肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系

目的研究肝癌荷瘤小鼠调节性T细胞数量的改变及其与肿瘤生长的关系。方法采用小鼠肝癌细胞系H22接种BALB／c小鼠,建立肝癌模型;采用流式细胞术方法检测CD4^＋ CD25^＋T／CD4^＋T细胞的比例;以RT-PCR和流式细胞术检测Foxp3基因的表达。以免疫磁珠分选法纯化CD4^＋CD25^＋T和CD4^＋CD25^-T细胞;在体外,用3H-TdR掺入法检测T细胞的增殖情况;在体内,观察荷瘤小鼠来源的CD4^＋CD25^＋T细胞对肿瘤生长的作用。结果（1）荷瘤小鼠在引流淋巴结中,CD4^＋CD25^＋T细胞占CD4＋T细胞（18．80％±0．06％）比例增高,与对照组（9．50％±0．03％）相比,差异有统计学意义（P〈0．01）;在非引流淋巴结（LN）和脾脏（SP）中,荷瘤小鼠CD4^＋CD25^＋T／CD4^＋T比例分别为16．28％±0．02％和17．28％±0．06％,与对照组9．50％±0．03％和11．08％±0．04％相比,差异有统计学意义（P〈0．01,P〈0．05）;同时,调节性T细胞特异性标志Foxp3 mRNA的表达也升高。在同一只荷瘤小鼠中,引流淋巴结中CD4^＋CD25^＋T细胞数量（18．8％±0．06％）较对侧非引流淋巴结（16．28％±0．02％）略有升高,但差异无统计学意义（P〉0．05）。（2）从荷瘤小鼠中纯化的CD4^＋CD25^＋T细胞,在体外对抗CD3单抗的刺激无反应,但能抑制CD4^＋CD25^-T细胞的增殖。（3）CD4^＋CD25^＋T／CD4＋T比例与肿瘤大小呈正相关,并且可以抑制CD4^＋CD25^-T细胞的抗肿瘤效应。结论肝癌细胞在小鼠体内的生长可以提高调节性T细胞的数量,其数量的高低与肿瘤的大小呈正相关,提示清除调节性T细胞将是肿瘤免疫治疗的策略之一。     

原发性肝细胞癌微环境中CD4^＋CD25^＋调节性T细胞分布与局部免疫的关系

目的通过检测原发性肝细胞癌（hepatocellular carcinoma,HCC）局部肝癌组织和癌旁组织中T细胞亚群的分布情况,探讨在肝癌发生、发展过程中CD4^＋CD25^＋调节性T细胞与局部免疫状态的关系。方法54例肝癌组织和癌旁组织及10例正常肝组织用免疫组织化学S—P法标记CD4^＋T细胞、CD8^＋T细胞及CD4^＋CD25^＋调节性T细胞（CD4^＋CD25^＋Tr细胞）,并对组织中CD4^＋CD25^＋Tr细胞与CD4^＋T、CD8^＋T及CD4^＋T／CD8^＋T值进行相关性分析。结果54例肝癌、癌旁组织中CD4^＋CD25^＋Tr细胞单个高倍视野平均数分别为5．6208±2．7235和3．8554±1．6018（P=0．001）;肝癌中CD4^＋CD25^＋Tr细胞数目与肿瘤大小、有无子灶有关,组间差异有显著性（P〈0．01）;肝癌中CD4^＋CD25^＋Tr数量与CD4^＋T细胞的数量以及CD4^＋T／CD8^＋T值呈显著负相关（r=-0．459,P=0．015;r=-0．563,P=0．011）,而与浸润性CD8^＋T细胞的数量分布无关（r=-0．485,P=0．072）。结论在肝癌微环境中CD4^＋T淋巴细胞表达降低,CD4^＋CD25^＋Tr细胞表达增高,后者可能通过抑制CD4^＋T淋巴细胞的增殖来抑制肝癌局部免疫,从而促进肿瘤的进展以及侵袭或转移。     

