Caspase-1 Responsive Nanoreporter for In Vivo Monitoring of Inflammasome Immunotherapy.

Inflammasomes are multimeric protein signaling complexes that are assembled in innate immune cells in response to a multitude of pathogen and damage-associated signals. They are essential for generating robust inflammatory responses to prevent pathogenic insults. However, inflammasome dysregulation can induce cascading immune responses, resulting in systemic toxicities and inflammatory disease. In this sense, there is a strong need to develop potent inflammasome inhibiting therapies as well as technologies to monitor their efficacy, yet current systems lack the ability to effectively image inflammasome activation and track therapy response early. To overcome these limitations, we report a novel nanoparticle system delivering both a caspase-1 cleavable inflammasome detecting probe and the NLRP3 inhibitor drug MCC-950, providing dual capabilities of monitoring and regulation of inflammasome activation in a biocompatible, tissue penetrating, and sustained release liposomal formulation. We observed this liposomal nanoreporter's ability to reduce and detect inflammasome activation both in vitro in immortalized bone marrow-derived macrophages and in vivo in a DSS-induced ulcerative colitis mouse model. Our results exhibited the nanoreporter's ability to penetrate inflammatory tissues and detect inflammasome activation early and in real-time for multiple days while alleviating inflammation in the groups coencapsulating imaging reporter and inflammasome inhibitor. Overall, the developed liposomal nanoreporter platform enables spatiotemporal delivery of imaging probe and inhibitor, captures early and sustained inflammasome detection, and induces inflammasome amelioration, thus establishing a novel tool for the real-time monitoring and treatment of inflammasome-mediated disease with high potential for clinical application.

Protein Corona Formation on Lipid Nanoparticles Negatively Affects the NLRP3 Inflammasome Activation.

The interaction between lipid nanoparticles (LNPs) and serum proteins, giving rise to a unique identification in the form of the protein corona, has been shown to be associated with novel recognition by cell receptors. The presence of the corona enveloping the nanoparticle strongly affects the interplay with immune cells. The immune responses mediated by protein corona can affect nanoparticle toxicity and targeting capabilities. But the intracellular signaling of LNPs after corona formation resulting in the change of nanoparticles' ability to provoke immune responses remains unclear. Therefore, a more systematic and delineated approach must be considered to present the correlation between corona complexes and the shift in nanoparticle immunogenicity. Here, we studied and reported the inhibiting effect of the absorbed proteins on the LNPs on the NLRP3 inflammasome activation, a key intracellular protein complex that modulates several inflammatory responses. Ionizable lipid as a component of LNP was observed to play an important role in modulating the activation of NLRP3 inflammasome in serum-free conditions. However, in the presence of serum proteins, the corona layer on LNPs caused a significant reduction in the inflammasome activation. Reduction in the lysosomal rupture after treatment with corona-LNPs significantly reduced inflammasome activation. Furthermore, a strong reduction of cellular uptake in macrophages after the corona formation was observed. On inspecting the uptake mechanisms in macrophages using transport inhibitors, lipid formulation was found to play a critical role in determining the endocytic pathways for the LNPs in macrophages. This study highlights the need to critically analyze the protein interactions with nanomaterials and their concomitant adaptability with immune cells to evaluate nano-bio surfaces and successfully design nanomaterials for biological applications.

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.

Nanoreporter for Real-Time Monitoring of Inflammasome Activity and Targeted Therapy.

Inflammasome activation is associated with a myriad of inflammatory diseases. However, existing methods provides a limited understanding of spatiotemporal kinetics of inflammasome activation, with restricted scope for early detection of associated treatment efficacy. This limitation offers an opportunity for the development of biocompatible in-vivo inflammasome monitoring tools with translational prospects. To achieve this, they report developing a pair of lipid-based nanoparticle systems, a reporter nanoparticle consisting of a caspase-1 activatable probe alone, and a theranostic nanoparticle combining the probe with an inflammasome-inhibiting drug. This biocompatible platform enhances the probe's residence time in circulation by preventing its opsonization and allowing its sustained release over time. Their results demonstrate the specificity of reporter nanoparticles towards caspase-1 activity and provides early-on monitoring of inflammasome activation both in-vitro as well as in-vivo. Additionally, the delivery of disulfiram, an inflammasome-inhibiting drug, along with reporter probe using theranostic nanoparticles enables real-time tracking of treatment efficacy in the gouty-arthritis inflammatory model. In summary, they report an unparalleled pair of the inflammasome-associated reporter and theranostic platforms suited not only for diagnostic applications but can also detect inflammasome-targeted treatment efficiency in real-time. These findings establish two novel, sensitive nanotools for non-invasive evaluation of inflammasome-targeted immunotherapy.

