Brain metastasis was a common metastasis site and leading cause of death in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors had improved survival of NSCLC patients with positive drive gene. It also brings good news to NSCLC patients with positive drive gene and brain metastases. However, there is still no effective treatment for NSCLC patients with drive gene-negative and brain metastases. In recent years, immunotherapy has made breakthrough progress and become important first and second line treatment options of NSCLC especially in patients with drive gene-negative. The role of immunotherapy in specific populations of NSCLC-brain metastasis patients, especially drive gene-negative patients has become the focus of attention. In this report, we review the research progress of immunotherapy in NSCLC with brain metastases, especially in driver-negative patients, analyze the limitations of existing research and future challenge.
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Brain Neoplasms ; immunology ; secondary ; therapy ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Humans ; Immunotherapy ; methods ; Lung Neoplasms ; genetics ; pathology ; Patient Selection

Cancer cell vaccine-based immunotherapy has received increasing interest in many clinical trials involving patients with breast cancer. Combining with appropriate adjuvants can enhance the weak immunogenic properties of tumor cell lysates (TCL). In this study, diphtheria toxin (DT) and two tandem repeats of mycobacterial heat shock protein 70 (mHSP70) fragment 407-426 (M2) were conjugated to TCL with glutaraldehyde, and the constructed cancer cell vaccine was named DT-TCL-M2. Subcutaneous injection of DT-TCL-M2 in mice effectively elicited tumor-specific polyclonal immune responses, including humoral and cellular immune responses. High levels of antibodies against TCL were detected in the serum of immunized mice with ELISA and verified with Western blot analyses. The splenocytes from immunized mice showed potent cytotoxicity on Ehrlich ascites carcinoma cells. Moreover, the protective antitumor immunity induced by DT-TCL-M2 inhibited tumor growth in a mouse breast tumor model. DT-TCL-M2 also attenuated tumor-induced angiogenesis and slowed tumor growth in a mouse intradermal tumor model. These findings demonstrate that TCL conjugated with appropriate adjuvants induced effective antitumor immunity in vivo. Improvements in potency could further make cancer cell vaccines a useful and safe method for preventing cancer recurrence after resection.
Adjuvants, Immunologic ; Animals ; Bacterial Proteins ; genetics ; immunology ; Cancer Vaccines ; immunology ; Carcinoma, Ehrlich Tumor ; immunology ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Diphtheria Toxin ; genetics ; immunology ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Immunoglobulin G ; immunology ; Immunotherapy ; Mice ; Neovascularization, Pathologic ; Peptide Fragments ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tandem Repeat Sequences

OBJECTIVETo construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice.

METHODSFifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry.

RESULTSComparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups.

CONCLUSIONSOur results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.

Animals ; Carcinoma, Hepatocellular ; blood ; pathology ; therapy ; Cell Line, Tumor ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Immunotherapy ; Interferon-gamma ; blood ; Interleukin-2 ; blood ; Interleukins ; genetics ; Killer Cells, Natural ; immunology ; Liver Neoplasms ; blood ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Plasmids ; therapeutic use ; Random Allocation ; Recombinant Proteins ; therapeutic use ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Burden

OBJECTIVETo prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.

METHODSTo construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2.

RESULTSThe pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05).

CONCLUSIONSGPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

Cancer Vaccines ; immunology ; Carcinoma, Transitional Cell ; immunology ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-2 ; metabolism ; Melanoma-Specific Antigens ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Urinary Bladder Neoplasms ; immunology ; metabolism ; pathology

OBJECTIVETo investigate apoptosis of tumor infiltrating dendritic cells (TIDC) and their expression of Fas/FasL (CD95/CD95L) in human endometrioid adenocarcinoma.

METHODSThe apoptotic rate of TIDC was measured in 45 cases of endometrioid adenocarcinoma and 20 cases of normal endometrium tissues (control) by double-label immunohistochemistry using the monoclonal antibody S-100 protein and TUNEL technique. The expressions of Fas and FasL in TIDCs were detected using double-label immunohistochemistry and imaging analysis.

