Evaluating tumor metastatic potential by imaging intratumoral acidosis via pH‐activatable near‐infrared fluorescent probe

Metastasis accounts for the vast majority of cancer deaths. To minimize metastasis‐associated mortality, it is crucially important to evaluate the metastatic potential (M.P.), that is, defined as a tendency of a primary tumor to colonize a distant organ. Dysregulated pH in solid tumors, especially the acidification of extracellular pH (pH_e) promotes dormant metastasis by driving protease‐mediated digestion, disrupting cell‐matrix interaction and increasing migration of cancer cells. Therefore, imaging intratumoral acidosis creates a unique opportunity to evaluate the M.P. In this work, a novel pH activatable probe was developed, in which two near‐infrared (NIR) fluorophores were conjugated via a flexible and acid liable linkage. While the fluorescence of this probe is quenched due to intramolecular dimeric aggregate under neutral environment, the cleavage of pH liable linkage with the concomitant disruption of aggregates in acidic tumor microenvironment results in a remarkable fluorescence enhancement. This probe not only visualized the primary tumors with high target to background (T/B) signal ratio in vivo, but also revealed the correlation between the M.P. and acidosis distribution pattern in tumor. While the acidosis locate dispersedly at tumor periphery in highly metastatic tumor, it distribute more widely in lowly metastatic tumor and the acidification degree increases substantially from the margin to core areas. This pH activatable NIR fluorescent probe holds the potential to evaluate the M.P., monitor the therapeutic response and predict the prognosis by delineating acidosis in tumors.     

In vivo imaging of membrane type‐1 matrix metalloproteinase with a novel activatable near‐infrared fluorescence probe

Membrane type‐1 matrix metalloproteinase (MT1‐MMP) is a protease activating MMP‐2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1‐MMP would be useful for tumor diagnosis, we developed a novel near‐infrared (NIR) fluorescence probe that can be activated following interaction with MT1‐MMP in vivo. MT1‐hIC7L is an activatable fluorescence probe comprised of anti‐MT1‐MMP monoclonal antibodies conjugated to self‐assembling polymer micelles that encapsulate NIR dyes (IC7‐1, λ_em: 858 nm) at concentrations sufficient to cause fluorescence self‐quenching. In aqueous buffer, MT1‐hIC7L fluorescence was suppressed to background levels and increased approximately 35.5‐fold in the presence of detergent. Cellular uptake experiments revealed that in MT1‐MMP positive C6 glioma cells, MT1‐hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti‐MT1‐MMP monoclonal antibody. In MT1‐MMP negative MCF‐7 breast adenocarcinoma cells, both MT1‐hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1‐hIC7L was suppressed by pre‐treatment with an MT1‐MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor‐bearing mice showed that MT1‐hIC7L specifically visualized C6 tumors (tumor‐to‐background ratios: 3.8 ± 0.3 [MT1‐hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF‐7 tumors. Together, these results show that MT1‐hIC7L would be a potential activatable NIR probe for specifically detecting MT1‐MMP‐expressing tumors.     

Preclinical evaluation of near‐infrared (NIR) fluorescently labeled cetuximab as a potential tool for fluorescence‐guided surgery

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence‐guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near‐infrared (NIR) fluorescently labeled as a new tool for fluorescence‐guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux‐Alexa‐647) and the co‐injected control human IgG Alexa Fluor 750 conjugate (hIgG‐Alexa‐750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC‐1 and MIA PaCa‐2 PDAC tumors. Cetux‐Alexa‐647, but not the control hIgG‐Alexa‐750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC‐1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux‐Alexa‐647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux‐Alexa‐750). Fluorescence‐guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux‐800CW), enabled a real‐time delineation of AsPC‐1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux‐800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence‐guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.     

人胃癌裸鼠原位移植瘤模型的建立及其活体荧光成像检测

目的:建立可动态实时监测的人胃癌裸鼠原位移植瘤模型.方法:将稳定表达荧光素酶的人胃癌细胞SGC-7901fLuc+注入裸鼠后肢根部皮下形成皮下移植瘤.待皮下移植瘤瘤块长至直径0.5 cm时,剥取肿瘤组织,应用叠合法将瘤块原位移植于裸鼠胃小弯处,形成原位移植瘤模型.利用小动物活体荧光成像系统,每4日监测移植瘤的进展情况.荷瘤3周后,解剖荧光成像阳性小鼠,利用H-E染色、光镜下观察移植瘤的形态.结果:应用小动物活体荧光成像系统,可在荷瘤裸鼠胃部检测到逐渐增强的荧光信号.荷瘤裸鼠胃部存在肿瘤包块贴附于胃壁,包块直径为0.5 ～1.0 cm,包块周围与相邻组织器官边界清晰,未见粘连,检查腹腔未见转移,未见腹水形成.应用叠合法构建人胃癌裸鼠原位移植瘤模型成功率为95％(19/20).对荷瘤胃组织H-E染色后,可见异常肿瘤细胞,肿瘤包膜完整,符合胃癌组织学特征.结论:成功建立了操作简便、成瘤率高的可动态监测的人胃癌裸鼠原位移植瘤模型,为研究胃癌发生机制、研发抗癌药物提供了理想的实验工具.     

An orthotopic implantation mouse model of human malignant pleural mesothelioma for in vivo photon counting analysis and evaluation of the effect of S‐1 therapy

Human malignant pleural mesothelioma (HMPM) is an aggressive neoplasm that is highly resistant to conventional therapies. We established 3 HMPM cell lines (TCC‐MESO‐1, TCC‐MESO‐2 and TCC‐MESO‐3) from Japanese patients; the first 2 from the primary and metastatic tumors of a patient with the epithelioid type of HMPM, and the third from a patient with biphasic characteristics of the tumor (epithelioid and sarcomatous phenotypes). The 3 cell lines resembled the original HMPMs in their morphological and biological features, including the genetic alterations such as lack of p16 expression and mutation of p53. Their tumorigenicity was determined in SCID mice by orthotopic implantation (20–46%). The tumorigenicity of the HMPM cell lines, which was relatively low, was enhanced by repeated subcultures and orthotopic implantations, and 3 competent tumorigenic sublines were produced (Me1Tu, Me2Tu and Me3Tu sublines from the TCC‐MESO‐1, TCC‐MESO‐2 and TCC‐MESO‐3 cell lines, respectively). The resultant HMPM sublines efficiently generated tumors in the SCID mice (100%) following orthotopic implantation. SCID mice implanted with the competent sublines, into one of which the luciferase gene was introduced, displayed quantitative fluctuation of the bioluminescence for the tumor volume in vivo. Oral administration of S‐1, an anticancer agent, suppressed the proliferation of the luciferase gene‐expressing Me1Tu subline in the mouse models in vivo, with a treated‐to‐control ratio of the mean tumor volume of 0.2. The orthotopic implantation mouse model proved to be useful for quantitative evaluation of the efficacy of novel anticancer drugs and also for studying the biology of HMPMs in vivo.