氯膦酸鹽脂質(zhì)體介導的腫瘤相關(guān)巨噬細胞(TAM)耗竭：一種新的高效抗血管生成治療方法

中文摘要：

腫瘤相關(guān)巨噬細胞 （TAM） 通過促進腫瘤血管生成在腫瘤生長和轉(zhuǎn)移中起關(guān)鍵作用。用包埋在脂質(zhì)體中的氯膦酸鹽 （Clodronate Liposomes） 治療有效地耗盡了小鼠 F9 畸胎癌和人 A673 橫紋肌肉瘤小鼠腫瘤模型中的這些吞噬細胞，導致腫瘤生長的顯著抑制，范圍為 75% 至 >92%，具體取決于治療和時間表。腫瘤抑制伴隨著腫瘤組織中血管密度的急劇降低。血管內(nèi)皮生長因子 （VEGF） 是腫瘤血管生成的主要誘導劑之一，也是巨噬細胞募集所必需的。氯膦鹽脂質(zhì)體和 VEGF 中和抗體的聯(lián)合治療觀察到效果為優(yōu)，而游離氯膦酸鹽沒有顯著活性。腫瘤的免疫組織學評估顯示 F4/80+ 和 MOMA-1+ 顯著耗竭，而 CD11b+ TAM 耗竭不太明顯。A673 模型中血管染色 （CD31） 和血管以及 TAM 和腫瘤相關(guān)樹突狀細胞 （TADC） 的定量顯示，即使在治療結(jié)束后 9 天，復位率也為 85% 至 >94%。此外，CD11c+ TADC 已被證明在腫瘤釋放的生長和分化因子刺激后可能分化為內(nèi)皮樣細胞，氯膦鹽脂質(zhì)體或抗體治療同樣降低了 CD11c+ TADC。這些結(jié)果驗證了氯膦酸鹽脂質(zhì)體 （Clodronate Liposomes） 療法與血管生成抑制劑聯(lián)合使用是一種很有前途的新型癌癥治療策略，用于針對刺激腫瘤生長和播散的造血前體細胞，并作為研究巨噬細胞和樹突狀細胞在腫瘤發(fā)生中的作用的工具。

英文摘要：

Tumour-associated macrophages, TAMs, play a pivotal role in tumour growth and metastasis by promoting tumour angiogenesis. Treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted these phagocytic cells in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth ranging from 75 to >92%, depending on therapy and schedule. Tumour inhibition was accompanied by a drastic reduction in blood vessel density in the tumour tissue. Vascular endothelial growth factor (VEGF) is one of the major inducers of tumour angiogenesis and is also required for macrophage recruitment. The strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Immunohistologic evaluation of the tumours showed significant depletion of F4/80+ and MOMA-1+ and a less pronounced depletion of CD11b+ TAMs. Blood vessel staining (CD31) and quantification of the vessels as well as TAMs and tumour-associated dendritic cells (TADCs) in the A673 model showed reduction rates of 85 to >94%, even 9 days after the end of therapy. In addition, CD11c+ TADCs, which have been shown to potentially differentiate into endothelial-like cells upon stimulation by tumour released growth and differentiation factors, were similarly reduced by clodrolip or antibody treatment. These results validate clodrolip therapy in combination with angiogenesis inhibitors as a promising novel strategy for an indirect cancer therapy aimed at the haematopoietic precursor cells that stimulate tumour growth and dissemination and as a tool to study the role of macrophages and dendritic cells in tumorigenesis.

論文信息：

論文題目： Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach

期刊名稱：British Journal of Cancer

時間期卷： volume 95, pages272–281 (2006)

在線時間：2006年7月11日

DOI： doi.org/10.1038/sj.bjc.6603240

Liposoma巨噬細胞清除劑氯膦酸鹽脂質(zhì)體Clodronate Liposomes見刊于BJC：

Liposoma巨噬細胞清除劑氯膦酸鹽脂質(zhì)體Clodronate Liposomes的材料和方法：

氯膦酸鹽脂質(zhì)體介導的腫瘤相關(guān)巨噬細胞(TAM)耗竭：一種新的高效抗血管生成治療方法，巨噬細胞清除解決方案

Tumour models and therapies-體內(nèi)實驗

Exponentially growing F9 teratocarcinoma (7 × 106?50?μl?1) or A673 rhabdomyosarcoma cells (6–8 × 106?50?μl?1 mixed 1?:?1, v?v?1 with Matrigel, Beckton Dickinson, Basel, Switzerland) were injected subcoutanously (s.c.) on the flanks of mice. Treatment was started 6?h after inoculation of F9 cells (female Sv129 mice) and 24?h after inoculation of A673 cells (female CD-1 nude mice), respectively. The mice (6–8/group) received clodronate dissolved in phosphate buffer (PB, 67?mM, pH 7.4) or clodrolip by intraperitoneal (i.p.) injection as initial dose of 2?mg?20?g?1 mouse body weight, followed by 1?mg for the subsequent doses. The Abs were given at 0.5?mg?20?g?1 mouse body weight in 100?μl PB by intravenous (i.v.) injection into the tail vein. Tumour growth was measured in a blinded fashion with a caliper and volumes were calculated using the equation: V=πab2/6 (a=largest tumour diameter, b=perpendicular diameter). Relative percentual tumour growth was normalised to day one. Mice were killed 8–22 days after onset of treatment and tumours and spleens removed for histology.

6-8周，20g小鼠，首劑量腹腔注射2mg，按Liposoma產(chǎn)品貨號CP-005-005，規(guī)格是5ml+5ml。濃度是5mg/ml。相當于注射了400ul，后期是1mg劑量維持，也就是注射200ul。

Cytotoxicity assay-體外實驗

The in vitro cytotoxicity of clodronate was assessed as described before . Briefly, cells were incubated in 96-well plates with liposomes, clodronate and clodrolip (6?h, 37°C, 1?mg clodronate?ml?1) and cell viability was determined by addition of WST-1 reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's recommendations.