RESULTSThe apoptotic rate of TIDCs in endometrioid adenocarcinoma were significantly higher than that in normal endormetrium [(13.02∓0.64)% vs (6.82∓0.53)%, P<0.05]. The expression levels of Fas in the TIDCs were significantly lower, whereas FasL expression significantly higher in endometrioid adenocarcinoma than in normal endormetrium (7.88∓1.05 vs 19.25∓3.03, P<0.05; 12.95∓2.25 vs 7.51∓1.14, P<0.05).

CONCLUSIONIncreased apoptosis of the TIDCs and abnormal expression of Fas/FasL in TIDCs in endometrioid adenocarcinoma may lead to tumor immune escape.

Apoptosis ; physiology ; Carcinoma, Endometrioid ; immunology ; metabolism ; pathology ; Case-Control Studies ; Dendritic Cells ; immunology ; Endometrial Neoplasms ; immunology ; metabolism ; pathology ; Fas Ligand Protein ; genetics ; metabolism ; Female ; Humans ; Lymphocytes, Tumor-Infiltrating ; immunology ; Tumor Escape ; fas Receptor ; genetics ; metabolism

OBJECTIVEDendritic cell vaccines are one of the important active immunotherapies for neoplasms. The aim of this study was to observe the killing effect of specific cytotoxic T lymphocytes (CTL) on liver carcinoma HepG2 and SMMC-7721 cells in vitro. The CTL was induced by human peripheral blood mononuclear cells-originated dendritic cells (DC) transfected by recombinant adeno-associated virus (rAAV) with hAFP gene fragment (137-145).

METHODSImmature DCs were generated from peripheral blood mononuclear cells of healthy volunteers and then transfected by rAAV with AFP gene fragment. The CTL was thereafter induced. The activities of DC and CTL were measured and the killing effect of the CTL on HepG2 cells was detected using M1Tr assay.

RESULTSThe mature DC, transfected or not, highly expressed CD40, CD86 and IL-12. IFN-gamma was highly expressed in the CTL. The DC-induced CTL could effectively recognize and destroy the HepG2 and SMMC-7721 cells.

CONCLUSIONDC transfected by rAAV can stimulate the proliferation and differentiation of lymphocytes and also induce the proliferation of CTL, and their own phenotypes are not significantly altered. The DC vaccine can be effectively used as an adjuvant immunotherapy for patients with hepatocellular carcinoma.

B7-2 Antigen ; metabolism ; CD40 Antigens ; metabolism ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; immunology ; metabolism ; Dependovirus ; genetics ; Hep G2 Cells ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; Peptide Fragments ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; pathology ; Transfection ; alpha-Fetoproteins ; genetics

OBJECTIVETo study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein.

METHODSGolph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5a. The expression of 6xHis and Golph2 fusion protein was induced by isopropylthio-beta-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose. The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively. Sandwich ELISA was used to detect the level of serum Golph2 protein.

RESULTSThere were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM177937 in Genbank, including 644(T-->C, L-->P) , 970 (G-->A, V-->I) and 802 G deletion. The sequence was completely reversed by PCR. The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value is more than or equal to 0.40.

CONCLUSIONThe polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.

Animals ; Antibodies, Monoclonal ; analysis ; immunology ; Base Sequence ; Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Cell Line, Tumor ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Membrane Proteins ; biosynthesis ; blood ; genetics ; immunology ; metabolism ; Molecular Sequence Data ; Polymerase Chain Reaction ; Rabbits

OBJECTIVETo investigate effect of tumor-specific T cell receptor gene transfection on memory T cell differentiation in vitro.

