Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells

Summary Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i. p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.     

Critical factors for liposome-incorporated tumour-associated antigens to induce protective tumour immunity to SL2 lymphoma cells in mice

Physical and immunogenic properties of reconstituted membranes designed for the presentation of tumour-associated antigens (TAA) to the immune system are described. Proteins and lipids of crude membranes of SL2 murine lymphosarcoma cells were partially solubilized with octylglucoside. Reconstituted membranes, consisting mainly of unilamellar vesicles with a diameter of 0.03–0.15 μm, were formed by detergent removal and were purified by floatation in a discontinuous sucrose gradient to remove non-lipid-bound protein. Subcutaneous immunization of syngeneic mice with reconstituted membranes or with purified reconstituted membranes induced protection against an intraperitoneal challenge with 10³ viable SL2 cells. Reconstituted membranes were more immunogenic than crude membranes in immunoprotection experiments when compared on the basis of protein dose. Detergent removal was required to obtain an immunogenic presentation form of SL2 membrane antigens and to avoid toxicity associated with the detergent. Reconstitution of SL2 membranes in the presence of exogenous phospholipid slightly increased the fraction of protein that associated with the reconstituted membranes. However, the immunogenicity of the solubilized membrane TAA was not significantly affected by the presence of exogenous phospholipid. The reconstitution procedure described may be useful in identifying membrane factors required for the induction of immune responses against TAA. The versatility of the system may be employed to develop safe alternatives for whole-cell vaccines.     

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF,VEGF and PDGF Signaling

MethodsIn vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl₄), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl₄, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.     

Tumoristatic Activity of Liposomes Reconstituted from Tumor Associated Antigens and Interleukin 2 for Immunotherapy of Fibrosarcomas in BALB/C MICE

Abstract

Human Interleukin 2 (IL 2) was entrapped into liposomes prepared from egg lecithin and plasma membranes of fibrosarcoma cells, including tumor associated antigens (TAA). The animal model was the Balb/c mouse. The efficiency of interleukin incorporation by the dehydration/rehydration technique was 51% measured with radiolabelled IL 2. The inhibition of tumor growth by the TAA-presenting liposomes was studied in syngeneic mice which had received the tumor subcutaneously. Peritumor injections of liposomes containing both IL 2 and TAA inhibited the tumor growth significantly.- With lower doses of this special liposomal IL 2, the inhibition was more pronounced than with IL 2 alone. In addition, systemic therapeutic effects on contralateral untreated tumors were discernible.     

The tumor-associated antigen 90K/Mac-2-binding protein secreted by human colon carcinoma cells enhances extracellular levels of promatrilysin and is a novel substrate of matrix metalloproteinases-2, -7 (matrilysin) and -9: Implications of proteolytic cleavage

Background

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein is expressed at elevated level in cancerous tissues and associated with poor prognosis. Since TAA90K has been implicated in the restructuring of the extracellular matrix, we examined the functional relationship between colon cancer cell-derived TAA90K and the matrix metalloproteinase (MMP) promatrilysin (proMMP-7), and also tested whether TAA90K is a novel substrate for MMPs-2, -7 and -9.

Methods

The effect of TAA90K on proMMP-7 levels in HT-29 conditioned media was quantified by enzyme-linked immunosorbent assays. Binding of TAA90K to MMPs, extracellular matrix proteins and galectin-3 was measured by solid-phase binding assays. Proteolytic cleavage of TAA90K by MMPs was documented by SDS-PAGE and protein sequencing analysis.

Results

TAA90K enhanced extracellular levels of proMMP-7 in HT-29 cells. In addition, TAA90K was cleaved by MMPs-2, -7 and -9. MMP-7-mediated cleavage of TAA90K did not affect its binding to MMP-7, laminin-1, collagen IV and galectin-3 but reduced its interaction with fibronectin and laminin-10, and lowered the levels of proMMP-7 in the HT-29 medium.

Conclusion

TAA90K is a novel substrate for MMPs-2, -7 and -9 and modulates proMMP-7 levels in colon cancer cells.

General significance

Proteolytic cleavage of TAA90K may have functional implications in colon cancer.     

