白细胞介素2锚定的exosomes的制备及其对膀胱癌细胞的诱导杀伤效应

目的 制备白细胞介素2(IL-2)锚定的肿瘤细胞来源的exosomes疫苗,探讨其体外诱导细胞毒性T淋巴细胞(CTL)的抗肿瘤效应.方法 构建含糖基化磷脂酰肌醇(GPI)信号肽序列与人IL-2的融合基因的pEGFP-N1-IL2gpi质粒,建立稳定表达GPI-IL-2的T24细胞系(T24/GPI-IL-2).采用超速和蔗糖梯度密度离心法提取Ex/GPI-IL-2(即T24/GPI-IL-2细胞培养液中提取的exosomes),并对其进行形态学观察和分子标志检测.通过混合淋巴细胞实验和细胞毒实验,观察Ex/GPI-IL-2对T细胞的增殖和诱导杀伤效应.结果 成功构建重组质粒pEGFP-N1-IL2gpi,建立了稳定表达GPI-IL-2蛋白的细胞系.Ex/GPI-IL-2为直径约30～90nm的类圆碟形小囊泡,表达细胞间黏附分子1(ICAM-1)、热休克蛋白70(HSP70)、MAGE-1和GPI-IL-2等免疫相关蛋白.Ex/GPI-IL-2经过树突状细胞负载后,能更有效地诱导T细胞的增殖和细胞毒效应(P＜0.05).结论 通过基因转染技术,可以将IL-2锚定到肿瘤细胞来源的exosomes上,获得的Ex/GPI-IL-2能够诱导CTL对肿瘤细胞产生更强的诱导杀伤效应,为以exosomes为基础的肿瘤免疫治疗提供了新的途径.

Abstract：

Objective To 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.Methods To 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 molecuule 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. Results The 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). Conclusions GPI-IL-2 gene-modified tumor cells can make the exosornes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder trnssitional cell tumors.     

Enhanced efficacy of therapeutic cancer vaccines produced by co-treatment with Mycobacterium tuberculosis heparin-binding hemagglutinin, a novel TLR4 agonist

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated na?ve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ, and induced T-cell-mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA(251-264)-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy.     

Distinct role of CD8 cells and CD4 cells in antitumor immunity triggered by cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir system

Immune cells can recognize tumor-associated antigens released from dead tumor cells, which elicit immune responses, potentially resulting in tumor regression. Tumor cell death induced by chemotherapy has also been reported to activate immunity. However, various studies have reported drug-induced immunosuppression or suppression of inflammation by apoptotic cells. Thus, this study aimed to investigate whether apoptotic tumor cells trigger antitumor immunity independent of anticancer treatment. Local immune responses were evaluated after direct induction of tumor cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. The inflammatory response was significantly altered at the tumor site after apoptosis induction. The expression of cytokines and molecules that activate and suppress inflammation simultaneously increased. The HSV-tk/GCV-induced tumor cell apoptosis resulted in tumor growth suppression and promoted T lymphocyte infiltration into tumors. Therefore, the role of T cells after inducing tumor cell death was explored. CD8 T cell depletion abrogated the antitumor efficacy of apoptosis induction, indicating that tumor regression was mainly dependent on CD8 T cells. Furthermore, CD4 T cell depletion inhibited tumor growth, suggesting the potential role of CD4 T cells in suppressive tumor immunity. Tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion to elucidate this immunological mechanism. Foxp3 and CTLA4, regulatory T-cell markers, decreased. Furthermore, arginase 1, an immune-suppressive mediator induced by myeloid cells, was significantly downregulated. These findings indicate that tumors accelerate CD8 T cell-dependent antitumor immunity and CD4 T cell-mediated suppressive immunity. These findings could be a therapeutic target for immunotherapy in combination with cytotoxic chemotherapy.     

Cancer and Immune Response: Old and New Evidence for Future Challenges

Cancer may occur as a result of abnormal host immune system tolerance. Recent studies have confirmed the occurrence of spontaneous and induced antitumor immune responses expressed as the presence of tumor‐infiltrating T cells in the tumor microenvironment in some cancer models. This finding has been recognized as a good prognostic factor in several types of tumors. Some chemotherapy agents, such as anthracyclines and gemcitabine, are effective boosters of the immune response through tumor‐specific antigen overexpression after apoptotic tumor cell destruction. Other strategies, such as GM‐CSF or interleukin‐2, are pursued to increase immune cell availability in the tumor vicinity, and thus improve both antigen presentation and T‐cell activation and proliferation. In addition, cytotoxic T lymphocyte antigen 4–blocking monoclonal antibodies enhance immune activity by prolonging T‐cell activation. Strategies to stimulate the dormant immune system against tumors are varied and warrant further investigation of their applications to cancer therapy in the future.     

