Targeted therapy for non-small-cell lung cancer: New insights into regulated cell death combined with immunotherapy.

Non-small-cell lung cancer (NSCLC), which has a high rate of metastatic spread and drug resistance, is the most common subtype of lung cancer. Therefore, NSCLC patients have a very poor prognosis and a very low chance of survival. Human cancers are closely linked to regulated cell death (RCD), such as apoptosis, autophagy, ferroptosis, pyroptosis, and necroptosis. Currently, small-molecule compounds targeting various types of RCD have shown potential as anticancer treatments. Moreover, RCD appears to be a specific part of the antitumor immune response; hence, the combination of RCD and immunotherapy might increase the inhibitory effect of therapy on tumor growth. In this review, we summarize small-molecule compounds used for the treatment of NSCLC by focusing on RCD and pharmacological systems. In addition, we describe the current research status of an immunotherapy combined with an RCD-based regimen for NSCLC, providing new ideas for targeting RCD pathways in combination with immunotherapy for patients with NSCLC in the future.

ETNK1 mutation occurs in a wide spectrum of myeloid neoplasms and is not specific for atypical chronic myeloid leukemia.

BACKGROUND: ETNK1 mutation has been suggested as a useful tool to support the diagnosis of atypical chronic myeloid leukemia. ETNK1 mutations, however, occur in other myeloid neoplasms. METHODS: The authors assessed the clinicopathologic and molecular genetic features of 80 ETNK1-mutated myeloid neoplasms. RESULTS: Thirty-seven neoplasms (46%) were classified as myelodysplastic syndrome, 17 (21%) were classified as myelodysplastic/myeloproliferative neoplasm, 14 (18%) were classified as acute myeloid leukemia, and 12 (15%) were classified as myeloproliferative neoplasm. ETNK1 mutations were detected at the first test in 96% of patients, suggesting that ETNK1 mutation is an early event in pathogenesis. ETNK1 mutations represented the dominant clone in 63% of patients and was persistently dominant in 93%. The variant allele frequencies were usually higher in acute myeloid leukemia and increased upon leukemic transformation. ETNK1 mutation was accompanied by coexisting mutations in all patients, with ASXL1 (50%), TET2 (25%), EZH2 (24%), RUNX1 (24%), and SRSF2 (24%) mutations being the most common. Neoplasms with ETNK1 mutations were associated with morphologic dysplasia, increased blasts, myelofibrosis, and noncomplex karyotypes. With a median follow-up of 16.5 months, 30 patients died, 44 had persistent disease, and four achieved complete remission after stem cell transplantation. CONCLUSIONS: ETNK1 mutation is present in various myeloid neoplasms, often as an early event and a dominant clone and always with concurrent mutations. It may play an important role in the pathogenesis and progression of myeloid neoplasms by causing DNA damage and inducing other mutations and genomic instability, and it may serve as a potential therapeutic target. ETNK1 mutation is not disease-specific and should be interpreted with caution to classify myeloid neoplasms.

Carboxypeptidase E Regulates Activity-Dependent TrkB Neuronal Surface Insertion and Hippocampal Memory.

