Anti-tumor efficacy of Calculus bovis: Suppressing liver cancer by targeting tumor-associated macrophages

Despite significant advances in our understanding of the molecular pathogenesis of liver cancer and the availability of novel pharmacotherapies,liver cancer remains the fourth leading cause of cancer-related mortality worldwide.Tumor relapse,resistance to current anti-cancer drugs,metastasis,and organ toxicity are the major challenges that prevent considerable improvements in patient survival and quality of life.Calculus bovis(CB),an ancient Chinese medicinal drug,has been used to treat various pathologies,including stroke,convulsion,epilepsy,pain,and cancer.In this editorial,we discuss the research findings recently published by Huang et al on the therapeutic effects of CB in inhibiting the development of liver cancer.Utilizing the comprehensive transcriptomic analyses,in vitro experiments,and in vivo studies,the authors demonstrated that CB treatment inhibits the tumor-promoting M2 phenotype of tumor-associated macrophages via downregulating Wnt pathway.While multiple studies have been performed to explore the molecular mechanisms regulated by CB,this study uniquely shows its role in modulating the M2 phenotype of macrophages present within the tumor microenvironment.This study opens new avenues of future investigations aimed at investigating this drug’s efficacy in various mouse models including the effects of combination therapy,and against drug-resistant tumors.

Autoantibodies against tumor-associated antigens for detection of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the most common tumors worldwide. The survival rate after the onset of symptoms is generally less than one year for the late presentation of HCC, and reliable tools for early diagnosis are lacking. Therefore, novel biomarkers for the early detection of HCC are urgently required. Recent studies show that the abnormal release of proteins by tumor cells can elicit humoral immune responses to self-antigens called tumor-associated antigens(TAAs). The corresponding autoantibodies can be detected before the clinical diagnosis of cancer. Therefore, there is growing interest in using serum autoantibodies as cancer biomarkers. In this review, we focus on the advances in research on autoantibodies against TAAs as serum biomarker for detection of HCC, the mechanism of the production of TAAs, and the association of autoantibodies with patients' clinical characteristics.

Multiplexed Biosensing Diagnostic Platforms Detecting Autoantibodies to Tumor-Associated Antigens from Exosomes Released by CRC Cells and Tissue Samples Showed High Diagnostic Ability for Colorectal Cancer

Colorectal cancer(CRC)is the second leading cause of cancer-related death worldwide.The five-year survival rate of CRC patients depends on the stage at diagnosis,being higher than 80%when CRC is diagnosed in the early stages but lower than 10%when CRC is diagnosed in advanced stages.Autoantibodies against specific CRC autoantigens(tumor-associated antigens(TAAs))in the sera of patients have been widely demonstrated to aid in early diagnosis.Thus,we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy.To that end,we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis.A total of 103 proteins were identified as potential autoantigens specific to CRC.After bioinformatics and meta-analysis,we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot(WB)and immunohistochemistry(IHC).We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients,along with an association of nine of these proteins with CRC prognosis.After validation,all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals,either by luminescence Halotag-based beads,or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection.Taken together,our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases;they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care(POC)device for CRC detection with high diagnostic ability.

Tumor-Associated Autoantibodies: Re-Optimization of EarlyCDT-Lung Diagnostic Performance and Its Application to Indeterminate Pulmonary Nodules

