Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer.

PURPOSE: Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer. METHODS: EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed. RESULTS: Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers. CONCLUSION: Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using a Fluorescently Labelled Anti-CEA Nanobody Probe: A Preclinical Study.

Molecular fluorescence-guided surgery using near-infrared light has the potential to improve the rate of complete resection of cancer. Typically, monoclonal antibodies are being used as targeting moieties, however smaller fragments, such as single-domain antibodies (i.e., Nanobodies®) improve tumor specificity and enable tracer injection on the same day as surgery. In this study, the feasibility of a carcinoembryonic antigen-targeting Nanobody (NbCEA5) conjugated to two zwitterionic dyes (ZW800-1 Forte [ZW800F] and ZW800-1) for visualization of pancreatic ductal adenocarcinoma (PDAC) was investigated. After site-specific conjugation of NbCEA5 to the zwitterionic dyes, binding specificity was evaluated on human PDAC cell lines with flow cytometry. A dose escalation study was performed for both NbCEA5-ZW800F and NbCEA5-ZW800-1 in mice with subcutaneously implanted pancreatic tumors. Fluorescence imaging was performed up to 24 h after intravenous injection. Furthermore, the optimal dose for NbCEA5-ZW800-1 was injected in mice with orthotopically implanted pancreatic tumors. A dose-escalation study showed superior mean fluorescence intensities for NbCEA5-ZW800-1 compared to NbCEA5-ZW800F. In the orthotopic tumor models, NbCEA5-ZW800-1 accumulated specifically in pancreatic tumors with a mean in vivo tumor-to-background ratio of 2.4 (SD = 0.23). This study demonstrated the feasibility and potential advantages of using a CEA-targeted Nanobody conjugated to ZW800-1 for intraoperative PDAC imaging.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. METHODS: A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. RESULTS: High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. CONCLUSIONS: Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

Side-by-Side Comparison of uPAR-Targeting Optical Imaging Antibodies and Antibody Fragments for Fluorescence-Guided Surgery of Solid Tumors.

PURPOSE: Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims to assess the feasibility of urokinase plasminogen activator receptor (uPAR) targeting antibody fragments for FGS in a direct comparison with their parent IgG in various relevant in vivo models. PROCEDURES: Humanized anti-uPAR monoclonal antibody MNPR-101 (uIgG) was proteolytically digested into F(ab')2 and Fab fragments named uFab2 and uFab. Surface plasmon resonance (SPR) and cell assays were used to determine in vitro binding before and after fluorescent labeling with IRDye800CW. Mice bearing subcutaneous HT-29 human colonic cancer cells were imaged serially for up to 120 h after fluorescent tracer administration. Imaging characteristics and ex vivo organ biodistribution were further compared in orthotopic pancreatic ductal adenocarcinoma (BxPc-3-luc2), head-and-neck squamous cell carcinoma (OSC-19-luc2-GFP), and peritoneal carcinomatosis (HT29-luc2) models using the clinical Artemis fluorescence imaging system. RESULTS: Unconjugated and conjugated uIgG, uFab2, and uFab specifically recognized uPAR in the nanomolar range as determined by SPR and cell assays. Subcutaneous tumors were clearly identifiable with tumor-to-background ratios (TBRs) > 2 after 72 h for uIgG-800F and 24 h for uFab2-800F and uFab-800F. For the latter two, mean fluorescence intensities (MFIs) dipped below predetermined threshold after 72 h and 36 h, respectively. Tumors were easily identified in the orthotopic models with uIgG-800F consistently having the highest MFIs and uFab2-800F and uFab-800F having similar values. In biodistribution studies, kidney and liver fluorescence approached tumor fluorescence after uIgG-800F administration and surpassed tumor fluorescence after uFab2-800F or uFab-800F administration, resulting in interference in the abdominal orthotopic mouse models. CONCLUSIONS: In a side-by-side comparison, FGS with uPAR-targeting antibody fragments compared with the parent IgG resulted in earlier tumor visualization at the expense of peak fluorescence intensity.

Prostate-Specific Membrane Antigen Targeted Pet/CT Imaging in Patients with Colon, Gastric and Pancreatic Cancer.

