Furthermore, ASF1B deficiency induced apoptosis and cell cycle arrest. Mechanistically, ASF1B knockdown reduced the expression of proliferating cell nuclear antigen (PCNA), cyclinB1, cyclinE2 and CDK9.Moreover, ASF1B interacted with CDK9 in HCC cells. Taken together, these results suggest that the oncogenic gene ASF1B could be a target for inhibiting hepatocellular carcinoma cell growth.Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high morbidity and mortality. Therefore, it is very important to find potential biomarkers that can effectively predict the prognosis and progression of HCC. Recent studies have shown that anti-silencing function 1B (ASF1B) may be a new proliferative marker for tumor diagnosis and prognosis. However, the expression and function of ASF1B in hepatocellular carcinoma remain to be determined. In this study, integrated analysis of the Cancer Genome Atlas (TCGA), genotypic tissue expression (GTEx), and Gene Expression Omnibus (GEO) databases revealed that ASF1B was highly expressed in HCC. Kaplan-Meier survival curve showed that elevated ASF1B expression was associated with poor survival in patients with liver cancer. Correlation analysis of immune infiltration suggested that ASF1B expression was significantly correlated with immune cell infiltration in HCC patients. Gene set enrichment analysis (GSEA) indicated that ASF1B regulated the cell cycle, DNA Replication and oocyte meiosis signaling. Our experiments confirmed that ASF1B was highly expressed in HCC tissues and HCC cell lines. Silence of ASF1B inhibited hepatocellular carcinoma cell growth  In global cancer statistics, the incidence of liver cancer ranks sixth and the mortality rate ranks third. In 2020, according to statistics, there are 905,677 new cases of liver cancer worldwide. It is estimated that by 2025, the incidence of liver cancer will exceed 1 million cases, which is a serious threat to human life and health . HepatocAnti-silence function 1 (ASF1), a chaperone protein of histone H3-H4, plays an important role in DNA replication, DNA damage repair and transcriptional regulation , 10. In in vitro. Our data implied that ASF1B was closely associated with proliferation and migration in HCC cells and might be a novel prognostic indicator and therapeutic target in HCC patients.In this study, we investigated the expression and prognosis of ASF1B in patients with HCC in The Cancer Genome Atlas (TCGA) and various public databases. Moreover, we analyzed the coexpression genes of ASF1B to explore the potential mechanisms of ASF1B in HCC. Experiments were performed to verify the expression of ASF1B in HCC tissues and cell lines. ASF1B knockdown in HCC cell lines was induced to detect the biological behavior changes https://portal.gdc.cancer.gov/), with the GTEx data  similarly being downloaded. GSE121248 and GSE33006 datasets was downloaded from Gene expression omnibus (GEO). Oncomine 4.5 database (https://www.oncomine.org/) and HCCDB database were also used in ASF1B expression analysis in pan-cancer and HCC.We downloaded RNA-seq gene expression data and clinical records from the TCGA database (http://kmplot.com) was used to generate survival curves, including overall survival (OS), progression-free survival (PFS), recurrence-free survival (RFS), and disease-specific survival (DSS), based on gene expression with the log-rank test and the Mantel-Cox test in liver cancer. TimeROC analysis  on HCC prognosis, univariate and multivariate Cox regression analysis and forest map were used to display the p value, HR and 95% CI of each variable through “forestplot” R package. The Kaplan-Meier (KM) survival curve analysis is implemented by R software package “Survival” and “Survminer”. Kaplan-Meier Plotter  was used to analyze the relationship between ASF1B transcriptional level and immune cell infiltration in patients with HCC. Differentially expressed genes associated with transcription of the ASF1B gene in HCC were analyzed based on the LinkedOmics (http://www.linkedomics.org/login.php) functional module.Tumor Immune Estimation Resource (TIMER) , and all patients signed a formal informed consent. Six pairs of HCC and paracancerous tissue samples were obtained from patients who underwent hepatocellular carcinoma resection in the First Affiliated Hospital of Zhejiang University School of Medicine during 2017-2019, and none of them received preoperative radiotherapy or preoperative chemotherapy. All specimens were anonymized in accordance with ethical and legal standards. The MHCC97H cell line was purchased from Guangzhou Cellcook Biology Co., Ltd. , while other cell lines, LO2, Hep3B, HepG2, and Huh7, were purchased from ATCC .Total protein was obtained with RIPA lysis buffer containing protease inhibitor  , and the protein was quantified with a BCA kit . The protein was quantified and then used to perform western blot as previously described . AntibodTo ensure the inhibition efficiency of ASF1B expression by siRNA, three different ASF1B small interfering RNA were designed.siASF1B-1 . siASF1B-2 . siASF1B-3  were obtained from Genomeditech Co. Ltd. .And 50 nmol/L siRNA was transfected into MHCC97H or Hep3B cells using INTERFERin  according to the manufacturer’s protocol.Total RNA was isolated using RNA fast 2000 kit . Reverse transcription of RNA to cDNA was obtained using PrimeScript™ RT Master Mix . qPCR was performed with QuantStudio 5 Detection System  in a 10 μl reaction mixture containing SYBR GreenII. Expression of different genes were normalized to GAPDH and were analyzed using the 2−ΔΔCT method.Cell viability was analyzed using cell counting kit-8  according to the manufacturer’s protocol. Briefly, MHCC97H and Hep3B were seeded in the 96-well plates with 5000 cells/well and incubated for overnight. At 24 h, 48 h, 72 h, and 120h, 10 µl CCK-8 solution was added to each well, and the cells were incubated for 90 min at 37°C. The absorbance at 450 nm was obtained using an IMARK microplate reader (BIO-RAD).5 cells were seeded into the upper chamber with 200µl serum-free DMEM. 500µl DMEM with 10% FBS was added to the lower chamber. The cells were cultured at 37°C in a 5% CO2 hydrosphere atmosphere. After 24 hours, the upper chamber was fixed with paraformaldehyde and stained with 0.5% crystal violet. Non-invading cells were removed, and the cells on the lower surface were counted microscopically.For cell invasion assay, Matrigel was diluted 1:8 with cold serum-free DMEM at 4°C and 50µl was carefully used to coat polycarbonate filters . Incubate at 37°C overnight. Next, 5 × 106 cells were washed with cold PBS. Cell pellets were suspended with 500µl PI working solution , and incubated for 30min at 4°C of light protection. Then the cells were dispersed by vortex oscillation and incubated at 37°C of darkness for 30min. After vortex blending, the cells were filtered by nylon screen for flow cytometry analysis. Cell apoptosis analysis was performed with a FITC Annexin V Apoptosis Detection Kit  according to the manufacturer’s protocol. In brief, after washing with cold PBS twice, the cells were resuspended in binding buffer. A total of 1 × 105 cells (100 µl) were transferred to the flow tube, and 5 µl of FITC Annexin V and 5 µl PI were added. After gentle vortexing and incubation for 15 min at room temperature in the dark, another 400 µl binding buffer was added and then analyzed by flow cytometry.To perform cell cycle assays, 1×10For co-immunoprecipitation, we used an IP/Co-IP kit from Absin . Co-IP was conducted according to the manufacturer’s protocol. Briefly, cells were lysed and incubated on ice, then centrifuged at 14000g for 10 minutes at 4°C. The supernatant was incubated with protein A/G agarose beads for pre-clean. Subsequently, it was immunoprecipitated with an antibody against ASF1B  or normal rabbit IgG  overnight at 4°C. Next day, the immunoprecipitated complexes were incubated with protein A/G agarose beads for 1 hour at 4°C. After incubation, the immunoprecipitated complexes were rinsed and analyzed by Western blotting. Input was used as positive control.p < 0.05 was the threshold of significance.Data were analyzed using R v 4.0.3. Wilcoxon tests were used to compare ASF1B expression levels in normal and tumor tissues, while Kruskal-Wallis tests were used to evaluate relationships between ASF1B expression and patient clinical stage. Kaplan-Meier curves were used to assess survival outcomes, and correlations were evaluated with Spearman’s correlation coefficients. A two-sided We used Oncomine to explore the expression levels of ASF1B in normal and various cancer tissues. The results showed that ASF1B was highly expressed in most tumors, and only a few tumors showed low expression , pT stage , pTNM stage  were significantly correlated with OS in HCC. ASF1B expression was also significant in multivariate cox regression analysis , suggesting that ASF1B may be an independent prognostic factor for HCC  and OS in HCC patients. Univariate Cox analysis showed that ASF1B expression  was estimated by purity-corrected partial