition potency of GTP-AuNPs that could find applications in the development of anti-angiogenic compounds.

Positional preferences in flavonoids for inhibition of ribonuclease A: Where "OH" where?

Flavonoids are a class of polyphenols that possess diverse properties. The structure-activity relationship of certain flavonoids and resveratrol with ribonuclease A (RNase A) has been investigated. The selected flavonoids have a similar skeleton and the positional preferences of the phenolic moieties toward inhibition of the catalytic activity of RNase A have been studied. The results obtained for RNase A inhibition by flavonoids suggest that the planarity of the molecules is necessary for effective inhibitory potency. Agarose gel electrophoresis and precipitation assay experiments along with kinetic studies reveal Ki values for the various flavonoids in the micromolar range. Minor secondary structural changes of RNase A were observed after interaction with the flavonoids. An insight into the specific amino acid involvement in the binding of the substrate using docking studies is also presented. The dipole moment of the flavonoids that depends on the orientation of the hydroxyl groups in the molecule bears direct correlation with the inhibitory potency against RNase A. The direct association of this molecular property with enzyme inhibition can be exploited for the design and development of inhibitors of proteins.

Design of configuration-restricted triazolylated ß-d-ribofuranosides: a unique family of crescent-shaped RNase A inhibitors.

Seven new carbohydrate-bistriazole hybrid molecules were designed taking into consideration the crescent shaped active site of ribonuclease A (RNase A). In this case, the ß-d-ribofuranose structure was used as the basic building unit; both the C1 and C4 arms protruding out towards the ß-face of the tetrahydrofuran moiety of the ribose sugar provided an overall "U" shape to the basic building block. Several combinations of bistriazole moieties were constructed on the two arms of this basic building block. These mono- and/or bis-substituted 1,2,3-triazole units were linked to acidic functional groups because of the overall basic nature of the hydrolytic site of RNase A. All these compounds were efficient competitive inhibitors of RNase A with inhibition constants (Ki) in the micromolar range. In contrast to the carboxylic acid-modified hybrid molecules, molecules carrying sulfonic acids were found to be more potent because of the stronger interactions with the positively charged active site. The most efficient inhibitor of the series was the disulfonic acid-functionalized carbohydrate-bis-triazole hybrid molecule. Docking studies disclosed that the molecule, because of its well defined "U" shape with flexible arms, fits effectively in the active site; moreover, in all cases, besides the acid groups, the triazole and sugar rings also actively participated in creating the hydrogen bonding network in the cavity of the enzyme active site.

RNase I Modulates Escherichia coli Motility, Metabolism, and Resistance.

Bacteria are constantly adapting to their environment by sensing extracellular factors that trigger production of intracellular signaling molecules, known as second messengers. Recently, 2',3'-cyclic nucleotide monophosphates (2',3'-cNMPs) were identified in Escherichia coli and have emerged as possible novel signaling molecules. 2',3'-cNMPs are produced through endonucleolytic cleavage of short RNAs by the T2 endoribonuclease, RNase I; however, the physiological roles of RNase I remain unclear. Our transcriptomic analysis suggests that RNase I is involved in modulating numerous cellular processes, including nucleotide metabolism, motility, acid sensitivity, metal homeostasis, and outer membrane morphology. Through a combination of deletion strain and inhibitor studies, we demonstrate that RNase I plays a previously unknown role in E. coli stress resistance by affecting pathways that are part of the defense mechanisms employed by bacteria when introduced to external threats, including antibiotics. Thus, this work provides insight into the emerging roles of RNase I in bacterial signaling and physiology and highlights the potential of RNase I as a target for antibacterial adjuvants.

Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor.

Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)32+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.

Nucleoside Tetra- and Pentaphosphates Prepared Using a Tetraphosphorylation Reagent Are Potent Inhibitors of Ribonuclease A.