Background: Low-dose computed tomography (CT) screening reduces lung cancer mortality but costs are prohibitive for most healthcare budgets due to high false positive rates. An adjunctive test able to distinguish malignant from benign pulmonary nodules would be hugely beneficial. EarlyCDT-Lung measures serum autoantibodies to tumor-associated antigens and has found clinical acceptance to aid early detection of lung cancer for high risk patients. However performance was optimized for screening. The construction of a receiver-operating characteristic (ROC) curve would enable optimization of performance for alternative settings, including nodule malignancy. Methods: A Monte-Carlo search method was used to construct a ROC curve using a case-control cohort, enabling high and low specificity versions of EarlyCDT-Lung to be determined. These were used for a theoretical evaluation of a nodule cohort, and positive predictive value (PPV) was calculated under the assumption of independence of risk source. Patients or their nodules are typically classified into three risk groups: low (0% - 10%), intermediate (10% - 65%) and high (>65%) risk of malignancy. The predicted shift in risk group by application of the high and low specificity versions, along with the current commercial EarlyCDT-Lung, was then estimated. Results: The ROC curve, with an area under the curve of 0.743, was constructed. The high specificity (98%), low specificity (49%) and current commercial (91% specificity) versions of EarlyCDT-Lung re-classified 27%, 23% and 26% of intermediate nodules, respectively, to either a higher (10%, 8% and 10%) or lower (17%, 15% and 16%) risk group. Conclusion: A ROC curve was constructed to allow performance prediction of EarlyCDT-Lung at different specificities in the indeterminate nodule setting. This enabled risk re-classification of intermediate risk nodules, and could therefore facilitate alternative more appropriate intervention. We have shown how a multivariate biomarker test can add to the interpretation of pulmonary nodules and therefore aid patient management.

Guiding function of positron emission tomographycomputed tomography examination in the diagnosis and treatment of ocular adnexal mucosa associated lymphoid tissue lymphoma

AIM:To explore the role of positron emission tomographycomputed tomography(PET-CT)examination in the diagnosis and treatment of ocular adnexal mucosa associated lymphoid tissue lymphoma(OAML).METHODS:The general clinical data,postoperative PET-CT results,treatment regimens,and the prognosis of 21 histopathologically confirmed OAML patients between October 2017 and September 2021 were collected.Among the 21 patients,five patients underwent surgical treatment alone,13 patients underwent surgical treatment combined with radiotherapy,and three patients underwent surgical treatment combined with chemotherapy.RESULTS:The follow-up period ranged from 8 to 79mo,with four cases of recurrence and no deaths.Through PETCT examination,two patients exhibited both local ocular metabolic elevation and systemic metastasis,and one of these patients had cervical lymph node metastasis,while the other had submandibular and parotid gland metastasis.Nine patients showed only local ocular metabolic elevation,while 10 patients had no abnormal metabolic activity locally.CONCLUSION:PET-CT examination plays a crucial role in detecting residual lesions and recurrence following tumor resection,aiding in precise disease staging,and facilitating the development of personalized treatment plans,ultimately improving patient prognosis.

Frequency, localization, and types of gastrointestinal stromal tumor-associated neoplasia

BACKGROUND In recent years,increasing evidence of second neoplasms associated with gastrointestinal stromal tumors(GIST)has been found.Numerous case reports,mostly retrospective studies and a few reviews,have been published.To our knowledge,however,no systematic review or meta-analysis of the existing data has been performed so far.AIM To prepare a compilation,as complete as possible,of all reported second tumor entities that have been described in association with GIST and to systematically analyze the published studies with regard to frequency,localization,and types of GIST-associated neoplasms.METHODS The MEDLINE and EBSCO databases were searched for a combination of the keywords GIST/secondary,synchronous,coincident/tumor,neoplasm,and relevant publications were selected by two independent authors.RESULTS Initially,3042 publications were found.After deletion of duplicates,1631 remained,and 130 papers were selected;22 of these were original studies with a minimum of 20 patients,and 108 were case reports.In the 22 selected studies,comprising a total number of 12050 patients,an overall rate of GIST-associated neoplasias of 20%could be calculated.Most second neoplasias were found in the gastrointestinal tract(32%)and in the male and female urogenital tract(30%).The specific risk scores of GISTs associated with other tumors were significantly lower than those without associated neoplasias.CONCLUSION In this first systematic review,we could confirm previously reported findings of a more than coincidental association between GIST and other neoplasias.The question whether there is an underlying causal association will need further investigation.Our data suggest that even GIST with a very low risk of disease progression should prompt screening for second neoplasia and subsequent frequent controls or extended staging.