Spearman’s rho. We found a slightly positive correlation between ASF1B expression and tumor purity . In addition, ASF1B expression was positively correlated with the abundance of all six immune infiltrates Table 2.To understand the biological significance of ASF1B in HCC, we used Linkedomics to analyze the co-expression network of ASF1B in the LIHC cohort. As shown in To investigate the role of ASF1B in HCC tumor progression, HCC tissue and corresponding adjacent tissue samples from 6 patients were tested by immunohistochemistry. As shown in According to the expression of ASF1B in HCC cells, we selected MHCC97H and Hep3B with relatively high expression of ASF1B for functional verification. First, we designed three types of siRNA  based on the ASF1B gene sequence. siRNAs were transfected into MHCC97H and Hep3B, and sicontrol was transfected into control group. The inhibition efficiency of the three siASF1B inhibitors on ASF1B in cells was detected by RT-PCR. The results showed that siASFB-2 had the best inhibition efficiency .Similarly, in Hep3B, the proportion of cells in FITC positive region of ASF1B knockout group was 18.67% higher than that of control group 17.08%. The results showed that more cells were apoptotic in ASF1B-siRNA HCC cells than in scrambled cells  to investigate the role of ASF1B in the biological function of HCC cells. The results confirmed that knocking down ASF1B impaired proliferation, induced cell apoptosis and altered cell cycle progression in HCC cells. These results suggest that ASF1B is crucial for maintaining tumorigenic activity of HCC cells lication . CyclinBlication . It has lication . CyclinElication . Unlike lication . Accordilication . We demolication , breast lication , acute mlication , hepatoclication . Shao etlication . CDK9 walication . These rIn conclusion, this study provided multi-level evidence for the significance of ASF1B in HCC development and its potential as a biomarker for HCC disease progression. We figured out the important role of ASF1B as a regulator in cell proliferation, apoptosis induction and cell cycle progression. Interference with ASF1B can significantly inhibit the growth of HCC by regulating cell cycle and apoptosis pathways. These findings proposed a potential target for the development of anti-cancer strategies in HCC.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Clinical Research Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. The patients/participants provided their written informed consent to participate in this study.LJL and DZ contributed to conception and design of the study. XO completed the experiment and analyzed the data. XO and LXL drafted the manuscript and prepared diagrams. YZ, FZ, QH, and ZL participated in the material preparation and manuscript review. All authors have read and approved the manuscript for publication.This study was funded by the National Key Research and Development Program of China  and by the Zhejiang Provincial Natural Science Foundation of China (LY17H030005).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor JC declared a shared parent affiliation with the authors at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The chemokine ligand CCL2 and its receptor CCR2 are implicated in the initiation and progression of various cancers. CCL2 can activate tumour cell growth and proliferation through a variety of mechanisms. By interacting with CCR2, CCL2 promotes cancer cell migration and recruits immunosuppressive cells to the tumour microenvironment, favouring cancer development. Over the last several decades, a series of studies have been conducted to explore the CCL2‐CCR2 signalling axis function in malignancies. Therapeutic strategies targeting the CCL2‐ CCR2 axis have also shown promising effects, enriching our approaches for fighting against cancer. In this review, we summarize the role of the CCL2‐CCR2 signalling axis in tumorigenesis and highlight recent studies on CCL2‐CCR2 targeted therapy, focusing on preclinical studies and clinical trials. The chemokine ligand CCL2 and its receptor CCR2 are implicated in the initiation and progression of various cancers. The CCL2‐CCR2 signalling axis plays a critical role in the promotion of pathological angiogenesis, the survival and invasion of tumour cells, and the recruitment of immune inhibitory cells. Therefore, CCL2 and CCR2 enable us to explore the sophisticated mechanisms underlying cancer development and provide potential options for treating malignant tumours. Structurally, the N‐terminal tail at the end of CCL2 is a significant determinant of CCR2 binding affinity and efficacy.CCR2 belongs to the GPCRs and contains an N‐terminal extracellular domain and seven conserved transmembrane domains. Based on the difference in terminal carboxyl tails, CCR2 is divided into CCR2A and CCR2B Figure , and theAs the mainly functional receptor of CCL2, CCR2 is widely expressed by different types of cells, including dendritic cells (DCs),3It has been revealed that the CCL2‐CCR2 signalling axis participated in the occurrence and progression of a wide range of malignancies, such as breast cancer,Tumorigenesis is a dynamic and complicated process that consists of three stages including initiation, progression and metastasis.3.1Primary tumour growth is a multi‐step and complicated process involving various growth factors and signalling pathways regulation. Previous studies have shown that both stromal‐ and tumour‐derived CCL2 could directly stimulate tumour cell growth and proliferation.Different signalling pathways activated by the CCL2‐CCR2 axis are involved in tumorigenesis Figure . It was +CCR2+ monocytes in tumour sites, thereby accelerating tumour development.One of the major issues with cancer eradication is the acquisition of resistance to both chemotherapies and radiotherapies in due course treatment. CCL2 can decrease drug‐induced cytotoxicity to support tumour cell growth by inhibiting proapoptotic autophagy and is regarded as an intervention target for chemotherapy resistance.CCL2 can also recruit various immune cells  to form an immunosuppressive microenvironment, which allows tumour cell to evade the body's immune surveillance and supports tumour cell proliferation.3.2Tumour angiogenesis is a critical process in tumour progression and consists of tightly regulated events that include basement membrane degradation, endothelial cell migration and proliferation, and capillary tube formation. Angiogenesis also involves various stimulatory factors such as vascular endothelial growth factor (VEGF).3 integrin. Furthermore, CCL2‐mediated Est‐1 activation was associated with the ERK1/2 cascade, and the inhibition of ERK1/2 with PD98059 obviously decreased Ets‐1 phosphorylation and then inhibited angiogenesis.CCL2 could bind to CCR2 expressed on vascular endothelial cells and directly stimulated angiogenesis in the absence of inflammatory infiltration. Treatment with a CCL2 neutralizing antibody showed obvious inhibition of the CCL2‐modulated angiogenic effect and reduced the metastatic lesions.On the other hand, CCL2 induced angiogenesis through indirect ways that were correlated with the recruitment of inflammatory cells.+ vascular endothelial cells and inflammatory cells and by stimulating the expression of angiogenic factors. Based on the finding that CCL2 inhibition limits tumour angiogenesis,In brief, CCL2 promotes tumour angiogenesis by recruiting CCR23.3Tumour metastasis is an integrated multi‐step process by which cancer cells disperse from the original tumour sites through the blood or lymphatic system to the surrounding tissues or distant organs, and remains the leading cause of death among cancer patients.3.3.1At the initial stage of metastasis, tumour cells acquire an invasive phenotype that can break down the extracellular matrix (ECM) to facilitate metastasis.Matrix metalloproteinases (MMPs) are a multigene family of metal‐dependent endopeptidases that play important roles in EMT and contribute to tumour metastasis.3.3.2−/− mice. Similarly, STAT5 and p38MAPK could not be activated in CCR2−/− tumour tissues after tumour cell injection, indicating that CCL2‐induced tumour cell migration had a close relationship with JAK2‐STAT5 and p38MAPK pathway activation.When tumour cells enter the lymphatic system or bloodstream, they become circulating tumour cells (CTCs) and disseminate along chemotactic gradients. In the new sites, tumour cells exude and continue to colonize. This step is associated with the interactions between the host factors in the target organs and the spreading tumour cells. In tumour cell extravasation, scientists found a population of cells derived from a subset of inflammatory monocyte precursors, known as metastasis‐associated macrophages (MAMs).3.3.3Stephen Paget proposed the ‘seed and soil’ hypothesis, which suggested that the spread of tumour cells was controlled by the interaction between the cancer cells (seed) and the host organ (soil).After malignant cell exudation from the circulatory system, their growth in secondary