MMP-9和CD4+/CD8+在乳腺癌淋巴结转移过程中的作用

目的探讨基质金属蛋白酶9（MMP-9）的表达变化以及转移肿瘤周围CD4^＋T细胞／CD8^＋T细胞数量和比值在乳腺癌淋巴结转移发展过程中的作用。方法收集92例乳腺癌患者的腋窝淋巴结293枚。采用免疫组化的方法行细胞角蛋白19（CK-19）以及肌动蛋白染色，筛选出含有微转移、孤立肿瘤细胞的淋巴结。对筛选出的淋巴结行MMP-9、CIM及CD8染色，观察不同转移情况下MMP-9、CD4及CD8的表达情况。结果293枚淋巴结中，明显转移的为84枚（28．67％），有微转移的为42枚（14．33％），孤立肿瘤细胞为22枚（7．51％）；所有明显转移灶周围均有肌动蛋白表达，34枚（80．95％）含有微转移的淋巴结中微转移灶周围有肌动蛋白表达；84枚有明显转移的淋巴结中，MMP-9均呈阳性表达，其中66枚（78．57％）淋巴结高表达（＋＋＋），18枚（21．43％）为中度表达（＋＋）；42枚含有微转移的淋巴结中，1枚（2．38％）呈高表达（＋＋＋），24枚（57．14％）淋巴结呈中度表达（＋＋），15枚（35．72％）淋巴结呈低表达（＋），2枚（4．76％）淋巴结无表达。22枚孤立肿瘤细胞中，3枚（13．64％）呈低表达，其余19枚（86．36％）淋巴结无表达。CD4^＋细胞表达率在有明显转移的淋巴结中明显高于微转移和无转移的淋巴结（P〈0．05）；CD8^＋细胞在有明显转移、微转移及无转移淋巴结之间没有明显差异（P〉0．05）。CD4^＋／CD8^＋在无转移的淋巴结明显低于有明显转移者（P〈0．05）。结论MMP-9的高表达可能是促进淋巴结内肿瘤细胞生长和侵袭的重要原因之一；肿瘤细胞可导致淋巴结癌转移灶局部CD4^＋T淋巴细胞明显增多，导致CD4^＋／CD8^＋比值升高，提示机体在淋巴结局部对肿瘤细胞的免疫应答改变可能是肿瘤细胞得以发展的原因之一。     

肿瘤患者CD4^＋CD25^＋调节性T细胞流式细胞术分析

目的：分析比较肿瘤患者和健康人外周血CD4^＋CD25^＋调节性T细胞的特点,为肿瘤免疫学研究及治疗探索新方法。方法：收集并分离30例肿瘤患者和32例健康人的外周血单个核细胞（PBMCs）,用荧光标记的抗人CD4及抗人CD25单抗标记肿瘤患者和健康人PBMCs细胞,FCM检测CD4^＋CD25^＋Treg细胞,分析CD4^＋CD25^＋Treg细胞在肿瘤患者和健康人中的差别。结果：肿瘤患者的CD4^＋CD25^＋Treg细胞百分数明显高于健康人（年龄〈55者62．4vs40．4;年龄≥55者53．1vs31．0,P〈0．05）。结论：肿瘤患者的CD4^＋CD25^＋Treg细胞高于健康对照,为肿瘤免疫治疗提供新策略,通过删除CD4^＋CD25^＋Treg细胞,有可能增强抗瘤免疫反应。     

hMIP-1β基因修饰瘤苗体内诱导抗肿瘤免疫效应的研究

目的：探讨人巨噬细胞炎性蛋白-1β（hMIP-1β）基因修饰瘤苗体内诱导的抗肿瘤免疫效应。方法：通过重组腺病毒载体介导将hMIP-1β基因导入CT26细胞中，X-gal染色法检测基因转染效率；ELISA法检测hMIP-1β基因修饰CT26细胞培养上清中hMIP-1β的含量；Boyden趋化小室法检测培养上清对CD4^＋T细胞、CD8^＋T细胞、NK细胞及未成熟树突状细胞（imDCs）的趋化作用；制备hMIP-1β基因修饰的CT26细胞瘤苗并免疫BALB／c小鼠，观察其诱导免疫细胞的杀伤活性和保护性免疫反应。结果：腺病毒载体可介导hMIP-1β基因转染CT26细胞和表达（X—gal染色的阳性率〉95％），培养上清中hMIP-1β水平为980pg／mL，并对CD4^＋T细胞、CD8^＋T细胞、NK细胞及imDCs有显著的趋化作用，与转染对照基因LacZ的CT26细胞及野生型CT26细胞比较差异均有统计学意义，P〈0．01。hMIP-1β基因修饰的CT26细胞瘤苗免疫小鼠能有效诱导肿瘤特异性CTL活性和非特异性NK活性，产生明显的免疫保护作用，可抵抗肿瘤细胞的再攻击，成瘤率降低，肿瘤生长速度减慢，小鼠生存期延长。结论：hMIP-1β基因修饰瘤苗可诱导产生有效的抗肿瘤免疫保护作用，有希望成为预防肿瘤转移与复发的有效手段。     

