Lighting up cancer aggressiveness: targeting the urokinase plasminogen activator receptor for intraoperative optical imaging

Cancers accounts for approximately one-in-six deaths worldwide. Despite the advances in treatment options over the past couple decades surgery remains the cornerstone for almost all solid tumors. Radical excision is crucial for curative treatment, however, intraoperative tumor identification is hampered by (1) fibrosis, inflammation and necrosis due to neoadjuvant therapy, and (2) an altered surgical field due to the adoption of laparoscopic and robotic procedures. In response, fluorescence guided surgery (FGS) has been introduced to the operating theater. FGS utilizes near-infrared light to visualize, an often intravenously administered, tumor-targeting tracer in real-time. In this thesis, uPAR was studied as target for FGS and uPAR-targeting FGS tracers were developed and compared in various cancer models. CHDR; MSB GoudaLUMC / Geneeskund

Targeting glycans and heavily glycosylated proteins for tumor imaging

Simple SummaryDistinguishing malignancy from healthy tissue is essential for oncologic surgery. Targeted imaging during an operation aids the surgeon to operate better. The present tracers for detecting cancer are directed against proteins that are overexpressed on the membrane of tumor cells. This review evaluates the use of tumor-associated sugar molecules as an alternative for proteins to image cancer tissue. These sugar molecules are present as glycans on glycosylated membrane proteins and glycolipids. Due to their location and large numbers per cell, these sugar molecules might be better targets for tumor imaging than proteins.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.Surgical oncolog

Glycan-based near-infrared fluorescent (NIRF) imaging of gastrointestinal tumors: a preclinical proof-of-conceptIn vivostudy

Purpose Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-conceptin vivostudy 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 Lewis(a/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 evaluatedin vitroon 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 imagedin vivoafter intravenous administration of 1 nmol (150 mu g) 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 analyzedex 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 Lewis(a/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 vivoanalysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. Conclusions Using a novel chimeric Lewis(a/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.Surgical oncolog

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.Surgical oncolog

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-31uc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-31uc2 orthotopic mouse model. InfliximabIRDye800CW 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. (C) 2021 The Author(s). Published by Elsevier Ltd.Surgical oncolog

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

Simple Summary Prostate-specific membrane antigen (PSMA)-targeted PET/CT imaging is increasingly being used for (re)staging in prostate cancer. Although PSMA suggests specificity to prostate cancer, previous preclinical studies and case reports have shown this protein to be overexpressed by multiple other tumor types. This study aims to investigate the applicability of a PSMA-targeted PET/CT tracer to detect gastrointestinal cancers, including colon, pancreatic and gastric 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 [F-18]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 [F-18]DCFPyL and [F-18]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 [F-18]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 [F-18]FDG PET/CT were significantly higher than on [F-18]DCFPyL PET/CT. In addition, no correlation between PSMA expression in the resection specimen and SUVmax on [F-18]DCFPyL PET/CT was found. In conclusion, the detection of several gastrointestinal cancers using [F-18]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, [F-18]FDG PET/CT was superior in detecting colon, gastric and pancreatic cancers.Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Optimale tijdsduur tot operatie bij een heupfractuur

De optimale tijdsduur tot operatie bij patiënten met een heupfractuur is nog steeds onderwerp van discussie en hierover is veel gepubliceerd. Hoewel ‘tijdsduur tot operatie’ sinds 2017 een kwaliteitsindicator is op de transparantiekalender van het Zorginstituut Nederland, bestaat er in de literatuur geen consensus over de tijdsduur waarbinnen patiënten met een heupfractuur geopereerd moeten zijn. Een analyse van de gegevens van IGJ-kwaliteitsindicator ‘percentage heupfractuur geopereerd binnen kalenderdag’ laat zien dat in Nederlandse ziekenhuizen gemiddeld 93% van de patiënten met ASA-klasse 1-2 en 86% van de patiënten met ASA-klasse > 2 binnen één kalenderdag na presentatie geopereerd werd. Uitstel van de operatie omwille van preoperatieve optimalisatie van de patiënt leidt niet tot een verhoogde mortaliteit. Uitstel van de operatie verhoogt wel het risico op complicaties als pneumonie en decubitus.  Trauma Surger

Molecular imaging of the urokinase plasminogen activator receptor: opportunities beyond cancer

The urokinase plasminogen activator receptor (uPAR) plays a multifaceted role in almost any process where migration of cells and tissue-remodeling is involved such as inflammation, but also in diseases as arthritis and cancer. Normally, uPAR is absent in healthy tissues. By its carefully orchestrated interaction with the protease urokinase plasminogen activator and its inhibitor (plasminogen activator inhibitor-1), uPAR localizes a cascade of proteolytic activities, enabling (patho)physiologic cell migration. Moreover, via the interaction with a broad range of cell membrane proteins, like vitronectin and various integrins, uPAR plays a significant, but not yet completely understood, role in differentiation and proliferation of cells, affecting also disease progression. The implications of these processes, either for diagnostics or therapeutics, have received much attention in oncology, but only limited beyond. Nonetheless, the role of uPAR in different diseases provides ample opportunity to exploit new applications for targeting. Especially in the fields of oncology, cardiology, rheumatology, neurology, and infectious diseases, uPAR-targeted molecular imaging could offer insights for new directions in diagnosis, surveillance, or treatment options

EGFR and alpha v beta 6 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 alpha v beta 6 and epidermal growth factor receptor (EGFR) on tumor cells. Normal epithelium stained less for alpha v beta 6 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 beta 3, carcinoembryonic antigen, epithelial cell adhesion molecule, or vascular endothelial growth factor A. In conclusion, alpha v beta 6 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 alpha v beta 6 as targets.Surgical oncolog

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

Surgical oncolog