Epilepsy associated tumors: Review article

Long-term epilepsy associated tumors(LEAT) represent a well known cause of focal epilepsies. Glioneuronaltumors are the most frequent histological type consisting of a mixture of glial and neuronal elements and most commonly ariseing in the temporal lobe. Cortical dysplasia or other neuronal migration abnormalities often coexist. Epilepsy associated with LEAT is generally poorly controlled by antiepileptic drugs while, on the other hand, it is high responsive to surgical treatment. However the best management strategy of tumor-related focal epilepsies remains controversial representing a contemporary issues in epilepsy surgery. Temporo-mesial LEAT have a widespread epileptic networkwith complex epileptogenic mechanisms. By using an epilepsy surgery oriented strategy LEAT may have an excellent seizure outcome therefore surgical treatment should be offered early, irrespective of pharmacoresistance, avoiding both the consequences of uncontrolled seizures as well as the side effects of prolonged pharmacological therapy and the rare risk of malignant transformation.

Comparisons of voided urine cytology, nuclear matrix protein-22 and bladder tumor associated antigen tests for bladder cancer of geriatric male patients in Taiwan, China

Aim： To compare the results of bladder tumor associated antigen （BTA TRAK）, nuclear matrix protein 22 （NMP 22） and voided urine cytology （VUC） in detecting bladder cancer. Methods： A total of 135 elderly male and 50 healthy volunteers enrolled in this study were classified into three groups： （i） 93 patients with bladder cancer; （ii） 42 patients with urinary benign conditions; and （iii） 50 healthy volunteers. BTA TRAK and NMP 22 kits were used to detect bladder cancer. Voided urine cytology was used to compare the sensitivity and specificity of the screening tests. Results： The sensitivity and specificity of cytology, BTA TRAK and NMP 22 were 24% and 97%, 51% and 73%, 78% and 73%, respectively. The level of NMP 22 increased with tumor grading. The BTA TRAK kit has the lowest sensitivity among the screening tests. The NMP 22 with the best sensitivity can be an adjunct to cytology for evaluating bladder cancer. Conclusion： The NMP 22 test has a better correlation with the grading of the bladder cancer than BTA TRAK. As cytology units are typically not available in hospitals or in outpatient clinics, NMP 22 might be a promising tool for screening bladder cancer.

Cancer-associated fibroblasts in digestive tumors

The significant influence of tumor stroma on malignant cells has been extensively investigated in this era of targeted therapy. The tumor microenvironment, as a dynamic system, is orchestrated by various cells including tumor vascular composing cells, inflammatory cells and fibroblasts. As a major and important component in tumor stroma, increasing evidence has shown that spindle-shaped cancer-associated fibroblasts (CAFs) are a significant modifier of cancer evolution, and promote tumorigenesis, tumor invasion and metastasis by stimulating angiogenesis, malignant cell survival, epithelial-mesenchymal transition (EMT) and proliferation via direct cell-to-cell contact or secretion of soluble factors in most digestive solid tumors. CAFs are thought to be activated, characterized by the expression of α-smooth muscle actin, fibroblast activated protein, fibroblast specific protein, vimentin, fibronectin, etc. They are hypothesized to originate from normal or aged fibroblasts, bone marrow-derived mesenchymal cells, or vascular endothelial cells. EMT may also be an important process generating CAFs, and most probably, CAFs may originate from multiple cells. A close link exists between EMT, tumor stem cells, and chemo-resistance of tumor cells, which is largely orchestrated by CAFs. CAFs significantly induce immunosuppression, and may be a prognostic marker in various malignancies. Targeted therapy toward CAFs has displayed promising anticancer efficacy, which further reinforces the necessity to explore the relationship between CAFs and their hosts.

