肿瘤微环境中相关细胞的研究进展

肿瘤的微环境对肿瘤的发生,发展具有重要的意义。实体瘤中除肿瘤细胞外存在大量的非肿瘤细胞,如肿瘤闻质细胞、成纤维细胞、血管内皮细胞、免疫细胞、脂肪细胞等等,这一系列的细胞与肿瘤细胞相互作用,通过一系列的因子分泌而促使肿瘤的进一步的恶化,目前传统的抗肿瘤药物研究往往局限于肿瘤细胞本身而忽略了肿瘤周围的细胞作用,使得肿瘤久治不愈。将来的药物开发应该围绕肿瘤细胞为主体的同时,兼顾微环境中的其他细胞,多靶点治疗肿瘤,真正实现肿瘤的治愈。     

Stat3 activity in melanoma cells affects migration of immune effector cells and nitric oxide-mediated antitumor effects

Infiltration of immune effector cells in tumors is critical for antitumor immune responses. However, what regulates immune cell infiltration of tumors remains to be identified. Stat3 is constitutively activated with high frequency in diverse cancers, promoting tumor cell growth and survival. Blocking Stat3 signaling in tumors in vivo results in tumor growth inhibition that involves killing of nontransfected tumor cells and infiltration of immune effector cells, suggesting that Stat3 activity in tumor cells might affect immune cell recruitment. However, dying tumor cells can also attract immune cells. In this study, we show in isogenic murine melanomas that natural Stat3 activity is associated with tumor growth and reduction of T cell infiltration. Blocking Stat3 signaling in the melanoma cells containing high Stat3 activity results in expression of multiple chemoattractants, leading to increased migration of lymphocytes, NK cells, neutrophils, and macrophages. In addition, blocking Stat3 triggers tumor cells to produce soluble factors capable of activating macrophage production of NO in vitro and in vivo. TNF-alpha and IFN-beta, which are secreted by Stat3-inhibited tumor cells, are able to activate macrophage NO production, whereas neutralizing TNF-alpha in the tumor supernatant from Stat3-blocked tumor cells abrogates nitrite production. Moreover, interrupting Stat3 signaling in tumor cells leads to macrophage-mediated, nitrite-dependent cytostatic activity against nontransduced tumor cells. These results suggest that tumor Stat3 activity affects recruitment of diverse immune effectors and it can be manipulated to activate the effector phase of innate immune responses.     

肿瘤微环境中相关细胞的研究进展

肿瘤的微环境对肿瘤的发生,发展具有重要的意义。实体瘤中除肿瘤细胞外存在大量的非肿瘤细胞,如肿瘤间质细胞、成纤维细胞、血管内皮细胞、免疫细胞、脂肪细胞等等,这一系列的细胞与肿瘤细胞相互作用,通过一系列的因子分泌而促使肿瘤的进一步的恶化,目前传统的抗肿瘤药物研究往往局限于肿瘤细胞本身而忽略了肿瘤周围的细胞作用,使得肿瘤久治不愈。将来的药物开发应该围绕肿瘤细胞为主体的同时,兼顾微环境中的其他细胞,多靶点治疗肿瘤,真正实现肿瘤的治愈。     

模式识别受体介导的肿瘤免疫耐受参与肿瘤转移

肿瘤细胞与宿主相互作用经历免疫警戒、免疫平衡和免疫逃逸过程,称为肿瘤免疫编辑。具有免疫逃逸能力是肿瘤细胞的标志性改变,也是肿瘤生长失控、转移和治疗失效的重要原因。病原微生物所含病原相关模式分子和肿瘤组织释放的损伤相关模式分子与肿瘤细胞及周围组织细胞和免疫细胞表达的模式识别受体相互作用,引起抑制性免疫微环境是导致肿瘤免疫耐受的关键。以肿瘤免疫耐受为药靶,使用小分子化合物或具有免疫刺激活性的生物制剂如单克隆抗体,可逆转肿瘤抑制性免疫微环境,打破肿瘤免疫耐受,抑制肿瘤细胞的生长和侵袭能力,从而降低肿瘤细胞转移和肿瘤致死率。     

The tumor microenvironment and its contribution to tumor evolution toward metastasis

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.     

Components of the hematopoietic compartments in tumor stroma and tumor-bearing mice