Adenosine and uridine 5'-tetra- and 5'-pentaphosphates were synthesized from an activated tetrametaphosphate ([PPN]2[P4O11], [PPN]2[1], PPN = bis(triphenylphosphine)iminium) and subsequently tested for inhibition of the enzymatic activity of ribonuclease A (RNase A). Reagent [PPN]2[1] reacts with unprotected uridine and adenosine in the presence of a base under anhydrous conditions to give nucleoside tetrametaphosphates. Ring opening of these intermediates with tetrabutylammonium hydroxide ([TBA][OH]) yields adenosine and uridine tetraphosphates (p4A, p4U) in 92% and 85% yields, respectively, from the starting nucleoside. Treatment of ([PPN]2[1]) with AMP or UMP yields nucleoside-monophosphate tetrametaphosphates (cp4pA, cp4pU) having limited aqueous stability. Ring opening of these ultraphosphates with [TBA][OH] yields p5A and p5U in 58% and 70% yield from AMP and UMP, respectively. We characterized inorganic and nucleoside-conjugated linear and cyclic oligophosphates as competitive inhibitors of RNase A. Increasing the chain length in both linear and cyclic inorganic oligophosphates resulted in improved binding affinity. Increasing the length of oligophosphates on the 5' position of adenosine beyond three had a deleterious effect on binding. Conversely, uridine nucleotides bearing 5' oligophosphates saw progressive increases in binding with chain length. We solved X-ray cocrystal structures of the highest affinity binders from several classes. The terminal phosphate of p5A binds in the P1 enzymic subsite and forces the oligophosphate to adopt a convoluted conformation, while the oligophosphate of p5U binds in several extended conformations, targeting multiple cationic regions of the active-site cleft.

Sulfonic nucleic acids (SNAs): a new class of substrate mimics for ribonuclease A inhibition.

Sulfonic nucleic acids were identified as inhibitors of ribonuclease A (RNase A). The incorporation of a strongly acidic group (sulfonic, -SO3H) at the 3'-end of pyrimidine nucleosides thymidine and uridine was prompted by the low inhibition constant (Ki) values recorded for carboxymethylsulfonyl (-SO2CH2CO2H) and -CO2H functionalized nucleosides. It was envisaged that the sulfonic acid-modified pyrimidines would bind effectively with the positively charged P1 site of ribonuclease A. Typical harsh conditions used for SO3H incorporation were replaced with milder reaction conditions. The uridine analogue showing a Ki value of 0.96 µM elicited a better result than the thymidine-modified inhibitor. Notably, it was also the best result among all modified non-phosphate acidic nucleosides reported and screened so far as RNase A inhibitors.

Blockade of angiogenin by thalidomide inhibits the tumorigenesis of murine hemangioendothelioma.

Thalidomide, a well-known immunomodulatory compound, has an anti-angiogenic activity, which may be utilized for the treatment of angiogenesis-related diseases such as hemangioendothelioma. The aim of the present study was to investigate both the antitumor role of thalidomide on hemangioendothelioma and the underlying mechanism. By using the xenograft mouse model, we found that thalidomide can inhibit the progression of hemangioendothelioma in vivo. Moreover, thalidomide shows no effect on the proliferation of hemangioendothelioma endothelial cell (EOMA), but significantly impairs the pro-angiogenic capacity of the EOMA cells in vitro. By qRT-PCR screening, we observed that the expression of angiogenin was downregulated by thalidomide treatment. We next performed tissue array analysis and found a positive correlation between angiogenin expression level and hemangioendothelioma occurrence in patients. Moreover, we confirmed that the antitumoral role of thalidomide is dependent on angiogenin expression both in vivo and in vitro. Taken together, we concluded that thalidomide can inhibit the progression of hemangioendothelioma by downregulating the expression of pro-angiogenic factor angiogenin and therefore can be used as a potent therapeutic to treat hemangioendothelioma.

Targeting the Tie2-αvß3 integrin axis with bi-specific reagents for the inhibition of angiogenesis.

BACKGROUND: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics. RESULTS: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and αvß3 integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg-Gly-Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone. CONCLUSIONS: Collectively, this study-the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin αvß3-targeting proteins for clinical use as imaging and therapeutic agents.

Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo.

