Automated monitoring of respiratory rate as a novel humane endpoint: A refinement in mouse metastatic lung cancer models.

In oncology research, while xenograft tumor models are easily visualized and humane endpoints can be clearly defined, metastatic tumor models are often based on more subjective clinical observations as endpoints. This study aimed at identifying objective non-invasive criteria for predicting imminent distress and mortality in metastatic lung tumor-bearing mice. BALB/c and C57BL/6 mice were inoculated with CT26 or B16F10 cells, respectively. The mice were housed in Vium smart cages to continuously monitor and stream respiratory rate and locomotion for up to 28 days until scheduled euthanasia or humane endpoint criteria were met. Body weight and body temperature were measured during the study. On days 11, 14, 17 and 28, lungs of subsets of animals were microCT imaged in vivo to assess lung metastasis progression and then euthanized for lung microscopic evaluations. Beginning at day 21, most tumor-bearing animals developed increased respiratory rates followed by decreased locomotion 1-2 days later, compared with the baseline values. Increases in respiratory rate did not correlate to surface tumor nodule counts or lung weight. Body weight measurement did not show significant changes from days 14-28 in either tumor-bearing or control animals. We propose that increases in respiratory rate (1.3-1.5 X) can be used to provide an objective benchmark to signal the need for increased clinical observations or euthanasia. Adoption of this novel humane endpoint criterion would allow investigators time to collect tissue samples prior to spontaneous morbidity or death and significantly reduce the distress of mice in the terminal stages of these metastatic lung tumor models.

Near-Infrared Dye-Labeled Anti-Prostate Stem Cell Antigen Minibody Enables Real-Time Fluorescence Imaging and Targeted Surgery in Translational Mouse Models.

PURPOSE: The inability to intraoperatively distinguish primary tumor, as well as lymphatic spread, increases the probability of positive surgical margins, tumor recurrence, and surgical toxicity. The goal of this study was to develop a tumor-specific optical probe for real-time fluorescence-guided surgery. EXPERIMENTAL DESIGN: A humanized antibody fragment against PSCA (A11 minibody, A11 Mb) was conjugated with a near-infrared fluorophore, IRDye800CW. The integrity and binding of the probe to PSCA were confirmed by gel electrophoresis, size-exclusion chromatography, and flow cytometry, respectively. The ability of the probe to detect tumor-infiltrated lymph nodes and metastatic lesions was evaluated in 2 xenograft models, as well as in transgenic mice expressing human PSCA (hPSCA). An invasive intramuscular model was utilized to evaluate the efficacy of the A11 Mb-IRDye800CW-guided surgery. RESULTS: A11 Mb was successfully conjugated with IRDye800CW and retained specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 48 hours. The A11 Mb-IRDye800CW specifically detected PSCA-positive primary tumors, tumor-infiltrated lymph nodes, and distant metastases with high contrast. Fluorescence guidance facilitated more complete tumor resection, reduced tumor recurrence, and improved overall survival, compared with conventional white light surgery. The probe successfully identified primary orthotopic tumors and metastatic lesions in hPSCA transgenic mice. CONCLUSIONS: Real-time fluorescence image-guided surgery with A11 Mb-IRDye800CW enabled detection of lymph node metastases and positive surgical margins, facilitated more complete tumor removal, and improved survival, compared with white light surgery. These results may be translatable into clinical practice to improve surgical and patient outcomes.

Pretargeted Positron Emission Tomography Imaging That Employs Supramolecular Nanoparticles with in Vivo Bioorthogonal Chemistry.

A pretargeted oncologic positron emission tomography (PET) imaging that leverages the power of supramolecular nanoparticles with in vivo bioorthogonal chemistry was demonstrated for the clinically relevant problem of tumor imaging. The advantages of this approach are that (i) the pharmacokinetics (PKs) of tumor-targeting and imaging agents can be independently altered via chemical alteration to achieve the desired in vivo performance and (ii) the interplay between the two PKs and other controllable variables confers a second layer of control toward improved PET imaging. In brief, we utilized supramolecular chemistry to synthesize tumor-targeting nanoparticles containing transcyclooctene (TCO, a bioorthogonal reactive motif), called TCO⊂SNPs. After the intravenous injection and subsequent concentration of the TCO⊂SNPs in the tumors of living mice, a small molecule containing both the complementary bioorthogonal motif (tetrazine, Tz) and a positron-emitting radioisotope ((64)Cu) was injected to react selectively and irreversibly to TCO. High-contrast PET imaging of the tumor mass was accomplished after the rapid clearance of the unreacted (64)Cu-Tz probe. Our nanoparticle approach encompasses a wider gamut of tumor types due to the use of EPR effects, which is a universal phenomenon for most solid tumors.

A small MRI contrast agent library of gadolinium(III)-encapsulated supramolecular nanoparticles for improved relaxivity and sensitivity.

We introduce a new category of nanoparticle-based T(1) MRI contrast agents (CAs) by encapsulating paramagnetic chelated gadolinium(III), i.e., Gd(3+)·DOTA, through supramolecular assembly of molecular building blocks that carry complementary molecular recognition motifs, including adamantane (Ad) and ß-cyclodextrin (CD). A small library of Gd(3+)·DOTA-encapsulated supramolecular nanoparticles (Gd(3+)·DOTA⊂SNPs) was produced by systematically altering the molecular building block mixing ratios. A broad spectrum of relaxation rates was correlated to the resulting Gd(3+)·DOTA⊂SNP library. Consequently, an optimal synthetic formulation of Gd(3+)·DOTA⊂SNPs with an r(1) of 17.3 s(-1) mM(-1) (ca. 4-fold higher than clinical Gd(3+) chelated complexes at high field strengths) was identified. T(1)-weighted imaging of Gd(3+)·DOTA⊂SNPs exhibits an enhanced sensitivity with a contrast-to-noise ratio (C/N ratio) ca. 3.6 times greater than that observed for free Gd(3+)·DTPA. A Gd(3+)·DOTA⊂SNPs solution was injected into foot pads of mice, and MRI was employed to monitor dynamic lymphatic drainage of the Gd(3+)·DOTA⊂SNPs-based CA. We observe an increase in signal intensity of the brachial lymph node in T(1)-weighted imaging after injecting Gd(3+)·DOTA⊂SNPs but not after injecting Gd(3+)·DTPA. The MRI results are supported by ICP-MS analysis ex vivo. These results show that Gd(3+)·DOTA⊂SNPs not only exhibits enhanced relaxivity and high sensitivity but also can serve as a potential tool for diagnosis of cancer metastasis.