树突状细胞体外诱导抗肿瘤免疫通过诱导肿瘤细胞凋亡抑制裸鼠移植瘤生长

研究树突状细胞（ＤＣ）体外诱导的细胞免疫抑制裸鼠移植瘤生长作用及其机理。方法：联合应用粒／巨噬细胞集落刺激因子ＫＭ－ＣＳＦ）及白介素－４（ＩＬ－４）直接从肝癌患者外周血中培养出ＤＣ,以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤抗原粗提物刺激ＤＣ,ＤＣ激活同源的Ｔ淋巴细胞,建立裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤模型,以被激活的Ｔ淋巴细胞治疗裸鼠ＨｅｐＧ２移植瘤并观察治疗效果,检测移植瘤标本肿瘤细胞凋亡情况。结果：ＤＣ诱导的Ｔ细胞免疫能够诱导裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤肿瘤细胞大量凋亡从而抑制裸鼠ＨｅｐＧ２移植瘤生长。结论：经肿瘤抗原激活的ＤＣ作为一新概念上的抗肿瘤疫苗有可能在肿瘤的治疗中发挥重要作用。     

Modulation of protective T cell immunity by complement inhibitor expression on tumor cells

Complement-inhibitory proteins expressed on cancer cells can provide protection from antitumor antibodies and may potentially modulate the induction of an immune response to tumor-associated antigens. In the current study, we investigated the consequences of complement inhibitor down-regulation on the effector and inductive phases of an immune response. Stable small interfering RNA-mediated down-regulation of the complement inhibitor Crry on MB49 murine bladder cancer cells increased their susceptibility to monoclonal antibody and complement in vitro. In a syngeneic model of metastatic cancer, the down-regulation of Crry on i.v.-injected MB49 cells was associated with a significant decrease in tumor burden and an increase in the survival of challenged mice. However, monoclonal antibody therapy had no additional benefit. There was an antitumor IgG response, but the response was not effected by Crry down-regulation on inoculated tumor cells. Down-regulation of Crry on MB49 cells resulted in an enhanced antitumor T-cell response in challenged mice (measured by lymphocyte IFN-gamma secretion), and CD8+ T cell depletion of mice prior to injection of MB49 cells completely abrogated the effect of Crry down-regulation on tumor burden and survival. Deficiency of C3 also abrogated the effect of Crry down-regulation on the survival of MB49-challenged mice, indicating a complement-dependent mechanism. These data indicate that complement inhibitors expressed on a tumor cell can suppress a T cell response and that enhancing complement activation on a tumor cell surface can promote protective T cell immunity.     

IL-24-Armed Oncolytic Vaccinia Virus Exerts Potent Antitumor Effects via Multiple Pathways in Colorectal Cancer

Colorectal cancer is an aggressive malignancy for which there are limited treatment options. Oncolytic vaccinia virus is being developed as a novel strategy for cancer therapy. Arming vaccinia virus with immunostimulatory cytokines can enhance the tumor cell-specific replication and antitumor efficacy. Interleukin-24 (IL-24) is an important immune mediator, as well as a broad-spectrum tumor suppressor. We constructed a targeted vaccinia virus of Guang9 strain harboring IL-24 (VG9-IL-24) to evaluate its antitumor effects. In vitro, VG9-IL-24 induced an increased number of apoptotic cells and blocked colorectal cancer cells in the G₂ /M phase of the cell cycle. VG9-IL-24 induced apoptosis in colorectal cancer cells via multiple apoptotic signaling pathways. In vivo, VG9-IL-24 significantly inhibited the tumor growth and prolonged the survival both in human and murine colorectal cancer models. In addition, VG9-IL-24 stimulated multiple antitumor immune responses and direct bystander antitumor activity. Our results indicate that VG9-IL-24 can inhibit the growth of colorectal cancer tumor by inducing oncolysis and apoptosis as well as stimulating the antitumor immune effects. These findings indicate that VG9-IL-24 may exert a potential therapeutic strategy for combating colorectal cancer.     

Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer

Despite advancements in the treatment of ovarian cancer, this disease continues to be a leading cause of cancer death in women. Adoptive transfer of tumor-reactive T cells is a promising antitumor therapy for many cancers. We designed a chimeric receptor linking NKG2D, a natural killer (NK) cell-activating receptor, to the CD3zeta chain of the T-cell receptor to target ovarian tumor cells. Engagement of chimeric NKG2D receptors (chNKG2D) with ligands for NKG2D, which are commonly expressed on tumor cells, leads to T-cell secretion of proinflammatory cytokines and tumor cytotoxicity. In this study, we show that >80% of primary human ovarian cancer samples expressed ligands for NKG2D on the cell surface. The tumor samples expressed MHC class I-related protein A, MICB, and UL-16 binding proteins 1 and 3. ChNKG2D-expressing T cells lysed ovarian cancer cell lines. We show that T cells from ovarian cancer patients that express chNKG2D secreted proinflammatory cytokines when cultured with autologous tumor cells. In addition, we show that chNKG2D T cells can be used therapeutically in a murine model of ovarian cancer. These data indicate that treatment with chNKG2D-expressing T cells is a potential immunotherapy for ovarian cancer.     

Immunomodulatory gene therapy with interleukin 12 and 4-1BB ligand: long- term remission of liver metastases in a mouse model

BACKGROUND: The success of immunomodulatory cancer therapy is frequently hampered by the transient nature of the antitumor immune response. We have shown previously in a mouse model that interleukin 12 (IL-12) generates a strong natural killer (NK) cell-mediated antitumor response and reduces liver metastases induced by a colon carcinoma cell line. However, only a small percentage of the treated animals developed the cytotoxic T-lymphocytic response required for a long-term systemic antitumor immunity. 4-1BB is a co-stimulatory molecule expressed on the surface of activated T cells. Interaction of 4-1BB with its natural ligand (4-1BBL) has been shown to amplify T-cell (especially CD8+)-mediated immunity. In this study, we investigated the effects of adenovirus-mediated gene therapy delivering both IL-12 and 4-1BBL genes on mice with hepatic metastases induced by colon cancer cells. METHODS: Syngeneic BALB/c mice received intrahepatic injection of poorly immunogenic MCA26 colon cancer cells. Various combinations of replication-defective adenoviruses expressing IL-12 and 4-1BBL genes were injected into the established liver tumors. Changes in tumor size and animal survival were then monitored. All statistical tests were two-sided. RESULTS: The long-term survival rate of mice treated with the combination of IL-12 and 4-1BBL was significantly improved over that of animals in the control group (P =.0001). In vivo depletion of NK cells or CD8+ T cells completely abolished the long-term survival advantage of the IL-12 plus 4-1BBL-treated animals (P<.002). Moreover, the systemic immunity induced by this combination treatment protected these animals against a subcutaneous challenge with parental MCA26 cells. CONCLUSION: Adenovirus-mediated transfer of IL-12 and 4-1BBL genes directly into liver tumors resulted in tumor regression that required both NK and CD8+ T cells and generated a potent, long-lasting antitumor immunity.     

Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells

Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.     

IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression

During malignant progression cancer cells tend to lose cell surface expression of MHC and other immune antigens, making them invisible to cytotoxic T cells and therefore inaccessible to tumor antigen-directed immunotherapy. Moreover, cancer cell variants that have lost antigen expression frequently contribute to deadly tumor relapses that occur following treatments that had been initially effective. In an effort to destroy antigen-loss cancer cells in tumors, we created a strategy that combines a chimeric antigen receptor (CAR)-redirected T-cell attack with an engineered local release of the cytokine interleukin 12 (IL-12), which recruits and reinforces macrophage function. Cytotoxic T cells were engineered to release inducible IL-12 upon CAR engagement in the tumor lesion, resulting in destruction of antigen-loss cancer cells that would normally escape. Importantly, elimination of the antigen-loss cancer cells was accompanied by an accumulation of activated macrophages that was critical to the antitumor response, because removing the macrophages abolished the response and restoring them reengaged it. Neutralizing TNF-α also abrogated the elimination of antigen-loss cancer cells, implying this proinflammatory factor in the process. Taken together, our results show how IL-12 supplementation by CAR T cells can target otherwise inaccessible tumor lesions, in a manner associated with reduced systemic toxicity, by recruiting and activating innate immune cells for a proinflammatory response.     

