Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.Damon Runyon Cancer Research Foundation (Damon Runyon-Rachleff Innovation Award

The Nucleocapsid Region of HIV-1 Gag Cooperates with the PTAP and LYPXnL Late Domains to Recruit the Cellular Machinery Necessary for Viral Budding

HIV-1 release is mediated through two motifs in the p6 region of Gag, PTAP and LYPXnL, which recruit cellular proteins Tsg101 and Alix, respectively. The Nucleocapsid region of Gag (NC), which binds the Bro1 domain of Alix, also plays an important role in HIV-1 release, but the underlying mechanism remains unclear. Here we show that the first 202 residues of the Bro1 domain (Broi) are sufficient to bind Gag. Broi interferes with HIV-1 release in an NC–dependent manner and arrests viral budding at the plasma membrane. Similar interrupted budding structures are seen following over-expression of a fragment containing Bro1 with the adjacent V domain (Bro1-V). Although only Bro1-V contains binding determinants for CHMP4, both Broi and Bro1-V inhibited release via both the PTAP/Tsg101 and the LYPXnL/Alix pathways, suggesting that they interfere with a key step in HIV-1 release. Remarkably, we found that over-expression of Bro1 rescued the release of HIV-1 lacking both L domains. This rescue required the N-terminal region of the NC domain in Gag and the CHMP4 binding site in Bro1. Interestingly, release defects due to mutations in NC that prevented Bro1 mediated rescue of virus egress were rescued by providing a link to the ESCRT machinery via Nedd4.2s over-expression. Our data support a model in which NC cooperates with PTAP in the recruitment of cellular proteins necessary for its L domain activity and binds the Bro1–CHMP4 complex required for LYPXnL–mediated budding

Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1 × 1.6 mm (3.4 mm(2)) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm(-1) used in conjunction with a 4 × 0.1 NA objective (v=0.165). This yielded an optical section thickness of 128 µm and an 8.9 × contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy

Modulation depth as a function of grid frequency for non-scattering phantoms and scattering phantoms (two µ_s′ levels).

<p>The points labeled A and B are referenced in the Discussion section.</p

Schematic demonstrating the method used to measure the optical sectioning strength of the imaging system.

<p>Also shown is a detailed diagram of the structure of the solid phantom used for measurement. Three separate phantoms were creating with increasing levels, µ_s′ = 0, 10, 20 cm⁻¹.</p

Detailed schematic of the SIM imaging system.

<p>The λ_ex peak was 480 nm and the λ_em peak was 520 nm. The spatial filter diameter was adjusted to allow only the 0 and +1 diffraction orders to pass. An image of group 7 of the USAF resolution target acquired by the system is also shown.</p

Optical section thickness measurements from solid phantoms.

<p>(<b>A</b>) Plot of the mean image intensity as a function of distance from focal plane used to determine optical section thickness. The circles represent data that was acquired on the µ_s′ = 0 cm⁻¹ phantom using a 4×NA = 0.1 objective with an illumination frequency of 19.6 mm⁻¹. The solid line represents the Stokseth approximation (Eq. 4) calculated using the same variables. (<b>B</b>) Plot relating optical section thickness to illumination grid frequency for a range of reduced scattering coefficients. The datapoints represent the measured values on phantoms, and the solid line represent the theoretical value calculated using the Stokseth approximation. The dotted arrows show how the optical section thickness was measured on the left and how it was placed on the corresponding plot on the right.</p

Fluorescent images of mouse sarcoma tissue processed using a high-pass filter (HPF) algorithm to isolate cell nuclei.

<p>The <i>in situ</i> sectioned image (acquired using f = 31.7 mm⁻¹) closely resembles the 50 µm tissue slice image (acquired from frozen sections), while the <i>in situ</i> uniform image failed to isolate the majority of cell nuclei.</p

Images of a 50 µm tissue slice demonstrating the correlation between images acquired by the SIM system and H&E histological micrograph.

<p>SIM images were taking using the 4×objective and a frequency of 31.7 mm⁻¹. H&E images were taken using a 2.5×objective and the images were cropped to match one another. Site imaged on tissue contains both cross sectional skeletal muscle (M) and sarcoma tumor (T). The arrows on each image point out a site where tumor tissue was invading into the normal skeletal muscle. Scale bars are 100 µm.</p