sites is also critical for cancer metastasis. Dating back to the 1980s, the English surgeon The PMN is considered as fertile soil for the survival and growth of metastatic tumour cell and represents an aberrant, tumour growth‐favouring microenvironment.In both the early and late stages of metastasis, the CCL2‐CCR2 signalling axis also recruits diverse immune cells to form an immunosuppressive TME that allows tumour cells to evade the body's immune surveillance, which will be discussed in the next section. Altogether, CCL2 and its receptor CCR2 are upregulated in tumours and engaged in tumour metastasis through the induction of EMT, the promotion of tumour cell extravasation and the establishment of a PMN.4In the past few decades, cancer researches mainly focused on malignant cells and related gene regulation. More recently, the concept of the tumour microenvironment (TME) is becoming widely accepted. The dynamic interplay between the tumour and its microenvironment has a significant influence on carcinogenesis. Other than tumour cells, the TME is composed of a great variety of nonmalignant cells such as fibroblasts, mesenchymal stem cells, haemolymph endothelial cells and numerous infiltrating leukocytes.4.1low IL‐23low IL‐10high TGF‐βhigh, whereas M1 macrophage is characterized by IL‐12high IL‐23high IL‐10low.As one of the leucocytes in the innate immune system, macrophages exist in nearly all organs and tissues. In the events of tissue injury, infection and cancer, monocytes are recruited to the related sites by chemotactic signals and further differentiate into macrophages to participate in the body's immune response.The CCL2‐CCR2 axis promotes monocytes' recruitment from the peripheral blood to the tumour sites, where these monocytes further differentiate into TAMs.4.2Neutrophils are derived from a common committed myeloid progenitor cell and are considered as effectors of the innate immune response. Neutrophils coordinate multiple immune processes including chronic inflammatory, autoimmunity and cancer.4.3Myeloid‐derived suppressor cells (MDSCs) were initially described in the 1980s and accumulated in the TME.+ MDSCs were recruited to the glioma microenvironment in response to CCL2, whereas MDSCs lacking CCR2 failed to maximally accumulate in the TME.+ T cell. Meanwhile, the CD8+T cell/MDSC ratio was increased within tumours derived from CCL2 deletion mice, and elevated levels of CD8+ T cells restored anti‐tumour immunity to inhibit tumour development.Previous studies showed that the CCL2‐CCR2 axis also played a key role in the recruitment of MDSCs into the TME, which protected tumour cells from immune‐mediated killing.4.4+ cytotoxic lymphocytes (CTL) that recognize peptides presented by MHC I and CD4+ helper (Th) cells that recognize peptides presented by MHC II.+ T cell, among which Th1 and Th2 cells are most common and important. Th1‐derived cytokines induce pro‐inflammatory responses and exhibit anti‐tumour effects, whereas Th2‐secreted cytokines produce anti‐inflammatory effects and support tumour progression.T cells are leukocytes that express T‐cell receptors (TCRs). T cells are divided into two types: CD8The secretion and expression of CCL2 is associated with Th2 polarization.+ T cell, and can foster tumour metastasis and progression by coordinating immunosuppressive effects.+ Treg.+ Treg represented high immunosuppressive activity and played a central role in suppressing anticancer responses. Although CCR2+ Treg accumulated in various cancers, it could be depleted by low‐dose cyclophosphamide, which allowed the definition of a novel target for improving chemotherapic efficacy.In nearly all cancers, the number of regulatory T cell (Treg) is typically increased. Treg is a type of CD44.5Cancer‐associated fibroblasts (CAFs) are one of the most abundant stromal components in the TME. Although their origin remains unclear, it is generally believed that CAFs might result from the activation of local tissue‐resident fibroblasts.Collectively, the above results suggest that the CCL2‐CCR2 axis is upregulated during cancer progression and has a close relationship with cancer initiation, progression and metastasis. Blockade of the CCL2‐CCR2 axis is a potential therapeutic option for cancer treatment.5The CCL2‐CCR2 signalling axis has a multifaceted involvement in a wide range of cancers, and accumulated experimental evidence is paving the way for future clinical research. Furthermore, blockade of the CCL2‐CCR2 axis has attracted extensive attention and represents promising anti‐tumour activity in both preclinical studies and clinical trials. Here, we mainly report the therapeutic strategies targeting the CCL2‐CCR2 axis.5.15.1.1A number of preclinical studies have used various CCL2 inhibitors or antibodies to delay tumour progression. Blockade of CCL2 achieves tumour suppression through diverse ways such as blocking CCL2‐mediated signalling pathways, inhibiting the immunosuppressive cell recruitment and increasing the number of tumour‐killing cells.+CD45+ TAMs and CD11b+Gr1+ MDSCs, inhibiting tumour proliferation and prolonging the survival of glioma‐bearing mice.+ T‐cell numbers and reduced tumour volume, suggesting that combining CCL2 antibody with the vaccine could be a promising tactic for treating cancer.C1142 is a rat/mouse chimeric monoclonal antibody that can specifically neutralize CCL2. This antibody has yielded encouraging results in different cancer models as a single agent or combination with standard chemotherapies.Bindarit is an anti‐inflammatory indazole derivative that can inhibit the synthesis of CCL2, with a potential inhibitory function in tumour development. Targeting CCL2 with bindarit induces tumour regression, which has been confirmed in some preclinical studies. Bindarit is able to impair the organization of endothelial cells in the formation of blood vessels to inhibit tumour angiogenesis.A CCL2 inhibitor, mNOX‐36 suppressed TAM recruitment in a rat glioblastoma multiforme model. Interestingly, the combination therapy of mNOX‐36 and bevacizumab reduced both the tumour size and blood volume.Curcuma longa) root. In a mouse model of colon cancer, plasma CCL2 was reduced after curcumin feedings.The expression and activity of CCL2 can be inhibited by curcumin, an active polyphenolic compound of the powdered turmeric  in tandem with N‐terminal truncated monocyte chemotactic protein‐1 (MCP1 6‐76).Teijin Compound 1(TC1) is a specific CCR2 antagonist and is developed to block the binding of human CCL2 to CCR2b.BMS CCR2 22 is a potent and high affinity CCR2 antagonist. In a metastatic liver tumour model (MC38), BMS CCR2 22 treatment decreased the infiltration of TAMs into tumour sites effectively and increased the efficacy of FOLFOX chemotherapy, thereby improving the survival time of tumour‐bearing mice.Abies georgei, named 747. 747 exhibited sensitivity and selectivity as a CCR2 antagonist and could block the CCL2‐CCR2 axis. 747 showed anti‐tumour activity by inhibiting TAM recruitment and elevating CD8+ T‐cell numbers. In addition, blockade of TAMs enhanced the anti‐tumour efficacy of sorafenib. The discovery of 747 provided a novel perspective on the development of CCR2 antagonists and enriched the therapeutic options against cancer.Recently, Yao et alCCX872, a small molecule antagonist of CCR2, can impede MDSC invasion into tumour sites.5.1.3+ TANs influx, which possibly limited the intended treatment efficacy.+ TAMs and CXCR2+ TANs restored anti‐tumour immunity and improved the chemotherapeutic effect, providing the theoretical basis for targeting both CCR2 and CXCR2 in future clinical trials.The pathogenesis is complicated in cancer. Blockade of the CCL2‐CCR2 axis alone may cause unexpected side effects. Therefore, combining inhibition therapy of CCL2‐CCR2 axis with current treatment tactics for different cancers is necessary. In pancreatic adenocarcinoma, TANs and TAMs are recruited to the TME, mediating immune escape and favouring tumour growth. In patients with pancreatic adenocarcinoma, blockade of CCR2 prevented the accumulation of TAM in tumour sites but led to a compensatory CXCR2+ DC maturation and suppressing CTL activity. Besides, CCR2−/− cancer cells had lower surface levels of PD‐L1 compared with CCR2+/+ cancer cells.The immune system is involved in almost every stage of cancer initiation and progression. Programmed cell death protein 1 (PD‐1) is an immune inhibitory receptor and is particularly expressed on cytotoxic T cells. PD‐1 plays a vital role in promoting self‐tolerance and inhibiting immune responses by binding to its ligand PD‐L1 or PD‐L2.5.2The efficacy of CCL2‐CCR2 axis inhibition has been widely investigated in numerous preclinical experiments. CCL2 and CCR2 can function as diagnostic biomarkers,5.2.1Targeting CCL2 is based on the assumption that CCL2 neutralizing antibodies can bind to CCL2 and block the recruitment and differentiation of macrophages. Carlumab (CNTO 888), a human anti‐CCL2 neutralizing antibody, can bind to human CCL2 and exhibits broad spectrum of preclinical anticancer activity.Collectively, although no significant adverse reaction was found in these patients, the therapeutic effects of carlumab were not satisfactory. In the future, how to permanently suppress CCL2 levels may be the focus of research.5.2.2Antibodies and inhibitors are used to interfere with CCR2 binding to its cognate ligand CCL2 and to block the activation of latent CCR2. Both steps are critical for CCR2‐induced pro‐tumorigenic effects and immune escape. CCR2‐associated targeted therapy has made progress in recent years.