白细胞介素12锚定修饰肿瘤源性外核体肾癌疫苗的制备及其体外抗肿瘤效应

目的 制备白细胞介素12(IL-12)锚定修饰肿瘤源性外核体(EXOs)的肾癌疫苗,探讨其体外对肾癌细胞特异性杀伤效应.方法 将精基化磷脂酰肌醇(GPI)信号肽序列与IL-12基因融合,构建真核双表达质粒pBIG,应用激光共聚焦显微镜和流式细胞仪检测融合蛋白(GPI-IL-12)在肾癌细胞中的表达.用超滤和蔗糖/重水密度梯度离心法分离EXOs,透射电镜鉴定其形态.采用Western blot检测其标志性分子热休克蛋白70(HSP70)、细胞间黏附分子(ICAM-1)、肾癌特异性抗原G250和GPI-IL-12的表达.应用酶联免疫吸附试验(ELISA)测定EXOs负载IL-12的量,干扰素γ(IFN-γ)释放试验检测IL-12修饰EXOs(EXO-IL-12)的功能,羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)和碘化丙啶(PI)双标的流式细胞仪分析其对肾癌细胞的诱导杀伤效应.结果 EXOs为类圆碟形、双层膜结构,直径30～80nm,表达HSP70、ICAM-1、G250和GPI-IL-12.ELISA测定结果显示,10μg/ml EXOs含约(80.0±9.6)pg/ml的IL-12.EXO-GPI-IL-12疫苗能显著地促进T淋巴细胞分泌IFN-γ,在体外对肾癌细胞的特异性杀伤率为53.7%,明显高于对膀胱癌细胞(9.8%)和结肠癌细胞的杀伤率(10.8%),差异有统计学意义(P<0.01).结论 EXO-GPI-IL-12疫苗能够在体外诱导T淋巴细胞产生较强的抗原特异性细胞毒作用,可望成为治疗肾癌的新疫苗.     

Regulatory T cells in tumor immunity

Recent studies have revealed that Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Tregs), which are physiologically engaged in the maintenance of immunological self‐tolerance, play critical roles for the control of antitumor immune responses. For example, a large number of Foxp3⁺Tregs infiltrate into tumors, and systemic removal of Foxp3⁺Tregs enhances natural as well as vaccine‐induced antitumor T‐cell responses. Tregs are recruited to tumor tissues via chemokines, such as CCL22 binding to CCR4 expressed by Tregs. They appear to expand and become activated in tumor tissues and in the draining lymph nodes by recognizing tumor‐associated antigens as well as normal self‐antigen expressed by tumor cells. These results indicate that cancer vaccines targeting tumor‐associated self‐antigens may potentially expand/activate Tregs and hamper effective antitumor immune responses, and that tumor immunity can therefore be enhanced by depleting Tregs, attenuating Treg suppressive function, or rendering effector T cells refractory to Treg‐mediated suppression. Recent attempts have indeed demonstrated that combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity and are more effective than a single monoclonal antibody therapy. Combination therapy targeting a variety of molecules expressed in antigen‐presenting cells, effector T cells and Tregs is envisaged to be a promising anticancer immunotherapy.     

Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target?

Regulatory T (Treg) cells suppress abnormal/excessive immune responses to self‐ and nonself‐antigens to maintain immune homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity. There are several Treg cell immune suppressive mechanisms: inhibition of costimulatory signals by CD80 and CD86 expressed by dendritic cells through cytotoxic T‐lymphocyte antigen‐4, interleukin (IL)‐2 consumption by high‐affinity IL‐2 receptors with high CD25 (IL‐2 receptor α‐chain) expression, secretion of inhibitory cytokines, metabolic modulation of tryptophan and adenosine, and direct killing of effector T cells. Infiltration of Treg cells into the tumor microenvironment (TME) occurs in multiple murine and human tumors. Regulatory T cells are chemoattracted to the TME by chemokine gradients such as CCR4‐CCL17/22, CCR8‐CCL1, CCR10‐CCL28, and CXCR3‐CCL9/10/11. Regulatory T cells are then activated and inhibit antitumor immune responses. A high infiltration by Treg cells is associated with poor survival in various types of cancer. Therefore, strategies to deplete Treg cells and control of Treg cell functions to increase antitumor immune responses are urgently required in the cancer immunotherapy field. Various molecules that are highly expressed by Treg cells, such as immune checkpoint molecules, chemokine receptors, and metabolites, have been targeted by Abs or small molecules, but additional strategies are needed to fine‐tune and optimize for augmenting antitumor effects restricted in the TME while avoiding systemic autoimmunity. Here, we provide a brief synopsis of these cells in cancer and how they can be controlled to achieve therapeutic outcomes.     