Current imaging modalities frequently misjudge disease stage in colorectal, gastric and pancreatic cancer. As treatment decisions are dependent on disease stage, incorrect staging has serious consequences. Previous preclinical research and case reports indicate that prostate-specific membrane antigen (PSMA)-targeted PET/CT imaging might provide a solution to some of these challenges. This prospective clinical study aims to assess the feasibility of [18F]DCFPyL PET/CT imaging to target and visualize primary colon, gastric and pancreatic cancer. In this prospective clinical trial, patients with colon, gastric and pancreatic cancer were included and underwent both [18F]DCFPyL and [18F]FDG PET/CT scans prior to surgical resection or (for gastric cancer) neoadjuvant therapy. Semiquantitative analysis of immunohistochemical PSMA staining was performed on the surgical resection specimens, and the results were correlated to imaging parameters. The results of this study demonstrate detection of the primary tumor by [18F]DCFPyL PET/CT in 7 out of 10 patients with colon, gastric and pancreatic cancer, with a mean tumor-to-blood pool ratio (TBR) of 3.3 and mean SUVmax of 3.6. However, due to the high surrounding uptake, visual distinction of these tumors was difficult, and the SUVmax and TBR on [18F]FDG PET/CT were significantly higher than on [18F]DCFPyL PET/CT. In addition, no correlation between PSMA expression in the resection specimen and SUVmax on [18F]DCFPyL PET/CT was found. In conclusion, the detection of several gastrointestinal cancers using [18F]DCFPyL PET/CT is feasible. However, low tumor expression and high uptake physiologically in organs/background hamper the clear distinction of the tumor. As a result, [18F]FDG PET/CT was superior in detecting colon, gastric and pancreatic cancers.

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma.

With a 5-year recurrence rate of 30-78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. METHODS: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. RESULTS: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. CONCLUSIONS: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.

Targeting Glycans and Heavily Glycosylated Proteins for Tumor Imaging.

Real-time tumor imaging techniques are increasingly used in oncological surgery, but still need to be supplemented with novel targeted tracers, providing specific tumor tissue detection based on intra-tumoral processes or protein expression. To maximize tumor/non-tumor contrast, targets should be highly and homogenously expressed on tumor tissue only, preferably from the earliest developmental stage onward. Unfortunately, most evaluated tumor-associated proteins appear not to meet all of these criteria. Thus, the quest for ideal targets continues. Aberrant glycosylation of proteins and lipids is a fundamental hallmark of almost all cancer types and contributes to tumor progression. Additionally, overexpression of glycoproteins that carry aberrant glycans, such as mucins and proteoglycans, is observed. Selected tumor-associated glyco-antigens are abundantly expressed and could, thus, be ideal candidates for targeted tumor imaging. Nevertheless, glycan-based tumor imaging is still in its infancy. In this review, we highlight the potential of glycans, and heavily glycosylated proteoglycans and mucins as targets for multimodal tumor imaging by discussing the preclinical and clinical accomplishments within this field. Additionally, we describe the major advantages and limitations of targeting glycans compared to cancer-associated proteins. Lastly, by providing a brief overview of the most attractive tumor-associated glycans and glycosylated proteins in association with their respective tumor types, we set out the way for implementing glycan-based imaging in a clinical practice.

Anti-GD2-IRDye800CW as a targeted probe for fluorescence-guided surgery in neuroblastoma.

Neuroblastoma resection represents a major challenge in pediatric surgery, because of the high risk of complications. Fluorescence-guided surgery (FGS) could lower this risk by facilitating discrimination of tumor from normal tissue and is gaining momentum in adult oncology. Here, we provide the first molecular-targeted fluorescent agent for FGS in pediatric oncology, by developing and preclinically evaluating a GD2-specific tracer consisting of the immunotherapeutic antibody dinutuximab-beta, recently approved for neuroblastoma treatment, conjugated to near-infrared (NIR) fluorescent dye IRDye800CW. We demonstrated specific binding of anti-GD2-IRDye800CW to human neuroblastoma cells in vitro and in vivo using xenograft mouse models. Furthermore, we defined an optimal dose of 1 nmol, an imaging time window of 4 days after administration and show that neoadjuvant treatment with anti-GD2 immunotherapy does not interfere with fluorescence imaging. Importantly, as we observed universal, yet heterogeneous expression of GD2 on neuroblastoma tissue of a wide range of patients, we implemented a xenograft model of patient-derived neuroblastoma organoids with differential GD2 expression and show that even low GD2 expressing tumors still provide an adequate real-time fluorescence signal. Hence, the imaging advancement presented in this study offers an opportunity for improving surgery and potentially survival of a broad group of children with neuroblastoma.

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

PURPOSE: 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 glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. PROCEDURES: Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewisa/c/x, was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluated in vitro on human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imaged in vivo after intravenous administration of 1 nmol (150 µg) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzed ex vivo. RESULTS: IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewisa/c/x-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 ± 0.3 (Pearl: 3.1 ± 0.8) was observed in the HT-29 tumors and a TBR of 1.8 ± 0.3 (Pearl: 1.9 ± 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week. Ex vivo analysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. CONCLUSIONS: Using a novel chimeric Lewisa/c/x-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors.

EGFR and αvß6 as Promising Targets for Molecular Imaging of Cutaneous and Mucosal Squamous Cell Carcinoma of the Head and Neck Region.

