Fluorescence‐ and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans‐cyclooctene (TCO) tag for future click chemistry (cRGD‐800CW‐TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC‐1 cells into the head of the pancreas, mice were injected with cRGD‐800CW‐TCO and imaged with fluorescence‐ and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD‐800CW‐TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole‐mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD‐800CW‐TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD‐800CW‐TCO within the tumor before and during surgical intervention.     

Detection of different tumor growth kinetics in single transgenic mice with oncogene‐induced mammary carcinomas by flat‐panel volume computed tomography

Transgenic mouse models offer an excellent opportunity for studying the molecular basis of cancer development and progression. Here we applied flat‐panel volume computed tomography (fpVCT) to monitor tumor progression as well as the development of tumor vasculature in vivo in a transgenic mouse model for oncogene‐induced mammary carcinogenesis (WAP‐T mice). WAP‐T mice develop multiple mammary carcinomas on oncogene induction within 3 to 5 months. Following induction, 3‐dimensional fpVCT data sets were obtained by serial single scans of entire mice in combination with iodine containing contrast agents and served as basis for precise measurements of tumor volumes. Thereby, we were able to depict tumors within the mammary glands at a very early stage of the development. Tumors of small sizes (0.001 cm³) were detected by fpVCT before being palpable or visible by inspection. The capability to determine early tumor onset combined with longitudinal noninvasive imaging identified diverse time points of tumor onset for each mammary carcinoma and different tumor growth kinetics for multiple breast carcinomas that developed in single mice. Furthermore, blood supply to the breast tumors, as well as blood vessels around and within the tumors, were clearly visible over time by fpVCT. Three‐dimensional visualization of tumor vessels in high resolution was enhanced by the use of a novel blood pool contrast agent. Here, we demonstrate by longitudinal fpVCT imaging that mammary carcinomas develop at different time points in each WAP‐T mouse, and thereafter show divergent growth rates and distinct vascularization patterns. © 2009 UICC     

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.     

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 a novel CEA‐targeting near‐infrared fluorescent tracer delineating colorectal and pancreatic tumors

Surgery is the cornerstone of oncologic therapy with curative intent. However, identification of tumor cells in the resection margins is difficult, resulting in nonradical resections, increased cancer recurrence and subsequent decreased patient survival. Novel imaging techniques that aid in demarcating tumor margins during surgery are needed. Overexpression of carcinoembryonic antigen (CEA) is found in the majority of gastrointestinal carcinomas, including colorectal and pancreas. We developed ssSM3E/800CW, a novel CEA‐targeted near‐infrared fluorescent (NIRF) tracer, based on a disulfide‐stabilized single‐chain antibody fragment (ssScFv), to visualize colorectal and pancreatic tumors in a clinically translatable setting. The applicability of the tracer was tested for cell and tissue binding characteristics and dosing using immunohistochemistry, flow cytometry, cell‐based plate assays and orthotopic colorectal (HT‐29, well differentiated) and pancreatic (BXPC‐3, poorly differentiated) xenogeneic human–mouse models. NIRF signals were visualized using the clinically compatible FLARE? imaging system. Calculated clinically relevant doses of ssSM3E/800CW selectively accumulated in colorectal and pancreatic tumors/cells, with highest tumor‐to‐background ratios of 5.1 ± 0.6 at 72 hr postinjection, which proved suitable for intraoperative detection and delineation of tumor boarders and small (residual) tumor nodules in mice, between 8 and 96 hr postinjection. Ex vivo fluorescence imaging and pathologic examination confirmed tumor specificity and the distribution of the tracer. Our results indicate that ssSM3E/800CW shows promise as a diagnostic tool to recognize colorectal and pancreatic cancers for fluorescent‐guided surgery applications. If successfully translated clinically, this tracer could help improve the completeness of surgery and thus survival.     

Miniaturized antibodies for imaging membrane type‐1 matrix metalloproteinase in cancers