Tumor-associated macrophages regulate gastric cancer cell invasion and metastasis through TGFβ2/NF-κB/Kindlin-2 axis

Objective: Recent studies have shown that tumor-associated macrophages(TAMs) play an important role in cancer invasion and metastasis. Our previous studies have reported that TAMs promote the invasion and metastasis of gastric cancer(GC) cells through the Kindlin-2 pathway. However, the mechanism needs to be clarified.Methods: THP-1 monocytes were induced by PMA/interleukin(IL)-4/IL-13 to establish an efficient TAM model in vitro and M2 macrophages were isolated via flow cytometry. A dual luciferase reporter system and chromatin immunoprecipitation(Ch IP) assay were used to investigate the mechanism of transforming growth factor β2(TGFβ2) regulating Kindlin-2 expression. Immunohistochemistry was used to study the relationships among TAM infiltration in human GC tissues, Kindlin-2 protein expression, clinicopathological parameters and prognosis in human GC tissues. A nude mouse oncogenesis model was used to verify the invasion and metastasis mechanisms in vivo.Results: We found that Kindlin-2 expression was upregulated at both m RNA and protein levels in GC cells cocultured with TAMs, associated with higher invasion rate. Kindlin-2 knockdown reduced the invasion rate of GC cells under coculture condition. TGFβ2 secreted by TAMs regulated the expression of Kindlin-2 through the transcription factor NF-кB. TAMs thus participated in the progression of GC through the TGFβ2/NF-κB/Kindlin-2 axis. Kindlin-2 expression and TAM infiltration were significantly positively correlated with TNM stage, and patients with high Kindlin-2 expression had significantly poorer overall survival than patients with low Kindlin-2 expression. Furthermore, Kindlin-2 promoted the invasion of GC cells in vivo.Conclusions: This study elucidates the mechanism of TAMs participating in GC cell invasion and metastasis through the TGFβ2/NF-κB/Kindlin-2 axis, providing a possibility for new treatment options and approaches.

Identifi cation of the differential expressive tumor associated genes in rectal cancers by cDNA microarray

AIM： To identify tumor associated genes of rectal cancer and to probe the application possibility of gene expression profiles for the classification of tumors.METHODS： Rectal cancer tissues and their paired normal mucosa were obtained from patients undergoing surgical resection of rectal cancer. Total RNA was extracted using Trizol reagents. First strand cDNA synthesis was indirectly labeled with aminoallyl-dUTP and coupled with Cy3 or Cy5 dye NHS mono-functional ester. After normalization to total spots, the genes which background subtracted intensity did not exceed 2 SD above the mean blank were excluded. The data were then sorted to obtain genes differentially expressed by ≥ 2 fold up or down in at least 5 of the 21 patients.RESULTS： In the 21 rectal cancer patients, 23 genes were up-regulated in at least 5 samples and 15 genes were down-regulated in at least 5 patients. Hierachical cluster analysis classified the patients into two groups according to the clinicopathological stage, with one group being all above stage Ⅱ and one group all below stage Ⅱ.CONCLUSION： The up-regulated genes and downregulated genes may be molecular markers of rectal cancer. The expression profiles can be used for classification of rectal cancer.

Clinical usefulness of endoscopic ultrasonography for the evaluation of ulcerative colitis-associated tumors

AIM:To evaluate the clinical usefulness of endoscopic ultrasonography(EUS) for the diagnosis of the invasion depth of ulcerative colitis-associated tumors.METHODS:The study group comprised 13 patients with 16 ulcerative colitis(UC)-associated tumors for which the depth of invasion was preoperatively estimated by EUS.The lesions were then resected endoscopically or by surgical colectomy and were examined histopathologically.The mean age of the subjects was 48.2 ± 17.1 years,and the mean duration of UC was 15.8 ± 8.3 years.Two lesions were treated by endoscopic resection and the other 14 lesions by surgical colectomy.The depth of invasion of UCassociated tumors was estimated by EUS using an ultrasonic probe and was evaluated on the basis of the deepest layer with narrowing or rupture of the colonic wall.RESULTS:The diagnosis of UC-associated tumors by EUS was carcinoma for 13 lesions and dysplasia for 3 lesions.The invasion depth of the carcinomas was intramucosal for 8 lesions,submucosal for 2,the muscularis propria for 2,and subserosal for 1.Eleven(69%) of the 16 lesions arose in the rectum.The macroscopic appearance was the laterally spreading tumor-non-granular type for 4 lesions,sessile type for 4,laterally spreading tumor-granular type for 3,semipedunculated type(Isp) for 2,type 1 for 2,and type 3 for 1.The depth of invasion was correctly estimated by EUS for 15 lesions(94%) but was misdiagnosed as intramucosal for 1 carcinoma with high-grade submucosal invasion.The 2 lesions treated by endoscopic resection were intramucosal carcinoma and dysplasia,and both were diagnosed as intramucosal lesions by EUS.CONCLUSION:EUS provides a good estimation of the invasion depth of UC-associated tumors and may thus facilitate the selection of treatment.