+ monocytes in patients was significantly decreased compared with that in the FOLFIRINOX alone group (1.06 vs. 6.46), which indicated that FOLFIRINOX plus PF‐04136309 could prevent CCR2+ monocyte egress from the bone marrow to the peripheral blood and might affect the anti‐tumour immunity.PF‐04136309 is a CCR2 inhibitor that exhibited anti‐tumour activity in an orthotopic model of murine pancreatic cancer.Another phase I study (NCT02732938) was initiated to evaluate the effect of administering PF‐04136309 combined with gemcitabine and nab‐paclitaxel in patients with metastatic pancreatic cancer. All patients (21) received PF04136309 at a starting dose of 500 mg or 750 mg twice daily (BID). The final result showed that 14 patients (66.7%) experienced treatment‐related serious adverse events (SAEs), which might attribute to at least one of the three exposure drugs . By the cut‐off date, the objective response rate (ORR) was 23.8% (95% CI = 8.2%‐47.2%) in all 21 patients who received the treatment, but the overall survival (OS) was not assessed in this trial. In the 500 mg BID group (n = 17), the median progression‐free survival (mPFS) was 5.3 months and the ORR was 29.4% (95% CI = 10.3%‐56.0%).MLN1202 is an anti‐CCR2 monoclonal antibody. In a phase II clinical trial, researchers assessed the efficacy of MLN1202 in forty‐four patients with bone metastases (NCT01015560). Among forty‐three eligible patients, forty‐one patients completed the study, and only 7.14% had severe adverse reactions. To some extent, MLN1202 was well tolerated in patients. After forty‐three days of MLN1202 treatment, the urinary N‐telopeptide value (a primary assessment indicator) was decreased in 14 patients, indicating some positive effects of MLN1202 for cancer treatment.Propagermanium (3‐oxygermylpropionic acid polymer) is an organogermanium compound that blocks CCR2 signalling.CCX872‐B is a specific CCR2 antagonist. A phase Ib trial was designed to investigate the safety and efficacy of CCX872‐B in patients with pancreatic adenocarcinoma (NCT02345408). The enrolled patients (50) received FOLFIRINOX  plus 150 mg CCX872 QD or BID for 12 weeks. From the perspective of survival improvement, all‐subjects overall survival (OS) at 18 months was 29%,BMS‐813160 is a dual CCR2/CCR5 antagonist. Several clinical trials are currently evaluating the combination therapy of BMS‐813160 and chemotherapies or immunotherapies for treating cancer.  The anti‐tumour effects of BMS‐813160 have not been disclosed.6Although CCL2 was initially identified as a mediator of inflammation, increasing evidence has linked CCL2 with human diseases, particularly cancer. Deeper insight into the mechanisms of the CCL2‐CCR2 axis would provide new directions for better understanding of malignant tumours. The CCL2‐CCR2 signalling axis sustains primary tumour cell survival and proliferation through angiogenesis and different signalling pathways. Moreover, the axis can induce immunosuppressive cell entry to the TME, accelerating tumour growth and metastasis. The expression levels of CCL2 and CCR2 can be regarded as a predictor of poor prognosis in patients with cancer. Nevertheless, researchers noticed that high CCL2 expression was associated with favourable overall survival and progression‐free survival in patients with lung squamous cell carcinoma (LUSC). They speculated that the possible reason is that most TAMs differentiated into the M1 subtype in LUSC.CCL2 neutralizing antibodies or CCR2 antagonists have been developed to potentiate anti‐tumour effects and have produced encouraging results in some preclinical studies. However, targeting CCL2 with carlumab did not appear to have a significant effect in clinical trials, suggesting that anti‐CCR2 may be a compensatory approach for the deficiency of targeting CCL2. In pancreatic cancer, CCR2 antagonists alone or combined with chemotherapeutic agents can control local tumours and are well tolerated in patients. Additionally, in several cancer models, scientists discovered that the CCL2‐CCR2 signalling axis could induce tumour immune evasion through PD‐1 signalling. Furthermore, the combined treatment of anti‐PD‐1 and inhibiting the CCL2‐CCR2 axis is more effective than monotherapy. It might be promising to move this combination therapy regimen into a clinical trial in the near future.In conclusion, the CCL2‐CCR2 signalling axis covers a wide range of tumour cells' activities and is involved in both the early and late steps of tumorigenesis. A series of therapeutic strategies targeting this axis have enriched our approaches for fighting cancer and have entered clinical trials. However, it is important to note that discontinuing CCL2 inhibition exacerbates tumour metastasis,The authors declare no competing financial interests.YQW and XWW offered main direction and significant guidance of this manuscript. MSX and YW drafted the manuscript. RLX illustrated the figures and tables for the manuscript. All authors approved the final manuscript.


The present paper discusses the case of a patient who was surgically treated for a rare calcifying epithelia/ odontogenic tumour (Pindborg tumour) at the Department of Maxillofacia/ Surgery, F. Chopin Clinical Voivodeship Hospital in Rzeszow. Ca/cifying Epithelia/ Odontogenic Tumour (CEOT) is a benign odontogenic tumour arising from the remnants of the dental lamina epithelium. The first three cases of this tumour were recognized by the Danish patho/ogist J.J. Pindborg in 1955. Since then Ca/cifying Epithelia/ Odontogenic Tumour has been commonly referred to as the Pindborg tumour. This type of neop/asm is relatively rare, since it occurs in approximately 0.4% of all odontogenic tumour cases. Due to a fairly common tendency of recurrence, estimated to be approximately 14% of all cases, the preferred o choice of treatment is radical surgical procedure and postoperative follow-up. Appropriate clinical and histopatho/ogical diagnosis is very important before applying the most suitable surgical treatment. Based on the case reviewed and the available literature, we can confirm the suitability of the therapeutic procedure course aligned with contemporary views, guidelines and established standards. Calcifying Epithelial Odontogenic Tumour (CEOT) was first recognized as a separate disease entity in 1955 by the Danish pathomorphologist Jens Jorgen Pindborg ,4. AccorThe aim of this article is to present the incidence, clinical and histopathological diagnosis and treatment of CEOT based on an overview of the literature and our own case of CEOT treated at the Department of Maxillofacial Surgery, E Chopin Clinical Voivodeship Hospital in Rzeszow.A 15-year-old patient was referred to the Maxillofacial Surgery Outpatient Clinic due to an epulis-like tumour localized on the alveolar process of the maxilla adjacent to teeth 11 and 12. The patient claimed that the lesion had appeared a few months before. She denied any other symptoms apart from discomfort and swelling in that area. Clinical examination revealed an exophytic gingival tumour, with a wide base, between teeth 11 and 12, firm texture, painless, overlying normal-appearing mucosa. During the first visit, an intraoral x-ray of the affected area was performed, which revealed damage to the bone of the alveolar process between teeth 11-12 .Due to the benign and slow-growing development of the tumour, the patient was enrolled for an excisional biopsy under local anaesthesia. Surgical procedure was performed and the surgical specimen was sent for a histopathological examination. Surgery was uneventful and the wound healed without complications. Histopathological examination of this primary excisional biopsy was performed at the hospital Pathology Department, and the specimen received for examination consisted of 4 small tissue fragments measuring from 3 to 13mm. The microscopic examination revealed islands and sheets of eosinophilic polyhedral epithelial cells  and 3 suThe literature refers to CEOT as a rare, slowly growing tumour, arising from the odontogenic epithelium, representing approximately 0.4% of all odontogenic tumours. It may occur over a wide age range , with a The above-mentioned case confirms the suitability of the therapeutic procedure undertaken, which is aligned with contemporary views, guidelines and established standards. Properly planned and conducted treatment enables therapeutic success and the subsequent prosthetic replacement offers the best aesthetic and functional results.


