Tumor infiltrating lymphocytes in ovarian cancer

The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment.     

MHC-dependent cytolysis of autologous tumor cells by lymphocytes infiltrating urothelial carcinomas

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3⁺. Although CD4⁺ and CD8⁺ T-cell sub-sets were grown in culture, CD4⁺ T-cell sub-sets predominated over CD8⁺ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4⁺ T cells surrounded the tumor islets, whereas CD8⁺ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4⁺ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8⁺ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8⁺ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response. Int. J. Cancer 71:585-594, 1997. © 1997 Wiley-Liss, Inc.     

Immunologic character of tumor infiltrating lymphocytes in ovarian carcinoma

Tumor-infiltratinglymphocytes(TIL)wasdirectlyisolatedfrompatient'stumortissues.ByrIL2activationandexpansioninvitro,TILwasagainimportedintothesamepatient'sbodytotreattumor,withapparenteffectsofanti-tumorandcomparativelessside-effect,withoutkillingothertumorcellsandnormalcells,allofwhichhavemadeitaeffectivewayoftreatingtumoratanadvancedstage.Inrecentyears,someresearchersfoundthattheeffectsofTIL'treatmentwerenotsofarapparent,withcomparativelyapparentdifference,whichmightbecloselyrelatedtothef…     

Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells

Seminoma lesions are characterized by a brisk inflammatory infiltrate containing both CD4 and CD8 T cells, which is of prognostic significance. However, whether seminoma cells express the HLA molecules required for classical T-cell recognition remains controversial. In the present study, we conducted a molecular, phenotypical and functional characterization of tumor infiltrating lymphocytes (TILs) from seminoma lesions. T-cell receptor clonotype mapping demonstrated the presence of clonally expanded T cells in the majority of the lesions. The cytotoxic capacity of TILs was indicated by expression of CD107a, which is a recently described surrogate marker for cytolytic activity. Indeed, the frequency of CD107a positive cells was substantially higher in TILs when compared to peripheral blood mononuclear cells. Moreover, fluorescence activated cell sorting of CD107a positive TILs allowed comparison of the clonotypic T-cell receptor fingerprint and demonstrated the ability of expanded clones to express this cytotoxic marker, suggesting cytotoxic activity at the tumor site. The cytotoxicity was confirmed by in situ granzyme B expression. Furthermore, by staining with multimeric HLA-peptide complexes, we could demonstrate the presence of Mage-3 specific T cells among TILs. In summary, specific and functional T-cell responses are operative in seminoma, indicating that the inflammatory infiltrate is indeed involved in the immunological control of the tumor.     

TIL细胞治疗恶性胸水的疗效观察

目的观察肿瘤浸润淋巴细胞(TIL)治疗恶性胸水的疗效。方法从30例恶性肿瘤胸水中的肿瘤细胞分离得到TIL,并经10%人AB血清RPM I-1640液培养,经rIL-2体外激活培养,随后胸腔注射。结果完全缓解12例(40.0%),有效15例(50.0%),无效3例(10.0%),总有效率90.0%(27/30)。结论 TIL治疗恶性胸腔积液疗效好,毒副作用小,能改善患者的生活质量。     

Chemotherapy sensitivity testing on ovarian cancer cells isolated from malignant ascites

Background: In epithelial ovarian cancer (EOC), 15–20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome.Materials and Methods: Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level.Results: Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence.Conclusions: Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.     

