Activatable Nanoreporters for Real-Time Tracking of Macrophage Phenotypic States Associated with Disease Progression.

Diagnosis of inflammatory diseases is characterized by identifying symptoms, biomarkers, and imaging. However, conventional techniques lack the sensitivities and specificities to detect disease early. Here, it is demonstrated that the detection of macrophage phenotypes, from inflammatory M1 to alternatively activated M2 macrophages, corresponding to the disease state can be used to predict the prognosis of various diseases. Activatable nanoreporters that can longitudinally detect the presence of the enzyme Arginase 1, a hallmark of M2 macrophages, and nitric oxide, a hallmark of M1 macrophages are engineered, in real-time. Specifically, an M2 nanoreporter enables the early imaging of the progression of breast cancer as predicted by selectively detecting M2 macrophages in tumors. The M1 nanoreporter enables real-time imaging of the subcutaneous inflammatory response that rises from a local lipopolysccharide (LPS) administration. Finally, the M1-M2 dual nanoreporter is evaluated in a muscle injury model, where an initial inflammatory response is monitored by imaging M1 macrophages at the site of inflammation, followed by a resolution phase monitored by the imaging of infiltrated M2 macrophages involved in matrix regeneration and wound healing. It is anticipated that this set of macrophage nanoreporters may be utilized for early diagnosis and longitudinal monitoring of inflammatory responses in various disease models.

Supramolecular nanotherapeutics enable metabolic reprogramming of tumor-associated macrophages to inhibit tumor growth.

Tumor-associated macrophages (TAMs) exist in multiple phenotypes across the spectrum, defined by an M1 antitumorigenic phenotype and an M2 pro-tumorigenic phenotype on two ends of the spectrum. A largely immunosuppressive tumor-microenvironment aids the polarization of the infiltrating macrophages to a pro-tumorigenic M2 phenotype that promotes tumor progression and metastasis. Recent developments in macrophage immunotherapy have focused on strategies to re-educate TAMs from an M2 to M1 phenotype. Recent findings in the realm of immuno-metabolism have indicated that distinct metabolic signatures accompany macrophages based on their polarization states (M1-Glycolysis and M2-TCA cycle). These metabolites are important drivers of cellular signaling responsible for acquiring these polarization states, with evidence showing that metabolism is essential to facilitate the energy requirements of immune cells and regulate immune cell response. We hypothesized that TAMs could be reprogrammed metabolically by co-delivery of drugs using a supramolecular nanoparticle system that could effectively rewire macrophage metabolism by simultaneous inhibition of the TCA cycle and upregulation of the glycolytic metabolic pathway. TLR7/8 agonist and Fatty Acid Oxidation (FAO) inhibitor loaded metabolic supramolecular nanoparticles (MSNPs) were synthesized. In vitro assays showed macrophages treated with MSNPs were reprogrammed from an M2 phenotype to an M1 phenotype while significantly upregulating phagocytosis. When injected in 4T1 tumor-bearing mice, MSNPs treatment reduced tumor growth progression more than other treatments. Hence, the delivery of TLR7/8 agonist combined with an FAO inhibitor can enhance antitumor efficacy through metabolic reprogramming of tumor-associated macrophages.

A perception bias of the gravitational vertical is confirmed in Adolescent Idiopathic Scoliosis.

PURPOSE: Adolescent Idiopathic Scoliosis (AIS) is the most frequent spine deformity in adolescence. The cause of AIS remains unknown. Several studies show that AIS can be associated with a perception bias of gravitational vertical. In particularly, AIS patients with a right thoracic convexity exhibit deviation of the Subjective Postural Vertical. The origin of this disturbance could be located in trunk proprioceptive graciveptors. We wanted to verify this result with a population of lumbar and thoracolumbar AIS with left convexity. METHODS: It was a multicenter, cross-sectional case-control study. Thirty adolescents with left lumbar or thoraco-lumbar AIS (age 14.3 ± 1.7 years; Cobb angle 27.6° ± 6.1°) and 30 controls matched for age (14.0 ± 1.5 years), were compared for Subjective Visual Vertical (SVV) measured in static and dynamic (optokinetic stimulation) conditions, and Subjective Postural Vertical (SPV). RESULTS: For SVV, there was no difference in the two groups, for static and dynamic conditions. The SPV was significantly different between the two groups (p < 0.0001). The SPV was shifted to the left for most of the AIS patients (med - 2.4°[- 3.6; - 1.7]) compared with controls (med 0°[- 0.5; 1.7]). Adolescents with AIS perceived verticality with significant greater uncertainty in postural modality than controls (p = 0.017). CONCLUSION: Our study confirms a significant directional bias in the orientation of SPV in left lumbar or thoraco-lumbar AIS. This confirmation paves the way to a new physiopathological model focused on trunk proprioception. LEVEL OF EVIDENCE: III.