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.

Rational combination of an immune checkpoint inhibitor with CSF1R inhibitor-loaded nanoparticle enhances anticancer efficacy.

Since the advent of immune checkpoint inhibitors, rapid strides have been made in the realm of cancer immunotherapy. Of the abundance of infiltrating immune cells in the tumor microenvironment (TME), macrophages contribute a significant portion and make up to 50% of the tumor mass. In addition to this, the relative plasticity of macrophages makes it an attractive target to modulate macrophage functions to initiate an anti-tumor response. However, many challenges hinder this strategy. Macrophage colony-stimulating factor (MCSF) secreted by cancer cells binds to the colony-stimulating factor receptor present on macrophages and negatively influences macrophage functions. MCSF, along with a cocktail of immunosuppressive cytokines present in the TME, polarizes macrophages to an immunosuppressive pro-tumorigenic M2-like phenotype. M2-like macrophages dampen tumor response and are known to be associated with increased tumor progression and metastasis. Indeed, clinical interventions aimed to reprogram macrophage response from an M2-like tumor aiding phenotype to an M1-like tumor-killing phenotype using small-molecule inhibitors of the CSF1R axis have gathered much attention in the recent past. However, poor response and systemic toxicities observed in these therapies necessitate alternative therapeutic strategies. Furthermore, another key signaling pathway that has been recently implicated in aiding the CSF1R signaling in TAMs is the PDL1 signaling axis. Hence, in this study, we designed a self-assembled lipid nanoparticle system encompassing a potent small-molecule inhibitor of the CSF1R signaling axis, while the surface of the nanoparticle was tethered with anti-PDL1 mAb. The purpose of this is twofold; the nanoparticles can deliver the cargo in a targeted manner to PDL1 expressing M2-like macrophages while simultaneously blocking the receptor. The resulting nanoparticle system termed α-PDL1-CSF-LNP showed enhanced repolarization of M2 like macrophages in vitro while also upregulating the phagocytic index. Furthermore, suboptimal dose administration of α-PDL1-CSF-LNP in an aggressive melanoma mouse model resulted in superior anti-tumor efficacy with minimal toxicities. These results were validated by ex vivo mechanistic analysis showing that TAMs have successfully been repolarized to a predominantly M1-like phenotype. This, along with increased tumor infiltration of CD8+ T cells, worked in synergy to provide an effective anti-tumor strategy.

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.

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.

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.

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.

Targeting macrophages: a novel avenue for cancer drug discovery.

Introduction: Tumor-associated macrophages (TAMs) make up a significant portion of the tumor microenvironment. Emerging clinical evidence indicate that cytokines present in the tumor microenvironment influence TAMs to play an immunosuppressive role by acquiring a pro-tumoral phenotype. However, TAMs are inherently plastic cells that can be phenotypically reprogrammed to elicit an anti-tumoral response. Therapeutic strategies that focus on targeting TAMs have opened new avenues for drug discoveries.Areas covered: This review discusses recent developments in TAM targeted immunotherapy in both preclinical and clinical settings. This article highlights the potential signaling pathways that can be targeted for macrophage reprogramming and discusses the progress of current clinical trials involved in TAMs targeting. Novel nanoparticle-based drug delivery strategies involved in macrophage-based cancer therapeutics and diagnostics are also discussed.Expert opinion: TAM targeted therapies have limited success in clinics due to reasons such as insufficient inhibition of signaling pathways, lower drug accumulation in the tumor, activation of feedback signaling pathways that induce resistance to monotherapies and systemic dose-related toxicities. Nanoparticle-based delivery platforms could overcome these challenges since they enable encapsulation of multiple drugs that target different signaling pathways and enhance intratumoral delivery and can enable delivery of imaging agents.

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.

Dual Inhibitors-Loaded Nanotherapeutics that Target Kinase Signaling Pathways Synergize with Immune Checkpoint Inhibitor.