细胞因子IL-21在慢性感染和T细胞衰竭中的作用

HBV、HCV和HIV等病毒的慢性感染一直以来困扰着免疫学家，病毒特异性CTL细胞衰竭被认为是机体不能有效清除病毒的重要机制——即CTL细胞功能耗竭，处于一种不反应的状态，不能增殖和分泌细胞因子（IL-2、IFN-γ、TNF—α等）或杀伤感染细胞。在这过程中，如果缺失了CD4^＋T细胞将导致更加严重的CTL细胞衰竭，然而其具体机制还不清楚。论文作者发现，在慢性感染中，CD4^＋T细胞分泌的IL-21能够作用于CD8^＋T细胞，促进其增殖，从而维持长期有效的抗病毒免疫。     

CD4^＋CD25^＋调节性T细胞在肿瘤免疫及化疗方面的研究进展

Treg（CD4^＋CD25^＋调节性T细胞）是具有独特免疫调节功能的T细胞亚群，抑制免疫反应，在机体免疫稳态维持、肿瘤免疫以及移植耐受等方面发挥着重要的作用影响其抑制作用的表面分子标记有Foxp3、CD127、GITR等越来越多的研究表明在实体肿瘤和血液恶性肿捕患者中Treg数目增多。在乳腺癌、卵巢癌、肺癌以及肝癌等多种实体肿瘤患者外周血和肿痛局部微环境中，Treg比例增高，且数目与患者肿瘸进展程度和预后、生存率呈负相关血液恶性肿瘤患者中Treg数目呈肿瘤发展阶段相关性增长。但是，Treg增高的机制尚不清楚。去除Treg或封闭其抑制功能，可以增强抗肿瘤免疫反应。化疗作为一种重要的治疗肿瘤的方法，对肿瘤患者外周血中的Treg数目可产生一定影响。化疗药物可能通过抑制肿瘤微环境中血管生成，促进Treg凋亡，从而起到有效控制肿瘤的作用，据报道环磷酰胺、多西紫杉醇和氟达拉滨等化疗药物均可使肿瘤明显退化，患者生存期延长但也有报道证明肿瘤患者接受化疗后，外周血中Treg数目增多。化疗究竞时Treg会产生怎样的影响，目前尚无定论，有待于进一步研究因此，Treg在肿瘤治疗方面的应用成为研究的热点，如何清除或逆转Treg的抑制作用是肿瘤免疫治疗的一个关键问题。     

放疗或化疗诱导淋巴细胞减少联合免疫重建和瘤苗免疫

目的利用淋巴细胞减少期T细胞发生增殖活化的原理,以全身照射或环磷酰胺引起淋巴细胞减少,联合免疫重建及肿瘤疫苗免疫,以增强机体的肿瘤特异性免疫反应。方法分别以放疗或化疗（环磷酰胺）引起小鼠淋巴细胞减少,然后输入同系小鼠的未致敏脾细胞,建立免疫重建的淋巴细胞减少小鼠模型（RLM）。用黑色素瘤细胞F10对前者行免疫．肿瘤攻击实验,并行T细胞亚群清除试验。而化疗-RLM-免疫模型的抗肿瘤免疫反应效果通过过继免疫治疗检测。免疫用瘤苗为GMCSF修饰的黑色素瘤细胞D6-G6。免疫后9～10d,采集肿瘤疫苗接种部位的引流淋巴结,制备效应T细胞,然后过继回输给荷瘤3d（D5）的小鼠。2周后处死小鼠,计数肺转移灶数目。结果63．2％的放疗-RLM-免疫组小鼠可对肿瘤攻击产生抵抗,显著高于正常-免疫组（16．7％,P〈0．0001）。CD8^＋T细胞是介导抗肿瘤保护性免疫的主要效应细胞。延长免疫重建和瘤苗接种之问的间隔可削弱保护性抗肿瘤免疫。化疗-RLM-免疫组效应T细胞的在体抗肿瘤活性显著强于正常．免疫组。结论在放、化疗引起的淋巴细胞减少期进行瘤苗免疫,有助于打破机体对肿瘤的免疫耐受,增强抗肿瘤免疫反应。     

