Development of a Metastatic Breast Cancer model in the nude rat for MRI Cell Tra

本研究的目的是开发一种大鼠乳腺癌转移性脑肿瘤模型，以使用磁共振成像（MRI）扫描仪和体内生物发光成像（BLI）的多模式成像方法监测疾病的自然史。MDA-MB 231 BR细胞系是一种脑寻找转移性乳腺癌细胞系，其被稳定转染以表达萤火虫荧光素酶cDNA（LUC），并用超顺磁性氧化铁纳米颗粒（SPION）离体磁性标记，用于通过MRI进行早期监测。裸大鼠心内输注SPION ferumoxides标记的MDAMB 231 BRLUC（231 BRL）细胞。FEPro标记的231 BRL细胞注射大鼠发生多发性脑和脊髓转移，所有脑损伤大鼠均发生多发性骨转移。普鲁士蓝阳性乳腺癌细胞可以检测到动物心内注射SPION标记的细胞后长达1周。BLI显示大鼠脑和骨中荧光素酶光子通量活性增加，而MRI显示在注射细胞后的前3天内脑中对应于SPION标记细胞的许多低信号区域。定量靶组织中标记干细胞的数量对于优化细胞治疗的剂量和时机非常重要。 我们采用了多梯度回波脉冲序列来计算T2* 弛豫时间，以提高体素内标记细胞的检测。 我们还研究了血氧水平依赖性（BOLD）MRI的使用，该MRI已用于评估转移性肿瘤的氧合和灌注。 目前尚不清楚BOLD是否可以用于诊断，除了其预后潜力。注射FEPro乳腺癌细胞后第1天，在整个大鼠脑的T2*w图像上检测到许多低信号区域，与脉管系统中的肿瘤细胞簇一致。 在注射细胞后第36天，在T2加权的离体MR图像上鉴定出总共34个皮质肿瘤。 将这些区域映射到体内图像。 在这些肿瘤中，26个在MRI前一周（第29天）的相应区域中有明显的BOLD降低，17个在MRI前2周（第22天）有BOLD降低。重要的是，当肿瘤小于500 μ m时，可以检测到BOLD效应。BOLD MRI是转移性肿瘤发展的早期标志物，其变化与未确诊的微转移性疾病的血管异常一致。 尽管很难将BOLD变化归因于氧合或灌注，但BOLD MRI可能有助于检测发展中的转移灶。 治疗乳腺癌脑转移的药理学方法已经取得了有限的成功。部分地，血脑屏障（BBB）的不渗透性阻碍了化学治疗剂向脑中转移性肿瘤的递送。血脑屏障渗透性化疗药物正在开发中，非侵入性评估这些药物的疗效将是重要的临床前和临床设置。我们使用动态对比增强（DCE）和扩散加权成像（DWI）的磁共振成像（MRI）技术来监测转移性乳腺癌在大鼠脑，肿瘤血管通透性和细胞。我们证明，在MB 231 BR模型中，脑和骨转移瘤具有不同的生理特征，如MRI所测量的。脑转移瘤的血脑屏障的渗透性有限，与周围脑相比，用DCE评估和DWI测量的表观扩散系数（ADC）增加。显微镜下，我们发现脑转移瘤是高度浸润性的，通过血管共生生长，并引起广泛的水肿和周围神经元及其树突的损伤。通过比较，位于软脑膜或骨中的转移瘤被证明具有高血管通透性和显著较低的ADC值，提示细胞过多。在组织学检查中，骨和软脑膜中的肿瘤是具有明显肿瘤边缘的实性肿块。这些组织部位的不同特征突出了微环境对转移性肿瘤生长的影响。这项工作的意义提出了DWI和DCE评估用于治疗增选脑转移瘤的化疗和抗血管生成药物的反应的适用性问题，而这些技术用于实体瘤肿块

The purpose of this study was to develop a metastatic brain tumor model of breast cancer in the rat to monitor the natural history of the disease using a multimodal imaging approach with magnetic resonance imaging (MRI) scanner and in vivo bioluminescence imaging (BLI). MDA-MB231BR cell line is a brain seeking metastatic breast cancer line that was stably transfected to express the firefly luciferase cDNA (LUC) and was magnetically labeled with superparamagnetic iron oxide nanoparticles (SPION) ex vivo for early monitoring by MRI. Nude rats underwent intracardiac infusion SPION ferumoxides labeled MDAMB231BRLUC (231BRL) cells. FEPro labeled 231BRL cells injected rats developed multiple brain and spinal cord metastasis and all rats with brain lesion had multiple skeletal metastasis. Prussian blue positive breast cancer cells could be detected in animals up to 1 week following intracardiac injection of SPION labeled cells. BLI demonstrated increase in luciferase photon flux activity in the brain and bones of the rats while MRI revealed numerous hypointense regions corresponding to SPION labeled cells in the brain within the first 3 days following injection of cell. Quantifying the number of labeled stem cells in target tissues is of great importance to optimize dose and timing of cellular therapy. We has employed a multi-gradient echo pulse sequence to calculate T2* relaxation times to improve the detection of labeled cells within the voxel. We also have investigated the use of Blood oxygen level dependant (BOLD) MRI has been used to evaluate metastatic tumor oxygenation and perfusion. It is unknown whether BOLD can be used for diagnosis in addition to its prognostic potential. Day 1 post injection of FEPro breast cancer cells, numerous hypointense regions were detected on T2*w images throughout rat brain consistent with clusters of tumor cells in the vasculature. At day 36 post injection of cells a total of 34 cortical tumors were identified on the T2-weighted ex vivo MR images. These regions were mapped to the in vivo images. Of these tumors, 26 had noticeable BOLD decreases in the corresponding region from the week prior (day 29) MRI, and 17 had BOLD decreases 2 weeks prior (day 22) MRI. Importantly, the BOLD effects could be detected when the tumor was less than 500 m. BOLD MRI was an early marker of metastatic tumor development, and the changes are consistent with vascular abnormalities from undiagnosed micrometastasis disease. Despite the difficulties in attributing BOLD changes to oxygenation or perfusion, BOLD MRI may aid the detection of developing metastases. Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. We used dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor metastatic breast cancer in the rat brain, tumor vascular permeability and cellularity. We demonstrate that brain and bone metastases in the MB 231BR model develop with distinct physiological characteristics as measured with MRI. Brain metastases had limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, we showed that brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone were demonstrated to have high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlighted the influence of the microenvironment on metastatic tumor growth. The implication of this work raises the issue of the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases in contrast to these techniques being used in solid tumor masses