Solid tumors are composed of cancerous cells and non-cancerous stroma. A better understanding of the tumor stroma could lead to new therapeutic applications. However, the exact compositions and functions of the tumor stroma are still largely unknown. Here, using a Lewis lung carcinoma implantation mouse model, we examined the hematopoietic compartments in tumor stroma and tumor-bearing mice. Different lineages of differentiated hematopoietic cells existed in tumor stroma with the percentage of myeloid cells increasing and the percentage of lymphoid and erythroid cells decreasing over time. Using bone marrow reconstitution analysis, we showed that the tumor stroma also contained functional hematopoietic stem cells. All hematopoietic cells in the tumor stroma originated from bone marrow. In the bone marrow and peripheral blood of tumor-bearing mice, myeloid populations increased and lymphoid and erythroid populations decreased and numbers of hematopoietic stem cells markedly increased with time. To investigate the function of hematopoietic cells in tumor stroma, we co-implanted various types of hematopoietic cells with cancer cells. We found that total hematopoietic cells in the tumor stroma promoted tumor development. Furthermore, the growth of the primary implanted Lewis lung carcinomas and their metastasis were significantly decreased in mice reconstituted with IGF type I receptor-deficient hematopoietic stem cells, indicating that IGF signaling in the hematopoietic tumor stroma supports tumor outgrowth. These results reveal that hematopoietic cells in the tumor stroma regulate tumor development and that tumor progression significantly alters the host hematopoietic compartment.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Tryptophan degradation in transplanted tumor cells undergoing rejection

The depletion of an essential amino acid, tryptophan, caused by indoleamine 2,3-dioxygenase induction in vitro, has been shown to be due to a mechanism that is used in self-defense against inhaled microorganisms and tumor growth. In this communication, we report the results of measuring dioxygenase activity in the peritoneal exudate cells and tumor cytotoxicity at the transplantation loci after in vivo transplantation of tumor cells into the peritoneal cavity of syngeneic or allogeneic strains of mice. The enzyme was induced only when the tumor cells were being rejected from allogeneic animals and no change was observed when the cells continued to grow in syngeneic animals. Furthermore, when the syngeneic tumor cells in a diffusion chamber were i.p. transplanted simultaneously with i.p. injection of allogeneic tumor cells, the enzyme was induced not only in allografted tumor cells but also in the syngeneic tumor cells. Under these conditions, the tumor cells in the diffusion chamber ceased to grow and 50% of the cells were rejected. To determine the type of cells containing the induced enzyme, the peritoneal exudate cells (tumor cells and host cells--mostly small lymphocytes) were separated into six fractions by sedimentation under gravity and by differential centrifugation. Approximately 80% of total enzyme activity was localized in a tumor-rich fraction (98.9% purity), whereas only 0.2% of the activity was found in a lymphocyte-rich fraction (99.5% purity). The localization of indoleamine 2,3-dioxygenase in the tumor cells was confirmed by complement-dependent lysis with specific antibodies against tumor and host cells.     

CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.     

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor

Summary Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAM-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the ⁵¹Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells. Thus, metastatic tumor cells within the spleen of tumor-bearing mice may have the potential to act as in situ stimulators for the generation of potent antitumor immunity which brings about the eradication of a large, metastatic tumorigenic load remaining after clearance of the drug from the circulation.Supported by United States Public Health Service Research Grants CA-30088 and CA-35761 from the National Cancer InstituteIn partial fulfillment of the requirements of the Graduate College for the Doctor of Philosophy degree for RCBSBE was visiting Fulbright Professor from the Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel, and was supported by the Council for International Exchange of Scholars     

Perspectives on immune checkpoint ligands: expression,regulation, and clinical implications

In the tumor microenvironment, immune checkpoint ligands (ICLs) must be expressed in order to trigger the inhibitory signal via immune checkpoint receptors (ICRs). Although ICL expression frequently occurs in a manner intrinsic to tumor cells, extrinsic factors derived from the tumor microenvironment can fine-tune ICL expression by tumor cells or prompt non-tumor cells, including immune cells. Considering the extensive interaction between T cells and other immune cells within the tumor microenvironment, ICL expression on immune cells can be as significant as that of ICLs on tumor cells in promoting anti-tumor immune responses. Here, we introduce various regulators known to induce or suppress ICL expression in either tumor cells or immune cells, and concise mechanisms relevant to their induction. Finally, we focus on the clinical significance of understanding the mechanisms of ICLs for an optimized immunotherapy for individual cancer patients.     

Sialylation of 3-methylcholanthrene-induced fibrosarcoma determines antitumor immune responses during immunoediting

Sialylation of tumor cells is involved in various aspects of their malignancy (proliferation, motility, invasion, and metastasis); however, its effect on the process of immunoediting that affects tumor cell immunogenicity has not been studied. We have shown that in mice with impaired immunoediting, such as in IL-1α(-/-) and IFNγ(-/-) mice, 3-methylcholanthrene-induced fibrosarcoma cells are immunogenic and concomitantly bear low levels of surface sialylation, whereas tumor cells derived from wild type mice are nonimmunogenic and bear higher levels of surface sialylation. To study immune mechanisms whose interaction with tumor cells involves surface sialic acid residues, we used highly sialylated 3-methylcholanthrene-induced nonimmunogenic fibrosarcoma cell lines from wild type mice, which were treated with sialidase to mimic immunogenic tumor cell variants. In vivo and in vitro experiments revealed that desialylation of tumor cells reduced their growth and induced cytotoxicity by NK cells. Moreover, sialidase-treated tumor cells better activated NK cells for IFN-γ secretion. The NKG2D-activating receptor on NK cells was shown to be involved in interactions with desialylated ligands on tumor cells, the nature of which is still not known. Thus, the degree of sialylation on tumor cells, which is selected during the process of immunoediting, has possibly evolved as an important mechanism of tumor cells with low intrinsic immunogenicity or select for tumor cells that can evade the immune system or subvert its function. When immunoediting is impaired, such as in IFN-γ(-/-) and IL-1α(-/-) mice, the overt tumor consists of desialylayed tumor cells that interact better with immunosurveillance cells.     