Activity-dependent insertion of the tropomyosin-related kinase B (TrkB) receptor into the plasma membrane can explain, in part, the preferential effect of brain-derived neurotrophic factor (BDNF) on active neurons and synapses; however, the underlying molecular mechanisms remain obscure. Here, we report a novel function for carboxypeptidase E (CPE) in controlling chemical long-term potentiation stimuli-induced TrkB surface delivery in hippocampal neurons. Total internal reflection fluorescence assays and line plot assays showed that CPE facilitates TrkB transport from dendritic shafts to the plasma membrane. The Box2 domain in the juxtamembrane region of TrkB and the C terminus of CPE are critical for the activity-dependent plasma membrane insertion of TrkB. Moreover, the transactivator of transcription TAT-CPE452-466, which could block the association between CPE and TrkB, significantly inhibited neuronal activity-enhanced BDNF signaling and dendritic spine morphologic plasticity in cultured hippocampal neurons. Microinfusion of TAT-CPE452-466 into the dorsal hippocampus of male C57BL/6 mice inhibited the endogenous interaction between TrkB and CPE and diminished fear-conditioning-induced TrkB phosphorylation, which might lead to an impairment in hippocampal memory acquisition and consolidation but not retrieval. These results suggest that CPE modulates activity-induced TrkB surface insertion and hippocampal-dependent memory and sheds light on our understanding of the role of CPE in TrkB-dependent synaptic plasticity and memory modulation.SIGNIFICANCE STATEMENT It is well known that BDNF acts preferentially on active neurons; however, the underlying molecular mechanism is not fully understood. In this study, we found that the cytoplasmic tail of CPE could interact with TrkB and facilitate the neuronal activity-dependent movement of TrkB vesicles to the plasma membrane. Blocking the association between CPE and TrkB decreased fear-conditioning-induced TrkB phosphorylation and led to hippocampal memory deficits. These findings provide novel insights into the role of CPE in TrkB intracellular trafficking as well as in mediating BDNF/TrkB function in synaptic plasticity and hippocampal memory.

Triplet pregnancy with hydatidiform mole: A report of two cases with literature review.

AIM: We present two cases of triplet pregnancy with complete hydatidiform mole (CHM) in contrasting outcomes and discuss the complications of mothers and outcomes of fetuses through a literature review, raising an important issue on the management of this special pregnancy. METHODS: We share our manage experience for two cases of triplet pregnancy with CHM and retrospectively analyze 18 similar pregnancies reported previously with different pregnancy outcomes. RESULTS: In our cases, one case receiving Clomiphene ovulation induction delivered two live fetuses by cesarean section at 30+ weeks without GTN (gestational trophoblastic neoplasia), unfortunately, the other case following ICSI-ET terminated the pregnancy in the setting of complications at 18+ weeks without GTN. No severe complications were detected during pregnancy and no pGTD was developed after delivery in neither of the pregnant. CONCLUSIONS: Co-existing complete hydatidiform mole in multiple pregnancies may become more common owing to the spreading use of ART. The decision for whether continue pregnancy depending on the personalized conditions including the complications of the pregnancy, the outcomes of the fetuses, the gestational age for delivery, and the potential progression of persistent gestational trophoblastic disease (pGTD). Furthermore, close monitor is necessary for the pregnant with triplet pregnancy with CHM who want to continue pregnancy.

Dynamics of the Bacterial Community's Soil During the In-Situ Degradation Process of Waste Chicken Feathers.

Bacteria play an important role in the biodegradation of feather waste. The exploration of the related microbial community structure and diversity is essential to improve the performance of feather waste treatment processes. In the present work, an in-situ soil sampled from a poultry farm was directly used to simulate and accelerate the natural degradation processes of feather waste under laboratory conditions, in which the dynamics of the microbial communities was further analyzed by Illumina HiSeq high-throughput 16S rRNA gene sequencing. Biochemical factors, including pH, feather degradation rate and soluble protein content were also determined in this study. The biochemical results showed that the in-situ soil exhibited an effective degradability on chicken feathers, and the degradation rate of feathers reached 57.95 ± 3.09% at 120 h of cultivation. Meanwhile, soluble protein content and pH reached 33.62 ± 1.45 mg/mL 8.99 ± 0.08, respectively. The results of bacterial diversity analysis showed that bacterial community structure and composition significantly varied in each phase of degradation. Additionally, the bacteria system with feather degradability might consist of Bacillus, Chryseobacterium, Lysobacter, Brevibacillus, and Stenotrophomonas genera. This system may include the following key pathways: carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, membrane transport, replication and repair, translation, signal transduction and energy metabolism. Moreover, the bacterial communities may occur community succession during the degradation processes of chicken feathers. In summary, the present work provided valuable insights into the understanding of microbial community and metabolic functions for feather degradation, although the in-situ biodegradation process was conducted under laboratory conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00996-6.

