Non-Covalently Pre-Assembled High-Performance Near-Infrared Fluorescent Molecular Probes for Cancer Imaging.

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvß3 or αvß5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.

High expression of integrin αvß3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors.

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvß3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvß3 receptors ([9.8 ±â€¯2.5] × 104/cell) compared to the well-characterized cell line U87-MG ([5.2 ±â€¯1.4] × 104/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvß3 receptor density is actually lower (11 ±â€¯3 versus 18 ±â€¯6 receptors/µm2). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (∼680 nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2 ±â€¯0.4 higher than the monovalent probe, which in turn was 2.2 ±â€¯0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvß3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.

A lower Instability Severity Index score threshold may better predict recurrent anterior shoulder instability after arthroscopic Bankart repair: a systematic review.

IMPORTANCE: The Instability Severity Index (ISI) score was developed to evaluate a patient's risk of recurrent shoulder instability following arthroscopic Bankart repair. While patients with an ISI score of >6 were originally recommended to undergo an open procedure (ie, Latarjet) to minimise the risk of recurrence, recent literature has called into question the utility of the ISI score. OBJECTIVE: The purpose of this systematic review was to evaluate the efficacy of the ISI score as a tool to predict postoperative recurrence among patients undergoing arthroscopic Bankart procedures. EVIDENCE REVIEW: Articles were included if study participants underwent arthroscopic Bankart repair for anterior shoulder instability and reported postoperative recurrence by ISI score at a minimum of 2 years of follow-up. Methodological study quality was assessed using the Methodological Index for Non-Randomized Studies criteria. Pearson's χ2 test was used to compare recurrence rates among patients above and below an ISI score of 4. Sensitivity, specificity, mean ISI scores and predictive value of individual factors of the ISI score were qualitatively reviewed. FINDINGS: Four studies concluded the ISI score was effective in predicting postoperative recurrence following arthroscopic Bankart repair; however, these studies found threshold values lower than the previously proposed score of >6 may be more predictive of recurrent instability. A pooled analysis of these studies found patients with an ISI score <4 to experience significantly lower recurrence rates when compared with patients with a score ≥4 (6.3% vs 26.0%, p<0.0001). The mean ISI score among patients who experienced recurrent instability was also significantly higher than those who did not. CONCLUSIONS AND RELEVANCE: The ISI score as constructed by Balg and Boileau may have clinical utility to help predict recurrent anterior shoulder instability following arthroscopic Bankart repair. However, this review found the threshold values published in their seminal article to be insufficient predictors of recurrent instability. Instead, a lower score threshold may provide as a better predictor of failure. The paucity of level I and II investigations limits the strength of these conclusions, suggesting a need for further large, prospective studies evaluating the predictive ability of the ISI score. LEVEL OF EVIDENCE: IV.