Yongbiao Li

Confocal reflectance mosaicing of basal cell carcinomas in Mohs surgical skin excisions

Precise removal of basal cell carcinomas (BCCs) with minimal damage to the surrounding normal skin is guided by the examination of frozen histology of each excision during Mohs surgery. The preparation of frozen histology is slow, requiring 20 to 45 min per excision. Confocal reflectance mosaicing may enable rapid detection of BCCs directly in surgical excisions, with minimal need for frozen histology. Soaking the excisions in acetic acid rapidly brightens nuclei and enhances BCC-to-dermis contrast. Clinically useful concentrations of acetic acid from 10 to 1% require 30 s to 5 min, respectively. A tissue fixture precisely controls the stability, flatness, tilt, and sag of the excisions, which enables mosaicing of 36x36 images to create a field of view of 12x12 mm. This simulates a 2x magnification view in light microscopes, which is routinely used by Mohs surgeons to examine frozen histology. Compared to brightfield, cross-polarization enhances contrast and detectability of BCCs in the papillary dermis but not in the reticular dermis. Comparison of mosaics to histology shows that nodular, micronodular, and superficial BCCs are easily detected. However, infiltrative and sclerosing BCCs tend to be obscured within the surrounding bright dermis. The mosaicing method currently requires 9 min, and thus may expedite Mohs surgery.

Confocal mosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology

Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12 x 12 mm (which is equivalent to a 2x-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.

Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology

Precise micro‐surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour‐intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10–1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5–5 min and enhances nuclear‐to‐dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 × 36 images to create a field of view of 12 × 12 mm. This large field of view displays the excision at 2× magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20–45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology‐at‐the‐bedside to expedite and guide surgery.

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue.

Abstract. Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12  mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called “strip mosaicing,” which was demonstrated on a 10-×-10  mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10  mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5  cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment

Targeting a cytokine on the surface of regulatory T cells inhibits their suppression of tumor cell killing by effector T cells. Blocking immunosuppression The antitumor effects of CD8+ T cells can be blocked in the tumor microenvironment, including through the suppressive function of regulatory T cells (Tregs). Standard in vitro systems fail to recapitulate the conditions that immune cells are exposed to in vivo. Budhu et al. used a three-dimensional, collagen-fibrin gel system to investigate the effects of CD8+ T cells on cocultured melanoma cells excised from mouse tumors. The antitumor activity of the CD8+ T cells was inhibited by the presence of tumor-derived Tregs, which depended on cell-cell contact or close proximity, required the cytokine TGF-β on the Treg cell surface, and resulted in the increased cell surface expression of the immune checkpoint receptor PD-1 on the CD8+ T cells. A blocking antibody against TGF-β prevented immunosuppression, suggesting a therapeutic strategy to inhibit Treg activity in tumors. Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor–β (TGF-β) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell–mediated killing, which depended on surface-bound TGF-β on the Tregs. Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-β–dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Endoscopic laser scalpel for head and neck cancer surgery

Minimally invasive surgical (MIS) techniques, such as laparoscopic surgery and endoscopy, provide reliable disease control with reduced impact on the function of the diseased organ. Surgical lasers can ablate, cut and excise tissue while sealing small blood vessels minimizing bleeding and risk of lymphatic metastases from tumors. Lasers with wavelengths in the IR are readily absorbed by water causing minimal thermal damage to adjacent tissue, ideal for surgery near critical anatomical structures. MIS techniques have largely been unable to adopt the use of lasers partly due to the difficulty in bringing the laser into the endoscopic cavity. Hollow waveguide fibers have been adapted to bring surgical lasers to endoscopy. However, they deliver a beam that diverges rapidly and requires careful manipulation of the fiber tip relative to the target. Thus, the principal obstacle for surgical lasers in MIS procedures has been a lack of effective control instruments to manipulate the laser in the body cavity and accurately deliver it to the targeted tissue. To overcome this limitation, we have designed and built an endoscopic laser system that incorporates a miniature dual wedge beam steering device, a video camera, and the control system for remote and /or robotic operation. The dual wedge Risley device offers the smallest profile possible for endoscopic use. Clinical specifications and design considerations will be presented together with descriptions of the device and the development of its control system.

Multimodal microscopy of immune cells and melanoma for longitudinal studies

Intravital microscopy of cancer is a well established tool that provides direct visualization of the tumor cycle. It traditionally involves one of several strategies: invasive subcutaneous (SC) implantation of tumors followed by surgical opening of skin flaps for imaging, techniques utilizing skin fold chambers and implanted optical windows or intradermal injections under 200μm from the skin surface. All of these techniques allow the use of fluorescent proteins as markers for biologically significant constituents. However, observation methods utilizing skin-flaps, skin-fold chambers and optical windows are invasive and tend to alter the immune environment of the tissue and/or limit the duration of studies that can be performed. If implanted correctly, intradermally injected tumors can be minimally invasive, will not require biopsies or surgical intervention to observe and are accessible for direct transdermal imaging with a number of in vivo modalities. We present our work in the development of a small animal intravital microscopy workstation that allows the acquisition of different contrast imaging modalities: reflectance confocal, wide field epifluorescence, multiphoton and second harmonic generation (SHG). The images are acquired pair-wise simultaneously and sequentially in time. The aim of our instrumentation is to gather all information generated by the single probing beam via the reflected or back-scattered signal, SHG signal and various fluorescence signals. Additionally, we also present our development of a microscopic tissue navigation technique to mark, label and track sites of interest. This technique enables us to revisit sites periodically and record, with different imaging contrasts, their biological changes over time.

Analysis of a Two-PMT System for Simultaneous Back- and Forward-Fluorescence Detection in Multiphoton Microscopy

A two-PMT system is analyzed as an opportunity to improve the S/N ratio in a multiphoton microscope. Conditions under which this arrangement is optimal and sub-optimal are presented and discussed with representative data.

Blockade of surface bound TGF-β abrogates Treg suppression of effector T cell function within the tumor microenvironment

Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that tumor-specific Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in Foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation had a similar effect, reducing the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs. This study was supported by Swim Across America; NIH grants R01CA56821, P01CA33049, and P01CA59350 (to J.W. and A.H.); D.S. and S.B. received support from the NIH/NCI Immunology Training GrantT32 CA09149-30.

Mobile large area confocal scanner for imaging tumor margins: initial testing in the pathology department

Surgical oncology is guided by examining pathology that is prepared during or after surgery. The preparation time for Mohs surgery in skin is 20-45 minutes, for head-and-neck and breast cancer surgery is hours to days. Often this results in incomplete tumor removal such that positive margins remain. However, high resolution images of excised tissue taken within few minutes can provide a way to assess the margins for residual tumor. Current high resolution imaging methods such as confocal microscopy are limited to small fields of view and require assembling a mosaic of images in two dimensions (2D) to cover a large area, which requires long acquisition times and produces artifacts. To overcome this limitation we developed a confocal microscope that scans strips of images with high aspect ratios and stitches the acquired strip-images in one dimension (1D). Our “Strip Scanner” can image a 10 x 10 mm2 area of excised tissue with sub-cellular detail in about one minute. The strip scanner was tested on 17 Mohs excisions and the mosaics were read by a Mohs surgeon blinded to the pathology. After this initial trial, we built a mobile strip scanner that can be moved into different surgical settings. A tissue fixture capable of scanning up to 6 x 6 cm2 of tissue was also built. Freshly excised breast and head-and-neck tissues were imaged in the pathology lab. The strip-images were registered and displayed simultaneously with image acquisition resulting in large, high-resolution confocal mosaics of fresh surgical tissue in a clinical setting.