There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient ‘vascular normalisation’. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP.We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP.FRT induced ‘vascular normalisation’ changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival.Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials. Moreover, FRT and ADT have significant side effects. Radical surgery (prostatectomy) is another treatment option for PCa,12 but this also has significant side effects, emphasising the clinical need for improved treatments.There is an unmet clinical need to improve treatment outcomes for clinically significant prostate cancer (PCa) and to reduce treatment side effects. Fractionated radiotherapy (FRT) combined with androgen deprivation therapy (ADT) is a treatment option for PCa, however, a third of cases recur,16 VTP is effective in focal ablation of low-risk low-volume PCa,19 and has been investigated as salvage therapy for radio-recurrent PCa.21 Despite interest in the use of focal ablation therapies such as VTP in higher-risk PCa, to date VTP has not been combined with other treatments as a multimodality therapy approach to high-risk PCa.Vascular-targeted photodynamic therapy (VTP) is a novel minimally invasive focal ablation surgical procedure, achieved by rapid free radical-mediated destruction of tumour vasculature.23 These ‘vascular normalisation’ changes arise from anti-angiogenesis effects and are defined by pruning of abnormal vessels, reduced vessel tortuosity, improved perivascular supporting cell coverage and function, restoration of the perivascular basement membrane and generation of a pressure gradient between the intravascular and interstitial compartments, leading to more ‘normal’ vessel function and improved perfusion.25 These ‘vascular normalisation’ changes may influence VTP effectiveness.VTP involves the intravenous administration of the soluble photosensitising agent WST-11, which conjugates to albumin and is focally activated upon near-infrared illumination in the presence of oxygen. VTP requires a functional tumour vasculature for WST-11 to be efficiently delivered to the target tumour tissue prior to activation by local near-infrared illumination, causing vascular and tumour destruction. Tumour vasculature is typically abnormal and characterised by blood vessel immaturity, increased permeability, increased interstitial pressure, reduced perivascular supporting cell number and function, aberrant vessel branching and inefficient blood flow. These properties impair therapeutic drug delivery and reduce treatment efficacy. FRT alters the tumour microenvironment, and can transiently restore tumour blood vessel function through ‘vascular normalisation’ changes.Whilst VTP has been investigated in small observation cohort studies of patients with radio-recurrent PCa, the application of VTP during a window of transient ‘vascular normalisation’ changes post-FRT has not been investigated. We investigated combined sequential FRT and VTP as multimodality treatment in a pre-clinical PCa model, to provide proof-of-concept to inform early phase clinical trials. We specifically tested the hypothesis that sequential delivery of FRT followed 7 days later by VTP, during transient post-FRT ‘vascular normalisation’, may improve tumour control compared to FRT or VTP alone.® CRL-2730™) and MyC-CaP (ATCC® CRL-3255™) cells purchased from American Type Culture Collection were cultured, as described.26 Human umbilical vein endothelial cells (HUVECs)  were cultured in EGM-2 medium .TRAMP-C1 . Cells were exposed to a single 2, 5 or 10 Gy dose of irradiation. Viable (trypan blue-negative) cells were counted over a 7-day time course to assess changes in HUVEC growth. Images of cells in situ, using a white-light microscope, were taken 48 h after RT.HUVECs were irradiated with a 137Caesium irradiator, HUVECs were seeded onto glass slides, and 48 h later stained with 4,6-diamidino-2-phenylindole . Slides were washed with PBS and mounted with Vectashield (Vector Labs). DNA damage was assessed using confocal microscopy  and 405-nm fluorescence excitation.Following 2, 5 or 10 Gy irradiation with a 137Caesium irradiator endothelial cell spheroids were generated by the hanging drop method as described.27Following irradiation of HUVECs with single 2, 5 or 10 Gy doses using a Animal procedures were performed according to UK Animal law (Scientific Procedures Act 1986) and ARRIVE guidelines, with local ethics and Home Office approval.26 In total, 2 × 106 TRAMP-C1 (C57BL/6 mice) or 1 × 106 MyC-CaP (FVB mice) cells in PBS and 1:1 high concentration phenol red-free Matrigel® (Corning) were injected into the flank of mice under isofluorane inhalational anaesthesia on a heat mat. Tumours were measured in the home cage pre- and post treatment using digital callipers three times per week (tumour volume = π/6 × length × width × height). When tumours reached 100–120 mm3, mice were assigned to treatment groups on a first come, first allocated basis using a randomly generated treatment list . All mice were culled by a schedule one procedure at end point in the morning using pentobarbitone injection overdose followed by cervical dislocation according to institutional guidelines.26Naive 6–8-week old male immunocompetent C57BL/6 and FVB mice  were housed in groups of six in a pathogen-free facility with 12-h light cycles, in individually ventilated cages on woodchip bedding, with access to water and food ad libitum, at 22 °C (range 21–24 °C) and 50% humidity (range 35–75%), with environmental enrichment and bedding material, and monitored for body weight changes twice weekly.26FRT was delivered as previously described.30 Dynamic contrast-enhanced MRI (DCE-MRI) was performed at 4.7 and 7.0 T scanners  using 25-mm inner diameter birdcage coils  as previously described.28 Respiratory-gated 3D gradient-echo scans  with an isotropic resolution of ∼420 μm and a respiratory rate-dependent frame acquisition time of ∼8–10 s were obtained.28 Fifty frames were acquired with a 25 µl bolus infusion of Gadolinium (Gd) solution (Omniscan GE HEALTHCARE) administered using a syringe pump  over 5 s commencing at the beginning of frame 11. Radiofrequency field inhomogeneities were accounted for using a respiratory-gated implementation of the Actual Flip Angle technique, and baseline T1 was measured using a variable flip angle sequence as reported previously.28 In order to analyse tumour perfusion, manual segmentation was initially performed from the average image of the DCE sequence using ITK-SNAP medical image segmentation software.31 The MR signal was then converted to Gd concentration, as previously described.32 The initial area under the Gd curve (iAUC) was measured by integrating the first 90 s after injection and used as an indicator of perfusion.Inhalational anaesthesia was induced and maintained with isofluorane (1–4% in air) in order to maintain a respiration rate of 40–60 breaths per minute, and the temperature was maintained at 35 °C using a homeothermic temperature maintenance system as previously described.2. A simple mechanical system and turning prism allowed the excitation and guide beam to be positioned over the area of interest. In addition, the enclosure was fitted with diffuse LEDs operating at 590 nm . The animal in the enclosure could be viewed at all times with the aid of a miniature high dynamic range monochrome camera mounted in the enclosure lid. The wavelength of the guide beam and the illumination (590 nm) was chosen as it was in the nadir of the WST-11 VTP spectral absorption. Similarly, WST-11 injections were performed under yellow light generated by an array of T1¾ 590-nm LEDs . Lyophilised WST-11 was a gift from Avigdor Scherz  and was reconstituted in sterile 5% dextran in water at 2 mg/mL under light protected conditions, and aliquots were stored at −20 °C in the dark and thawed on the day of VTP treatment and sterile filtered through a 0.2-μm disc syringe filter. Mice with 120-mm3 tumours received VTP treatment in the morning under isofluorane inhalational anaesthesia with physiological monitoring. Anaesthetised mice received an intravenous infusion of 7 or 9 mg/kg WST-11 via the tail vein followed immediately by 10-min laser excitation of the subcutaneous flank tumour at 120 mW/cm2 using a collimating lens. The optical irradiation light field was arranged to cover the entire subcutaneous flank tumour area plus a 1-mm rim. Mice were returned to the home cage following recovery from anaesthesia and underwent post-procedure health monitoring and tumour measurements using digital callipers.A bespoke optical excitation system was constructed to administer VTP. A small enclosure , and fixed with 4% dilution electron microscopy grade paraformaldehyde (Electron Microscopy Sciences) in PBS for 30 min at room temperature in a Coplin jar. Tissue section slides were then washed with PBS in a Coplin jar prior to a combined blocking and permeabilisation step using 3% BSA in 0.3% triton/PBS for 90 minutes at room temperature. Tissue sections were then encircled with a hydrophobic PAP marking pen, and 200 µl of primary antibody  diluted in blocking solution was added for incubation in a humidified chamber at 4 °C overnight, with this and all subsequent steps under light protection. After removal of primary antibody, a 300 nM DAPI nuclear counterstain in PBS was applied for 30 min incubation prior to washes in PBS, and mounting with coverslips sealed with Wirosil dental silicone. αSMA and CD31 sample images were acquired with a Plan Apo 20 × 0.75 NA objective on a Nikon Ti Widefield Microscope equipped with a Lumencor SpectraX LED light source and an Andor Neo Zyla Camera.Tumour CD31-positive blood vessel quantification was performed with classical image processing methods. Following image pre-processing, image segmentation was performed to identify tissue areas and CD31-positive blood vessels in an automated fashion. The vessel segmentation was analysed using the Euclidian distance transformation to obtain the local thickness of all segmented objects in the images. The combined binary masks indicating the presence of tissue and CD31-positive blood vessel masks were used to compute the vessel density and the distance from any tissue pixel to the closest vessel.33 as these are relatively poorly defined heterogeneous perivascular cell types, without highly specific markers available for their identification.34 We used computer-assisted image annotation to identify αSMA-positive pericytes by overlapping the image channels corresponding to αSMA (red) and CD31 (green), and selecting αSMA-positive cells adjacent to, or overlapping, CD31-positive blood vessels. To assist annotation, αSMA-positive cells with a distance of more than 10 pixels, or 3.2 μm, from CD31-positive blood vessels were suppressed. Annotation was performed using the Annotation of Image Data by Assignments (AIDA) web application platform  developed in-house for the annotation of large microscopy images. Here, relevant regions of interest in the tumour were selected, and the annotators then marked αSMA-positive cells and CD31-positive blood vessels in these regions. Areas in the αSMA channel selected by at least three of four independent observers were accepted as αSMA-positive pericytes. In order to investigate the extent of αSMA-positive pericyte coverage of CD31-positive blood vessels, the fraction of blood vessels in each region with a spatially adjacent αSMA-positive cell was computed. Control untreated samples were compared against FRT-treated samples.