Supramolecular Nanotherapeutics for Macrophage Immunotherapy.

Tumor-associated macrophages are recruited in high abundance in the tumor microenvironment and are implicated in the various stages of tumorigenesis, such as tumor proliferation, enhanced angiogenesis, metastasis, and immune escape. However, inherent macrophage plasticity and ability of macrophages to switch their phenotype and function from tumor-promoting (M2 phenotype) to tumor-eliminating capacities (M1 phenotype) make them ideal for therapeutic targeting. This spotlight on applications summarizes our recent efforts in designing supramolecular nanotherapeutics for macrophage immunotherapy, specifically, the strategies that can repolarize the M2 tumor-associated macrophages to M1-phenotype by sustained inhibition of key signaling pathways. With exciting recent developments in the field of macrophage immunotherapy, the ability to harness the innate inflammatory response of these macrophages in aiding tumor regression offers an avenue for cancer immunotherapy.

Engineered Multifunctional Nano- and Biological Materials for Cancer Immunotherapy.

Cancer immunotherapy is set to emerge as the future of cancer therapy. However, recent immunotherapy trials in different cancers have yielded sub-optimal results, with durable responses seen in only a small fraction of patients. Engineered multifunctional nanomaterials and biological materials are versatile platforms that can elicit strong immune responses and improve anti-cancer efficacy when applied to cancer immunotherapy. While there are traditional systems such as polymer- and lipid-based nanoparticles, there is a wide variety of other materials with inherent and additive properties that can allow for more potent activation of the immune system. By synthesizing and applying multifunctional strategies, it allows for a more extensive and more effective repertoire of tools to use in the wide variety of situations that cancer presents itself. Here, several types of nanoscale and biological material strategies and platforms that provide their inherent benefits for targeting and activating multiple aspects of the immune system are discussed. Overall, this review aims to provide a comprehensive understanding of recent advances in the field of multifunctional cancer immunotherapy and trends that pave the way for more diverse and tactical regression of tumors through soliciting responses by either the adaptive or innate immune system, and even both simultaneously.

A Nitric Oxide (NO) Nanoreporter for Noninvasive Real-Time Imaging of Macrophage Immunotherapy.

Macrophage-centered therapeutic approaches that rely on immune modulation of tumor associated macrophages (TAMs) from a pro-tumorigenic phenotype (M2) to an anti-tumorigenic phenotype (M1) have facilitated a paradigm shift in macrophage immunotherapy. However, limited clinical success has been achieved due to the low response rates observed in different types of cancers. The ability to measure immune response in real time is critical in order to differentiate responders from non-responders; however, there are currently no platforms to monitor real-time macrophage immunotherapy response. Hence, there is an immediate need to develop imaging techniques that can longitudinally monitor macrophage immunotherapy response. Nitric oxide (NO) produced as a result of activation of macrophages to an anti-tumorigenic state is considered as a hallmark of M1 and can be a direct indication of response. In this study, a NO nanoreporter (NO-NR) is reported that enables real-time monitoring of macrophage immunotherapy drugs in vitro and in vivo. Furthermore, it is observed that sustained inhibition of colony stimulating factor 1 receptor (CSF1R) using a CSF1R inhibitor-NO-NR system leads to enhanced efficacy and better imaging signal. In conclusion, a first-of-its-kind NO nanoreporter tool is reported that can be used as an activatable imaging agent to monitor macrophage immunotherapy response in real time.

Lipid-based phagocytosis nanoenhancer for macrophage immunotherapy.

