A flexible electronic strain sensor for the real-time monitoring of tumor regression.

Assessing the efficacy of cancer therapeutics in mouse models is a critical step in treatment development. However, low-resolution measurement tools and small sample sizes make determining drug efficacy in vivo a difficult and time-intensive task. Here, we present a commercially scalable wearable electronic strain sensor that automates the in vivo testing of cancer therapeutics by continuously monitoring the micrometer-scale progression or regression of subcutaneously implanted tumors at the minute time scale. In two in vivo cancer mouse models, our sensor discerned differences in tumor volume dynamics between drug- and vehicle-treated tumors within 5 hours following therapy initiation. These short-term regression measurements were validated through histology, and caliper and bioluminescence measurements taken over weeklong treatment periods demonstrated the correlation with longer-term treatment response. We anticipate that real-time tumor regression datasets could help expedite and automate the process of screening cancer therapies in vivo.

In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma.

PURPOSE: Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model. PROCEDURES: TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections. RESULTS: The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%). CONCLUSIONS: We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

Whole-body PET Imaging of T-cell Response to Glioblastoma.

PURPOSE: Immunotherapy is a promising approach for many oncological malignancies, including glioblastoma, however, there are currently no available tools or biomarkers to accurately assess whole-body immune responses in patients with glioblastoma treated with immunotherapy. Here, the utility of OX40, a costimulatory molecule mainly expressed on activated effector T cells known to play an important role in eliminating cancer cells, was evaluated as a PET imaging biomarker to quantify and track response to immunotherapy. EXPERIMENTAL DESIGN: A subcutaneous vaccination approach of CpG oligodeoxynucleotide, OX40 mAb, and tumor lysate at a remote site in a murine orthotopic glioma model was developed to induce activation of T cells distantly while monitoring their distribution in stimulated lymphoid organs with respect to observed therapeutic effects. To detect OX40-positive T cells, we utilized our in-house-developed 89Zr-DFO-OX40 mAb and in vivo PET/CT imaging. RESULTS: ImmunoPET with 89Zr-DFO-OX40 mAb revealed strong OX40-positive responses with high specificity, not only in the nearest lymph node from vaccinated area (mean, 20.8%ID/cc) but also in the spleen (16.7%ID/cc) and the tumor draining lymph node (11.4%ID/cc). When the tumor was small (<106 p/sec/cm2/sr in bioluminescence imaging), a high number of responders and percentage shrinkage in tumor signal was indicated after only a single cycle of vaccination. CONCLUSIONS: The results highlight the promise of clinically translating cancer vaccination as a potential glioma therapy, as well as the benefits of monitoring efficacy of these treatments using immunoPET imaging of T-cell activation.

A miniaturized optoelectronic biosensor for real-time point-of-care total protein analysis.

A miniaturized optoelectronic sensor is demonstrated that measures total protein concentration in serum and urine with sensitivity and accuracy comparable to gold-standard methods. The sensor is comprised of a vertical cavity surface emitting laser (VCSEL), photodetector and other custom optical components and electronics that can be hybrid packaged into a portable, handheld form factor. In conjunction, a custom fluorescence assay has been developed based on the protein-induced fluorescence enhancement (PIFE) phenomenon, enabling real-time sensor response to changes in protein concentration. Methods are described for the following:•Standard curves: Used to determine the sensitivity, dynamic range, and linearity of the VCSEL biosensor/PIFE assay system in buffer as well as in human blood and urine samples.•Comparison of VCSEL biosensor performance with a benchtop fluorimetric microplate reader.•Accuracy of the VCSEL biosensor/PIFE assay system: Evaluated by comparing sensor measurements with gold-standard clinical laboratory measurements of total protein in serum and urine samples from patients with diabetes.

Molecular imaging of a fluorescent antibody against epidermal growth factor receptor detects high-grade glioma.

The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

Real-time point-of-care total protein measurement with a miniaturized optoelectronic biosensor and fast fluorescence-based assay.

Measurement of total protein in urine is key to monitoring kidney health in diabetes. However, most total protein assays are performed using large, expensive laboratory chemistry analyzers that are not amenable to point-of-care analysis or home monitoring and cannot provide real-time readouts. We developed a miniaturized optoelectronic biosensor using a vertical cavity surface-emitting laser (VCSEL), coupled with a fast protein assay based on protein-induced fluorescence enhancement (PIFE), that can dynamically measure protein concentrations in protein-spiked buffer, serum, and urine in seconds with excellent sensitivity (urine LOD = 0.023 g/L, LOQ = 0.075 g/L) and over a broad range of physiologically relevant concentrations. Comparison with gold standard clinical assays and standard fluorimetry tools showed that the sensor can accurately and reliably quantitate total protein in clinical urine samples from patients with diabetes. Our VCSEL biosensor is amenable to integration with miniaturized electronics, which could afford a portable, low-cost, easy-to-use device for sensitive, accurate, and real-time total protein measurements from small biofluid volumes.