α-SMA is often used to identify pericytes,3 to reach the end-point size of 400 mm3 compared against control untreated tumours, a single mouse being considered an experimental unit. All results are mean ±  standard error of the mean. P < 0.05 was considered to be a statistically significant difference.Statistical analysis was performed using GraphPad Prism 8 . For in vitro work, ordinary one-way ANOVA tests were performed with Dunnett’s or Tukey’s post hoc adjustment for multiple comparisons. For in vivo tumour growth delay experiments, ordinary one-way ANOVA was performed using Tukey’s test for multiple comparisons following Brown–Forsythe’s test for equality of the means. Tumour growth delay was defined as a significant increase in time (days) for a tumour treated at 100–120 mm26 To similarly investigate antitumour effects of VTP in this model and subsequently combine FRT and VTP as multimodality therapy, we developed an enclosed optical irradiation system to deliver VTP to flank allograft tumours  was lower for FRT-treated tumours than for untreated control tumours at 7 days post initiation of FRT   Fig. . Experim88) Fig. .Fig. 3FRThe in vivo findings in TRAMP-C1 flank tumour allografts 7-days post-FRT demonstrated that CD31-positive tumour vessels were larger in size, fewer in number, further apart, with enhanced αSMA-positive pericyte coverage, and these tumours had enhanced vascular perfusion on DCE-MRI. This indicated that FRT had induced ‘vascular normalisation’ changes by 7-days post-FRT in TRAMP-C1 flank tumours. To investigate the effects of irradiation on endothelial cell sprouting and angiogenesis in vitro, HUVECs were exposed to a single 2, 5 or 10 Gy dose. This demonstrated that an increasing irradiation dose reduced the HUVEC cell number over time following treatment, compared against untreated control cells Fig. . Using a3 compared to 9 mg/kg WST-11 VTP alone or FRT alone . However, around a third of patients with high-risk PCa treated with these modalities develop disease recurrence, and these treatments have significant side effects, reducing the quality of life in long-term survivors. There is increasing interest in the role of multimodality therapy for PCa.40 VTP has not yet been clinically evaluated in a multimodality therapy approach, or for high-risk PCa. Evidence from several clinical trials in low-risk low-volume disease demonstrates VTP is safe and tolerable.19 Given that the treatment parameters for VTP have been established for optimal targeted tumour ablation, whilst FRT for PCa is well-established, it could be feasible, based on pre-clinical data, to combine these treatments in early and subsequent late phase clinical trials. Herein, we provide evidence that initial moderate hypofractionated FRT ahead of VTP, with VTP being delivered during a window of time when the irradiated PCa tumour displays ‘vascular normalisation’ changes, leads to enhanced tumour growth delay, with a resultant survival benefit in mice and the possibility of complete tumour cure in some cases. It may be possible, and indeed it is attractive, to combine neoadjuvant FRT with VTP in treating PCa. This may obviate the need for neoadjuvant and/or concomitant ADT, and reduce the dose of conventional FRT administered. This new multimodality therapy approach will require formal clinical evaluation, and in particular will require determination of the optimal dose and fractionation schedule of FRT in patients, combined with the feasibility of performing VTP during the window of ‘vascular normalisation’ changes.VTP is a minimally invasive focal ablation precision surgical technique, largely evaluated as monotherapy for low-risk low-volume PCa.42 VTP has advantages as a focal therapy; VTP has been evaluated in a phase-III randomised clinical trial,19 and can anatomise treatment to conform to the malignant lesion along with a margin of normal tissue within the prostate gland, preserving urinary and sexual function. Pre-clinical PCa studies demonstrate that VTP may be successfully combined with ADT,43 a 111In-DOTA-AR bombesin antagonist,44 and anti-macrophage colony-stimulating factor (anti-CSFR1),45, however, VTP has not been investigated in close sequential combination with FRT in pre-clinical or clinical studies. VTP has been demonstrated to be safe and effective in early phase clinical trials in a small number of patients with recurrent PCa following radiotherapy, however, there was a considerable interval of several years between radiotherapy and VTP. The evidence presented herein suggests that sequential combined FRT and VTP within a short interval enhances antitumour control, and warrants evaluation in clinical trials.The concept of organ-sparing PCa treatment strategies, directed towards a focal lesion or using a hemi-gland approach to deliver effective tumour control and/or cure with reduced toxicity versus whole-gland treatment, is an increasing focus of PCa research. This approach may have less detrimental effects on quality of life versus the current standard of care of whole-gland therapy.28 The dose and fraction size of FRT, along with the window of time in which to look for ‘vascular normalisation’ changes, are key issues as it is known that large irradiation doses rapidly destroy the tumour vasculature, rather than transiently enhancing vascular function and perfusion.50 The results of in vivo xenograft pre-clinical model experiments show that the size and number of irradiation fractions determine whether transient induction of enhanced tumour vascularity occurs. In general: 5–10 Gy fractions cause transient blood flow increase, which returns to normal; 10–15 Gy fractions cause tumour blood flow decrease, which then recovers; and 15–20 Gy fractions cause blood flow to rapidly decrease, and vessels deteriorate.51 It is important for future clinical translation to consider these aspects of FRT delivery. Hypofractionated RT is increasingly used clinically,35 and it would be feasible to use FRT schedules that are likely to induce ‘vascular normalisation’ changes ahead of VTP in a multimodality approach.The observation that VTP delivered shortly after neoadjuvant FRT increases delay in tumour growth is intriguing and suggests that neoadjuvant FRT delivered in the correct fraction size and dose ahead of subsequent VTP improves the efficacy of this focal therapy. One possible explanation is that transient ‘vascular normalisation’ changes induced by moderate hypofractionated RT transiently increase tumour vessel perfusion, thereby enhancing WST-11 delivery and subsequent tumour ablation by VTP. Experiments with VTP delivered during concomitant FRT showed no benefit from combination, whereas a benefit was seen with VTP delivered 7 days following initiation of FRT, suggesting that the FRT-induced increased tumour perfusion may be important in the efficacy of sequential combination treatment. We observed that FRT enhances tumour perfusion as observed on DCE-MRI 7 days post-FRT, consistent with other studies using similar fractions and doses of FRT.We observed that 7 mg/kg WST-11 resulted in a more pronounced reduction in tumour growth following FRT than 9 mg/kg WST-11. One might speculate that near-infrared illumination of 7 mg/kg WST-11 ablates fewer tumour vessels than 9 mg/kg WST-11, leaving sufficient tumour vessels available for immune cells to infiltrate the tumour and elicit an antitumour immune response, thereby enhancing the observed antitumour effects. This intriguing hypothesis warrants investigation in future research.53 potentially rendering them susceptible to targeted therapy with molecular agents. In the case of VTP, which requires a functional vasculature, it is possible that prior enhancement of vascular function through ‘vascular normalisation’ changes22 improves antitumour effects of subsequent VTP treatment. A variety of vascular targeting agents have been evaluated in pre-clinical cancer research, including inhibitors of angiogenesis and vascular disrupting agents (VDAs). Some VDAs have been investigated in combination with FRT,59 where the VDA was administered following initial FRT. Combining VDAs with conventional therapies such as FRT may improve treatment outcomes by enhancing antitumour efficacy, with non-overlapping toxicities, and spatial cooperation. VTP may have particular clinical benefit as a VDA compared to drugs such as DMXAA, CA4DP and ZD6126 investigated in other studies. WST-11 used for VTP has minimal toxicity as it is focally activated by near-infrared illumination in the tumour vasculature rather than being active systemically. VTP is therefore a more attractive clinical agent than other VDAs in combination with FRT.The concept of targeting the tumour vasculature, rather than the cancer cells, has been explored in multiple pre-clinical and clinical studies. Tumour vessels are often functionally abnormal26 and VTP16 each induce immunological changes within tumours including PCa, and the effects of sequential FRT and VTP on the tumour immune microenvironment will be investigated and reported separately. Secondly, this work was performed on the TRAMP-C1 flank tumour allograft model of PCa in C57BL/6 mice, which is the most commonly used in vivo pre-clinical PCa model. It would be helpful to validate the benefit of combined sequential FRT and VTP in an alternative model of PCa, although the feasibility of combining FRT and VTP in an orthotopic mouse model would be challenging due to concerns over rectal and urethral toxicity of combined treatment. Thirdly, it is an important relative limitation of the reported experiments that they have not incorporated ADT, which is conventionally used in the neoadjuvant clinical setting prior to FRT. ADT could modulate the effect of sequentially combined FRT and VTP, as it is recognised that ADT can affect the tumour vasculature. An extension of the work reported in this manuscript incorporating ADT with combined FRT and VTP will be an important next step to evaluate this multimodality treatment approach. Fourthly, although we demonstrate the growth kinetics of TRAMP-C1 allograft tumours are slower post-VTP following prior FRT, compared to VTP monotherapy, it will be important to investigate the microenvironment of recurrent tumours, as recurrence following combination therapy may promote aggressive disease. Fifthly, we administered a single VTP treatment following FRT, whereas VTP can be repeatedly administered. Finally, given that a functional tumour vasculature and viable endothelial cells after FRT are conventionally considered to be undesirable post-treatment effects as they can promote tumour recurrence and metastasis, the clinical safety of delivering VTP during a window of ‘vascular normalisation’ changes following sublethal FRT requires evaluation.This study has several limitations. Firstly, it has not investigated the immune response to sequential FRT and VTP, and this will be a focus of future research. FRTThese findings demonstrate that combined sequential FRT and VTP may be a promising clinical strategy to treat PCa, paving the way for the development of future early phase clinical trials. This approach may also be a useful strategy for other solid organ cancers where improved therapy is desirable.Supplementary Figure LegendsSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3NC3Rs ARRIVE Checklist