Expression and association of IL-21, FBXL20 and tumour suppressor gene PTEN in laryngeal cancer

ObjectiveTo study the expression of three genes IL-21, FBXL20 and tumour suppressor gene PTEN in laryngeal cancer; analyse the differences in their expression in laryngeal cancer and adjacent tissues; by using pEGFP-N1-IL21 and pGPU/GFP/Neo-FBXL20 expression vectors, to analyse the characteristics in their expression in laryngeal cancer cells outside the body as well as the associations among them.MethodsThe expression of the three genes in laryngeal cancer and adjacent tissues from 30 cases and in normal laryngeal tissues from 20 healthy persons was detected with the RT-PCR; laryngeal cancer cell line (HEp-2 cells) transfection was also performed with the pEGFP-N1-IL21 and pGPU/GFP/Neo-FBXL20 expression vectors we constructed, to detect the mRNA expression of the three genes. Cell proliferation, apoptosis and cell cycle were measured by the MTT assay.ResultsThe results of RT-PCR showed that the expression of IL-21 and FBXL20 was up-regulated in laryngeal cancer, while the expression of tumour suppressor gene PTEN was significantly decreased (p < 0.01). In HEp-2 cells transfected with pGPU/GFP/Neo-IL-21 and pGPU/GFP/Neo-FBXL20 expression vectors, the mRNA expression of PTEN was restored to some extent (p < 0.05); in addition, the ability of HEp-2 cells in proliferation and invasion was also reduced.ConclusionsIL-21 and FBXL20 genes are important in the occurrence and development of laryngeal cancer; the expression of PTEN gene can suppress laryngeal cancer, and there's a certain association among IL-21, FBXL20 and PTEN.     

Decursin promotes HIF-1α proteasomal degradation and immune responses in hypoxic tumour microenvironment

BackgroundHypoxia and HIF-1α are important regulators of tumour growth and angiogenesis and could be attractive targets for cancer therapeutics. Decursin is an active compound extracted from the roots of Angelica gigas and has been shown to have potent anti-cancer and anti-angiogenic activities. However, whether decursin regulates HIF-1α activity and immune responses under hypoxic conditions is not yet understood.PurposeThe aim of this study was to identify whether decursin exhibits anti-cancer activity by targeting HIF-1α.Study designWe investigated whether decursin regulates HIF-1α protein stability and increases its degradation. In addition, we determined if decursin increases immune responses in tumour microenvironment to identify its hypoxia-associated anti-cancer activities.Materials and methodsWe performed the hypoxia-responsive element promoter–reporter assay, Western blot analysis, immune-fluorescence assay, semi-quantitative RT-PCR and ELISA for VEGF secretion, CCK-8 assay for cell proliferation, TUNEL assay for apoptosis and invasion assay in A549 human lung cancer or HCT116 human colon cancer cells. In vivo Lewis lung carcinoma (LLC) allograft mouse model was used to check tumour growth and immune responses in tumour microenvironment by immunohistochemistry analysis.ResultsWe observed that decursin inhibited HIF-1 activation under hypoxia by down-regulating the protein level of its subunit HIF-1α. It increased oxygen-dependant hydroxylation and ubiquitination of HIF-1α to promote HIF-1α degradation. Decursin also decreased mRNA expression of HIF-1α target genes. Decursin suppressed cancer cell proliferation, induced apoptosis and inhibited cancer cell invasion under hypoxia in cancer cells. In the allograft mouse tumour model, decursin reduced the hypoxic area and HIF-1α and PD-L1 expression. Infiltrating T cells (CD3+), helper T cells (CD4+) and cytotoxic (CD8+) T cells were accumulated, but regulatory T cells (Foxp3) and myeloid-derived suppressor cell-mediated immune suppressors (Arg1) were attenuated by decursin.ConclusionOur results suggest that decursin is a novel HIF-1α inhibitor that functions by promoting its proteasomal degradation and that it also helps improve T cell activation in tumour microenvironment; these findings provide new explanations about its anti-cancer and anti-angiogenic activity mechanisms.     