High-resolution X-ray structure of three microtubule-stabilizing agents in complex with tubulin provide a rationale for drug design.

Microtubule is a key component of cytoskeleton and has been considered as an important target for the treatment of cancer. In particular, the tubulin taxane-site inhibitors such as taxol analogs and epothilones have achieved great success in clinical trials. However, the structural basis of many taxane-site inhibitors is still lacking in exploring their mechanism of action. We here reported crystal complex structures for three taxane-site inhibitors, Ixabepilone, Epothilone B, and Epothilone D, which were determined to 2.4 Å, 2.4 Å, and 2.85 Å, respectively. The crystal structures revealed that these taxane-site inhibitors possess similar binding modes to that of Epothilone A at the taxane site, e.g. making critical hydrogen-bonding interactions with multiple residues on the M-loop, which facilitating the tubulin polymerization. Furthermore, we summarized the binding modes of almost all taxane-site inhibitors and identified novel taxane-site ligands with simpler chemical structures through virtual screening. On this basis, new derivatives with higher binding affinity to tubulin were designed and developed, which can form additional hydrogen bond interactions with tubulin. Overall, this work determined the mechanism of action of epothilones and provided a structural basis to design reasonably novel taxane-site inhibitors with simpler structure and improved pharmacokinetic properties.

Magnetic hydrogel with long in situ retention time for self-regulating temperature hyperthermia.

Aim: Magnetic hydrogels (MHGs) have been proposed to avoid the redistribution and loss of magnetic nanoparticles (MNPs) when administrated by intratumoral injection. However, the requirement of complex cooling systems and temperature monitoring systems still hinder the clinical application of MHGs. This study investigates the feasibility of developing an MHG to realize the self-regulation of hyperthermia temperature. Methods: The MHG was developed by dispersing the MNPs with self-regulating temperature property into the temperature-sensitive hydrogel through physical crosslinking. The MHG's gelation temperature was tested by measuring the storage modulus and loss modulus on a rotational rheometer. The biocompatibility of the MHG and MNPs was characterized by CCK-8 assay against HaCaT cells. The in vivo magnetic heating property was examined through monitoring the temperature in the MHG on mice back upon the application of the alternating magnetic field (400 ± 5 Oe, 100 ± 5 kHz) every week for successive six weeks. Results: The gelation temperature of the MHG falls in 28.4°C-37.4°C. At in vivo applied concentration of 80 mg/mL, the MHG exhibits over 80% cell viability after 72 h, significantly higher than 50% cell viability of the MNPs (p<0.001). The MHG's stable magnetic hyperthermia temperatures in vivo are in the range of 43.4°C-43.8°C. Conclusions: The developed MHG can be injected using a syringe and will solidify upon body temperature. The biocompatibility is improved after the MNPs being made into MHG. The MHG can self-regulate the temperature for six weeks, exhibiting application potential for self-regulating temperature hyperthermia.

Antifungal Action of Antifungalmycin N2 Against Rhizoctonia solani by Disrupting Cell Membrane and Inhibiting Succinate Dehydrogenase.

Antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) was a novel structural antifungal metabolite produced by Streptomyces sp. strain N2. Our previous study reported that the antagonistic interaction between antifungalmycin N2 and Rhizoctonia solani was accompanied by an oxidative stress in R. solani cell, indicating a probable damage occurred in the cell membranes and mitochondria. To verify this, the present study focused on investigating the effects of antifungalmycin N2 on the structure and function of cell membranes and mitochondria of R. solani. Morphological observations in transmission electron microscopy and fluorescence microscope showed that cell membranes of R. solani were damaged, and its cytoplasmic organelles were disorganized when treated with antifungalmycin N2. Meanwhile, the kinetics of membrane-related physiological and biochemical parameters, such as the increased malondialdehyde level, dropped ergosterol formation, and enhanced electrical conductivity in R. solani mycelia, further confirmed that antifungalmycin N2 would disrupt the cell membrane structure and function. More significantly, antifungalmycin N2 had a significantly inhibitory effect on the succinate dehydrogenase (SDH) activity of R. solani, and indicated that the mode and site of action of antifungalmycin N2 against R. solani might be similar to the existing succinate dehydrogenase inhibitors fungicides by binding in the ubiquinone-binding site. In conclusion, the above results demonstrated that the mode and site of action of antifungalmycin N2 targeted to cell membrane and SDH of R. solani, thus exerting the antifungal activity by damaging cell membrane structure and function, together with inhibiting the SDH activity.

Discovery of novel quinazolines as potential anti-tubulin agents occupying three zones of colchicine domain.

A series of novel quinazolines as tubulin inhibitors occupying three zones of colchicine domain have been designed and synthesized inspired by the recently disclosed crystal structure of verubulin analogue 6 with tubulin. Among the newly synthesized compounds, 19c showed noteworthy potency against K562, HepG2, KB, HCT-8 and MDB-MB-231 cancer cells. In vitro microtubule polymerization assays identified 19c as a potent tubulin assembly inhibitor, the binding mode of which with tubulin was confirmed by molecular modeling studies to occupy three zones of tubulin domain. Furthermore, 19c disrupted the intracellular microtubule network, caused G2/M phase arrest, induced cell apoptosis and depolarized mitochondria of K562 cells. 19c also reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 19c significantly and dose dependently inhibited tumor growth in H22 liver cancer xenograft mouse model. All these results suggested that 19c deserves further research as a novel and potential anti-tubulin agent for the treatment of cancers.

Synthesis, molecular properties prediction and biological evaluation of indole-vinyl sulfone derivatives as novel tubulin polymerization inhibitors targeting the colchicine binding site.

Twenty-two novel indole-vinyl sulfone derivatives were designed, synthesized and evaluated as tubulin polymerization inhibitors. The physicochemical and drug-likeness properties of all target compounds were predicted by Osiris calculations. All compounds were evaluated for their antiproliferative activities, among them, compound 7f exhibited the most potent activity against a panel of cancer cell lines, which was 2-7 folds more potent than our previously reported compound 4. Especially, 7f displayed about 8-fold improvement of selective index as compared with compound 4, indicating that 7f might have lower toxicity. Besides, 7f inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Further investigations showed that compound 7f effectively disrupted microtubule network, caused cell cycle arrest at G2/M phase and induced cell apoptosis in K562 cells. Moreover, 7f reduced the cell migration and disrupted capillary-like tube formation in HUVEC cells. Importantly, the in vivo anti-tumor activity of 7f was validated in H22 liver cancer xenograft mouse model without apparent toxicity, suggesting that 7f is a promising anti-tubulin agent for cancer therapy.

Antagonistic activity and mechanism of an isolated Streptomyces corchorusii stain AUH-1 against phytopathogenic fungi.

The various diseases that occur during the growth of plants usually cause a significant reduction in production and quality of agricultural products. Actinomycetes, especially Streptomyces spp., become a valuable biological control resource due to their preponderant abilities to produce various secondary metabolites with novel structure and remarkable biological activity. The present work aimed to isolate an effective antagonistic actinomycete against various soilborne phytopathogenic fungi. By dual culture with Fusarium oxysporum f. sp. niveum, an antagonistic actinomycete named Streptomyces corchorusii stain AUH-1 was screened out from 26 soil samples. The in vitro bioassay results showed that S. corchorusii stain AUH-1 had a broad-spectrum antagonistic activity against a range of fungal plant pathogens, such as F. oxysporum f. sp. niveum, Phytophthora parasitica var. nicotianae, Rhizoctonia solani, P. capsica, Botryosphaeria dothidea, F. oxysporum f. sp. vasinfectum, Verticillium dahliae, and F. oxysporum f. sp. cucumerinum. According to the morphological observations in scanning electron microscopy (SEM) and fluorescence microscope (FM), it was found that the cell membranes of F. oxysporum f. sp. niveum were damaged when treated with the antifungal metabolite form S. corchorusii stain AUH-1. Meanwhile, the dropped ergosterol formation and increased malondialdehyde levels further confirmed that S. corchorusii strain AUH-1 exerted its antagonistic activity against F. oxysporum f. sp. niveum via damaging the structure and function of cell membranes. In conclusion, S. corchorusii strain AUH-1 showed a promising prospect for the development of biological agent, especially due to its broad-spectrum and effective antagonist on various soil-borne plant pathogens.

