An end-to-end workflow for nondestructive 3D pathology.

Recent advances in 3D pathology offer the ability to image orders of magnitude more tissue than conventional pathology methods while also providing a volumetric context that is not achievable with 2D tissue sections, and all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis, however, is not trivial and requires careful attention to a series of details during tissue preparation, imaging and initial data processing, as well as iterative optimization of the entire process. Here, we provide an end-to-end procedure covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. Although 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol focuses on the use of a fluorescent analog of hematoxylin and eosin, which remains the most common stain used for gold-standard pathological reports. We present our guidelines for a broad range of end users (e.g., biologists, clinical researchers and engineers) in a simple format. The end-to-end workflow requires 3-6 d to complete, bearing in mind that data analysis may take longer.

Synergizing Thermal Ablation Modalities with Immunotherapy: Enough to Induce Systemic Antitumoral Immunity?

Thermal ablation modalities (cryoablation, radiofrequency ablation, and microwave ablation) have long been noted to occasionally induce a systemic antitumoral response. With the widespread use of checkpoint inhibitors, there is a significant interest in whether thermal ablation can promote immune system tumor recognition and increase checkpoint inhibitor response rates. In this review, we examine the current state of preclinical and clinical evidence examining the combination of checkpoint inhibitor therapies and thermal ablation modalities as well as discuss remaining the unanswered questions and directions for future research.

An end-to-end workflow for non-destructive 3D pathology.

Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.

Comprehensive Surface Histology of Fresh Resection Margins With Rapid Open-Top Light-Sheet (OTLS) Microscopy.

OBJECTIVE: For tumor resections, margin status typically correlates with patient survival but positive margin rates are generally high (up to 45% for head and neck cancer). Frozen section analysis (FSA) is often used to intraoperatively assess the margins of excised tissue, but suffers from severe under-sampling of the actual margin surface, inferior image quality, slow turnaround, and tissue destructiveness. METHODS: Here, we have developed an imaging workflow to generate en face histologic images of freshly excised surgical margin surfaces based on open-top light-sheet (OTLS) microscopy. Key innovations include (1) the ability to generate false-colored H&E-mimicking images of tissue surfaces stained for < 1 min with a single fluorophore, (2) rapid OTLS surface imaging at a rate of 15 min/cm2 followed by real-time post-processing of datasets within RAM at a rate of 5 min/cm2, and (3) rapid digital surface extraction to account for topological irregularities at the tissue surface. RESULTS: In addition to the performance metrics listed above, we show that the image quality generated by our rapid surface-histology method approaches that of gold-standard archival histology. CONCLUSION: OTLS microscopy has the feasibility to provide intraoperative guidance of surgical oncology procedures. SIGNIFICANCE: The reported methods can potentially improve tumor-resection procedures, thereby improving patient outcomes and quality of life.

A path toward the clinical translation of nano-based imaging contrast agents.

Recently, nanoparticles have evolved ubiquitously in therapeutic applications to treat a range of diseases. Despite their regular use as therapeutic agents in the clinic, we have yet to see much progress in their clinical translation as diagnostic imaging agents. Several clinical and preclinical studies support their use as imaging contrast agents, but their use in the clinical setting has been limited to off-label imaging procedures (i.e., Feraheme). Since diagnostic imaging has been historically used as an exploratory tool to rule out disease or to screen patients for various cancers, nanoparticle toxicity remains a concern, especially when introducing exogenous contrast agents into a potentially healthy patient population, perhaps rationalizing why several nano-based therapeutic agents have been clinically translated before nano-based imaging agents. Another potential hindrance toward their clinical translation could be their market potential, as most therapeutic drugs have higher earning potential than small-molecule imaging contrast agents. With these considerations in mind, perhaps a clinical path forward for nano-based imaging contrast agents is to help guide/manage therapy. Several studies have demonstrated the ability of nanoparticles to produce more accurate imaging preoperatively, intraoperatively, and postoperatively. These applications illustrate a more reliable method of cancer detection and treatment that can prevent incomplete tumor resection and incorrect assessment of tumor progression following treatment. The aim of this review is to highlight the research that supports the use of nanoparticles in biomedical imaging applications and offer a new perspective to illustrate how nano-based imaging agents have the potential to better inform therapeutic decisions. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging.