Tumor associated macrophages (TAMs) play an important role in initiating the immunosuppressive environment that negatively impacts the immunotherapy efficacy and has long been linked with cancer progression. On the other hand, activated macrophages display immense phagocytic potential and can be used as an effector cell for cancer therapy. But, activating TAMs to effectively phagocytose cancer cells is challenging. Cancer cells upregulate CD47, a "don't eat me" receptor that ligates with SIRPα present on macrophages to downregulate the phagocytosis. Since phagocytosis is a physical phenomenon based on engulfment of aberrant cells, we hypothesized that the phagocytic function of macrophages can be enhanced by blocking both CD47 and SIRPα in tandem and at the same time, engaging both macrophages and cancer cells can favor increased macrophage-cancer cellular interactions. Here, we demonstrate that a simple approach of anti-CD47 and anti-SIRPα antibodies conjugated lipid-based phagocytosis nanoenhancer (LPN) can perform both of these functions. The LPNs were stable in both physiological and biologically relevant conditions, bound to both macrophages and cancer cells and significantly enhanced phagocytosis of cancer cells as compared to combination of free antibodies. LPN treatment showed significant tumor growth inhibition and increased survival in B16F10 melanoma tumor bearing mice with no systemic toxicity. Mechanistic analysis of efficacy revealed an increase in intra-tumoral infiltration of effector T cells and NK cells. Cytokine analysis revealed increased secretion of intracellular iNOS, a hallmark of activated macrophages. This study shows that LPN can simultaneously block both CD47 and SIRPα and can effectively engage macrophage and cancer cell in close proximity. Combining these facets provide a simple approach to enhance phagocytosis and improve anti-cancer macrophage immunotherapy.

Dual inhibition of CSF1R and MAPK pathways using supramolecular nanoparticles enhances macrophage immunotherapy.

Among the numerous immune interactions, or lack-thereof, that occur during cancer progression, tumor-associated macrophages (TAMs) - cancer cell interactions have been shown to play an important role in modulating the tumor-microenvironment to an immune suppressive mode, promoting accelerated tumor growth, survival and metastatic spread. TAMs are predominantly polarized to a pro-tumorigenic M2-phenotype through macrophage colony stimulating factor 1 (MCSF) cytokines that bind to the colony-stimulating factor 1 receptor (CSF1R), a class III receptor tyrosine kinase. This MCSF-CSF1R interaction results in autophosphorylation of CSF1R and subsequent phosphorylation and activation of downstream signaling pathways including mitogen-activated protein kinase (MAPK) pathway leading to proliferation, survival and functional activity of M2 TAMs. Therapeutic inhibition of CSF1R and MAPK signaling could effectively re-polarize M2 macrophages to an anti-tumorigenic M1 phenotype; however, this is challenging. In this study, we demonstrate that concurrent and sustained inhibition of the CSF1R and MAPK signaling pathways using dual-kinase inhibitor-loaded supramolecular nanoparticles (DSNs) enhance repolarization of pro-tumorigenic M2 macrophages to the anti-tumorigenic M1 phenotype. The supramolecular nanoparticles exhibited physical stability of over 7 days during storage conditions at 4 °C and over 24 h in human serum, released the inhibitors in a sustained manner and showed significantly higher internalization and accumulation of inhibitors in the M2 macrophages even at longer time points. When tested in a highly aggressive 4T1 breast cancer model, the supramolecular nanoparticles accumulated in TAMs at a significantly higher concentration, increased M1-like phenotype at significantly higher proportion and improved anti-tumor efficacy as compared to combination of single-inhibitor nanoparticles or the small molecule inhibitors. Our data suggests that concurrent, vertical inhibition of multiple intracellular kinase signaling pathways is important for repolarization of M2 macrophages to M1 phenotype, and by utilizing dual-inhibitor loaded supramolecular nanoparticles, further increase the ability to produce more M1 macrophages as compared to M2 macrophages in the tumor microenvironment. This results in enhanced tumor growth inhibition and reduced toxicity. Therefore, vertical, co-inhibition of CSF1R and downstream signaling pathways like MAPK could be a promising macrophage immunotherapy strategy for aggressive cancers.

Supramolecular Polysaccharide Nanotheranostics that Inhibit Cancer Cells Growth and Monitor Targeted Therapy Response.