R0 resection is paramount in cutaneous squamous cell carcinoma (CSCC) and head and neck squamous cell carcinoma (HNSCC). However, in the setting of recurrence, immunocompromised patients, or non-keratinizing squamous cell carcinoma (SCC) with a spindle growth pattern, tumor borders are difficult, if not impossible, to determine. Fluorescence-guided surgery (FGS) aids in this differentiation. Potential targets for FGS of CSCC and HNSCC were evaluated. Most sections stained intensely for αvß6 and epidermal growth factor receptor (EGFR) on tumor cells. Normal epithelium stained less for αvß6 than for EGFR. In addition, soft tissue and stroma stained negative for both, allowing for clear discrimination of the soft tissue margin. Tumor cells weakly expressed urokinase plasminogen activator receptor (uPAR) while expression on stromal cells was moderate. Normal epithelium rarely expressed uPAR, resulting in clear discrimination of superficial margins. Tumors did not consistently express integrin ß3, carcinoembryonic antigen, epithelial cell adhesion molecule, or vascular endothelial growth factor A. In conclusion, αvß6 and EGFR allowed for precise discrimination of SSC at the surgically problematic soft tissue margins. Superficial margins are ideally distinguished with uPAR. In the future, FGS in the surgically challenging setting of cutaneous and mucosal SCC could benefit from a tailor-made approach, with EGFR and αvß6 as targets.

Small Molecules for Multi-Wavelength Near-Infrared Fluorescent Mapping of Regional and Sentinel Lymph Nodes in Colorectal Cancer Staging.

Assessing lymph node (LN) status during tumor resection is fundamental for the staging of colorectal cancer. Current guidelines require a minimum of 12 LNs to be harvested during resection and ultra-staging regional lymph nodes by sentinel lymph node (SLN) assessment is being extensively investigated. The current study presents novel near-infrared (NIR) fluorescent dyes for simultaneous pan lymph node (PanLN; regional) and SLN mapping. PanLN-Forte was intravenously injected in mice and assessed for accumulation in regional LNs. SLN800 was injected intradermally in mice, after which the collection and retention of fluorescence in SLNs were measured using indocyanine green (ICG) and its precursor, SLN700, as references. LNs in the cervical, inguinal, jejunal, iliac, and thoracic basins could clearly be distinguished after a low dose intravenous injection of PanLN-Forte. Background fluorescence was significantly lower compared to the parent compound ZW800-3A (p < 0.001). SLN700 and SLN800 specifically targeted SLNs with fluorescence being retained over 40-fold longer than the current clinically used agent ICG. Using SLN700 and SLN800, absolute fluorescence in SLN was at least 10 times higher than ICG in second-tier nodes, even at 1 hour post-injection. Histologically, the fluorescent signal localized in the LN medulla (PanLN-Forte) or sinus entry (SLN700/SLN800). PanLN-Forte and SLN800 appear to be optimal for real-time NIR fluorescence imaging of regional and SLNs, respectively.

[Optimal timing of a hip fracture operation]. / Optimale tijdsduur tot operatie bij een heupfractuur.

There is an ongoing discussion about optimal timing of hip fracture surgery and much has been published about this subject. There is no literature-based consensus regarding the timeframe in which a hip fracture patient should be operated on; nonetheless, the National Health Care Institute in the Netherlands has been using 'time to operation' as a quality indicator since 2017. Analysis of the data from the Health and Youth Care Inspectorate on the quality indicator 'Percentage of patients operated on within one calendar day' showed that in Dutch hospitals 93% of the ASA grade 1-2 patients and 86% of the ASA grade > 2 patients were operated on within one calendar day. Delay of surgery due to preoperative optimisation of the patient is not associated with an increased mortality. The chance of complications, such as pneumonia or pressure sores, does increase with delay.

Prognostic Impact of Urokinase Plasminogen Activator Receptor Expression in Pancreatic Cancer: Malignant Versus Stromal Cells.

The urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for various malignancies. The aim of this study is to assess the prognostic value of uPAR expression in neoplastic and stromal cells of patients with pancreatic adenocarcinoma. Urokinase plasminogen activator receptor expression was determined by immunohistochemistry in 122 pancreatic ductal adenocarcinomas. Kaplan-Meier and Cox regression analyses were used to determine the association with survival. Respectively 66%, 82% and 62% of patients with pancreatic cancer expressed uPAR in neoplastic cells, stromal, and in both combined. Multivariate analysis showed a significant inverse association between uPAR expression in both neoplastic and stromal cells and overall survival. The prognostic impact of uPAR in stromal cells is substantial, but not as pronounced as that of uPAR expression in neoplastic cells. This study suggests a role for uPAR as a biomarker to single out higher risk subgroups of patients with pancreatic cancer.