A multispectral 3D live organoid imaging platform to screen probes for fluorescence guided surgery.

Achieving complete tumor resection is challenging and can be improved by real-time fluorescence-guided surgery with molecular-targeted probes. However, pre-clinical identification and validation of probes presents a lengthy process that is traditionally performed in animal models and further hampered by inter- and intra-tumoral heterogeneity in target expression. To screen multiple probes at patient scale, we developed a multispectral real-time 3D imaging platform that implements organoid technology to effectively model patient tumor heterogeneity and, importantly, healthy human tissue binding.

Evaluation of Potential Targets for Fluorescence-Guided Surgery in Pediatric Ewing Sarcoma: A Preclinical Proof-of-Concept Study.

Fluorescence-guided surgery (FGS), based on fluorescent tracers binding to tumor-specific biomarkers, could assist surgeons to achieve complete tumor resections. This study evaluated potential biomarkers for FGS in pediatric Ewing sarcoma (ES). Immunohistochemistry (IHC) was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. LINGO-1 and GD2 evaluation did not work on the acquired tissue. Based on the immunoreactive scores, anti-CD99 and anti-CD117 were evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. Anti-GD2, a tracer in the developmental phase, was also tested. These three tracers were topically applied to a freshly resected ES tumor and adjacent healthy tissue. IHC demonstrated moderate/strong CD99 and CD117 expression in ES tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117 and low GD2 expression, in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient. In conclusion, CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients.

Sentinel Lymph Node Procedure in Pediatric Patients with Melanoma, Squamous Cell Carcinoma, or Sarcoma Using Near-Infrared Fluorescence Imaging with Indocyanine Green: A Feasibility Trial.

BACKGROUND: Standard sentinel lymph node procedure (SNP) in pediatric cancer consists of a preoperative injection with 99mtechnetium nanocolloid in combination with an optional intraoperative injection with blue dye. However, blue dye has disadvantages, and the detection rate is low, with only 60% of sentinel lymph nodes (SLNs) staining blue. In adult oncology, fluorescence imaging using indocyanine green (ICG) has been shown to be a safe and accurate method for visual detection of SLNs, with a higher sensitivity (up to 97%) compared with blue dye. Therefore, our aim is to determine the feasibility of the addition of ICG to 99mtechnetium nanocolloid (ICG-TC) for visual detection of SLN in pediatric patients. METHODS: A total of 15 pediatric patients with melanoma, squamous cell carcinoma, and sarcoma were prospectively included. Preoperatively, patients were injected with ICG-TC and imaging with lymphoscintigraphy and single-photon emission computed tomography- computed tomography was performed. Intraoperatively, SLN was detected with fluorescence and the gamma probe. Postoperatively, fluorescence was quantified by tumor-to-background ratio (TBR) and surgeons evaluated the use of ICG using a standardized questionnaire. RESULTS: In 10/15 (67%) patients, SLNs were visible transcutaneously. Of all intraoperatively detected SLNs, 35/37 (95%) were fluorescent and 37/37 (100%) were radioactive. Furthermore, ICG-TC led to the identification of six additional SLNs as compared with preoperative imaging. The median TBR in vivo was 6.5 (IQR 5.3). The surgical evaluation showed that ICG assisted in SLN detection and was easy to use. CONCLUSIONS: ICG-TC for the SNP is a feasible procedure in pediatric patients. It showed an accurate detection rate, was helpful for visual guidance, and no adverse events occurred.

Indocyanine green versus technetium-99m with blue dye for sentinel lymph node detection in early-stage cervical cancer: A systematic review and meta-analysis.

