Inserting "OFF-to-ON" BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells.

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives-unlike IL-33-GFP constructs-exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

Development of the Life-Worldly Communication Scale to Improve Quality of Life: Daily Conversation as Care for Older Adults.

The lack of daily conversation may lead to the deterioration of quality of life and cognitive function in older adults requiring long-term care. This study aimed to develop a scale to measure daily conversation among them, the Life-Worldly Communication Scale: LWCS, and to test its structural, convergent, and discriminant validity. The subjects were 539 older adults requiring long-term care in facilities and at home. A 24-item provisional scale was created, using a panel of experts. Structural validity of LWCS was examined from exploratory factor analysis to establish the factor structure, two confirmatory factor analyses for cross-validation, and measurement invariance between the institutional and home setting. Convergent validity was examined from the average variance extracted: AVE, composite reliability: CR, and simple regression analysis between LWCS and Interdependent Happiness Scale: IHS. Discriminant validity was examined using the heterotrait-monotrait ratio of correlations: HTMT. Multiple imputations were used to deal with missing data on these scales. The results showed that the goodness of fit of the three-factor, 11-item model obtained from the two-step CFA was SRMR = .043, RMSEA = .059, CFI = .978, and AGFI = .905. The model was confirmed for structural validity by measurement invariance tests: configural invariance (CFI=.973, RMSEA = .047), metric invariance (ΔCFI= .001, ΔRMSEA=-.004), scalar invariance (ΔCFI =-0.002, RMSEA = -0.003). Convergent validity was confirmed by AVE = .503~.772, CR = .801~.910, and simple regression analysis between LWCS and IHS (adjusted r2=.18, p < .001). Discriminant validity was also confirmed among the three factors (HTMT=.496~.644). LWCS can contribute to the assessment of daily conversation in geriatric settings and research regarding its promotion.

Tumour-associated macrophages as a potential target to improve natural killer cell-based immunotherapies.

Adoptive transfer of natural killer (NK) cells has been proposed as a novel immunotherapy for malignant tumours resistant to current therapeutic modalities. Several clinical studies have demonstrated that the NK cell-infusion is well tolerated without severe side effects and shows promising results in haematological malignancies. However, patients with malignant solid tumours do not show significant responses to this therapy. Such disappointing results largely arise from the inefficient delivery of infused NK cells and the impairment of their functions in the tumour microenvironment (TME). Tumour-associated macrophages (TAMs) are the most abundant stromal cells in the TME of most solid tumours, and a high TAM density correlates with poor prognosis of cancer patients. Although our knowledge of the interactions between TAMs and NK cells is limited, many studies have indicated that TAMs suppress NK cell cytotoxicity against cancer cells. Therefore, blockade of TAM functions can be an attractive strategy to improve NK cell-based immunotherapies. On the other hand, macrophages are reported to activate NK cells under certain circumstances. This essay presents our current knowledge about mechanisms by which macrophages regulate NK cell functions and discusses possible therapeutic approaches to block macrophage-mediated NK cell suppression.

Use of multi-color flow cytometry for canine immune cell characterization in cancer.

Although immunotherapy is becoming a standard approach of human cancer treatment, only a small but critical fraction of patients responds to the therapy. It is therefore required to determine the sub-populations of patients who will respond to immunotherapies along with developing novel strategies to improve efficacy of anti-tumor immune reactions. Current development of novel immunotherapies relies heavily on mouse models of cancer. These models are important for better understanding of mechanisms behind tumor immune escape and investigation of novel strategies to overcome it. Nevertheless, the murine models do not necessarily represent the complexity of spontaneously occurring cancers in humans. Dogs spontaneously develop a wide range of cancer types with an intact immune system under similar environment and exposure to humans, which can serve as translational models in cancer immunotherapy research. To date though, there is still a relatively limited amount of information regarding immune cell profiles in canine cancers. One possible reason could be that there are hardly any established methods to isolate and simultaneously detect a range of immune cell types in neoplastic tissues. To date only a single manuscript describes characterization of immune cells in canine tumour tissues, concentrating solely on T-cells. Here we describe a protocol for multi-color flow cytometry to distinguish immune cell types in blood, lymph nodes, and neoplastic tissues from dogs with cancer. Our results demonstrate that a 9-color flow cytometry panel enables characterization of different cell subpopulations including myeloid cells. We also show that the panel allows detection of minor/aberrant subsets within a mixed population of cells in various neoplastic samples including blood, lymph node and solid tumors. To our knowledge, this is the first simultaneous immune cell detection panel applicable for solid tumors in dogs. This multi-color flow cytometry panel has the potential to inform future basic research focusing on immune cell functions in translational canine cancer models.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies.