CD8 T cell help for innate antitumor immunity

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.     

Tumor structure and extracellular matrix as a possible barrier for therapeutic approaches using immune cells or adenoviruses in colorectal cancer

In this article we report about the role that tumor structure and extracellular matrix (ECM) may play in immunotherapy and in gene therapy using adenoviruses. We performed studies in a rat model for colorectal cancer, CC531, and in specimens of human colorectal cancer. The tumors were composed of two compartments, tumor cell nests surrounded by stromal cells. ECM proteins were expressed in the stromal part, where the blood vessels were also located. Furthermore, in several tumors, the tumor cell nests were surrounded by basal membrane-like structures. Therefore, in vascular approaches to treat cancer, therapeutic agents on their route to tumor cells may be hampered by ECM to reach tumor cells. We found that immune cells were abundantly present in tumors from colorectal origin. These cells were, however, not found in direct contact with tumor cells, but mainly in the stromal part of the tumor. Adenoviruses, when intravascularly injected, did not reach tumor cells in the CC531 rat model. Tumor cells were only infected, and even then in limited numbers, in cases of intratumoral injection. We hypothesize that ECM in a tumor is a barrier both for immune cells and for adenoviruses to make direct contact with these tumor cells, and thus limits colorectal tumor therapy.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

Overexpression of Calcium-Permeable Glutamate Receptors in Glioblastoma Derived Brain Tumor Initiating Cells

Glioblastoma multiforme is the most malignant type of primary brain tumor with a poor prognosis. These tumors consist of a heterogeneous population of malignant cells, including well-differentiated tumor cells and less differentiated cells with stem cell properties. These cancer stem cells, known as brain tumor initiating cells, likely contribute to glioma recurrence, as they are highly invasive, mobile, resistant to radiation and chemotherapy, and have the capacity to self-renew. Glioblastoma tumor cells release excitotoxic levels of glutamate, which may be a key process in the death of peritumoral neurons, formation of necrosis, local inflammation, and glioma-related seizures. Moreover, elevated glutamate levels in the tumor may act in paracrine and autocrine manner to activate glutamate receptors on glioblastoma tumor cells, resulting in proliferation and invasion. Using a previously described culturing condition that selectively promotes the growth of brain tumor initiating cells, which express the stem cell markers nestin and SOX-2, we characterize the expression of α-amino-3-hydroxy-5-methyl-4-isozolepropionic acid (AMPA)-type glutamate receptor subunits in brain tumor initiating cells derived from glioblastomas. Here we show for the first time that glioblastoma brain tumor initiating cells express high concentrations of functional calcium-permeable AMPA receptors, compared to the differentiated tumor cultures consisting of non-stem cells. Up-regulated calcium-permeable AMPA receptor expression was confirmed by immunoblotting, immunocytochemistry, and intracellular calcium imaging in response to specific agonists. Our findings raise the possibility that glutamate secretion in the GBM tumor microenvironment may stimulate brain tumor derived cancer stem cells.     

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.     

抗原负载DCs诱导的CIK细胞对B16黑色素瘤抑制作用的形态学观察

目的:探讨抗原负载树突状细胞(dentritic cells,DCs)诱导的CIK(cytokine induced killer)细胞对B16黑色素瘤的抑瘤作用。方法:分离、培养DC和CIK细胞,取部分DC进行肿瘤抗原负载,将其与CIK细胞按1:10的比例共培养3d,即为抗原负载的DC-CIK。建立B16黑色素瘤小鼠模型,分别于瘤周围皮下注射经Brdu标记的CIK、DC-CIK、抗原负载DC-CIK。按注射细胞进行分组,测量注射前后各组小鼠的瘤体积,计算抑瘤率,比较其抑瘤作用。应用免疫组化方法和透射电镜观察抗原负载DC-CIK细胞在皮肤中的分布及杀伤肿瘤细胞的形态学表现。结果:抗原负载DC诱导的CIK(细胞组抑瘤率(86.57%)高于CIK细胞组(33.34%,P<0.05)和DC-CIK细胞组(61.08%,P<0.05);光镜下抗原负载DC-CIK细胞主要分布在皮下组织,癌组织周围,特别是癌巢周边。透射电镜下抗原负载DC-CIK细胞体积大,核有切迹,细胞质内细胞器丰富,粗面内质网扩张。细胞表面有突起,与肿瘤细胞密切接触。大量肿瘤细胞凋亡、坏死。结论:CIK细胞经抗原负载DC诱导后抑瘤作用明显强于单纯CIK细胞和DC-CIK细胞。