BACKGROUND: Graphene oxide (GO) has attracted intensive interest in biological and medical fields in recent years due to its unique physical, chemical, and biological properties. In our previous work, we proved that GO could deliver small interfering RNA (siRNA) into cells and downregulate the expression of the desired gene. METHODS: This study investigated the potential of a modified GO nanocarrier for co-delivery of siRNA and doxorubicin (DOX) for enhanced cancer therapy. Fourier transform infrared spectroscopy, laser particle size analyzer, UV-visible spectroscopy, gel electrophoresis retardation, and in vitro release assay were studied. RESULTS: The results of real-time polymerase chain reaction revealed that the expression of vascular endothelial growth factor (VEGF) mRNA was decreased 46.84%±3.72% (mean ± SD). Enzyme-linked immunosorbent assay indicated that the expression of VEGF protein was down-regulated to 52.86%±1.10% (mean ± SD) in vitro. In vivo tumor growth assay GO-poly-l-lysine hydrobromide/folic acid (GPF)/DOX/siRNA exhibited gene silencing and tumor inhibition (66.95%±2.35%, mean ± SD) compared with naked siRNA (1.62%±1.47%, mean ± SD) and DOX (33.63%±5.85%, mean ± SD). GPF/DOX/siRNA exhibited no testable cytotoxicity. CONCLUSION: The results indicated that co-delivery of siRNA and DOX by GPF could be a promising application in tumor clinical therapy.

HBV Facilitated Hepatocellular Carcinoma Cells Proliferation by Up-Regulating Angiogenin Expression Through IL-6.

BACKGROUND/AIMS: Patients with hepatitis B virus (HBV) infection are at a high risk of developing hepatocellular carcinoma (HCC). In this study, we aim to investigate the roles of HBV on angiogenin (ANG), as well as the effects on cell proliferation in presence of ANG down-regulation. METHODS: Serum ANG was determined by ELISA. The expression of ANG mRNA and protein in HCC cell lines with or without HBV/HBx were determined. Western blot and ELISA were conducted to determine the effects of HBV/HBx on IL-6 expression. The role of IL-6 on ANG was evaluated by IL-6 recombinant protein or IL-6 neutralizing antibody. Immunofluorescence staining was used to detect the nuclear translocation of ANG. MTT was performed to evaluate the relative inhibition ratio. RESULT: In vivo experiments showed elevation of serum ANG in patients infected with HBV. In vitro experiments showed HBV and HBx contributed to the transcription and translation of ANG. ANG expression showed increase after IL-6 stimulation, and ANG protein decreased in the presence of IL-6 blocking with its antibody. HBV promoted nuclear translocation of ANG. Inhibiting ANG expression or blocking of nuclear transfer of ANG attenuated the 45S rRNA synthesis and cell proliferation. CONCLUSION: HBV and HBx protein can increase the level of ANG through IL-6. HBV and HBx contributed to the nuclear translocation of ANG. Cell proliferation was inhibited after inhibiting the expression or nuclear transfer of ANG.

Evidence for Novel Action at the Cell-Binding Site of Human Angiogenin Revealed by Heteronuclear NMR Spectroscopy, in†silico and in†vivo Studies.

A member of the ribonuclease A superfamily, human angiogenin (hAng) is a potent angiogenic factor. Heteronuclear NMR spectroscopy combined with induced-fit docking revealed a dual binding mode for the most antiangiogenic compound of a series of ribofuranosyl pyrimidine nucleosides that strongly inhibit hAng's angiogenic activity in vivo. While modeling suggests the potential for simultaneous binding of the inhibitors at the active and cell-binding sites, NMR studies indicate greater affinity for the cell-binding site than for the active site. Additionally, molecular dynamics simulations at 100 ns confirmed the stability of binding at the cell-binding site with the predicted protein-ligand interactions, in excellent agreement with the NMR data. This is the first time that a nucleoside inhibitor is reported to completely inhibit the angiogenic activity of hAng in vivo by exerting dual inhibitory activity on hAng, blocking both the entrance of hAng into the cell and its ribonucleolytic activity.

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe.

In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 µM (Ba2+), 90.9 µM (Co2+), 110.6 µM (Pb2+), 171.5 µM (As3+), and 165.1 µM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

1,5-Disubstituted 1,2,3-triazole linked disaccharides: Metal-free syntheses and screening of a new class of ribonuclease A inhibitors.

1,5-Regioisomeric triazole linked disaccharides have been synthesized and screened for their inhibitory properties against ribonuclease A (RNase A). The angular constraint-driven 'crescent shaped' inhibitors accommodated themselves into the enzyme active site. An improved enzyme inhibition was observed with increased H-bonding ability of polar functional groups in the modified disaccharides. In this series, introduction of two carboxyl groups in the furanose rings elicited the best result with an inhibition constant of 50 ±â€¯3 µM. This is the first ever report on the use of disaccharides as RNase A inhibitors.