放疗联合细胞因子诱导杀伤细胞对鼻咽癌患者穿孔素和颗粒酶水平的影响

肿瘤的发生、发展与机体的免疫功能有很大的关系，尤其与CD8^＋T细胞关系密切，具有肿瘤杀伤效应的CD8^＋T细胞增多则机体的免疫力强。本研究对50例早期鼻咽非角化性癌患者采用放疗联合细胞因子诱导的杀伤细胞（cytokine induced killer cells，CIK）进行治疗，随后检测患者体内CD8^＋T细胞及其分泌的细胞因子穿孔素和颗粒酶水平，并与单纯进行放疗的患者相比较，发现CIK细胞能明显提高鼻咽非角化性癌患者中CD8^＋T细胞，与具有杀伤肿瘤细胞作用的细胞因子穿孔素和颗粒酶的水平。     

CD4+CD25+调节性T细胞及其在肿瘤免疫治疗中的意义

CD4^＋ CD25^＋调节性T细胞是具有独特免疫调节功能的T细胞亚群。近年来研究发现各种恶性肿瘤患者外周血及肿瘤环境中该细胞比例增加，去除CD4^＋ CD25^＋调节性T细胞或封闭其抑制功能可以增强抗肿瘤免疫反应。CD4^＋ CD25^＋调节性T细胞成为肿瘤免疫治疗的新靶点。     

Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics

The application of tumor necrosis factor-alpha (TNF) for the treatment of solid tumors is limited by its severe, life-threatening, toxicity. Therefore, only low dosages of this cytokine can be applied systemically, which results in poor tumor response. It has been demonstrated previously that administration of high-dose TNF in a so-called isolated perfusion system markedly improved tumor response when combined with chemotherapy. It appeared that TNF had a major impact specifically on the tumor-associated vasculature. At these high concentrations, endothelial cell death is induced by TNF, resulting in complete collapse of the tumor vascular bed. Strikingly, this effect alone is not enough to induce a tumor response, but addition of a chemotherapeutic drug is mandatory to obtain an anti-tumor effect. We showed that TNF has no anti-tumor effect by itself but augmented drug accumulation mainly in the tumor, most likely by enhancing vascular leakage. It seems that enhanced vascular leakage, but not endothelial cell death, explains the interaction between TNF and the co-administered drug. We hypothesized that in a low-dose setting TNF could induce tumor accumulation of chemotherapeutic drugs and consequently improve tumor response. We demonstrate that free TNF has a strong effect on the pharmacokinetics of co-administered Doxil in B16BL6 melanoma-bearing mice, resulting in strongly augmented drug accumulation in the tumor and improved tumor response. Co-injection of Stealth liposomal TNF with Doxil resulted in comparable or less pronounced tumor responses as compared to free TNF. These results imply that systemic application of clinically tolerable doses of TNF may improve drug distribution and tumor response and could be useful in a number of anti-cancer therapies.     

The interaction between recombinant human tumor necrosis factor and radiation in 13 human tumor cell lines

Tumor necrosis factor (TNF) was cytotoxic at concentrations of 10 to 1000 units/ml to 12 of 14 human tumor cell lines. Synergistic or additive cell killing between TNF and radiation was observed in 7 of 10 tumor cell lines, while independent tumor cell killing by each agent occurred in two tumor cell lines. The maximum synergistic effect was observed when TNF was added 4-12 hr prior to irradiation. This interaction was absent when TNF was added after irradiation. TNF also reduced potentially lethal damage repair in 3 of 5 cell lines tested. Possible mechanisms of interaction of TNF and X rays including induction of hydroxyl radicals and subsequent DNA damage by TNF and radiation are discussed.     

