The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8+ (CD8+) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1+ tumor cells and CD8+ T cells simultaneously in vivo, surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.

In this work, we demonstrate the targeted diagnosis of immunomarker programmed death ligand 1 (PD-L1) and simultaneous detection of epidermal growth factor receptor (EGFR) in breast cancer tumors in vivo using gold nanostars (AuNS) with multiplexed surface enhanced Raman spectroscopy (SERS). Real-time longitudinal tracking with SERS demonstrated maximum accumulation of AuNS occurred 6 h post intravenous (IV) delivery, enabling detection of both biomarkers simultaneously. Raman signal correlating to both PD-L1 and EGFR decreased by ∼30% in control tumors where receptors were pre-blocked prior to AuNS delivery, indicating both the sensitivity and specificity of SERS in distinguishing tumors with different levels of PD-L1 and EGFR expression. Our in vivo study was combined with the first demonstration of ex vivo SERS spatial maps of whole tumor lesions that provided both a qualitative and quantitative assessment of biomarker status with near cellular-level resolution. High resolution SERS maps also provided an overview of AuNS distribution in tumors which correlated well with the vascular density. Mass spectrometry showed AuNS accumulation in tumor and liver, and clearance via spleen, and electron microscopy revealed AuNS were endocytosed in tumors, Kupffer cells in the liver, and macrophages in the spleen. This study demonstrates that SERS-based diagnosis mediated by AuNS provides an accurate measure of multiple biomarkers both in vivo and ex vivo, which will ultimately enable a clinically-translatable platform for patient-tailored immunotherapies and combination treatments.

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Thermal analysis techniques were utilized to investigate the thermal properties of two soils and a lignite coal obtained from the International Humic Substances Society (IHSS), and sediment obtained from The Netherlands. Differential scanning calorimetry (DSC) revealed glass transition behavior of each sample at temperatures ranging from 52 degrees C for Pahokee peat (euic, hyperthermic Lithic Medisaprists), 55 degrees C for a Netherlands (B8) sediment, 64 degrees C for Elliott loam (fine, illitic, mesic Aquic Arguidolls), to 70 degrees C for Gascoyne leonardite. Temperature-modulated differential scanning calorimetry (TMDSC) revealed glass transition behavior at similar temperatures, and quantified constant-pressure specific heat capacity (Cp) at 0 degrees C from 0.6 J g(-1) degrees C(-1) for Elliott loam and 0.8 J g(-1) degrees C(-1) for the leonardite, to 1.0 J g(-1) degrees C(-1) for the peat and the sediment. Glass transition behavior showed no distinct correlation to elemental composition, although Gascoyne Leonardite and Pahokee peat each demonstrated glass transition behavior similar to that reported for humic acids derived from these materials. Thermomechanical analysis (TMA) revealed a large thermal expansion followed by a matrix collapse for each sample between 20 and 30 degrees C, suggesting the occurrence of transition behavior of unknown origin. Thermal transitions occurring at higher temperatures more representative of glass transition behavior were revealed for the sediment and the peat.