Colorectal cancer vaccines: Tumor-associated antigens vs neoantigens

Therapeutic options for the treatment of colorectal cancer(CRC) are diverse but still not always satisfying. Recent success of immune checkpoint inhibition treatment for the subgroup of CRC patients suffering from hypermutated tumors suggests a permanent role of immune therapy in the clinical management of CRC. Substantial improvement in treatment outcome could be achieved by development of efficient patient-individual CRC vaccination strategies. This mini-review summarizes the current knowledge on the two general classes of targets: tumor-associated antigens(TAAs) and tumorspecific antigens. TAAs like carcinoembryonic antigen and melanoma associated antigen are present in and shared by a subgroup of patients and a variety of clinical studies examined the efficacy of different TAA-derived peptide vaccines. Combinations of several TAAs as the next step and the development of personalized TAA-based peptide vaccines are discussed. Improvements of peptidebased vaccines achievable by adjuvants and immunestimulatory chemotherapeutics are highlighted. Finally, we sum up clinical studies using tumor-specific antigens-in CRC almost exclusively neoantigens-which revealed promising results; particularly no severe adverse events were reported so far. Critical progress for clinical outcomes can be expected by individualizing neoantigen-based peptide vaccines and combining them with immunestimulatory chemotherapeutics and immune checkpoint inhibitors. In light of these data and latest developments, truly personalized neoantigen-based peptide vaccines can be expected to fulfill modern precision medicine's requirements and will manifest as treatment pillar for routine clinical management of CRC.

Polarization of M1 tumor associated macrophage promoted by the activation of TLR3 signal pathway

Objective: To investigate the correlation between activation of toll-like receptors 3(TLR3) signaling pathway and tumor-associated macrophage and its effect on the tumor growth. Methods: The mice Lewis lung cancer cell lines 3LL and melanoma B16H10 were used to construct the subcutaneous transplantation tumor models and then they were treated with Poly-ICLC. The curative effect was observed and then the T cell and macrophage phenotypes infiltrated in local tumor were detected by flow cytometry. After the in vitro culture of mouse bone marrow-derived macrophage, the real-time PCR and western blot were applied to detect the expression of macrophage activation markers and the activation of intracellular signaling pathways. Results: The survival time of mice with brown tumor treated with Poly-ICLC significantly increased and the tumor growth was inhibited. The ratio of local tumor-infiltrated Treg decreased, while the ratio of CD8+ T cell increased significantly. The macrophages surface CD206 expression was down-regulated while the expression of i NOS increased. The Poly-ICLC could promote the expression of M1 markers(IL-1毬, TNF-α毩 and i NOS) in bone marrow-derived macrophage and inhibited the expression of M2 molecules(Arg-1, YM-1 and CD206). The phosphorylation level of downstream p65, TBK1 and IRF3 increased significantly. Conclusions: The Poly-ICLC can activate the TLR3 downstream signaling pathway to induce a M1 polarization of tumor associated macrophage, thereby inhibiting the tumor growth.