Immune cells and cytokines - their role in cancer-immunotherapy (review)

Conventional cancer therapies such as surgery, chemotherapy and/or radiotherapy, are not always successful in providing long-term survival for cancer patients. One of the major problems with conventional cancer therapies is their inability to eradicate residual/metastatic tumor cells that are resistant to therapy. Therefore, it is necessary to develop new methods for treating such cancer cells in order to improve the clinical outcome of these patients. Despite antitumor effector mechanisms working against cancer cells in the host's body, tumor-cell-induced immunosuppression and or antigenic modulation by the tumor cells often help tumor cells escape host defense mechanisms. Therefore, one approach for treating residual cancer would be to enhance the host's own immunological/antitumor defense mechanisms. Immune cells that have a significant role in mediating antitumor responses include: T lymphocytes; natural killer (NK) cells; macrophages; and B lymphocytes. The ability of these immune cells to effectively destroy malignant cells is carefully governed by chemical mediators in the form of proteins otherwise known as cytokines. Many cytokines (interleukins, interferons, and tumor necrosis factor) have been shown to enhance in vitro and in vivo effector cell antitumor cytotoxic activities. Utilization of cytokines in conjunction with effector cells can also mediate significant antitumor responses in both animal models and cancer patients. One of the major problems associated with systemic treatment with cytokines is the development of dose limiting toxicities. Currently, attempts to reduce this problem include developing techniques to allow for the preferential release of cytokines in proximity to the tumor cell. In this regard, effector cells or tumor cells that have been genetically engineered to secrete cytokine(s) may be useful in localizing an immune response, preferably at the tumor site. Clinical trial using cytokine gene transfected cells for treating cancer are currently under investigation. With the availability of recombinant lymphokines and with our ability to genetically modify effector cells and tumor cells this hopefully will allow us to improve current therapeutic modalities for treating cancer.     

IL-24-Armed Oncolytic Vaccinia Virus Exerts Potent Antitumor Effects via Multiple Pathways in Colorectal Cancer

Colorectal cancer is an aggressive malignancy for which there are limited treatment options. Oncolytic vaccinia virus is being developed as a novel strategy for cancer therapy. Arming vaccinia virus with immunostimulatory cytokines can enhance the tumor cell-specific replication and antitumor efficacy. Interleukin-24 (IL-24) is an important immune mediator, as well as a broad-spectrum tumor suppressor. We constructed a targeted vaccinia virus of Guang9 strain harboring IL-24 (VG9-IL-24) to evaluate its antitumor effects. In vitro, VG9-IL-24 induced an increased number of apoptotic cells and blocked colorectal cancer cells in the G₂/M phase of the cell cycle. VG9-IL-24 induced apoptosis in colorectal cancer cells via multiple apoptotic signaling pathways. In vivo, VG9-IL-24 significantly inhibited the tumor growth and prolonged the survival both in human and murine colorectal cancer models. In addition, VG9-IL-24 stimulated multiple antitumor immune responses and direct bystander antitumor activity. Our results indicate that VG9-IL-24 can inhibit the growth of colorectal cancer tumor by inducing oncolysis and apoptosis as well as stimulating the antitumor immune effects. These findings indicate that VG9-IL-24 may exert a potential therapeutic strategy for combating colorectal cancer.Key words: Oncolytic vaccinia virus, Interleukin-24 (IL-24), Colorectal cancer (CRC), Immunotherapy, Apoptosis     