Since membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays pivotal roles in tumor progression and metastasis and holds great promise as an early biomarker for malignant tumors, a method of evaluating MT1‐MMP expression levels would be valuable for molecular biological and clinical studies. Although we have previously developed a ^99mTc‐labeled anti‐MT1‐MMP monoclonal IgG (^99mTc‐MT1‐mAb) as an MT1‐MMP imaging probe by nuclear medical techniques for this purpose, slow pharmacokinetics were a problem due to its large molecular size. Thus, in this study, our aim was to develop miniaturized antibodies, a single chain antibody fragment (MT1‐scFv) and a dimer of two molecules of scFv (MT1‐diabody), as the basic structures of MT1‐MMP imaging probes followed by in vitro and in vivo evaluation with an ¹¹¹In radiolabel. Phage display screening successfully provided MT1‐scFv and MT1‐diabody, which had sufficiently high affinity for MT1‐MMP (K_D = 29.8 and 17.1 nM). Both ¹¹¹In labeled miniaturized antibodies showed higher uptake in MT1‐MMP expressing HT1080 cells than in non‐expressing MCF7 cells. An in vivo biodistribution study showed rapid pharmacokinetics for both probes, which exhibited >20‐fold higher tumor to blood radioactivity ratios (T/B ratio), an index for in vivo imaging, than ^99mTc‐MT1‐mAb 6 h post‐administration, and significantly higher tumor accumulation in HT1080 than MCF7 cells. SPECT images showed heterogeneous distribution and ex vivo autoradiographic analysis revealed that the radioactivity distribution profiles in tumors corresponded to MT1‐MMP‐positive areas. These findings suggest that the newly developed miniaturized antibodies are promising probes for detection of MT1‐MMP in cancer cells.     

Potent and specific antitumor effect of CEA‐targeted photoimmunotherapy

Conventional photodynamic therapy (PDT) for cancer is limited by the insufficient efficacy and specificity of photosensitizers. We herein describe a highly effective and selective tumor‐targeted PDT using a near‐infrared (NIR) photosensitizer, IRDye700DX, conjugated to a human monoclonal antibody (Ab) specific for carcinoembryonic antigen (CEA). The antitumor effects of this Ab‐assisted PDT, called photoimmunotherapy (PIT), were investigated in vitro and in vivo. The Ab‐IRDye conjugate induced potent cytotoxicity against CEA‐positive tumor cells after NIR‐irradiation, whereas CEA‐negative cells were not affected at all, even in the presence of excess photoimmunoconjugate. We found an equivalent phototoxicity and a predominant plasma membrane localization of Ab‐IRDye after both one and six hours of incubation. Either no or little caspase activation and membrane peroxidation were observed in PIT‐treated cells and a panel of scavengers for reactive oxygen species showed only partial inhibition of the phototoxic effect. Strikingly, Ab‐IRDye retained significant phototoxicity even under hypoxia. We established a xenograft model, which allowed us to sensitively investigate the therapeutic efficacy of PIT by non‐invasive bioluminescence imaging. Luciferase‐expressing MKN‐45‐luc human gastric carcinoma cells were subcutaneously implanted into both flanks of nude mice. NIR‐irradiation was performed for only the tumor on one side. In vivo imaging and measurement of the tumor size revealed that a single PIT treatment, with intraperitoneal administration of Ab‐IRDye and subsequent NIR‐irradiation, caused rapid cell death and significant inhibition of tumor growth, but only on the irradiated side. Together, these data suggest that Ab‐IRDye‐mediated PIT has great potential as an anticancer therapeutics targeting CEA‐positive tumors.     

Tumor Angiogenesis Phenotyping by Nanoparticle-facilitated Magnetic Resonance and Near-infrared Fluorescence Molecular Imaging

One of the challenges of tailored antiangiogenic therapy is the ability to adequately monitor the angiogenic activity of a malignancy in response to treatment. The α_vβ₃ integrin, highly overexpressed on newly formed tumor vessels, has been successfully used as a target for Arg-Gly-Asp (RGD)-functionalized nanoparticle contrast agents. In the present study, an RGD-functionalized nanocarrier was used to image ongoing angiogenesis in two different xenograft tumor models with varying intensities of angiogenesis (LS174T > EW7). To that end, iron oxide nanocrystals were included in the core of the nanoparticles to provide contrast for T₂*-weighted magnetic resonance imaging (MRI), whereas the fluorophore Cy7 was attached to the surface to enable near-infrared fluorescence (NIRF) imaging. The mouse tumor models were used to test the potential of the nanoparticle probe in combination with dual modality imaging for in vivo detection of tumor angiogenesis. Pre-contrast and post-contrast images (4 hours) were acquired at a 9.4-T MRI system and revealed significant differences in the nanoparticle accumulation patterns between the two tumor models. In the case of the highly vascularized LS174T tumors, the accumulation was more confined to the periphery of the tumors, where angiogenesis is predominantly occurring. NIRF imaging revealed significant differences in accumulation kinetics between the models. In conclusion, this technology can serve as an in vivo biomarker for antiangiogenesis treatment and angiogenesis phenotyping.     