INTRODUCTION: Immune checkpoint inhibitors that boost cytotoxic T cell-based immune responses have emerged as one of the most promising approaches in cancer treatment. However, it is increasingly being realized that T cell activation needs to be rationally combined with molecularly targeted therapeutics for a maximal anti-tumor outcome. Currently, two oncogenic drivers, MAPK and PI3K-mTOR have emerged as the two main molecular targets for combining with immunotherapy. However, there are major challenges in enabling such combinations: first, such combinations can result in high rates of toxicity. Second, while, these molecular targets could be driving tumor progression, they are essential for activation of the immune cells. So, the kinase inhibitors and immunotherapy can antagonize each other. OBJECTIVES: We rationalized that the synergistic combination of kinase inhibitors and immunotherapy could be enabled by dual inhibitors-loaded supramolecular nanotherapeutics (DiLN) that can co-deliver PI3K- and MAPK-inhibitors to the cancer cells and activate immune response by T cell-modulating immunotherapy, resulting in greater anti-tumor efficacy while minimizing toxicity. METHODS: We engineered DiLNs by designing the amphiphilic building blocks (both drugs and co-lipids) that enables supramolecular nanoassembly. DiLNs were tested for their physiochemical properties including size, morphology, stability and drug release kinetics profiles. The efficacy of DiLNs was tested in drug-resistant cells such as BRAFV600E melanoma (D4M), Clear cell ovarian carcinoma (TOV21G) cells. The tumor inhibition efficiency of DiLNs in combination with immune checkpoint inhibitor antibody was studied in syngeneic D4M animal model. RESULTS: DiLNs were stable for over a month and released the drugs in a sustained manner. In vitro cytotoxicity studies in D4M and TOV21G cells showed that DiLNs were significantly more effective than free drugs. In vivo studies showed that the combination of DiLNs with anti PD-L1 antibody resulted in superior antitumor effect and survival. CONCLUSION: This study shows that the rational combination of DiLNs that target multiple oncogenic signaling pathways with immune checkpoint inhibitors could emerge as an effective strategy to improve immunotherapeutic response against drug resistant tumors.

CSF1R- and SHP2-Inhibitor-Loaded Nanoparticles Enhance Cytotoxic Activity and Phagocytosis in Tumor-Associated Macrophages.

Immune modulation of macrophages has emerged as an attractive approach for anti-cancer therapy. However, there are two main challenges in successfully utilizing macrophages for immunotherapy. First, macrophage colony stimulating factor (MCSF) secreted by cancer cells binds to colony stimulating factor 1 receptor (CSF1-R) on macrophages and in turn activates the downstream signaling pathway responsible for polarization of tumor-associated macrophages (TAMs) to immunosuppressive M2 phenotype. Second, ligation of signal regulatory protein α (SIRPα) expressed on myeloid cells to CD47, a transmembrane protein overexpressed on cancer cells, activates the Src homology region 2 (SH2) domain -phosphatases SHP-1 and SHP-2 in macrophages. This results in activation of "eat-me-not" signaling pathway and inhibition of phagocytosis. Here, it is reported that self-assembled dual-inhibitor-loaded nanoparticles (DNTs) target M2 macrophages and simultaneously inhibit CSF1R and SHP2 pathways. This results in efficient repolarization of M2 macrophages to an active M1 phenotype, and superior phagocytic capabilities as compared to individual drug treatments. Furthermore, suboptimal dose administration of DNTs in highly aggressive breast cancer and melanoma mouse models show enhanced anti-tumor efficacy without any toxicity. These studies demonstrate that the concurrent inhibition of CSF1-R and SHP2 signaling pathways for macrophage activation and phagocytosis enhancement could be an effective strategy for macrophage-based immunotherapy.

A designer self-assembled supramolecule amplifies macrophage immune responses against aggressive cancer.

Effectively activating macrophages that can 'eat' cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumourigenic M2 phenotype. Also, cancer cells can express CD47, an 'eat me not' signal that ligates with the signal regulatory protein alpha (SIRPα) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPα-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPα on macrophages, blocking the CD47-SIRPα signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances the M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47-SIRPα and MCSF-CSF-1R signalling axes may constitute a promising immunotherapy.