Cytokine-induced senescence for cancer surveillance

The immune response is a first-line systemic defense to curb tumorigenesis and metastasis. Much effort has been invested to design antitumor interventions that would boost the immune system in its fight to defeat or contain cancerous growth. Tumor vaccination protocols, transfer of tumor-associated-antigen-specific T cells, T cell activity-regulating antibodies, and recombinant cytokines are counted among a toolbox filled with immunotherapeutic options. Although the mechanistic underpinnings of tumor immune control remain to be deciphered, these are studied with the goal of cancer cell destruction. In contrast, tumor dormancy is considered as a dangerous equilibrium between cell proliferation and cell death. There is, however, emerging evidence that tumor immune control can be achieved in the absence of overt cancer cell death. Here, we propose cytokine-induced senescence (CIS) by transfer of T helper-1 cells (T_H1) or by recombinant cytokines as a novel therapeutic intervention for cancer treatment. Immunity-induced senescence triggers a stable cell cycle arrest of cancer cells. It engages the immune system to construct defensive, isolating barriers around tumors, and prevents tumor growth through the delivery or induction of T_H1-cytokines in the tumor microenvironment. Keeping cancer cells in a non-proliferating state is a strategy, which directly copes with the lost homeostasis of aggressive tumors. As most studies show that even after efficient cancer therapies minimal residual disease persists, we suggest that therapies should include immune-mediated senescence for cancer surveillance. CIS has the goal to control the residual tumor and to transform a deadly disease into a state of silent tumor persistence.     

High frequencies of functional tumor-reactive T cells in bone marrow and blood of pancreatic cancer patients

Pancreatic cancer is characterized by aggressive growth and treatment resistance. New approaches include immunotherapeutic strategies but the type and extent of spontaneous immune responses against tumor antigens remains unclear. A dominance of TH2 cytokines in patients' sera reported previously suggests systemic tumor-induced immunosuppression, potentially inhibiting the induction of tumor-reactive T cells. We characterized the localization, frequencies, and functional potential of spontaneously induced memory T cells specific for individual tumor antigens or the tumor-associated antigen mucin-1 in the peripheral blood and bone marrow of 41 pancreatic cancer patients. We found high numbers of tumor-reactive T cells in all bone marrow samples and in 50% of the blood samples. These cells secreted the TH1 cytokine IFN-gamma rather than TH2 cytokines upon stimulation with tumor antigens. Although consistently induced during pancreatic cancer, T cells specific for pancreatic antigens were not detected during chronic pancreatitis, suggesting that their evaluation may be of diagnostic use in both diseases. Freshly isolated T cells from cancer patients recognized autologous tumor cells and rejected them in vitro and in a xenotransplant model in vivo, suggesting their therapeutic potential. Thus, tumor antigen-specific T cell responses occur regularly during pancreatic cancer disease and lead to enrichment of tumor cell-reactive memory T cells in the bone marrow. The bone marrow can therefore be considered an important organ for antitumor immune responses in pancreatic cancer.     

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF

Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment.     

Improvement of DC vaccine with ALA-PDT induced immunogenic apoptotic cells for skin squamous cell carcinoma

Dendritic cell (DC) based vaccines have emerged as a promising immunotherapy for cancers. However, most DC vaccines so far have achieved only limited success in cancer treatment. Photodynamic therapy (PDT), an established cancer treatment strategy, can cause immunogenic apoptosis to induce an effective antitumor immune response. In this study, we developed a DC-based cancer vaccine using immunogenic apoptotic tumor cells induced by 5-aminolevulinic acid (ALA) mediated PDT. The maturation of DCs induced by PDT-treated apoptotic cells was evaluated using electron microscopy, FACS, and ELISA. The anti-tumor immunity of ALA-PDT-DC vaccine was tested with a mouse model. We observed the maturations of DCs potentiated by ALA-PDT treated tumor cells, including morphology maturation (enlargement of dendrites and increase of lysosomes), phenotypic maturation (upregulation of surface expression of MHC-II, DC80, and CD86), and functional maturation (enhanced capability to secrete IFN-γ and IL-12, and to induce T cell proliferation). Most interestingly, PDT-induced apoptotic tumor cells are more capable of potentiating maturation of DCs than PDT-treated or freeze/thaw treated necrotic tumor cells. ALA-PDT-DC vaccine mediated by apoptotic cells provided protection against tumors in mice, far stronger than that of DC vaccine obtained from freeze/thaw treated tumor cells. Our results indicate that immunogenic apoptotic tumor cells can be more effective in enhancing a DC-based cancer vaccine, which could improve the clinical application of PDT-DC vaccines.