Interaction between human angiogenin and the p53 TAD2 domain and its implication for inhibitor discovery.

Interaction between angiogenin and the p53 TAD2 domain in cancer cells can inhibit the function of the p53 tumor suppressor and promote cell survival. Based on a model structure using NMR and mutational analysis, positively charged 31 RRR33 and 50 KRSIK54 motifs of human angiogenin were identified as p53-binding sites that could interact with negatively charged D48/E51 and E56 residues of the p53 TAD2 domain, respectively. These results suggest that 31 RRR33 and 50 KRSIK54 motifs of human angiogenin might play a critical role in the regulation of p53-mediated apoptosis and angiogenesis in cancer cells. This study identifies potential target sites for screening angiogenin-specific inhibitors that could not only inhibit p53 binding but could also simultaneously inhibit cell binding, internalization, DNA binding, and nuclear translocation of human angiogenin.

Normalization of Tumor Vessels by Tie2 Activation and Ang2 Inhibition Enhances Drug Delivery and Produces a Favorable Tumor Microenvironment.

A destabilized tumor vasculature leads to limited drug delivery, hypoxia, detrimental tumor microenvironment, and even metastasis. We performed a side-by-side comparison of ABTAA (Ang2-Binding and Tie2-Activating Antibody) and ABA (Ang2-Blocking Antibody) in mice with orthotopically implanted glioma, with subcutaneously implanted Lewis lung carcinoma, and with spontaneous mammary cancer. We found that Tie2 activation induced tumor vascular normalization, leading to enhanced blood perfusion and chemotherapeutic drug delivery, markedly lessened lactate acidosis, and reduced tumor growth and metastasis. Moreover, ABTAA favorably altered the immune cell profile within tumors. Together, our findings establish that simultaneous Tie2 activation and Ang2 inhibition form a powerful therapeutic strategy to elicit a favorable tumor microenvironment and enhanced delivery of a chemotherapeutic agent into tumors.

The ammonium sulfate inhibition of human angiogenin.

In this study, we investigate the inhibition of human angiogenin by ammonium sulfate. The inhibitory potency of ammonium sulfate for human angiogenin (IC50 = 123.5 ± 14.9 mm) is comparable to that previously reported for RNase A (119.0 ± 6.5 mm) and RNase 2 (95.7 ± 9.3 mm). However, analysis of two X-ray crystal structures of human angiogenin in complex with sulfate anions (in acidic and basic pH environments, respectively) indicates an entirely distinct mechanism of inhibition. While ammonium sulfate inhibits the ribonucleolytic activity of RNase A and RNase 2 by binding to the active site of these enzymes, sulfate anions bind only to peripheral substrate anion-binding subsites of human angiogenin, and not to the active site.

Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages.

Glioblastomas (GBMs) rapidly become refractory to anti-VEGF therapies. We previously demonstrated that ectopic overexpression of angiopoietin-2 (Ang-2) compromises the benefits of anti-VEGF receptor (VEGFR) treatment in murine GBM models and that circulating Ang-2 levels in GBM patients rebound after an initial decrease following cediranib (a pan-VEGFR tyrosine kinase inhibitor) administration. Here we tested whether dual inhibition of VEGFR/Ang-2 could improve survival in two orthotopic models of GBM, Gl261 and U87. Dual therapy using cediranib and MEDI3617 (an anti-Ang-2-neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth and increasing U87 necrosis, effectively reducing viable tumor burden. Consistent with their vascular-modulating function, the dual therapies enhanced morphological normalization of vessels. Dual therapy also led to changes in tumor-associated macrophages (TAMs). Inhibition of TAM recruitment using an anti-colony-stimulating factor-1 antibody compromised the survival benefit of dual therapy. Thus, dual inhibition of VEGFR/Ang-2 prolongs survival in preclinical GBM models by reducing tumor burden, improving normalization, and altering TAMs. This approach may represent a potential therapeutic strategy to overcome the limitations of anti-VEGFR monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells.

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival.

Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.