Gliomas are hard to treat. Their prognosis has improved little over the past few decades. Fundamental therapeutic challenges such as treatment resistance, malignant progression, and tumour recurrence persist. New strategies are needed to advance the management and treatment of gliomas. Here, we focus on where those new strategies could emerge. We consider how recent advances in our understanding of the biology of adult gliomas are informing new approaches to their treatment. Gliomas are the most frequent primary brain tumour in adults . Unlike Two features of gliomas mean that they are particularly hard to treat and make them prone to recurrence. First, the majority of gliomas are highly invasive . ConsequIn this review, we focus on emerging strategies to manage and treat adult gliomas. The 2016 WHO classification of brain tumours has transformed brain tumour diagnosis by combining molecular and histological features of gliomas. Since then, there have been several excellent recent reviews on different types of gliomas –17, stanAdvances in our understanding of gliomagenesis have led to new strategies for treating gliomas. Two influential ideas have been clonal evolution and the glioma stem cell hypothesis, which is an example of the more general cancer stem cell hypothesis.Clonal evolution is thought to be a feature of many cancers , 19. EssThe glioma stem cell hypothesis is a more recent idea. Glioma stem cells are a small, sub-population of the glioma cells, which are able to proliferate, self-renew, and propagate the entire tumour when transplanted . They haClonal variation and the cancer stem cell hypothesis were initially thought to be distinct explanations for tumour heterogeneity , but othKnowledge of the genetic mutations and the resulting tumour proteins implicated in gliomagenesis has opened avenues for the development of new precision medicine treatments, some of which are undergoing trials. For example, mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified as driver mutations in many gliomas , 31. SmaImmunotherapies have taken advantage of our molecular understanding of gliomas too. Gliomas present fragments of tumour-specific proteins called neoantigens on their cell surface. Peptide vaccines have been developed to prime the immune system to attack cells presenting tumour neoantigens. Neoantigens derived from mutant IDH1 were an early target in preclinical models . More reThe glioma stem cell hypothesis suggests further approaches to treating gliomas. It implies that glioma growth would stop if all the glioma stem cells were killed . The theThe ability to study the molecular profiles of large numbers of single cells enables heterogeneity within gliomas to be probed in detail. Primary glioblastomas (WHO grade IV) are highly aggressive tumours. Molecular profiling using genomic, epigenomic, and single-cell transcriptomic information indicates that individual glioblastoma cells converge on one of four main cellular states: astrocyte-like, mesenchymal-like, oligodendrocyte-like, and neural progenitor-like . Each glO6-methylguanine DNA methyltransferase (MGMT). The methylation status of the MGMT gene promoter controls MGMT protein expression and, therefore, the response of gliomas to alkylating chemotherapy agents [MGMT promoter is measured to determine whether Temozolomide should be given. However, the methylation status of single cells varies within individual gliomas [Glioma cell heterogeneity has led to a re-evaluation of treatment biomarkers. Temozolomide is an alkylating agent that is the backbone of chemotherapy treatment for gliomas. The effect of Temozolomide is reversed by the endogenous enzyme, y agents . The met gliomas . Importa gliomas . These f gliomas . This prGlioma cells change their cellular state and molecular profile in response to the microenvironment within and around the tumour . The gliThe neocortex around gliomas becomes hyperexcitable leading to seizures , 52. It The idea of a direct interaction between a glioma and the surrounding brain has developed further with the finding that neurons form putative synapses on glioma cells. Ultrastructural analysis of human glioma cells transplanted into mouse brains revealed that the glioma cells form structures with neighbouring axons –57. ThesThe evidence above considered how neural circuitry in the brain affects gliomas. Newly emerging evidence suggests that the reverse happens; that is, gliomas modify neural circuitry. An early idea was that gliomas caused seizures, because glutamate release from glioma cells resulted in greater excitotoxic cell death of inhibitory interneurons than of excitatory pyramidal cells in the peritumoural cortex , 52, 61.Collectively, the recent evidence suggests that a subset of gliomas forms a positive feedback loop with the surrounding peritumoural cortex. The therapeutic challenge now is to break this feedback loop in a way that does not disrupt neural activity and impair cognition.Epileptic seizures are the most common presenting symptom in patients with glioma , 65. ThiSome gliomas have an indolent course initially and then behave more aggressively. The terminology around this has been re-evaluated. Malignant transformation was used to describe low-grade (WHO grade I–II) gliomas developing features of high-grade (WHO grade III–IV) tumours. The practise has been undermined by molecular profiling of gliomas, which gives a more accurate prognosis . IncreasWe do not understand what causes malignant progression and where it occurs. Genetic changes associated with malignant progression have been identified , 72. HowMalignant progression is a key clinical issue in the management of gliomas, particularly as lower grade gliomas commonly affect young adults. Malignant progression is frequently detected neuroradiologically by contrast enhancement or increased perfusion on MRI scans. However, these findings are not always present Fig. . The issTissue biopsies give a snapshot of a glioma. However, the molecular profile of glioma cells is not static. It evolves. Ideally, treatment would change to mirror progression of the glioma. A minimally invasive biomarker based on the molecular profile of glioma cells could provide valuable information about tumour evolution. Such a biomarker could also be used to diagnose chemoradiotherapy-induced changes in brain imaging termed pseudoprogression and to monitor response of the glioma to treatment.There has been huge interest in studying body fluids, such as blood or cerebrospinal fluid, to detect evidence of cancer. This is referred to as liquid biopsy. Blood biomarkers include circulating tumour cells, extracellular vesicles, and cell-free tumour DNA (ctDNA) . Low freBlood biomarkers have the huge advantage of ease of collection. However, the blood–brain barrier limits access of tumour tissue to the blood. Tests of blood-derived extracellular vesicles and circulating tumour cells have suffered with low sensitivity . As a reLiquid biopsy based on cerebrospinal fluid offers an approach that circumvents the blood–brain barrier. Analysis of ctDNA in the CSF successfully identified glioma in approximately half of patients , 79. TheLiquid biomarkers offer a minimally invasive alternative to tissue biopsies. Currently, blood- and CSF-derived biomarkers lack diagnostic specificity, due to the short half-life, penetrance, and dilution of the biomarker . These lLiquid biopsy could be used to diagnose glioma mimics. A common diagnostic problem for neuro-oncology services is to differentiate glioblastoma from primary CNS lymphoma. Current practise is that tumour tissue is required from both glioblastoma and primary CNS lymphoma to diagnose the condition and to plan treatment . AcquiriThere has been considerable progress in our understanding of the biology of gliomas. However, many outstanding questions remain (Box 1). First, the finding that glioma growth and invasion is facilitated by aberrant neuronal activity suggests that effective control of seizures is vital for glioma patients. Novel agents to diminish neuronal hyperexcitability will need careful titration to avoid inhibiting neuronal plasticity, as this could impair cognitive function. Enzyme-inducing anticonvulsants need to be avoided as they can increase metabolism of some chemotherapy agents. Blood and CSF liquid biopsy have great potential as methods to diagnose glioma mimics and tumour pseudoprogression, and to monitor tumour progression. However, new approaches are needed to increase the sensitivity of liquid biopsies. What joins all the outstanding questions is the overwhelming need to translate the new knowledge about gliomas into novel strategies for their diagnosis and management.Does control of seizure activity limit glioma growth and invasion?Is killing glioma stem cells sufficient to reduce glioma growth or must all of the glioma cellular states be attacked?Can new investigations predict which lower grade gliomas will progress into more malignant tumours?Can the sensitivity and specificity of liquid biopsy be improved through interventions that increase release of glioma tissue fragments into the blood or CSF?