A colorful approach towards developing new nano-based imaging contrast agents for improved cancer detection.

Providing physicians with new imaging agents to help detect cancer with better sensitivity and specificity has the potential to significantly improve patient outcomes. Development of new imaging agents could offer improved early cancer detection during routine screening or help surgeons identify tumor margins for surgical resection. In this study, we evaluate the optical properties of a colorful class of dyes and pigments that humans routinely encounter. The pigments are often used in tattoo inks and the dyes are FDA approved for the coloring of foods, drugs, and cosmetics. We characterized their absorption, fluorescence and Raman scattering properties in the hopes of identifying a new panel of dyes that offer exceptional imaging contrast. We found that some of these coloring agents, coined as "optical inks", exhibit a multitude of useful optical properties, outperforming some of the clinically approved imaging dyes on the market. The best performing optical inks (Green 8 and Orange 16) were further incorporated into liposomal nanoparticles to assess their tumor targeting and optical imaging potential. Mouse xenograft models of colorectal, cervical and lymphoma tumors were used to evaluate the newly developed nano-based imaging contrast agents. After intravenous injection, fluorescence imaging revealed significant localization of the new "optical ink" liposomal nanoparticles in all three tumor models as opposed to their neighboring healthy tissues (p < 0.05). If further developed, these coloring agents could play important roles in the clinical setting. A more sensitive imaging contrast agent could enable earlier cancer detection or help guide surgical resection of tumors, both of which have been shown to significantly improve patient survival.

A Raman Imaging Approach Using CD47 Antibody-Labeled SERS Nanoparticles for Identifying Breast Cancer and Its Potential to Guide Surgical Resection.

Raman spectroscopic imaging has shown great promise for improved cancer detection and localization with the use of tumor targeting surface enhanced Raman scattering (SERS) nanoparticles. With the ultrasensitive detection and multiplexing capabilities that SERS imaging has to offer, scientists have been investigating several clinical applications that could benefit from this unique imaging strategy. Recently, there has been a push to develop new image-guidance tools for surgical resection to help surgeons sensitively and specifically identify tumor margins in real time. We hypothesized that SERS nanoparticles (NPs) topically applied to breast cancer resection margins have the potential to provide real-time feedback on the presence of residual cancer in the resection margins during lumpectomy. Here, we explore the ability of SERS nanoparticles conjugated with a cluster of differentiation-47 (CD47) antibody to target breast cancer. CD47 is a cell surface receptor that has recently been shown to be overexpressed on several solid tumor types. The binding potential of our CD47-labeled SERS nanoparticles was assessed using fluorescence assisted cell sorting (FACS) on seven different human breast cancer cell lines, some of which were triple negative (negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2)). Xenograft mouse models were also used to assess the ability of our Raman imaging system to identify tumor from normal tissue. A ratiometric imaging strategy was used to quantify specific vs. nonspecific probe binding, resulting in improved tumor-to-background ratios. FACS analysis showed that CD47-labeled SERS nanoparticles bound to seven different breast cancer cell lines at levels 12-fold to 70-fold higher than isotype control-labeled nanoparticles (p < 0.01), suggesting that our CD47-targeted nanoparticles actively bind to CD47 on breast cancer cells. In a mouse xenograft model of human breast cancer, topical application of CD47-targeted nanoparticles to excised normal and cancer tissue revealed increased binding of CD47-targeted nanoparticles on tumor relative to normal adjacent tissue. The findings of this study support further investigation and suggest that SERS nanoparticles topically applied to breast cancer could guide more complete surgical resection during lumpectomy.