METHODSTCRVbeta7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vbeta7.1 was detected by flow cytometry before and after the transfection. Memory T cell differentiation was induced by stimulation with the hepatocarcinoma cell line BEL-7402 in vitro. The expression of surface molecules CD45RO, CD45RA and CCR7 was analyzed by flow cytometry to identify the phenotype and subsets of the memory T cells. Fluorescence-activated cell sorting was performed to detect the apoptosis of the tumor cells, and enzyme-linked immunoabsorbent assay was used to determine the production of interferon-gamma (IFN-gamma) for assessing the immune function of the memory T cells.

RESULTSFlow cytometry showed that TCRVbeta7.1 gene was efficiently expressed after transfection. After stimulation by the tumor cells in vitro, the expression of CD45RO in TCRVbeta7.1 gene-modified T cells increased gradually, and analysis of the coexpression of CD45RA and CCR7 revealed that the effector memory T cells constituted the majority of the differentiated memory T cells. The apoptotic rate of the tumor cells induced by the T cells increased significantly with also obviously increased INF-gamma secretion in the memory T cells.

CONCLUSIONTumor-specific TCRVbeta7.1 gene transfection can promote the differentiation of the memory T cells, the majority of which belongs to effector memory T cells that perform immune functions by inducing apoptosis and cytokine secretion.

Adult ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Genes, T-Cell Receptor alpha ; genetics ; Humans ; Immunologic Memory ; immunology ; Interferon-gamma ; metabolism ; Leukocyte Common Antigens ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Transfection

Antibodies, Neoplasm ; blood ; immunology ; Carcinoma, Hepatocellular ; pathology ; Cell Differentiation ; Female ; Humans ; Liver Neoplasms ; pathology ; Male ; Tumor Suppressor Protein p53 ; blood ; genetics ; immunology

OBJECTIVETo study the clinicopathologic features and immunophenotype of renal cell carcinomas, and to discuss their diagnostic value.

METHODSThe clinicopathologic features of 114 cases of renal cell carcinoma were reviewed and categorized on the basis of 2004 WHO classification. Immunohistochemical study for a panel of antibodies (including CK, CD10, vimentin, CD117, AMACR, CK7 and TFE3) was carried out. The follow-up data, if available, were also analyzed.

RESULTSThe cases were reclassified into 5 subtypes, including 77 cases (67.5%) of clear cell carcinoma (CCRCC), 11 cases (9.6%) of papillary renal cell carcinoma (PRCC), 14 cases (12.3%) of chromophobe renal cell carcinoma (chrRCC), 10 cases (8.8%) of renal carcinoma associated with Xp11.2 translocations/TFE3 gene fusions (Xp11.2RCC) and 2 cases (1.8%) of unclassified renal cell carcinoma (unRCC). Immunohistochemical study showed that the expression rates of CK, CD10 and vimentin in CCRCC were 93.5% (72/77), 93.5% (72/77) and 75.3% (58/77), respectively. On the other hand, all the 11 cases of PRCC studied were positive for AMACR. The expression rate of CD117 in chrRCC was 78.5% (11/14). In the 10 cases of Xp11.2 RCC studied, the expression rates of TFE3, AMACR, CD10 and CK were 100% (10/10), 100% (10/10), 90% (9/10) and 70% (7/10), respectively.

CONCLUSIONSThe various subtypes of renal cell carcinomas are heterogeneous in histologic appearance and demonstrate distinctive immunophenotype. The expressions of CD10, vimentin, CD117, AMACR, CK7 and TFE3 are helpful in the differential diagnosis.

Adenocarcinoma, Clear Cell ; pathology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; immunology ; metabolism ; Biomarkers, Tumor ; analysis ; genetics ; Carcinoma, Papillary ; immunology ; pathology ; Carcinoma, Renal Cell ; immunology ; metabolism ; pathology ; Female ; Gene Fusion ; Humans ; Immunophenotyping ; Kidney Neoplasms ; immunology ; metabolism ; pathology ; Male ; Middle Aged ; Neprilysin ; analysis ; Racemases and Epimerases ; analysis ; genetics ; Translocation, Genetic ; Vimentin ; analysis ; World Health Organization ; Young Adult