ARHGEF19 interacts with BRAF to activate MAPK signaling during the tumorigenesis of non-small cell lung cancer.

Rho guanine nucleotide exchange factors (RhoGEFs) are proteins that activate Rho GTPases in response to extracellular stimuli and regulate various biologic processes. ARHGEF19, one of RhoGEFs, was reported to activate RhoA in the Wnt-PCP pathway controlling convergent extension in Xenopus gastrulation. The goal of our study was to identify the role and molecular mechanisms of ARHGEF19 in the tumorigenesis of non-small cell lung cancer (NSCLC). ARHGEF19 expression was significantly elevated in NSCLC tissues, and ARHGEF19 levels were significantly associated with lymph node status, distant metastasis and TNM stage; Patients with high ARHGEF19 levels had poor overall survival (OS) and progression-free survival (PFS). Our investigations revealed that ARHGEF19 overexpression promoted the cell proliferation, invasion and metastasis of lung cancer cells, whereas knockdown of this gene inhibited these processes. Mechanistically, ARHGEF19 activated the mitogen-activated protein kinase (MAPK) pathway in a RhoA-independent manner: ARHGEF19 interacted with BRAF and facilitated the phosphorylation of its downstream kinase MEK1/2; both the Dbl homology (DH) and Pleckstrin homology (PH) domains of ARHGEF19 were indispensable for the phosphorylation of MEK1/2. Furthermore, downregulation of miR-29b was likely responsible for the increased expression of ARHGEF19 in lung cancer tissues and, consequently, the abnormal activation of MAPK signaling. These findings suggest that ARHGEF19 upregulation, due to the low expression of miR-29 in NSCLC tissues, may play a crucial role in NSCLC tumorigenesis by activating MAPK signaling. ARHGEF19 could serve as a negative prognostic marker as well as a therapeutic target for NSCLC patients.

Protein tyrosine phosphatases: emerging role in cancer therapy resistance.

BACKGROUND: Tyrosine phosphorylation of intracellular proteins is a post-translational modification that plays a regulatory role in signal transduction during cellular events. Dephosphorylation of signal transduction proteins caused by protein tyrosine phosphatases (PTPs) contributed their role as a convergent node to mediate cross-talk between signaling pathways. In the context of cancer, PTP-mediated pathways have been identified as signaling hubs that enabled cancer cells to mitigate stress induced by clinical therapy. This is achieved by the promotion of constitutive activation of growth-stimulatory signaling pathways or modulation of the immune-suppressive tumor microenvironment. Preclinical evidences suggested that anticancer drugs will release their greatest therapeutic potency when combined with PTP inhibitors, reversing drug resistance that was responsible for clinical failures during cancer therapy. AREAS COVERED: This review aimed to elaborate recent insights that supported the involvement of PTP-mediated pathways in the development of resistance to targeted therapy and immune-checkpoint therapy. EXPERT OPINION: This review proposed the notion of PTP inhibition in anticancer combination therapy as a potential strategy in clinic to achieve long-term tumor regression. Ongoing clinical trials are currently underway to assess the safety and efficacy of combination therapy in advanced-stage tumors.