Exposure to the electrofusion process can increase the immunogenicity of human cells

The cellular products obtained following electrofusion (EF) of dendritic cells (DC) and tumour cells have shown promise as cancer vaccines. The immunogenicity of these preparations has been attributed to the presence of small numbers of DC-tumour hybrids and the contribution of the non-hybrid tumour cells present has received little attention. In this report, we investigated the effect of the EF process on the immunogenicity of allogeneic human cells, in particular the colorectal cell line, SW620. EF conditions were optimised to yield the maximum number of DC-SW620 hybrids co-expressing tumour associated antigen (TAA) and DC associated antigens. Exposure of SW620 to EF induced significant increases (P<0.05) in apoptosis and necrosis. Pre-exposure of SW620 to the EF buffer alone [0.3 M glucose, 0.1 mM Ca(CH₃COO)₂ and 0.5 mM Mg(CH₃COO)₂] resulted in significant increases in TAA uptake by DC during co-culture (P<0.05). DC phenotype was, however, not altered by exposure to EF treated tumour cells. In co-cultures of PBMC responders with SW620, the levels of IFN release and cytotoxic activity were significantly increased (P<0.05) by pre-exposure of the SW620 to EF. Pre-exposure of allogeneic non-T cells, the colorectal cell line Lovo and a breast cancer cell line (MCF7) to EF also significantly (P<0.05) increased the levels of IFN release by responding PBMC. These results demonstrate that the EF process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation. These findings suggest that EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells.BH and GR contributed equally as senior authors     

Ginsenoside Rh2 mitigates doxorubicin‐induced cardiotoxicity by inhibiting apoptotic and inflammatory damage and weakening pathological remodelling in breast cancer‐bearing mice

ObjectivesThere are presently a few viable ways to reduce cardiotoxicity of doxorubicin (Dox). The combination of chemotherapy agents with natural compounds delivers greater efficacy and reduces adverse effects in recent researches for cancer treatment. Here, we examined the potential effect of ginsenoside Rh2 on a Dox‐based regimen in chemotherapy treatment.Materials and MethodsHuman breast tumour (MDA‐MB‐231) xenograft nude mice, human cardiac ventricle fibroblasts, and human umbilical vein endothelial cells (HUVEC) were employed in the present study. Histology, immunohistochemistry, immunofluorescence, western blot, antibody array, and RNA‐sequencing analyses were utilized to assess the protective effect of Rh2 on cardiotoxicity induced by Dox and the underlying mechanisms.ResultsRh2‐reduced cardiotoxicity by inhibiting the cardiac histopathological changes, apoptosis and necrosis, and consequent inflammation. Pathological remodelling was attenuated by reducing fibroblast to myofibroblast transition (FMT) and endothelial–mesenchymal transition (EndMT) in hearts. RNA‐sequencing analysis showed that Dox treatment predominantly targets cell cycle and attachment of microtubules and boosted tumour necrosis, chemokine and interferon‐gamma production, response to cytokine and chemokine, and T cell activation, whereas Rh2 regulated these effects. Intriguingly, Rh2 also attenuated fibrosis via promoting senescence in myofibroblasts and reversing established myofibroblast differentiation in EndMT.ConclusionsRh2 regulates multiple pathways in the Dox‐provoked heart, proposing a potential candidate for cancer supplement and therapy‐associated cardiotoxicity.

Doxorubicin is extensively reported to induce severe cardiotoxicity in clinical applications. Our work proposed a natural herbal compound, ginsenoside Rh2, as a potential candidate for attenuating this side effect. Rh2 significantly inhibited cardiac apoptosis and necrosis, inflammation, and pathological remodelling in Dox‐challenged hearts.     

Reduced tumour progression and angiogenesis in 1,2-dimethylhydrazine mice treated with NS-398 is associated with down-regulation of cyclooxygenase-2 and decreased beta-catenin nuclear localisation

Abstract

Cyclooxygenase (COX)-2 is a key molecular target of colon cancer prevention. However, the mechanisms by which COX-2 inhibitors confer protective effects against tumour development are not completely understood. The aim of this study was to elucidate the effects of NS-398 in the 1,2-dimethylhydrazine (DMH) mouse model with respect to alteration in the expression of COX-2 and E-cadherin-catenin complex. Alterations in cell proliferation, apoptosis, and vascular density were investigated. NS-398 showed reduced COX-2 immunoreactivity in adenomas with a decrease in vascular density in non-dysplastic mucosa. Adenomas revealed increased E-cadherin and beta-catenin reactivity. NS-398 reduced the percentages of tumour cells with nuclear localisation of beta-catenin and cyclin D1. Bromodeoxyuridine (BrdUrd) index in adenomas was significantly higher in untreated animals. NS-398 resulted in significant increase in apoptosis in adenomas. Our results suggest a protective role of NS-398 on tumour development associated with reduced COX-2 expression, reduced vascular density and perturbation of beta-catenin signalling pathway.     