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

Metastasis-associated macrophages constrain antitumor capability of natural killer cells in the metastatic site at least partially by membrane bound transforming growth factor ß.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of the immunosuppressive tumor environment is required to improve its efficacy. The effects of this "metastatic" tumor environment on NK cells, however, remain largely unknown. Previous studies, including our own, have demonstrated that metastasis-associated macrophages (MAMs) are one of the most abundant immune cell types in the metastatic tumor niche in mouse models of metastatic breast cancer. We thus investigated the effects of MAMs on antitumor functions of NK cells in the metastatic tumor microenvironment. METHODS: MAMs were isolated from the tumor-bearing lung of C57BL/6 mice intravenously injected with E0771-LG mouse mammary tumor cells. The effects of MAMs on NK cell cytotoxicity towards E0771-LG cells were evaluated in vitro by real-time fluorescence microscopy. The effects of MAM depletion on NK cell activation, maturation, and accumulation in the metastatic lung were evaluated by flow cytometry (CD69, CD11b, CD27) and in situ hybridization (Ncr1) using colony-stimulating factor 1 (CSF-1) receptor conditional knockout (Csf1r-cKO) mice. Finally, metastatic tumor loads in the chest region of mice were determined by bioluminescence imaging in order to evaluate the effect of MAM depletion on therapeutic efficacy of endogenous and adoptively transferred NK cells in suppressing metastatic tumor growth. RESULTS: MAMs isolated from the metastatic lung suppressed NK cell-induced tumor cell apoptosis in vitro via membrane-bound transforming growth factor ß (TGF-ß) dependent mechanisms. In the tumor-challenged mice, depletion of MAMs increased the percentage of activated (CD69+) and mature (CD11b+CD27-) NK cells and the number of Ncr1+ NK cells as well as NK cell-mediated tumor rejection in the metastatic site. Moreover, MAM depletion or TGF-ß receptor antagonist treatment significantly enhanced the therapeutic efficacy of NK cell infusion in suppressing early metastatic tumor outgrowth. CONCLUSION: This study demonstrates that MAMs are a main negative regulator of NK cell function within the metastatic tumor niche, and MAM targeting is an attractive strategy to improve NK cell-based immunotherapy for metastatic breast cancer.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

Does Everyday Conversation Contribute to Cognitive Functioning? A Comparison of Brain Activity During Task-Oriented and Life-Worldly Communication Using Near-Infrared Spectroscopy.

This study aimed to explore differences in frontal lobe brain activity associated with two types of communication: task-oriented and life-worldly, the latter of which largely overlaps with everyday conversation. Using near-infrared spectroscopy, we explored differences by comparing oxygenated hemoglobin concentrations associated with periods of rest and conversation in two experimental groups comprising older and younger adults. Artifacts were removed from the signals using discrete wavelet transforms. Paired t-tests were used to compare the resulting data for the two types. The results showed that oxygenated hemoglobin levels during life-worldly communication were significantly higher than at baseline or during task-oriented communication, particularly for the older adult group. In addition, during life-worldly communication, relatively high levels of brain activity were found in the upper part of the Broca area and in the premotor cortex. These results, which suggest that life-worldly communication generates more activity in the frontal lobe, could potentially contribute to improving how caregivers communicate with older patients/residents in hospitals and nursing homes.

Generation of mouse bone marrow-derived macrophages using tumor coculture assays to mimic the tumor microenvironment.

Macrophages are one of the key immune cells within the tumor microenvironment that encourage the growth of tumors at the primary site as well as contributing to all parts of the metastatic cascade. Although it is possible to isolate macrophages directly from the tumor, this can be a laborious process and due to their plasticity, it is not possible to maintain their in vivo phenotype in vitro. For this reason, differentiating macrophages from bone marrow is an attractive alternative. Here we present robust methods to study in vitro derived macrophages including (i) the isolation and generation of macrophages from bone marrow, (ii) differentiation/characterization of classically activated, alternatively activated and tumor-conditioned macrophages, as well as (iii) in vitro co-culturing assays for tumor cell-macrophage interaction/transmigration.

Mammary Tumor Cells with High Metastatic Potential Are Hypersensitive to Macrophage-Derived HGF.

Metastasis-associated macrophages (MAM) promote persistent growth of breast cancer cells at the metastatic site and are, thus, an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer-related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Using mouse models of metastatic breast cancer, we showed that MAMs uniquely expressed hepatocyte growth factor (HGF) in metastatic tumors. We also demonstrated that a selected population of cancer cells with high metastatic potential (cancer cells that can establish metastatic tumors in mice with higher number and incidence than parental cells) had higher expression of HGF receptor, MNNG HOS transforming gene (MET), and were more responsive to HGF released from macrophages compared with the parental cells. Blockade of MET signaling in cancer cells suppressed metastatic tumor expansion, in part, through activation of natural killer cells. Results from this study suggest an approach to prevent life-threatening metastatic tumor formation using blockade of MAM-induced MET signal activation in metastatic cancer cells.

A Fluorescent Activatable AND-Gate Chemokine CCL2 Enables In Vivo Detection of Metastasis-Associated Macrophages.

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND-gates since they emit only after receptor binding and intracellular activation, showing enhanced selectivity over existing agents. We have applied this strategy to produce mCCL2-MAF as the first probe for in vivo detection of metastasis-associated macrophages in a preclinical model of lung metastasis. This strategy will accelerate the preparation of new chemokine-based probes for imaging immune cell function in tumours.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured naïve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.