Small molecule inhibitors targeting m6A regulators.

As the most common form of epigenetic regulation by RNA, N6 methyladenosine (m6A) modification is closely involved in physiological processes, such as growth and development, stem cell renewal and differentiation, and DNA damage response. Meanwhile, its aberrant expression in cancer tissues promotes the development of malignant tumors, as well as plays important roles in proliferation, metastasis, drug resistance, immunity and prognosis. This close association between m6A and cancers has garnered substantial attention in recent years. An increasing number of small molecules have emerged as potential agents to target m6A regulators for cancer treatment. These molecules target the epigenetic level, enabling precise intervention in RNA modifications and efficiently disrupting the survival mechanisms of tumor cells, thus paving the way for novel approaches in cancer treatment. However, there is currently a lack of a comprehensive review on small molecules targeting m6A regulators for anti-tumor. Here, we have comprehensively summarized the classification and functions of m6A regulators, elucidating their interactions with the proliferation, metastasis, drug resistance, and immune responses in common cancers. Furthermore, we have provided a comprehensive overview on the development, mode of action, pharmacology and structure-activity relationships of small molecules targeting m6A regulators. Our aim is to offer insights for subsequent drug design and optimization, while also providing an outlook on future prospects for small molecule development targeting m6A.

Targeted regulated cell death with small molecule compounds in colorectal cancer: Current perspectives of targeted therapy and molecular mechanisms.

Colorectal cancer (CRC), a tumor of the digestive system, is characterized by high malignancy and poor prognosis. Currently, targeted therapy of CRC is far away from satisfying. The molecular mechanisms of regulated cell death (RCD) have been clearly elucidated, which can be intervened by drug or genetic modification. Numerous studies have provided substantial evidence linking these mechanisms to the progression and treatment of CRC. The RCD includes apoptosis, autophagy-dependent cell death (ADCD), ferroptosis, necroptosis, and pyroptosis, and immunogenic cell death, etc, which provide potential targets for anti-cancer treatment. For the last several years, small-molecule compounds targeting RCD have been a well concerned therapeutic strategy for CRC. This present review aims to describe the function of small-molecule compounds in the targeted therapy of CRC via targeting apoptosis, ADCD, ferroptosis, necroptosis, immunogenic dell death and pyroptosis, and their mechanisms. In addition, we prospect the application of newly discovered cuproptosis and disulfidptosis in CRC. Our review may provide references for the targeted therapy of CRC using small-molecule compounds targeting RCD, including the potential targets and candidate compounds.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

Design, Synthesis, and Biological Evaluation of Dual Inhibitors of EGFRL858R/T790M/ACK1 to Overcome Osimertinib Resistance in Nonsmall Cell Lung Cancers.

Activation of the alternative pathways and abnormal signaling transduction are frequently observed in third-generation EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors)-resistant patients. Wherein, hyperphosphorylation of ACK1 contributes to EGFR-TKIs acquired resistance. Dual inhibition of EGFRL858R/T790M and ACK1 might improve therapeutic efficacy and overcome resistance in lung cancers treatment. Here, we identified a EGFRL858R/T790M/ACK1 dual-targeting compound 21a with aminoquinazoline scaffold, which showed excellent inhibitory activities against EGFRL858R/T790M (IC50 = 23 nM) and ACK1 (IC50 = 263 nM). The cocrystal and docking analysis showed that 21a occupied the ATP binding pockets of EGFRL858R/T790M and ACK1. Moreover, 21a showed potent antiproliferative activities against the H1975 cells, MCF-7 cells and osimertinib-resistant cells AZDR. Further, 21a showed significant antitumor effects and good safety in ADZR xenograft-bearing mice. Taken together, 21a was a potent dual inhibitor of EGFRL858R/T790M/ACK1, which is deserved as a potential lead for overcoming acquired resistance to osimertinib during the EGFR-targeted therapy.

Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.