Tebipenem pivoxil hydrobromide, an orally bioavailable prodrug of tebipenem, is currently in development for the treatment of complicated urinary tract infections. The spectrum of tebipenem activity against Enterobacterales is consistent with intravenous carbapenems such as ertapenem and meropenem including extended spectrum beta-lactamase (ESBL) and AmpC producing organisms. The goal of these studies was to determine the tebipenem exposure required to prevent on-therapy resistance and to evaluate the tebipenem regimen utilized in the recent Phase 3 clinical trial.Escherichia coli clinical isolates  (tebipenem MIC = 0.008 to 0.03 mg/L) were subjected to concentration-time profiles simulating free-drug plasma concentrations after oral administration of tebipenem regimens in a hollow-fiber in vitro infection model. Dose-ranging studies were completed using two isolates at an initial burden of 108 CFU/mL and with exposures for tebipenem regimens ranging from 4.69 to 1200 mg administered every eight hours (q8h). An additional isolate was subjected to only the tebipenem 600 mg q8h regimen evaluated in the Phase 3 trial. Samples were collected for the enumeration of bacterial burdens and evaluation of the simulated pharmacokinetic profile. Each sample for bacterial enumeration was suspended onto drug-free and tebipenem-supplemented agar plates in order to observe the density of the total- and drug-resistant subpopulations over the 10-day period.Three E. coli isolates. Tebipenem consistently lowered bacterial burdens below that of the initial inoculum at a dose of 600 mg q8h for all three E. coli isolates. Amplification of resistance was observed intermittently for all regimens, but was never equal to that of the total population at the 600 mg q8h clinical dose. Tebipenem MIC values for isolates collected from the drug-supplemented agar ranged from 0.06 to 0.25 mg/L, representing four two-fold dilutions from baseline.A full dose response, ranging from treatment failure to reductions in bacterial burden from baseline, were observed in the dose-ranging studies for the two These data support the selection of tebipenem dosing regimens that minimize the potential for on-therapy drug-resistance amplification.Brian D. VanScoy, B.S., 3-V Biosciences (Grant/Research Support)Achogen (Grant/Research Support)Amplyx Pharmaceuticals, Inc. (Grant/Research Support)Arixa Pharmaceuticals (Grant/Research Support)Arsanis Inc. (Grant/Research Support)B. Braun Medical Inc. (Grant/Research Support)Basilea Pharmaceutica (Grant/Research Support)BLC USA (Grant/Research Support)Boston Pharmaceuticals (Grant/Research Support)Bravos Biosciences, LLC (Grant/Research Support)Cidara Therapeutics Inc. (Grant/Research Support)Cipla, USA (Grant/Research Support)Corcept Therapeutics (Grant/Research Support)Cumberland Pharmaceuticals (Grant/Research Support)Debiopharm International SA (Grant/Research Support)Discuva Limited (Grant/Research Support)Emerald Lake Technologies (Grant/Research Support)Enhanced Pharmacodynamics (Grant/Research Support)Entasis Therapeutics (Grant/Research Support)E-Scape Bio (Grant/Research Support)Genentech (Grant/Research Support)Geom Therapeutics, Inc. (Grant/Research Support)GlaxoSmithKline (Grant/Research Support)Hoffmann-La Roche (Grant/Research Support)Horizon Orphan LLC (Grant/Research Support)ICPD Biosciences, LLC (Grant/Research Support)Indalo Therapeutics (Grant/Research Support)Insmed Inc. (Grant/Research Support)Institute for Clinical Pharmacodynamics (Employee)Iterum (Grant/Research Support)KBP Biosciences USA (Grant/Research Support)Kyoto Biopharma, Inc. (Grant/Research Support)Matinas (Grant/Research Support)Meiji Seika Pharma Co., Ltd. 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Fluid warming devices using an uncoated aluminium containing heating element have been reported to liberate aluminium resulting in critical aluminium concentrations in heated fluids. We investigated saline solution (0.9%), artificially spiked with organic acids to determine the influence of fluid composition on aluminium release using the uncoated enFlow® device. Additionally, the Level1® as a high volume fluid warming device and the ThermoSens® device were investigated with artificial spiked fluid at high risk for aluminum release and a clinically used crystalloid solution.Fluid warming, recommended for fluid rates of > 500 ml hSaline solution spiked with lactate more than acetate, especially at a non neutral pH, led to high aluminium release. Next to the enFlow® device, aluminium release was observed for the Level1® device, but not for the coated ThermoSens®-device.Uncoated aluminium containing fluid warming devices lead to potentially toxic levels of aluminium in heated fluids, especially in fluids with non-neutral pH containing organic acids and their salts like balanced electrolyte solutions.The online version contains supplementary material available at 10.1186/s12871-021-01378-7. Aluminium release by anodized und uncoated fluid warming devices may lead to concerning aluminium concentration in intravenous fluids. The chemical background of this problem is addressed in this investigation with six fluids and two different fluid warming devices.− 1 ) µmol l− 1) after 60 min. Highest aluminium concentrations were observed when saline was spiked with 50 mmol lactate at a pH 9 induced by sodium hydroxide. The aluminium concentrations were determined to 2462.4 (2109.6-2954.3 [1921.1-3379.8]) µg l− 1 (91.36 (78.27–109.6 [71.27-125.39]) µmmol l− 1) and 1692 (433.1-3152.4 [282.8-3938.6]) µg l− 1(62.77 (16.07-116.95 [10.49-146.12]) µmol l− 1). In lactate spiked saline at a pH of 4 induced by hydrochloric acid the aluminium concentration was determined to 369.4 (157.1-624.7 [29.6-1134.7]) µg l− 1 (13.7 (5.83–23.18 [1.1–42.1]) µmol l− 1) after 60 min. Aluminium concentration in saline spiked with 50 mmol acetate was determined to 210.6 (0-450.2 [0-537]) µg l− 1 (7.81 (0-16.7 [0-19.92]) µmol l− 1) after 60 min  µmol l− 1) and titrated to a pH of 9 with sodium hydroxide 2798.8 (691.7-3050.7 [6.9-3645.7]) µg l− 1 (103.84 (25.66-113.18 [0.26-135.26]) µmol l− 1)  was determined to 2609.3 (2135.6–3043 [1891.8-3172.2]) µg l− 1 9.02 (2.42–14.84 [0.4-96.78]) µmol l− 1) for the Level1® and 102.6 (80.6-209.6 [35.8-371.8]) µg l− 1 (3.81 (2.99–7.78 [1.33–13.79]) µmol l− 1) for the enFlow® device  after flowing 60 min through the three tested devices revealed to aluminium concentrations of 243 (65.2–400 [10.9-2608.6]) µg lThe main result of our study is that lactate with non-neutral pH is most effective in aluminium release with uncoated heating devices. It was confirmed that sodium chloride solution itself has only very limited capacity of aluminum release. However, aluminium concentrations determined by analysing acetate buffered sodium solutions or commercial balanced electrolyte solutions, which are common in clinical routine, are reasonable.− 1 (0.04–0.07 µmol l− 1), with a transferrin binding of > 90 % [Aluminium is one of the most studied toxic metals and associated with many diseases  like encf > 90 % . Althougf > 90 % .− 1 d− 1 is a risk for encephalopathy, impaired neurologic development and osteomalacia [− 1 [− 1) for aluminium content of parenteral nutrition which is also noted by the United States FDA [One possible safety threshold is the recommended threshold for parenteral nutrition as this threshold reflects an intravenous administration. For preterm infants with need of parenteral nutrition a repeated intravenous aluminum supply of > 4–5 µg kgomalacia , 9. The cia [− 1   in mean using a balanced, lactate buffered electrolyte solution at a flow rate of 4 ml min− 1 [− 1 (162.5 µmol l− 1) using lactated saline solution and 6027.9 µg l− 1 (223.2 µmol l− 1) for Plasma-Lyte 148 [− 1. An investigation using the high flow device Level 1® using Lactated Ringer’s solution at a higher flow rate of 30 ml min− 1 described an aluminium concentration of 278.5 µg l− 1 (10.3 µmol l− 1) [− 1 does not reflect the range in which high volume fluid warming devices are typically used. However, even is concentration might be significant lower under influence of increased flow rate, the effect on total amount of leached aluminium would be less. The influence of flow rate on resulting aluminium concentration  an aspect for future investigations.Even if these values are inconsistent, it can be assumed that any kind of aluminium infusion is inadvertent and a dosage of more than 1000 µg for adults is unsafe. Regarding the results of this investigation not only the concentration of aluminium in the warmed fluid alone is relevant. For the estimation of safety, the concentration must be multiplied with the administrated volume. Fluid warming is indicated and recommended fluid rates of more than 500 ml h-1. For bleeding patients e.g., after major trauma high volume fluid demands of more than 5 l.h-1 are reasonable. In consequence the observed concentrations of aluminium liberated by uncoated fluid warming devices as enFlow® and Level1® are both not safe and bear a potential risk of aluminium intoxication and maybe long-term effects like enzephalopathia , 19. Ourol l− 1) . Higher ol l− 1)  may be eol l− 1) . The dif3(OH)45+, Al13(OH)327+) [3+ is present and Al(OH)2+ exists in the pH range between 4 and 6, while Al(OH)4− is predominant at pH > 8. At a pH of 9, aluminium hydroxide species is formed and this is coordinated to the lactic acid or the corresponding salt [4−Ions is relevant for clinical settings, as buffered electrolyte solutions with pH > 7 are in daily routine widespread used.In the previous study  the incrOH)327+) . The moling salt . At a hiThe strength of a bench investigation is the standardized examination of aluminium release. However, a problem of any bench investigation is that no resulting plasma concentrations of aluminium can be reported. Assessment of any patient outcomes such as cognitive dysfunction induced by the infused aluminium is impossible. However, the exposition of patients resulting from uncoated fluid warming devices using aluminium as heating element can be estimated and bear a potential risk of toxic levels.In conclusion, uncoated fluid warming devices using aluminium as heating element bear the risk of aluminium release in a potential toxic amount. Organic acids and their salts like lactate and acetate are compounds provoking the reaction, especially under non-neutral conditions. Test protocols for leaching of aluminium from fluid warming devices should apply lactate spiked saline better than saline solution. In clinical context the use of balanced electrolyte fluids with uncoated fluid warming devices using aluminium heating elements is associated with a risk of inadvertent aluminium exposition of patients.Additional file 1