Matriptase‐2 gene (TMPRSS6) variants associate with breast cancer survival,and reduced expression is related to triple‐negative breast cancer

Matriptase‐2 (TMPRSS6) has been identified as a breast cancer risk factor. Here, we examined relationships between TMPRSS6 genetic variations and breast cancer risk and survival, and determined the gene and protein expressions in breast tumors and assessed their clinical importance. Thirteen TMPRSS6 polymorphisms were genotyped in 462 invasive breast cancer cases and 458 controls. Gene expression was analyzed from 83 tumors and protein expression from 370 tumors. We then assessed the statistical significance of associations among genotypes, clinicopathological characteristics and survival. The TMPRSS6 variant rs2543519 was associated with breast cancer risk (p = 0.032). Multivariate analysis showed that four variants had effects on survival—rs2543519 (p = 0.017), rs2235324 (p = 0.038), rs14213212 (p = 0.044) and rs733655 (p = 0.021)—which were used to create a group variable that was associated with poorer prognosis correlating with more alleles related to reduced survival (p = 0.006; risk ratio, 2.375; 95% confidence interval, 1.287–4.382). Low gene expression was related to triple‐negative breast cancer (p = 0.0001), and lower protein expression was detected in undifferentiated (p = 0.019), large (p = 0.014) and ductal or lobular tumors (p = 0.036). These results confirm the association of TMRRSS6 variants with breast cancer risk and survival. Matriptase‐2 levels decrease with tumor progression, and lower gene expression is seen in poor‐prognosis‐related triple‐negative breast cancers. Our study is the first to show that matriptase‐2 gene variants are related to breast cancer prognosis, supporting matriptase‐2 involvement in tumor development.     

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

Background

The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression.

Methods

A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-κB-responsive promoter (κB- lacZ) exhibiting constitutive activity of β-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40- lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels.

Results

SV40- lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels.

Conclusion

SV40- lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.     

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual‐modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1‐specific contrast agent (MN‐EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran‐coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1‐specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2‐weighted MRI and optical imaging before and 24 hr after injection of the MN‐EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT‐PCR. We observed that the average delta‐T2 in the gemcitabine‐treated group was significantly reduced compared to the control group indicating lower accumulation of MN‐EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine‐treated group compared to the control, which was confirmed by qRT‐PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN‐EPPT‐enhanced in vivo MR and optical imaging. These results suggest that the uMUC1‐targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.     

Clostridium perfringens enterotoxin C‐terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy‐resistant ovarian cancer

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin‐3‐ and ‐4‐overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy‐naïve and chemotherapy‐resistant human ovarian cancer xenografts or patient‐derived xenografts (PDXs) were treated with the carboxyl‐terminal binding domain of the Clostridium perfringens enterotoxin (c‐CPE) conjugated to FITC (FITC‐c‐CPE) or the near‐infrared (NIR) fluorescent tag IRDye CW800 (CW800‐c‐CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC‐c‐CPE and the CW800‐c‐CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800‐c‐CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor‐to‐background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c‐CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c‐CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real‐time detection of micrometastatic ovarian disease overexpressing the Claudin‐3 and ‐4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.     

Morphologic Changes of Mammary Carcinomas in Mice over Time as Monitored by Flat-Panel Detector Volume Computed Tomography

Noninvasive methods are strongly needed to detect and quantify not only tumor growth in murine tumor models but also the development of vascularization and necrosis within tumors. This study investigates the use of a new imaging technique, flat-panel detector volume computed tomography (fpVCT), to monitor in vivo tumor progression and structural changes within tumors of two murine carcinoma models. After tumor cell inoculation, single fpVCT scans of the entire mice were performed at different time points. The acquired isotropic, high-resolution volume data sets enable an accurate real-time assessment and precise measurements of tumor volumes. Spreading of contrast agent-containing blood vessels around and within the tumors was clearly visible over time. Furthermore, fpVCT permits the identification of differences in the uptake of contrast media within tumors, thus delineating necrosis, tumor tissues, and blood vessels. Classification of tumor tissues based on the decomposition of the underlying mixture distribution of tissue-related Hounsfield units allowed the quantitative acquisition of necrotic tissues at each time point. Morphologic alterations of the tumor depicted by fpVCT were confirmed by histopathologic examination. Concluding, our data show that fpVCT may be highly suitable for the noninvasive evaluation of tumor responses to anticancer therapies during the course of the disease.     

In vivo subcellular imaging of tumors in mouse models using a fluorophore‐conjugated anti‐carcinoembryonic antigen antibody in two‐photon excitation microscopy

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro‐lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore‐conjugated anti‐carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio‐conjugation of Alexa Fluor 594 to the anti‐CEA antibody allowed visualization of tumor mass consisting of CEA‐expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two‐photon excitation microscope and the same fluorescent antibody resulted in subcellular‐resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two‐photon excitation microscopy in conjunction with fluorophore‐conjugated antibodies could be widely adapted to detection of cancer‐specific cell‐surface molecules, both in cancer research and in clinical applications.     