Modification of cell cholesterol content of rat tumour cells

Summary Among the different constituents of the cell membrane, lipids have been poorly studied with respect to their role in the immunogenicity of tumour cells and their influence on the expression on tumour-associated antigens. Since liposome-associated antigens are more potent immunogens when the lipid matrix is in a rigid state, we have modified the lipid composition of rat hepatoma cells by incorporation of cholesteryl hemisuccinate (CH) into the lipid matrix, and studied its effect on the tumorigenicity and immunogenicity of these tumour cells in syngeneic animals. A slight and significant decrease of tumorigenicity of CH-enriched D23 cells was observed when 2×10³ cells were injected SC, whereas with a higher tumour cell challenge there was no difference in the tumorigenicity of untreated or treated cells. The immunogenicity of CH-treated cells was tested by IP immunization with 10⁷ or 10⁶ cells followed 1 week later by an SC challenge with 2×10⁴ viable D23 cells. No statistical difference was observed between the immunogenicity of CH-enriched cells and that of control cells on either tumour incidence or tumour growth rate. In addition, similar experiments performed with the spontaneous mammary carcinoma SP4 showed that CH-enriched SP4 cells were of lower immunogenicity and unable to induce a significant memory immunity. This lack of effect of the CH treatment on the immunogenicity was not related to the absence of incorporation of CH, since the CH treatment increased the cell lipid rigidity as determined by the increase of fluorescence anisotropy of the diphenyl hexatriene probe. These results obtained in two weak immunogenic tumour models underlined the need for further studies before such a lipid modification of cancer cells is applied in human immunotherapy trials.Attaché de Recherche au CNRS, Fellow of the Royal Society (European Science Exchange Programm) from 1. 4. 1981 to 30. 9. 1981     

Unraveling the regulatory role of endoplasmic-reticulum-associated degradation in tumor immunity

Abstract

During malignant transformation and cancer progression, tumor cells face both intrinsic and extrinsic stress, endoplasmic reticulum (ER) stress in particular. To survive and proliferate, tumor cells use multiple stress response pathways to mitigate ER stress, promoting disease aggression and treatment resistance. Among the stress response pathways is ER-associated degradation (ERAD), which consists of multiple components and steps working together to ensure protein quality and quantity. In addition to its established role in stress responses and tumor cell survival, ERAD has recently been shown to regulate tumor immunity. Here we summarize current knowledge on how ERAD promotes protein degradation, regulates immune cell development and function, participates in antigen presentation, exerts paradoxical roles on tumorigenesis and immunity, and thus impacts current cancer therapy. Collectively, ERAD is a critical protein homeostasis pathway intertwined with cancer development and tumor immunity. Of particular importance is the need to further unveil ERAD’s enigmatic roles in tumor immunity to develop effective targeted and combination therapy for successful treatment of cancer.     

Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol

PurposeColon cancer is a major public health problem. Cannabis-based medicines are useful adjunctive treatments in cancer patients. Here, we have investigated the effect of a standardized Cannabis sativa extract with high content of cannabidiol (CBD), here named CBD BDS, i.e. CBD botanical drug substance, on colorectal cancer cell proliferation and in experimental models of colon cancer in vivo.MethodsProliferation was evaluated in colorectal carcinoma (DLD-1 and HCT116) as well as in healthy colonic cells using the MTT assay. CBD BDS binding was evaluated by its ability to displace [³H]CP55940 from human cannabinoid CB₁ and CB₂ receptors. In vivo, the effect of CBD BDS was examined on the preneoplastic lesions (aberrant crypt foci), polyps and tumours induced by the carcinogenic agent azoxymethane (AOM) as well as in a xenograft model of colon cancer in mice.ResultsCBD BDS and CBD reduced cell proliferation in tumoral, but not in healthy, cells. The effect of CBD BDS was counteracted by selective CB₁ and CB₂ receptor antagonists. Pure CBD reduced cell proliferation in a CB₁-sensitive antagonist manner only. In binding assays, CBD BDS showed greater affinity than pure CBD for both CB₁ and CB₂ receptors, with pure CBD having very little affinity. In vivo, CBD BDS reduced AOM-induced preneoplastic lesions and polyps as well as tumour growth in the xenograft model of colon cancer.ConclusionsCBD BDS attenuates colon carcinogenesis and inhibits colorectal cancer cell proliferation via CB₁ and CB₂ receptor activation. The results may have some clinical relevance for the use of Cannabis-based medicines in cancer patients.     

Hepatoprotective effects of methanolic extract of green tea against Thioacetamide-Induced liver injury in Sprague Dawley rats

Ethnopharmacological relevanceSince ancient times, herbal medicines have been applied in the treatment of cancer. Tea, derivative from the dried leaves of Camellia sinensis (L.) Kuntze plant is the most popular beverage globally after water and is available in various forms. Green tea has been expansively investigated for its beneficial properties of cancer prevention and therapy. The goal of the research: The current study was conducted to evaluate the hepaprotective character of methanolic green tea extract and its mechanism of action contrary to thioacetamide (TAA)-produced liver fibrosis of Sprague Dawley rats.Materials and MethodsThirty rodents were equally placed in 5 clusters including normal control, TAA group as a positive control, silymarin as standard drug control, and treatment groups consisting of high dose and a low dose Camellia sinensis. Rats in experimental clusters by mouth fed with C. sinensis at 250 mg/kg or 500 mg/kg daily for 2 months. After 60 days, all rats were sacrificed. Blood specimens were gathered for liver biochemical examination. Livers of all groups were dissected out and subjected to histopathological examination through the Hematoxylin and Eosin stain, Masson trichrome, and immunohistochemistry stains (PCNA). Liver tissue homogenate was also analyzed for antioxidant activity parameters.ResultsGross morphological examination showed a regular liver architecture in C. sinensis fed collections compared to the TAA sets. Histology of rat’s liver fed with C. sinensis showed an important decrease in the liver index with hepatic cells propagation, mild cellular injury, and immunostaining showed significant down-expression of proliferating cell nuclear antigen (PCNA). TAA produced liver fibrosis through a significant increase in serum alanine transferase, aspartate aminotransferase, alkaline phosphatase, and bilirubin. Total protein and albumin also decreased in the TAA group. Moreover, the reduction of antioxidant enzyme activity including superoxide dismutase and catalase as well as the increase in malondialdehyde was detected in the TAA control group. Meanwhile, an abnormal level of liver biochemical parameters was restored closer to the normal levels in serum of the C. sinensis-fed clusters. In addition, C. sinensis fed assemblies showed elevated antioxidative enzymes activity with a reduction in malondialdehyde level comparable to the levels in silymarin-treated rats.ConclusionsGreen tea potentially inhibited the progression of liver cirrhosis, down -regulation of PCNA proliferation, prevented oxidation of hepatocytes, recovered SOD and CAT enzymes, condensed MDA and reduced cellular inflammation.     

Evaluating the Association between Anesthesia Type and Postoperative Complications for Patients Receiving Total Ankle Arthroplasty

BackgroundTotal ankle arthroplasty (TAA) is performed for ankle arthritis and there has been interest investigating which anesthetic method is the best choice in order to optimize perioperative outcomes. In this study, we compared postoperative complications after TAA for patients receiving either 1) general anesthesia alone or 2) general anesthesia plus regional anesthesia.MethodsPatients undergoing primary TAA from 2007 to 2018 were identified in a national database. Patients were stratified into 2 cohorts: general anesthesia and general anesthesia combined with regional anesthesia. In this analysis, 30-day wound, cardiac, pulmonary, renal, thromboembolic, and sepsis complications, as well mortality, postoperative transfusion, urinary tract infection, extended length of stay, and reoperation were assessed. Bivariate analyses and multivariable logistical regression were performed.ResultsOf 1,084 total patients undergoing TAA, 878 patients (81.0%) had general anesthesia and 206 (19.0%) had general anesthesia combined with regional anesthesia. Following adjustment, there were no increased risk of postoperative complications in the combined general and regional anesthesia group compared to those who only underwent general anesthesia.ConclusionCompared to general anesthesia alone, the addition of regional anesthesia to general anesthesia for TAA is not associated with increased risk of complications in the perioperative period. Level of Evidence: III     

