Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells.

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.     

Glycan-Based Near-infrared Fluorescent (NIRF) Imaging of Gastrointestinal Tumors: a Preclinical Proof-of-Concept In Vivo Study

Molecular Imaging and Biology - Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-concept in vivo study aims to validate the use of aberrant Lewis...     

Intraoperative Pancreatic Cancer Detection using Tumor-Specific Multimodality Molecular Imaging

Background

Operative management of pancreatic ductal adenocarcinoma (PDAC) is complicated by several key decisions during the procedure. Identification of metastatic disease at the outset and, when none is found, complete (R0) resection of primary tumor are key to optimizing clinical outcomes. The use of tumor-targeted molecular imaging, based on photoacoustic and fluorescence optical imaging, can provide crucial information to the surgeon. The first-in-human use of multimodality molecular imaging for intraoperative detection of pancreatic cancer is reported using cetuximab-IRDye800, a near-infrared fluorescent agent that binds to epidermal growth factor receptor.

Methods

A dose-escalation study was performed to assess safety and feasibility of targeting and identifying PDAC in a tumor-specific manner using cetuximab-IRDye800 in patients undergoing surgical resection for pancreatic cancer. Patients received a loading dose of 100 mg of unlabeled cetuximab before infusion of cetuximab-IRDye800 (50 mg or 100 mg). Multi-instrument fluorescence imaging was performed throughout the surgery in addition to fluorescence and photoacoustic imaging ex vivo.

Results

Seven patients with resectable pancreatic masses suspected to be PDAC were enrolled in this study. Fluorescence imaging successfully identified tumor with a significantly higher mean fluorescence intensity in the tumor (0.09?±?0.06) versus surrounding normal pancreatic tissue (0.02?±?0.01), and pancreatitis (0.04?±?0.01; p?<?0.001), with a sensitivity of 96.1% and specificity of 67.0%. The mean photoacoustic signal in the tumor site was 3.7-fold higher than surrounding tissue.

Conclusions

The safety and feasibilty of intraoperative, tumor-specific detection of PDAC using cetuximab-IRDye800 with multimodal molecular imaging of the primary tumor and metastases was demonstrated.

     

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.     

Intraoperative imaging using fluorescence

Intraoperative imaging using fluorescence is an experimental technique by which specific tissue structures can be visualised. A preoperatively administered optical contrast agent with fluorescence properties is detected during the operation using a light-sensitive camera system. Using this technique, vital anatomical structures such as blood vessels, bile ducts and ureters are rendered visible to the surgeon. The technique can also serve as a detection method for sentinel lymph nodes. Furthermore, tumour-specific fluorescent tracers are being developed to delineate tumours from surrounding tissue. The aim of this is to increase the number of radical cancer operations and reduce iatrogenic tissue damage. Currently, clinical studies are being conducted to investigate the value and feasibility of this technique for different surgical specialties.     

Toward Optimization of Imaging System and Lymphatic Tracer for Near-Infrared Fluorescent Sentinel Lymph Node Mapping in Breast Cancer

Background

Near-infrared (NIR) fluorescent sentinel lymph node (SLN) mapping in breast cancer requires optimized imaging systems and lymphatic tracers.

Materials and Methods

A small, portable version of the FLARE imaging system, termed Mini-FLARE, was developed for capturing color video and two semi-independent channels of NIR fluorescence (700 and 800 nm) in real time. Initial optimization of lymphatic tracer dose was performed using 35-kg Yorkshire pigs and a 6-patient pilot clinical trial. More refined optimization was performed in 24 consecutive breast cancer patients. All patients received the standard of care using ^99mTechnetium-nanocolloid and patent blue. In addition, 1.6 ml of indocyanine green adsorbed to human serum albumin (ICG:HSA) was injected directly after patent blue at the same location. Patients were allocated to 1 of 8 escalating ICG:HSA concentration groups from 50 to 1000 μM.

Results

The Mini-FLARE system was positioned easily in the operating room and could be used up to 13 in. from the patient. Mini-FLARE enabled visualization of lymphatic channels and SLNs in all patients. A total of 35 SLNs (mean = 1.45, range 1–3) were detected: 35 radioactive (100%), 30 blue (86%), and 35 NIR fluorescent (100%). Contrast agent quenching at the injection site and dilution within lymphatic channels were major contributors to signal strength of the SLN. Optimal injection dose of ICG:HSA ranged between 400 and 800 μM. No adverse reactions were observed.

Conclusions

We describe the clinical translation of a new NIR fluorescence imaging system and define the optimal ICG:HSA dose range for SLN mapping in breast cancer.     

Translational optical imaging in diagnosis and treatment of cancer

In cancer imaging, many different modalities are used that each have their specific features, leading to the combined use of different techniques for the detection, staging and treatment evaluation of cancer. Optical imaging using near-infrared fluorescence light is a new imaging modality that has recently emerged in the field of cancer imaging. After extensive preclinical research, the first steps of translation to the clinical practice are currently being made. In this article, we discuss the preclinical and clinical results of near-infrared optical imaging for non-invasive detection and classification of tumors, therapy monitoring, sentinel lymph node procedures, and image-guided cancer surgery. Widespread availability of imaging systems and optical contrast agents will enable larger studies on their clinical benefit and can help establish a definitive role in clinical practice.