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 αvß3-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 αvß3-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.

Multimodal molecular imaging of integrin αvß3 for in vivo detection of pancreatic cancer.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease. Late detection of then nonresectable or metastasized tumors emphasizes the need for novel imaging approaches. Here, we report on so far nonexploited potentials of αvß3 integrin-targeted molecular imaging technologies for detection of PDAC using genetically engineered mouse models. METHODS: Immunohistochemistry and Western blot were used for characterization of αvß3 expression in murine and human PDAC. We applied IntegriSense 680 fluorescence molecular tomography, intraoperative fluorescence imaging, and (68)Ga-NODAGA-RGD PET for αvß3 integrin molecular in vivo imaging of spontaneous PDAC occurring in Ptf1a(+/Cre);Kras(+/LSL-G12D);p53(LoxP/LoxP) mice. (NODAGA is 1,4,7-triazacyclononane-1,4-bis[acetic acid]-7-[2-glutaric acid] and RGD is arginine-glycine-aspartic acid.) RESULTS: αvß3 integrin is expressed in tumor cells of human and murine PDAC. IntegriSense fluorescence molecular tomography and (68)Ga-NODAGA-RGD PET enabled faithful visualization of PDAC. Furthermore, intraoperative optical imaging with IntegriSense 680 allowed good delineation of tumor borders. CONCLUSION: Imaging approaches targeting αvß3 integrin expand the potential of molecular imaging for identification of αvß3-positive PDAC with potential implications in early detection, fluorescence-guided surgery, and therapy monitoring.

Optical and opto-acoustic interventional imaging.

Many clinical interventional procedures, such as surgery or endoscopy, are today still guided by human vision and perception. Human vision however is not sensitive or accurate in detecting a large range of disease biomarkers, for example cellular or molecular processes characteristic of disease. For this reason advanced optical and opto-acoustic (photo-acoustic) methods are considered for enabling a more versatile, sensitive and accurate detection of disease biomarkers and complement human vision in clinical decision making during interventions. Herein, we outline developments in emerging fluorescence and opto-acoustic sensing and imaging techniques that can lead to practical implementations toward improving interventional vision.

Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results.

The prognosis in advanced-stage ovarian cancer remains poor. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and thereby improve prognosis. The overexpression of folate receptor-α (FR-α) in 90-95% of epithelial ovarian cancers prompted the investigation of intraoperative tumor-specific fluorescence imaging in ovarian cancer surgery using an FR-α-targeted fluorescent agent. In patients with ovarian cancer, intraoperative tumor-specific fluorescence imaging with an FR-α-targeted fluorescent agent showcased the potential applications in patients with ovarian cancer for improved intraoperative staging and more radical cytoreductive surgery.

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment.

BACKGROUND AND PURPOSE: The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure. MATERIALS AND METHODS: Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo. RESULTS: The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 °C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 °C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection. CONCLUSIONS: In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

Enhancing surgical vision by using real-time imaging of αvß3-integrin targeted near-infrared fluorescent agent.

BACKGROUND: This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon's vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome. METHODS: Mice bearing xenograft human tumors were injected with αvß3-integrin receptor-targeted fluorescent probe and in vivo visualized by using a novel, real-time, multispectral fluorescence imaging system. Confirmatory ex vivo imaging, bioluminescence imaging, and histopathology were used to validate the in vivo findings. RESULTS: Fluorescence images were all in good correspondence with the confirming bioluminescence images in respect to signal colocalization. Fluorescence imaging detected all tumors and successfully guided total tumor excision by effectively detecting small tumor residuals, which occasionally were missed by the surgeon. Tumor tissue exhibited target-to-background ratio of ~4.0, which was significantly higher compared with white-light images representing the visual contrast. Histopathology confirmed the capability of the method to identify tumor negative margins with high specificity and better prediction rate compared with visual inspection. CONCLUSIONS: Real-time multispectral fluorescence imaging using tumor specific molecular probes is a promising modality for tumor excision by offering real time feedback to the surgeon in the operating room.

Imaging the bio-distribution of fluorescent probes using multispectral epi-illumination cryoslicing imaging.