Tumor idiotype vaccines

In this study, the contribution of idiotype-positive antitumor antibodies (anti-Id) in protective tumor immunity was investigated. We have previously shown that among various anti-Id generated and typed as the internal-image Ab2 of the tumor-associated antibody (TAA) gp52, only 2F10 antibody induces protective immunity. Increase of the 2F10 idiotope in sera of tumor-bearing mice correlated with long-term survival, while in mice with short survival the circulating 2F10 idiotype decreased. 2F10⁺ Ig were purified from sera of tumor-bearing mice with longterm survival and the amount of 2F10⁺ anti-TAA antibodies was determined. Only about 3% of 2F10⁺ antibodies are 2F10⁺ anti-TAA⁺. Hybridomas were generated from a 2F10 high mouse with spontaneous tumor regression. Only 2 out of 52 tumor-specific hybridomas were 2F10⁺. These results suggest that the protective effect induced by 2F10 vaccination may not be directly mediated by 2F10⁺ antibodies but indirectly through the stimulation of a 2F10-specific cellular immune response.Supported in part by a grant from N.I.H. no. CA51434     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.     

Single‐cell sequencing reveals the heterogeneity and intratumoral crosstalk in human endometrial cancer

BackgroundEndometrial cancer (EC) is one of the most common gynecologic malignancies with increasing morbidity. Cell–cell and cell‐matrix interactions within the tumour microenvironment (TME) exert a powerful influence over the progression of EC. Therefore, a comprehensive exploration of heterogeneity and intratumoral crosstalk is essential to elucidate the mechanisms driving EC progression and develop novel therapeutic approaches.Methods4 EC and 2 normal endometrium samples were applied for single‐cell RNA sequencing (scRNA‐seq) analysis. In addition, we also included the public database to explore the clinical benefits of the single cell analysis.Results9 types of cells were identified with specific expression of maker genes. Both the malignant epithelial cells and cells comprising the immune microenvironment displayed a high degree of intertumoral heterogeneity. Notably, the proliferation T cells also showed an exhausted feature. Moreover, the malignant cells may induce an immunosuppressive microenvironment through TNF‐ICOS pair. Cancer‐associated fibroblasts (CAFs) were divided into four subsets with distinct characteristics and they maintained frequent communications with malignant cells which facilitating the progression of EC. We also found that the existence of vascular CAF (vCAF) may indicate a worse prognosis for EC patients through integrating TCGA database.ConclusionThe TME of human EC remains highly heterogeneous. Out finding that malignant cells interact closely with immune cells and vCAFs identifies potential therapeutic targets.

In order to comprehensively explore the heterogeneity and intra‐tumoral crosstalk of human endometrial cancer, we profiled 41,358 single cells of human endometrial cancer and normal endometrial tissues. The results showed that the tumour microenvironment of human ECs remains highly heterogeneity. Malignant cells interact closely with immune cells and vCAFs which may indicate potential therapeutic targets.     

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies. Financial support and conflicts of interest: This study was supported by grants from Medinet (Japan), and Suncorp Metway and Gallipoli Research Foundation (Australia). No financial or commercial interests arise from this study. Informed consent: This study was approved by Human Research Ethics Committees of the University of Queensland and Greenslopes Private Hospital and informed consent was obtained from all subjects.     

Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP⁻) or TAP1-transfected (TAP1⁺) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP⁻ and TAP1⁺ B7.1 expressing CMT.64 cells depending on the doses of γ-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1⁺ cells rejected TAP⁻ tumors, only high dose immunization with B7.1-expressing TAP⁻ cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP⁻ tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of γ-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP⁻ cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.