Engineering of a novel subnanomolar affinity fibronectin III domain binder targeting human programmed death-ligand 1.

The programmed death-ligand 1 (PD-L1) is a major checkpoint protein that helps cancer cells evade the immune system. A non-invasive imaging agent with rapid clearance rate would be an ideal tool to predict and monitor the efficacy of anti-PD-L1 therapy. The aim of this research was to engineer a subnanomolar, high-affinity fibronectin type 3 domain (FN3)-based small binder targeted against human PD-L1 (hPD-L1) present on tumor cells. A naive yeast G4 library containing the FN3 gene with three binding loop sequences was used to isolate high-affinity binders targeted to purified full-length hPD-L1. The selected binder clones displayed several mutations in the loop regions of the FN3 domain. One unique clone (FN3hPD-L1-01) with a 6x His-tag at the C-terminus had a protein yield of >5 mg/L and a protein mass of 12 kDa. In vitro binding assays on six different human cancer cell lines (MDA-MB-231, DLD1, U87, 293 T, Raji and Jurkat) and murine CT26 colon carcinoma cells stably expressing hPD-L1 showed that CT26/hPD-L1 cells had the highest expression of hPD-L1 in both basal and IFN-γ-induced states, with a binding affinity of 2.38 ± 0.26 nM for FN3hPD-L1-01. The binding ability of FN3hPD-L1-01 was further confirmed by immunofluorescence staining on ex vivo CT26/hPD-L1 tumors sections. The FN3hPD-L1-01 binder represents a novel, small, high-affinity binder for imaging hPD-L1 expression on tumor cells and would aid in earlier imaging of tumors. Future clinical validation studies of the labeled FN3hPD-L1 binder(s) have the potential to monitor immune checkpoint inhibitors therapy and predict responders.

Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection.

Purpose: Stem cell transplants are an effective approach to repair large bone defects. However, comprehensive techniques to monitor the fate of transplanted stem cells in vivo are lacking. Such strategies would enable corrective interventions at an early stage and greatly benefit the development of more successful tissue regeneration approaches. In this study, we designed and synthesized a dual-modality imaging probe (Feru-AFC) that can simultaneously localize transplanted stem cells and diagnose immune rejection-induced apoptosis at an early stage in vivo. Methods: We used a customized caspase-3 cleavable peptide-dye conjugate to modify the surface of clinically approved ferumoxytol nanoparticles (NPs) to generate the dual-modality imaging probe with fluorescence "light-up" feature. We labeled both mouse mesenchymal stem cells (mMSCs, matched) and pig mesenchymal stem cells (pMSCs, mismatched) with the probe and transplanted the labeled cells with biocompatible scaffold at the calvarial defects in mice. We then employed intravital microscopy (IVM) and magnetic resonance imaging (MRI) to investigate the localization, engraftment, and viability of matched and mismatched stem cells, followed by histological analyses to evaluate the results obtained from in vivo studies. Results: The Feru-AFC NPs showed good cellular uptake efficiency in the presence of lipofectin without cytotoxicity to mMSCs and pMSCs. The fluorescence of Feru-AFC NPs was turned on inside apoptotic cells due to the cleavage of peptide by activated caspase-3 and subsequent release of fluorescence dye molecules. Upon transplantation at the calvarial defects in mice, the intense fluorescence from the cleaved Feru-AFC NPs in apoptotic pMSCs was observed with a concomitant decrease in the overall cell number from days 1 to 6. In contrast, the Feru-AFC NP-treated mMSCs exhibited minimum fluorescence and the cell number also remained similar. Furthermore, in vivo MRI of the Feru-AFC NP-treated mMSC and pMSCs transplants could clearly indicate the localization of matched and mismatched cells, respectively. Conclusions: We successfully developed a dual-modality imaging probe for evaluation of the localization and viability of transplanted stem cells in mouse calvarial defects. Using ferumoxytol NPs as the platform, our Feru-AFC NPs are superparamagnetic and display a fluorescence "light-up" signature upon exposure to activated caspase-3. The results show that the probe is a promising tool for long-term stem cell tracking through MRI and early diagnosis of immune rejection-induced apoptosis through longitudinal fluorescence imaging.

Longitudinal Monitoring of Antibody Responses against Tumor Cells Using Magneto-nanosensors with a Nanoliter of Blood.

Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.