Sensitivity of resistant human tumor cell lines to tumor necrosis factor and adriamycin used in combination: correlation between down-regulation of tumor necrosis factor-messenger RNA induction and overcoming resistance.

A number of human tumor cell lines of various histological origin were examined for their sensitivity and resistance to tumor necrosis factor-alpha (TNF) and Adriamycin (ADR). Six ovarian lines, and one each of a renal, lung, and B-cell line, were tested for putative mechanisms of resistance to these agents. Cytotoxicity resulting from TNF or ADR showed no overall correlation in these lines. The combination of TNF and ADR produced enhanced cytotoxicity against these tumor lines and furthermore resulted in overcoming the resistance of TNF or ADR alone or in combination. A proposed mechanism of TNF resistance in tumor cells is the endogenous production of TNF mRNA and protein. There was a positive correlation between resistance to TNF and the constitutive production of TNF mRNA and protein. The TNF-resistant lines that did not constitutively produce TNF mRNA and protein and the three TNF-sensitive tumor lines exhibited up-regulation of their TNF mRNA in the presence of TNF or phorbol myristate acetate/ionophore, but did not secrete any detectable protein. Due to the enhanced cytotoxicity seen with the combination of TNF and ADR, the effect of this combination on the level of TNF mRNA was examined. ADR alone reduced the constitutive level of TNF mRNA and in combination with TNF reduced the level of induction produced by TNF. This down-regulation of TNF mRNA by ADR may play a role in the enhanced cytotoxicity seen with the combination of these 2 agents.     

Tumor vasculature‐targeted delivery of tumor necrosis factor‐α*

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor‐α (TNF‐α) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF‐α, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF‐α targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno‐associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF‐α (AAVP‐TNF‐α). The activity of AAVP‐TNF‐α was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP‐TNF‐α infection of human melanoma cells resulted in high levels of TNF‐α expression. Systemic administration of targeted AAVP‐TNF‐α to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF‐α, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF‐α specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF‐α is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy. Cancer 2009. Published 2008 by the American Cancer Society.     

Mechanism of the cytotoxic effect of tumor necrosis factor

The mechanism of murine tumor necrosis factor (TNF) cytotoxicity against tumor cell lines (L929, HeLa, K562) was investigated. Electron microscopic observation revealed that most of the organellas of L929 cells incubated with partially purified murine TNF underwent almost complete lysis with no drastic disruption of the cytoplasmic membrane, while injection of the TNF into the cytoplasm or nuclei of L929 cells caused no apparent morphological change or growth inhibition. Preincubation of the TNF with tumor cells (L929, HeLa, K562) resulted in a decrease in cytotoxic activity which was proportional to their susceptibility to TNF, thus indicating their absorption of TNF. The susceptibility of L929 tumor cells to TNF was apparently suppressed by treatment with proteases, suggesting the existence of protease-sensitive recognition sites for TNF on the tumor cell.     

Nanotherapeutics for enhancing thermal therapy of cancer.

PURPOSE: The current work describes the synergistic enhancement of hyperthermic cancer therapy by selective thermal sensitization and induction of vascular injury at the tumor site. The specificity of this response was mediated by CYT-6091: a pegylated colloidal gold-based nanotherapeutic designed to selectively deliver an inflammatory cytokine, tumor necrosis factor alpha (TNF), to solid tumors. MATERIALS AND METHODS: FSaII murine fibrosarcoma-bearing C3H mice received an intravenous injection of either soluble TNF or CYT-6091 (50-250 microg/kg TNF). Four hours later the tumors were exposed to localized heating (42.5 or 43.5 degrees C, 60 min). Tumor responses were assessed by growth delay and/or perfusion. RESULTS: Both soluble TNF and CYT-6091 reduced tumor perfusion by 80% of control (no treatment), 4 hours post administration. However, soluble TNF was toxic to the tumor burdened mice and resulted in 40% mortality alone and 100% mortality when combined with hyperthermia. Conversely, no toxicities were noted with CYT-6091 alone or when combined with hyperthermia. Additionally, CYT-6091 combined with heat yielded significant tumor regression in vivo as compared to heat or CYT-6091 alone as demonstrated by tumor growth delay. Pretreatment with soluble TNF or CYT-6091 followed by heating reduced in vitro tumor and endothelial cell survival by 40-50% (TNF) and 70-75% (CYT-6091) of the control cell (i.e. tumor and endothelial) values, respectively. CONCLUSIONS: CYT-6091, by selectively delivering TNF to solid tumors, improves the safety of TNF treatment. In addition, the targeted delivery of TNF augments cancer thermal therapy efficacy possibly by inducing a tumor-localized inflammatory response.     