Targeted anticancer therapies directed against specific molecular drivers of tumors are emerging as effective treatment strategies, however, monitoring their response is still challenging. Current clinical imaging techniques that measure either morphological or metabolic changes in the tumor are not always indicative of clinical outcome due to delayed or variable responses. Here, dual-stage polysaccharide-based supramolecular nanotheranostics (SPN) were designed that enable co-delivery of kinase inhibitor and an activatable imaging probe. Methods: The SPNs were prepared by supramolecular assembly of two components, polysaccharide construct conjugated with kinase inhibitor-function activatable probe and kinase inhibitor- ß-cyclodextrin conjugate. Physiochemical characterization of SPNs including size, stability, zeta potential and pH-responsiveness of the assembly was performed. The efficacy of SPNs in inducing cancer cell death by inhibition of kinase signaling and imaging the response was evaluated in murine BRAFV600E melanoma (D4M) and triple-negative breast cancer (4T1) cell lines. Finally, the in vivo efficacy was investigated in D4M melanoma tumor model. Results: The polysaccharide-constructs along with kinase inhibitor- ß-cyclodextrin conjugates self-assemble to produce SPNs of around 200 nm in diameter and were stable for over a week under physiologically relevant conditions. The SPNs exhibited enhanced cytotoxic effect and significant inhibition of kinase signaling as compared to the free inhibitor. In vitro imaging studies confirmed their enzyme-activatable therapy response tracking abilities both in cancer cells and tumor spheroids. Furthermore, SPN treated mice exhibited better tumor growth inhibition as compared to the control groups and therapy response could be imaged at both early (24-48h) and later time points. Conclusion: These findings demonstrate that the supramolecular polysaccharide nanotheranostics can not only inhibit kinase signaling pathway in aggressive tumor, but also monitor targeted therapy response early.

Granzyme B nanoreporter for early monitoring of tumor response to immunotherapy.

Despite recent advancements in cancer immunotherapy, accurate monitoring of its efficacy is challenging due to heterogeneous immune responses. Conventional imaging techniques lack the sensitivity and specificity for early response assessment. In this study, we designed a granzyme B (GrB) nanoreporter (GNR) that can deliver an immune checkpoint inhibitor to the tumor and track time-sensitive GrB activity as a direct way to monitor initiation of effective immune responses. Anti-programmed death-ligand 1 (PD-L1) antibody-conjugated GNRs inhibited PD-1/PD-L1 interactions efficiently and induced T cell-mediated GrB release that can be imaged using activatable imaging probe. GNRs enabled real-time immunotherapy response monitoring in a tumor-bearing mice model and distinguished between highly responsive and poorly responsive tumors. Furthermore, increasing doses resulted in a better response and enhanced sensitivity in poorly responsive tumors. These findings indicate that GNR has the potential to serve as a tool for sensitive and noninvasive evaluation of immunotherapy efficacy.

Do Adolescents With Idiopathic Scoliosis Have an Erroneous Perception of the Gravitational Vertical?

STUDY DESIGN: Multicenter, case-control study. OBJECTIVES: Demonstrate altered perception of verticality in AIS compared with matched controls. SUMMARY OF BACKGROUND DATA: The cause of adolescent idiopathic scoliosis (AIS) remains to be found. AIS is associated with neurosensorial anomalies, in particular, altered control of orthostatic posture. During kinetic activity, the upright posture, in humans, is determined in reference to the gravitational vertical (GV). We hypothesized that in AIS, there is a discordance in the perception of the GV and the true GV. In AIS, the longitudinal axis of the body would thus be misoriented because of an erroneous perception of the GV. METHODS: Thirty adolescents with right thoracic AIS (age 14.23 ± 1.75 years; Cobb angle 31.97°± 12.83°) and 30 controls matched for age (13.93 ± 1.85 years), body mass index, Tanner stage, and handedness were compared for subjective visual vertical (SVV) measured in static and dynamic (optokinetic stimulation) conditions, and subjective postural vertical (SPV). RESULTS: There was no difference in the two groups, AIS and controls, for SVV. The SPV was significantly different between the two groups (p = .00023). The SPV was shifted to the right for most of the AIS patients (2.13°± 2.22°) compared with controls (-0.08°±1.40°). There was a significant correlation between SPV and clinical frontal tilt in the AIS patients. CONCLUSION: Our findings demonstrate that patients with right thoracic AIS have an erroneous perception of the GV. In most AIS patients, SPV was shifted to the right, with no alteration of the SVV. AIS might be the consequence of a reoriented longitudinal body axis aligned with an erroneous vertical reference. The underlying mechanism might involve dysfunction of trunk graviceptors. The primary or secondary nature of this dysfunction remains an open question.