Biochemical composition of malignant ascites determines high aggressiveness of undifferentiated ovarian tumors

Although undifferentiated tumors are the most lethal among all ovarian cancer histotypes, the exact reasons for this situation are unclear. This report was aimed at investigating whether the high aggressiveness of undifferentiated ovarian cancer may be associated with a biochemical composition of malignant ascites accumulating in the peritoneal cavity. We analyzed ascites from patients with undifferentiated, high-grade serous, endometrioid and clear-cell ovarian cancers, and from non-cancerous patients with respect to a group of soluble agents involved in cancer cell progression. Moreover, the effect of these fluids on proliferation and migration of ovarian cancer cells (A2780, OVCAR-3 and SKOV-3) was evaluated. The study showed that the level of all tested proteins in malignant ascites was higher than in the benign fluids. Concentration of 9/11 agents (CCL2, CXCL1, CXCL5, CXCL8, CXCL12, HGF, PAI-1, TGF-β1 and VEGF) was the greatest in the fluids from undifferentiated cancer, while the level of remaining 2 (IL-6 and uPA) was the highest in ascites from serous carcinoma. Proliferation of cancer cells was the most effective when they were subjected to ascites from patients with undifferentiated and serous cancer, whereas the migration was the highest in the case of undifferentiated tumors. Our findings indicate that the aggressiveness of undifferentiated ovarian tumors may be associated with the composition of malignant ascites, in particular the concentration of specific proinflammatory, cancer-promoting agents.     

卵巢癌腹水来源ｅｘｏｓｏｍｅｓ的免疫分子检测

目的　通过检测卵巢癌腹水中ｅｘｏｓｏｍｅｓ免疫相关分子的表达,探讨以ｅｘｏｓｏｍｅｓ为基础的肿瘤免疫治疗的可行性。方法　采用离心超滤联合蔗糖密度梯度离心法分离出４例恶性腹水中的ｅｘｏｓｏｍｅｓ,经透射电镜观察其超微结构。通过免疫胶体金技术和Ｗｅｓｔｅｒｎ杂交技术鉴定和确定ｅｘｏｓｏｍｅｓ的ＨＳＰ７０、ＨＬＡ-Ⅰ、ＨＬＡ-Ⅱ、ＮＹ-ＥＳＯ-１、ＭＡＧＥ-Ａ１及ＭＡＧＥ-Ｃ２等抗原分子表达。结果　卵巢癌患者恶性腹水中ｅｘｏｓｏｍｅｓ为直径３０～８０ｎｍ的膜性囊泡结构,圆形或椭圆形,腔内为低电子密度成分;经上述相应抗原的抗体 胶体金免疫电镜标记,囊外膜及腔内可见颗粒状电子致密物沉积。Ｗｅｓｔｅｒｎ杂交也证实上述抗原的存在,但在４例卵巢癌恶性腹水来源的ｅｘｏｓｏｍｅｓ中,各类免疫相关分子的表达谱或表达量是不同的。结论　卵巢癌腹水来源的ｅｘｏｓｏｍｅｓ富含各类肿瘤抗原、ＨＳＰ７０、ＨＬＡ-Ⅰ和ＨＬA-Ⅱ等免疫相关分子,并且在不同的卵巢个体存在明显的异质性,通过对腹水中ｅｘｏｓｏｍｅｓ免疫相关分子检测,有可能为以ｅｘｏｓｏｍｅｓ为基础的肿瘤免疫治疗提供帮助。     

Intraperitoneal recombinant interferon-alpha 2b for recurrent malignant ascites due to ovarian cancer

Thirteen patients with malignant ascites due to recurrent ovarian cancer were treated with intraperitoneal interferon 2b (5 mu/m2 escalating to 15 mu/m2, administered twice weekly by indwelling intraperitoneal catheter). The study population included eight patients with ascites only and five patients with ascites plus tumor masses greater than 2 cm in diameter. Five of 13 responded to treatment. All the responses were seen in patients with ascites only. Although tumor mass was an important determinant of response there was also a correlation between clinical response and suppression of colony growth on soft agar after in vitro exposure of tumor cells to interferon-alpha (IFN alpha). The intraperitoneal use of IFN alpha should be explored further in tumors with a predominant intraperitoneal location and microimplantation growth pattern.     