Early recognition of lung cancer by integrin targeted imaging in K‐ras mouse model

Non‐small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin‐targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K‐ras mutation. We conducted ex vivo and fluorescence molecular tomography‐X‐ray computed tomography (FMT‐XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin‐targeted fluorescent signal significantly differed between wild type lung tissue and K‐ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3‐integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4‐week‐old animals could be noninvasively detected in vivo on the fluorescence channel of FMT‐XCT. Our findings demonstrated αvβ3‐integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K‐ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.     

In vivo tracking of the tropism of mesenchymal stem cells to malignant gliomas using reporter gene‐based MR imaging

Mesenchymal stem cells (MSCs) have emerged as a promising cellular vehicle for gene therapy of malignant gliomas due to their property of tumor tropism. However, MSCs may show bidirectional and divergent effects on tumor growth. Therefore, a robust surveillance system with a capacity for noninvasive monitoring of the homing, distribution and fate of stem cells in vivo is highly desired for developing stem cell‐based gene therapies for tumors. In this study, we used ferritin gene‐based magnetic resonance imaging (MRI) to track the tumor tropism of MSCs in a rat orthotopic xenograft model of malignant glioma. MSCs were transduced with lentiviral vectors expressing ferritin heavy chain (FTH) and enhanced green fluorescent protein (eGFP). Intra‐arterial, intravenous and intertumoral injections of these FTH transgenic MSCs (FTH‐MSCs) were performed in rats bearing intracranial orthotopic C6 gliomas. The FTH‐MSCs were detected as hypointense signals on T2‐ and T2*‐weighted images on a 3.0 T clinical MRI. After intra‐arterial injection, 17% of FTH‐MSCs migrated toward the tumor and gradually diffused throughout the orthotopic glioma. This dynamic process could be tracked in vivo by MRI up to 10 days of follow‐up, as confirmed by histology. Moreover, the tumor tropism of MSCs showed no appreciable impact on the progression of the tumor. These results suggest that FTH reporter gene‐based MRI can be used to reliably track the tropism and fate of MSCs after their systemic transplantation in orthotopic gliomas. This real‐time in vivo tracking system will facilitate the future development of stem cell‐based therapies for malignant gliomas.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Near-infrared fluorescent imaging of tumor apoptosis

Noninvasive imaging using radioactive annexin V is an emerging strategy for the assessment of cell death in vivo (F. G. Blankenberg, and H. W. Strauss. Apoptosis, 6: 117-123, 2001.). Therefore, we investigated whether annexin V labeled with the fluorophore Cy5.5 (Cy) could serve as a probe for imaging of tumor apoptosis using near infrared fluorescence (NIRF). We prepared active Cy-annexin (an equimolar dye:protein ratio) that bound to apoptotic Jurkat T cells and an inactive Cy-annexin probe (>2 dyes/mol protein) that did not. Active Cy annexin was used to image a 9L gliosarcoma, constitutively expressing green fluorescent protein marker, and the CR8 variant of Lewis lung carcinoma, stably transfected to express DsRed2. The expression of transfected fluorescent protein provided an indication of tumor margins and a means of defining tumor-associated NIRF signal intensity with both tumor models. Tumors were imaged with and without cyclophosphamide treatment. In both tumor models active Cy-annexin V tumor NIRF signal increased two to three times after the treatment. Tumor NIRF signal developed by 75 min after active Cy-annexin injection and remained for a 20-h observation period. Inactive annexin V was used as a control in the CR8 carcinoma experiments and resulted in a low nonspecific signal. With the 9L gliomosacrcoma model, active Cy-annexin V bound to both tumor cells (Cy-annexin V staining only) and endothelial cells (costained with Cy-annexin V and antibody to the endothelial marker CD31). Our results demonstrate that active Cy-annexin can be used as a NIRF probe to image apoptosis from outside an intact living animal and may provide nonradioactive method of measuring the antiproliferative effects of cancer chemotherapeutic regimens.     

Fluorescence‐based endoscopic imaging of Thomsen–Friedenreich antigen to improve early detection of colorectal cancer

Thomsen–Friedenreich (TF) antigen belongs to the mucin‐type tumor‐associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF‐associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio‐imaging index for the probe. The nanobeacon exhibited specificity for TF‐associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio‐imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.