BACKGROUND: The fluorescent dye indocyanine green (ICG) has emerged as a promising tracer for intraoperative detection of sentinel lymph nodes (SLNs) in early-stage cervical cancer. Although researchers suggest the SLN detection of ICG is equal to the more conventional combined approach of a radiotracer and blue dye, no consensus has been reached. AIMS: We aimed to assess the differences in overall and bilateral SLN detection rates with ICG versus the combined approach, the radiotracer technetium-99m (99m Tc) with blue dye. METHODS AND RESULTS: We searched MEDLINE, Embase, and the Cochrane Library from inception to January 1, 2020 and included studies reporting on a comparison of SLN detection with ICG versus 99m Tc with blue dye in early-stage cervical cancer. The overall and bilateral detection rates were pooled with random-effects meta-analyses. From 118 studies retrieved seven studies (one cross-sectional; six retrospective cohorts) were included, encompassing 589 patients. No significant differences were found in the pooled overall SLN detection rate of ICG versus 99m Tc with blue dye. Meta-analyses of all studies showed ICG to result in a higher bilateral SLN detection rate than 99m Tc with blue dye; 90.3% (95%CI, 79.8-100.0%) with ICG versus 73.5% (95%CI, 66.4-80.6%) with 99mTc with blue dye. This resulted in a significant and clinically relevant risk difference of 16.6% (95%CI, 5.3-28.0%). With sensitivity analysis, the risk difference of the bilateral detection rate maintained in favor of ICG but was no longer significant (13.2%, 95%CI -0.8-27.3%). CONCLUSION: ICG appears to provide higher bilateral SLN detection rates compared to 99m Tc with blue dye in patients with early-stage cervical cancer. However, in adherence with the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) guidelines, the quality of evidence is too low to provide strong recommendations and directly omit the combined approach of 99m Tc with blue dye.

Introducing Fluorescence-Guided Surgery for Pediatric Ewing, Osteo-, and Rhabdomyosarcomas: A Literature Review.

Sarcomas are a rare heterogeneous group of malignant neoplasms of mesenchymal origin which represent approximately 13% of all cancers in pediatric patients. The most prevalent pediatric bone sarcomas are osteosarcoma (OS) and Ewing sarcoma (ES). Rhabdomyosarcoma (RMS) is the most frequently occurring pediatric soft tissue sarcoma. The median age of OS and ES is approximately 17 years, so this disease is also commonly seen in adults while non-pleiomorphic RMS is rare in the adult population. The mainstay of all treatment regimens is multimodal treatment containing chemotherapy, surgical resection, and sometimes (neo)adjuvant radiotherapy. A clear resection margin improves both local control and overall survival and should be the goal during surgery with a curative intent. Real-time intraoperative fluorescence-guided imaging could facilitate complete resections by visualizing tumor tissue during surgery. This review evaluates whether non-targeted and targeted fluorescence-guided surgery (FGS) could be beneficial for pediatric OS, ES, and RMS patients. Necessities for clinical implementation, current literature, and the positive as well as negative aspects of non-targeted FGS using the NIR dye Indocyanine Green (ICG) were evaluated. In addition, we provide an overview of targets that could potentially be used for FGS in OS, ES, and RMS. Then, due to the time- and cost-efficient translational perspective, we elaborate on the use of antibody-based tracers as well as their disadvantages and alternatives. Finally, we conclude with recommendations for the experiments needed before FGS can be implemented for pediatric OS, ES, and RMS patients.

Value of the Sentinel Node Procedure in Pediatric Extremity Rhabdomyosarcoma: A Systematic Review and Retrospective Cohort Study.

BACKGROUND: Our aim is to show whether the sentinel node procedure (SNP) is recommendable for pediatric patients with extremity rhabdomyosarcoma (RMS). Lymph node metastases are an important prognostic factor in pediatric patients with extremity RMS. Accurate nodal staging is necessary to treat the patient accordingly. An alternative to the current recommended lymph node sampling is the sentinel node procedure (SNP). METHODS: A systematic review was performed summarizing all published cases of SNP in addition to 13 cases from our hospital and 8 cases from two other hospitals that have not been published before. RESULTS: For all patients (n = 55), at least one SLN was identified, but the SNP technique used was not uniform. The SNP changed the nodal classification of eight patients (17.0%) and had a false-negative rate of 10.5%. CONCLUSIONS: The SNP is recommendable for pediatric patients with extremity RMS. It can change lymph node status and can be used to sample patients in a more targeted way than nodal sampling alone. Therefore, we recommend use of the SNP in addition to clinical and radiological nodal assessment for pediatric patients with extremity RMS.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.

BACKGROUND: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. RESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. CONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.