Promotion of Sema4D expression by tumor-associated macrophages: Significance in gastric carcinoma

AIM To study the role of semaphorin 4 D(Sema4 D) expression promoted by tumor-associated macrophages(TAMs) in gastric carcinoma cells and its clinical significance in the invasion and metastasis of gastric carcinoma.METHODS CD68 and Sema4 D expression was analyzed in gastric carcinoma and adjacent normal tissues from 290 patients using the immunohistochemical streptavidinperoxidase method, and their relationships with clinicopathological features were evaluated. Human M2 macrophages were induced in vitro and co-cultured in non-contact with gastric carcinoma SGC-7901 cells. Changes in the secretory Sema4 D level in the SGC-7901 cell supernatant were measured using an enzymelinked immunosorbent assay. The effects of TAMs on SGC-7901 cell invasion and migration were assessed with invasion and migration assays, respectively.RESULTS CD68 and Sema4 D protein expression was significantly higher in gastric carcinoma tissues than in adjacent normal tissues(71.7% vs 33.8% and 74.5% vs 42.8%, respectively; P < 0.01). CD68 and Sema4 D protein expression was significantly associated with histological differentiation, TNM stage, and lymph node metastasis(P < 0.05), and their expression levels were positively correlated with one another(r = 0.467, P < 0.01). In the in vitro experiment, secretory Sema4 D protein expression was significantly increased in the supernatant of SGC-7901 cells co-cultured with TAMs compared with the blank control(1224.13 ± 29.43 vs 637.15 ± 33.84, P < 0.01). Cell invasion and metastasis were enhanced in the Transwell invasion and migration assays(P < 0.01).CONCLUSION TAMs promote the invasion and metastasis of gastric carcinoma cells possibly through upregulated secretory Sema4 D protein expression. Combined detection of TAM markers, CD68 and Sema4 D, in gastric carcinoma tissue shows potential to predict the trend of gastric carcinoma progression.

Clinical significance of tumor-associated macrophage infiltration in supraglottic laryngeal carcinoma

Tumor-associated macrophages(TAMs) can elicit contrasting effects on tumor progression,depending on different tumor microenvironment.This study aimed to explore the correlation between TAM infiltration and clinicopathologic characteristics,metastasis,and prognosis of supraglottic laryngeal carcinoma.TAMs in intratumoral and peritumoral regions of 84 specimens of supraglottic laryngeal carcinoma tissues were detected by immunohistochemical staining with monoclonal CD68 antibody.The density of peritumoral CD68+ TAMs in recurrence cases(9/11) and in dead cases(17/23) were significantly higher than those in non-recurrence cases(33/73) and in survival cases(25/61),with significant differences(P = 0.024 and 0.007,respectively).The Kaplan-Meier survival analysis showed a significant relationship between the infiltration of both intratumoral and peritumoral CD68 + TAMs and the overall survival of patients.The 5year survival rate was significantly lower in the group with a high density of intratumoral CD68+ TAMs than in the group with a low density(39.6% vs.82.5%,P < 0.05).Similarly,the 5-year survival rate was significantly lower in the group with a high density of peritumoral CD68+ TAMs than in the group with a low density(50.6% vs.73.1%,P < 0.05).Cox regression analysis revealed that T classification,distant metastasis,and intratumoral or peritumoral CD68 + TAMs were independent factors for disease-free survival,whereas T classification and intratumoral CD68 + TAMs were independent factors for overall survival.The results indicate that TAM infiltration in supraglottic laryngeal carcinoma can be used to predict metastasis and prognosis and is an independent factor for prognosis.

Update on a tumor-associated NADH oxidase in gastric cancer cell growth

Gastric cancer is one of the most common human malignancies, and its prevalence has been shown to be well-correlated with cancer-related deaths worldwide. Regrettably, the poor prognosis of this disease is mainly due to its late diagnosis at advanced stages after the cancer has already metastasized. Recent research has emphasized the identification of cancer biomarkers in the hope of diagnosing cancer early and designing targeted therapies to reverse cancer progression. One member of a family of growth-related nicotinamide adenine dinucleotide(NADH or hydroquinone) oxidases is tumor-associated NADH oxidase(t NOX; ENOX2). Unlike its counterpart CNOX(ENOX1), identified in normal rat liver plasma membranes and shown to be stimulated by growth factors and hormones, t NOX activity purified from rat hepatoma cells is constitutively active. Its activity is detectable in the sera of cancer patients but not in those of healthy volunteers, suggesting its clinical relevance. Interestingly, t NOX expression was shown to be present in an array of cancer cell lines. More importantly, inhibition of t NOX was well correlated with reduced cancer cell growth and induction of apoptosis. RNA interference targeting t NOX expression in cancer cells effectively restored non-cancerous phenotypes, further supporting the vital role of t NOX in cancer cells. Here, we review the regulatory role of t NOX in gastric cancer cell growth.

Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages

Background： Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages （TAMs） and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors （TLRs） are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology. Methods： To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytoldnes in lung cancer cells, we established an in vitro coculture system using TAMs and human non- small cell lung cancer （NSCLC） cell line SPC-A1. Levels of interleukin （IL）-113, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-Al were further confirmed by RT-PCR and western blot. The level changes of IL-1β, IL-6 and IL-8 in SPC-Al were also detected after the stimulation of TLRs agonists. Results： We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-β-acetate （PMA）. Higher mRNA and supernate secretion levels of IL-1β, IL-6 and IL-8 were detected in SPC-A1 after being eocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1β, IL-6 and IL-8. Conclusions： These findings demonstrated that TAMs may enhance IL-1β, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.

HCA520, A NOVEL TUMOR ASSOCIATED ANTIGEN, INVOLVED IN CELL PROLIFERATION AND APOPTOSIS

Objective: Tumor associated antigen encoding gene HCA520 (AF146019) was identified by screening a human hepatocellular carcinoma expressing cDNA library using SEREX technique. In this experiment we studied the effect of HCA520 on cell proliferation and apoptosis. Methods: Gene HCA520 was gained by PCR and transfected into 293 cells. The stable expression cells were obtained by G418 selection. The cell proliferation was measured by [3H]-TdR uptake and apoptosis assay was measured by FACS. Results: Eukaryotic expression plasmid pcDNA3-HCA520 was constructed and its stable transfectants were obtained. Overexpression of HCA520 inhibited the cell proliferation and enhanced cell apoptosis after serum deprivation. Conclusion: HCA520 is a novel tumor associated antigen that can affect cell proliferation and apoptosis.

Combretastatin A-1 phosphate, a microtubulin inhibitor, induces hepatocellular carcinoma cells and tumor-associated macrophage apoptosis by uncoupling tubulin and p-AKT

Combretastatin A-1 phosphate （CA1P） is a tubulin polymerization inhibitor that binds to the colchicine- binding site of tubulin and shows potential anti-tumor activity to acute myelocytic leukemia as reported. We demon- strated that CA1P also showed an outstanding anti-cancer effect on hepatocellular carcinoma （HCC） in vivo and in vitro. As determined by DCFH-DA dye and Western blot, CA1P induced ROS accumulation and apoptosis in HepG2 cells with the down-regulation of Mcl-1. Additonal western blot and immunofluorescence assays further indi- cated that CA1P inhibited Wnt/β-catenin pathway through GSK-3β activition with an increasing of Mcl phosphoryl- ation and subsequent degradation mediated by tubulin-dynactin p l50-AKT signaling pathway axis. Apoptosis of HepG2 cells induced by CA1P was reversed by the GSK-3β inhibitor （ CHIR-99021 ）. Furthermore, determined by immunohistochemistry of an orthotopic HCC tumor model, CA1P showed a significantly effect on tumor associated macrophage （TAM） apoptosis in vitro and eliminated TAM in tumor microenviroment in vivo, while the infiltration of Treg cells and expression of TGF-β were also altered. Adoptive transfer of macrophages reinstated tumor growth treated by CA1P. These results indicated that CA1P presented potent potential on the regulation of hepatocellular carcinoma cells and TAMs, and also revealed a novel anti-HCC mechanism of CA1P, which acted on both cancer cells and tumor microenvironment. The findings would be beneficial for exploring new application of anti-microtubu- lar drugs on oncotherapy.