IL-6 trans-signaling in formation and progression of malignant ascites in ovarian cancer

Classic signaling by the proinflammatory cytokine interleukin 6 (IL-6) involves its binding to target cells that express the membrane-bound IL-6 receptor α. However, an alternate signaling pathway exists in which soluble IL-6 receptor (sIL-6Rα) can bind IL-6 and activate target cells that lack mIL-6Rα, such as endothelial cells. This alternate pathway, also termed trans-signaling, serves as the major IL-6 signaling pathway in various pathologic proinflammatory conditions including cancer. Here we report that sIL-6Rα is elevated in malignant ascites from ovarian cancer patients, where it is associated with poor prognosis. IL-6 trans-signaling on endothelial cells prevented chemotherapy-induced apoptosis, induced endothelial hyperpermeability, and increased transendothelial migration of ovarian cancer cells. Selective blockade of the MAPK pathway with ERK inhibitor PD98059 reduced IL-6/sIL-6Rα-mediated endothelial hyperpermeability. ERK activation by the IL-6/sIL-6Rα complex increased endothelial integrity via Src kinase activation and Y685 phosphorylation of VE-cadherin. Selective targeting of IL-6 trans-signaling in vivo reduced ascites formation and enhanced the taxane sensitivity of intraperitoneal human ovarian tumor xenografts in mice. Collectively, our results show that increased levels of sIL-6Rα found in ovarian cancer ascites drive IL-6 trans-signaling on endothelial cells, thereby contributing to cancer progression. Selective blockade of IL-6 trans-signaling may offer a promising therapeutic strategy to improve the management of patients with advanced ovarian cancer.     

Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma

Lymphoid cells infiltrating into human tumors can be expanded in vitro in medium containing interleukin-2 (IL-2). Adoptive transfer of these tumor-infiltrating lymphocytes (TIL) mediates potent antitumor effects in murine tumor models. Clinical trials to evaluate the efficacy of these cells in patients with advanced cancer are underway. We have investigated whether infused TIL labeled with indium 111 (111In) oxine can traffic and localize to metastatic deposits of tumor. Six patients with metastatic malignant melanoma who had multiple sites of subcutaneous, nodal, and/or visceral disease were the subjects of the study. The patients received cyclophosphamide 36 hours before receiving the intravenous (IV) infusion of TIL followed by IL-2 IV every eight hours. The distribution and localization of the TIL were evaluated using serial whole body gamma camera imaging, serial blood and urine samplings, and serial biopsies of tumor and normal tissue. 111In-labeled TIL localized to lung, liver, and spleen within two hours after the infusion of activity. Activity in the lung diminished within 24 hours. As early as 24 hours after injection of 111In-labeled TIL, localization of TIL to sites of metastatic deposits was demonstrated in all six patients using either imaging studies or biopsy specimens or both. 111In activity in tumor tissue biopsies ranged from three to 40 times greater than activity in normal tissue. A progressive increase in the radioactive counts at sites of tumor deposit was seen. This study shows that labeled TIL can localize preferentially to tumor, and provides information concerning the possible mechanism of the therapeutic effects of TIL.     

Increased expression of dipeptidyl peptidase IV in human mesothelial cells by malignant ascites from ovarian carcinoma patients

Cell surface aminopeptidases play an important role in biological processes through degradation of small peptides. There are many bioactive peptides in ascites and these peptides are involved in carcinoma cell dissemination and infiltration. In human mesothelial cells dipeptidyl peptidase IV (DPPIV) shows the highest expression mostly in four cell surface aminopeptidases: aminopeptidase A, neutral endopeptidase 24-11, aminopeptidase N and DPPIV. Since mesothelial cells are always in contact with ascites, we examined the influence of malignant ascites on DPPIV. DPPIV enzyme activity in mesothelial cells was enhanced by the addition of ascites obtained from ovarian carcinoma patients in a time- and concentration-dependent manner, and flow cytometry and immunocytochemistry also revealed an increased expression of DPPIV on the cell surface of mesothelial cells. The <3-kD fraction of malignant ascites increased the DPPIV enzyme activity to the same level as the total ascites. Northern hybridization demonstrated that DPPIV mRNA was increased 3-fold by the addition of the <3-kD malignant ascites. In conclusion, DPPIV is highly expressed in human mesothelial cells and was regulated by ascites.     

