Adib Keikhosravi

Second-harmonic generation imaging of cancer

The last 30 years has seen great advances in optical microscopy with the introduction of sophisticated fluorescence-based imaging methods such as confocal and multiphoton laser scanning microscopy. There is increasing interest in using these methods to quantitatively examine sources of intrinsic biological contrast including autofluorescent endogenous proteins and light interactions such as second-harmonic generation (SHG) in collagen. In particular, SHG-based microscopy has become a widely used quantitative modality for imaging noncentrosymmetric proteins such as collagen in a diverse range of tissues. Due to the underlying physical origin of the SHG signal, it is highly sensitive to collagen fibril/fiber structure and, importantly, to collagen-associated changes that occur in diseases such as cancer, fibrosis, and connective tissue disorders. An overview of SHG physics background and technologies is presented with a focused review on applications of SHG primarily as applied to cancer.

Highly aligned stromal collagen is a negative prognostic factor following pancreatic ductal adenocarcinoma resection

Risk factors for pancreatic ductal adenocarcinoma (PDAC) progression after surgery are unclear, and additional prognostic factors are needed to inform treatment regimens and therapeutic targets. PDAC is characterized by advanced sclerosis of the extracellular matrix, and interactions between cancer cells, fibrillar collagen, and other stromal components play an integral role in progression. Changes in stromal collagen alignment have been shown to modulate cancer cell behavior and have important clinical value in other cancer types, but little is known about its role in PDAC and prognostic value. We hypothesized that the alignment of collagen is associated with PDAC patient survival. To address this, pathology-confirmed tissues from 114 PDAC patients that underwent curative-intent surgery were retrospectively imaged with Second Harmonic Generation (SHG) microscopy, quantified with fiber segmentation algorithms, and correlated to patient survival. The same tissue regions were analyzed for epithelial-to-mesenchymal (EMT), α-SMA, and syndecan-1 using complimentary immunohistostaining and visualization techniques. Significant inter-tumoral variation in collagen alignment was found, and notably high collagen alignment was observed in 12% of the patient cohort. Stratification of patients according to collagen alignment revealed that high alignment is an independent negative factor following PDAC resection (p = 0.0153, multivariate). We also found that epithelial expression of EMT and the stromal expression of α-SMA and syndecan-1 were positively correlated with collagen alignment. In summary, stromal collagen alignment may provide additional, clinically-relevant information about PDAC tumors and underscores the importance of stroma-cancer interactions.

Comparison of Picrosirius Red Staining With Second Harmonic Generation Imaging for the Quantification of Clinically Relevant Collagen Fiber Features in Histopathology Samples.

Stromal collagen alignment has been shown to have clinical significance in a variety of cancers and in other diseases accompanied by fibrosis. While much of the biological and clinical importance of collagen changes has been demonstrated using second harmonic generation (SHG) imaging in experimental settings, implementation into routine clinical pathology practice is currently prohibitive. To translate the assessment of collagen organization into routine pathology workflow, a surrogate visualization method needs to be examined. The objective of the present study was to quantitatively compare collagen metrics generated from SHG microscopy and commonly available picrosirius red stain with standard polarization microscopy (PSR-POL). Each technique was quantitatively compared with established image segmentation and fiber tracking algorithms using human pancreatic cancer as a model, which is characterized by a pronounced stroma with reorganized collagen fibers. Importantly, PSR-POL produced similar quantitative trends for most collagen metrics in benign and cancerous tissues as measured by SHG. We found it notable that PSR-POL detects higher fiber counts, alignment, length, straightness, and width compared with SHG imaging but still correlates well with SHG results. PSR-POL may provide sufficient and additional information in a conventional clinical pathology laboratory for certain types of collagen quantification.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Quantification of collagen organization in histopathology samples using liquid crystal based polarization microscopy

A number of histopathology studies have utilized the label free microscopy method of Second Harmonic Generation (SHG) to investigate collagen organization in disease onset and progression. Here we explored an alternative label free imaging approach, LC-PolScope that is based on liquid crystal based polarized light imaging. We demonstrated that this more accessible technology has the ability to visualize all fibers of interest and has a good to excellent correlation between SHG and LC-PolScope measurements in fibrillar collagen orientation and alignment. This study supports that LC-PolScope is a viable alternative to SHG for label free collagen organization measurements in thin histology sections.

PolScope based imaging and quantification of fibrillar collagen organization (Conference Presentation)

Collagen organization plays an integral role in many diseases including cancer. Here we introduce a low-cost, open-access collagen imaging and image analysis platform for quantifying fibrillar collagen organization. LC-PolScope was used as the imaging modality that incorporates a precision universal compensator made of two computer controlled liquid crystal variable retarders. This imaging system can be easily implemented on standard microscopes as a cost-effective alternative to second harmonic generation (SHG) imaging and staining on a wide range of available pathology slide formats, including the most commonly used HE 2) Use an iterative intensity-based image registration algorithm to find the affine transform that registers the collagen extracted image to the SHG image at different resolutions. Then, the registered bright field H&E image was used as a guide to evaluate collagen organization near any biological structure such as blood vessels, tumors etc. These algorithms have been implemented in our open source collagen analysis software tool “CurveAlign” package that has been widely used for collagen feature extraction, including detection of tumor associated collagen signatures. As a proof of concept, we are now using this platform to investigate collagen organization in metastatic pancreatic cancer vs non-metastatic pancreatic cancer.