PURPOSE: The increasing availability of fluorescent probes for in vivo optical imaging enables the interrogation of complex biological processes in small animals serving as models for human-like tissue function and disease. However, the validation of probe bio-distribution during their development or the study of different disease models, in support of in vivo imaging studies, is not straightforward. PROCEDURES: The imaging system developed consists of a customized multispectral planar imager that has been adapted on a commercial cryomicrotome and provides a powerful modality for ex vivo imaging of small animals. RESULTS: The ability to capture 3D anatomical (color) and fluorescence volumetric distributions of multiple fluorescent markers in high resolution is showcased. CONCLUSIONS: Serving both as a method for accurately imaging the bio-distribution of multiple fluorescent agents inside organisms and as a modality for the validation of non-invasive methods, multispectral cryoslicing imaging offers useful insights to ex vivo optical imaging of molecular probes.

Intraoperative multispectral fluorescence imaging for the detection of the sentinel lymph node in cervical cancer: a novel concept.

PURPOSE: Real-time intraoperative near-infrared fluorescence (NIRF) imaging is a promising technique for lymphatic mapping and sentinel lymph node (SLN) detection. The purpose of this technical feasibility pilot study was to evaluate the applicability of NIRF imaging with indocyanin green (ICG) for the detection of the SLN in cervical cancer. PROCEDURES: In ten patients with early stage cervical cancer, a mixture of patent blue and ICG was injected into the cervix uteri during surgery. Real-time color and fluorescence videos and images were acquired using a custom-made multispectral fluorescence camera system. RESULTS: Real-time fluorescence lymphatic mapping was observed in vivo in six patients; a total of nine SLNs were detected, of which one (11%) contained metastases. Ex vivo fluorescence imaging revealed the remaining fluorescent signal in 11 of 197 non-sentinel LNs (5%), of which one contained metastatic tumor tissue. None of the non-fluorescent LNs contained metastases. CONCLUSIONS: We conclude that lymphatic mapping and detection of the SLN in cervical cancer using intraoperative NIRF imaging is technically feasible. However, the technique needs to be refined for full applicability in cervical cancer in terms of sensitivity and specificity.

Multispectral real-time fluorescence imaging for intraoperative detection of the sentinel lymph node in gynecologic oncology.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases. Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema. As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor. In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix. The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid. Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion. It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used. The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Imaging performance of a hybrid x-ray computed tomography-fluorescence molecular tomography system using priors.

PURPOSE: The performance is studied of two newly introduced and previously suggested methods that incorporate priors into inversion schemes associated with data from a recently developed hybrid x-ray computed tomography and fluorescence molecular tomography system, the latter based on CCD camera photon detection. The unique data set studied attains accurately registered data of high spatially sampled photon fields propagating through tissue along 360 degrees projections. METHODS: Approaches that incorporate structural prior information were included in the inverse problem by adding a penalty term to the minimization function utilized for image reconstructions. Results were compared as to their performance with simulated and experimental data from a lung inflammation animal model and against the inversions achieved when not using priors. RESULTS: The importance of using priors over stand-alone inversions is also showcased with high spatial sampling simulated and experimental data. The approach of optimal performance in resolving fluorescent biodistribution in small animals is also discussed. CONCLUSIONS: Inclusion of prior information from x-ray CT data in the reconstruction of the fluorescence biodistribution leads to improved agreement between the reconstruction and validation images for both simulated and experimental data.

Hybrid system for simultaneous fluorescence and x-ray computed tomography.

A hybrid imaging system for simultaneous fluorescence tomography and X-ray computed tomography (XCT) of small animals has been developed and presented. The system capitalizes on the imaging power of a 360 ( degrees )-projection free-space fluorescence tomography system, implemented within a microcomputed tomography scanner. Image acquisition is based on techniques that automatically adjust a series of imaging parameters to offer a high dynamic range dataset. Image segmentation further allows the incorporation of structural priors in the optical reconstruction problem to improve the imaging performance. The functional system characteristics are showcased, and images from a brain imaging study are shown, which are reconstructed using XCT-derived priors into the optical forward problem.