肿瘤浸润B细胞与免疫治疗

B细胞是肿瘤微环境的重要成分,然而,B细胞对肿瘤进展及治疗响应的影响目前仍然存在争议,这反映了肿瘤浸润B细胞(Tumor infiltrating B cells,TIL-Bs)亚群的异质性。事实上,这些TIL-Bs随着所处的免疫微环境向不同的方向极化进而获得不同的表型及功能,同时也反向塑造着微环境的组成及类型,最终导致了截然不同的临床预后。本文总结了TIL-Bs的组成特征及形成机制。进一步结合临床试验及临床前研究,我们尝试解释TIL-Bs的异质性对肿瘤免疫治疗效果的影响。阐明肿瘤中B细胞亚群的组成、产生和相关免疫网络的性质对于全面了解肿瘤中的B细胞反应,设计更有效的癌症免疫疗法具有重要意义。     

卵巢癌组织及腹腔积液中肿瘤干细胞的分离与培养

目的:从人卵巢癌组织和腹腔积液中分离培养肿瘤干细胞,分析细胞表型和化疗药物敏感性。方法:取卵巢浆腺癌患者的原发肿瘤组织及腹腔积液,通过无血清培养形成悬浮生长的球体。采用FCM及免疫荧光法检测干细胞标志物的表达。体外药敏实验检测获得的球体细胞和分化细胞对紫杉醇和卡铂的药物敏感性。结果:卵巢癌组织及腹腔积液中的卵巢癌细胞经无血清悬浮培养可以形成球体,后者具有CD44+CD24-表型并表达Oct4、Nanog及Sox2等干细胞标志物;腹腔积液及组织来源的球体细胞经血清培养液培养3周后发生分化,CD44+CD24-细胞比例分别下降至47%和3%;分化细胞对紫杉醇和卡铂的敏感性均显著高于球体细胞(P<0.01)。结论:卵巢癌的肿瘤干细胞属于CD44+CD24-细胞,后者表达Oct4等干细胞标志并具有显著耐药性。     

癌性腹水对卵巢癌细胞增殖及迁移的影响

目的 探讨癌性腹水对卵巢癌腹水中肿瘤细胞和卵巢癌细胞系(SKOV3)的形态学特征、增殖和迁移能力的影响。方法 卵巢癌腹水中提取的肿瘤细胞在体外用DMEM高糖培养液培养;卵巢癌细胞系(SKOV3)分别用DMEM高糖培养液和DMEM高糖培养液中加入不同比例癌性腹水制成的混合培养液培养;分别通过光学显微镜和电子显微镜观察各组细胞的形态学特点;用CCK试剂盒检测各组细胞的增殖能力;用划痕实验检测癌性腹水对卵巢癌细胞系SKOV3迁移能力的影响。结果 卵巢癌腹水中肿瘤细胞与卵巢癌细胞系(SKOV3)形态学特征明显不同;腹水中的肿瘤细胞离开腹水微环境后增殖能力明显减弱;混合培养液培养的SKOV3肿瘤细胞比DMEM高糖培养液培养的SKOV3肿瘤细胞的增殖及迁移能力明显增强。结论 卵巢癌腹水中的肿瘤细胞发生了更有利于其增殖及迁移的形态学改变;癌性腹水对卵巢癌细胞系(SKOV3)的增殖及迁移具有促进作用。     

Palliation of malignant ascites

Malignant ascites (MA) carries a poor prognosis. It can have a significant impact on quality of life (QOL), with increasing abdominal distention, pain, and dyspnea. Diuretics typically do not work well for MA. Paracentesis is effective in providing temporary symptom relief but requires frequent repeat procedures. Options for durable symptom management include indwelling catheters, peritoneal ports, peritoneovenous shunts, intraperitoneal (i.p.) catumaxomab, and hyperthermic i.p. chemotherapy. These interventions do not necessarily improve overall survival but may improve QOL.