TNF expressed by tumor-associated macrophages, but not microglia, can eliminate glioma

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF.     

Induction of intratumoral tumor necrosis factor (TNF) synthesis and hemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF knockout mice.

5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.     

Nanotherapeutics for enhancing thermal therapy of cancer

Purpose: The current work describes the synergistic enhancement of hyperthermic cancer therapy by selective thermal sensitization and induction of vascular injury at the tumor site. The specificity of this response was mediated by CYT-6091: a pegylated colloidal gold-based nanotherapeutic designed to selectively deliver an inflammatory cytokine, tumor necrosis factor alpha (TNF), to solid tumors.

Materials and methods: FSaII murine fibrosarcoma-bearing C3H mice received an intravenous injection of either soluble TNF or CYT-6091 (50–250?µg/kg TNF). Four hours later the tumors were exposed to localized heating (42.5 or 43.5°C, 60?min). Tumor responses were assessed by growth delay and/or perfusion.

Results: Both soluble TNF and CYT-6091 reduced tumor perfusion by 80% of control (no treatment), 4 hours post administration. However, soluble TNF was toxic to the tumor burdened mice and resulted in 40% mortality alone and 100% mortality when combined with hyperthermia. Conversely, no toxicities were noted with CYT-6091 alone or when combined with hyperthermia. Additionally, CYT-6091 combined with heat yielded significant tumor regression in vivo as compared to heat or CYT-6091 alone as demonstrated by tumor growth delay. Pretreatment with soluble TNF or CYT-6091 followed by heating reduced in vitro tumor and endothelial cell survival by 40–50% (TNF) and 70–75% (CYT-6091) of the control cell (i.e. tumor and endothelial) values, respectively.

Conclusions: CYT-6091, by selectively delivering TNF to solid tumors, improves the safety of TNF treatment. In addition, the targeted delivery of TNF augments cancer thermal therapy efficacy possibly by inducing a tumor-localized inflammatory response.     

Augmentation by tumor necrosis factor alpha of the systemic therapeutic effect of lymphokine-activated killer cells in adoptive immunotherapy of murine tumor

The therapeutic effect of a combined modality of lymphokine-activated killer (LAK) cells and tumor necrosis factor alpha (TNF alpha) on MBL-2 tumor in C57BL/6 mice was studied. Murine LAK cells induced from splenocytes by interleukin 2 (IL2) could lyse MBL-2 target cells in vitro. but no enhancement of the LAK activity was found by the treatment of LAK cells with TNF alpha in vitro. However, the treatment of MBL-2 with TNF alpha enhanced the sensitivity to LAK cells. Moreover, administration of TNF alpha to mice bearing solid MBL-2 tumor led to increased tumor vascular permeability within 1 h, and resulted in the enhanced accumulation of systemically transferred LAK cells in tumor tissue. Based on these results, we treated MBL-2-bearing mice with TNF alpha and then with LAK cells 1 h later. No therapeutic effect was observed when tumor-bearing mice were treated with TNF alpha alone or LAK cells plus IL2. However, adoptive immunotherapy using LAK cells and TNF alpha had therapeutic effects, i.e., growth inhibition of tumor nodules and prolongation of survival. These results indicated that appropriately timed pretreatment of tumor-bearing mice with TNF alpha augmented the anti-tumor efficacy of LAK cells.