Targeting tumor associated macrophages: The new challenge for nanomedicine

Seminars in Immunology 34 (2017) 103–113

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Review

Targeting tumor associated macrophages: The new challenge for nanomedicine

T

⁎

Fernando Torres Andóna,b, , Elisabeth Digificoa,c, Akihiro Maedaa, Marco Errenia, Alberto Mantovania,c, María José Alonsob,d,e, Paola Allavenaa a

Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Via A. Manzoni 113, 20089 Rozzano, Milan, Italy Center for Research in Molecular Medicine & Chronic Diseases (CIMUS), University of Santiago de Compostela, 15706 Campus Vida, Santiago de Compostela, Spain c Humanitas University, Via A. Manzoni 113, 20089 Rozzano, Milan, Italy d Pharmacy & Pharmaceutical Technology Department, School of Pharmacy, University of Santiago de Compostela, 15705 Campus Vida, Santiago de Compostela, Spain e Health Research Institute of Santiago de Compostela (IDIS), 15706 Santiago de Compostela, Spain b

A R T I C L E I N F O

A B S T R A C T

Keywords: Nanomedicine Tumor associated macrophages Cancer immunotherapy Mononuclear phagocyte system Drug delivery Tumor microenvironment

The engineering of new nanomedicines with ability to target and kill or re-educate Tumor Associated Macrophages (TAMs) stands up as a promising strategy to induce the effective switching of the tumor-promoting immune suppressive microenvironment, characteristic of tumors rich in macrophages, to one that kills tumor cells, is anti-angiogenic and promotes adaptive immune responses. Alternatively, the loading of monocytes/ macrophages in blood circulation with nanomedicines, may be used to profit from the high infiltration ability of myeloid cells and to allow the drug release in the bulk of the tumor. In addition, the development of TAMtargeted imaging nanostructures, can be used to study the macrophage content in solid tumors and, hence, for a better diagnosis and prognosis of cancer disease. The major challenges for the effective targeting of TAM with nanomedicines and their application in the clinic have already been identified. These challenges are associated to the undesirable clearance of nanomedicines by, the mononuclear phagocyte system (macrophages) in competing organs (liver, lung or spleen), upon their intravenous injection; and also to the difficult penetration of nanomedicines across solid tumors due to the abnormal vasculature and the excessive extracellular matrix present in stromal tumors. In this review we describe the recent nanotechnology-base strategies that have been developed to target macrophages in tumors.

1. Introduction In the field of oncology, the use of nanotechnology has provided a broad range of nanostructures designed to improve the delivery of therapeutic compounds towards cancer cells [1]. Different nanostructures have been used to encapsulate pharmacological compounds allowing to overcome common problems of solubility and stability, to reduce their side-effects, to extend their circulating half-time and, in some cases, to enable their controlled release towards the target cell [2–7]. Despite their underlined significant potential to improve the efficacy and to reduce the toxicity of antitumoral drugs, improvements in overall survival of patients are still modest [8–10]. Important hindrance point towards the complexity of the tumor microenvironment (TME), due to its physicochemical and cellular heterogeneity, with a major contribution of the abnormal tumoral stroma and the presence of immunosuppressive cells (majorly tumoral associated macrophages,

TAM) [11,12]. In this review article, we provide an overview of the most recent investigations involving the development of nanomedicines for the targeting of macrophages in tumors with therapeutic or diagnostic purposes, and also the results of recent efforts intended to use monocytes/macrophages loaded with nanomedicines as live cell-mediated drug delivery systems (LCDDS) to transport drugs into the bulk of the tumor. Furthermore, we recapitulate the major challenges (i.e. mononuclear phagocytic system and tumor microenvironment) which need to be addressed for the improvement of the actual efficacy of nanomedicines to reach the center of solid tumors and provide an overview of possible solutions and future perspectives in the field of nanomedicine for the treatment of cancer.

⁎ Corresponding author at: Laboratory of Cellular Immunology, Istituto Clinico Humanitas, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Via A. Manzoni 113, 20089 Rozzano, Milan, Italy. E-mail address: [email protected] (F.T. Andón).

http://dx.doi.org/10.1016/j.smim.2017.09.004 Received 31 July 2017; Received in revised form 15 September 2017; Accepted 15 September 2017 Available online 21 September 2017 1044-5323/ © 2017 Elsevier Ltd. All rights reserved.

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Fig. 1. Pro-tumoral functions of Tumor Associated Macrophages (TAM): they induce the proliferation and survival of cancer cells; stimulate the invasion and metastatic properties of cancer cells; enhance abnormal tumor angiogenesis; support tissue remodelling, fibrosis and extracellular matrix deposition in the tumor; contribute to genomic instability; and suppress adaptive immunity, mainly through the inhibition of anti-tumor T cell responses.

Fig. 2. Nanomedicines to target and impact on TAM. Shown on the upper left therapeutic nanostructures to inhibit the recruitment of TAM towards the tumor, on the up right traditional clodronate liposomes and new mannose-targeted-NPs loaded with doxorubicin to kill TAM. In the lower section two NPs engineered to reach and re-educate TAM, from an M2-like pro-tumoral phenotype into M1-like macrophages with antitumor functions, such as direct killing of tumor cells and activation of cytotoxic T cells (see main text for more info).

2. Opportunities offered by macrophages for the application of nanomedicine in cancer

increased CD8+ T-cell infiltration in the tumor. These antitumor immune responses were significantly enhanced by the combination of the CCL5-siRNA-loaded-MSVs with the CCR5 inhibitor Maraviroc® [18]. Another target of interest to inhibit TAM recruitment is the colonystimulating factor receptor (CSF-1R). Several CSF-1R inhibitors have demonstrated promising antitumoral effects in different murine tumor models, and some of them are now progressing in clinical trials [19–21]. Despite these positive results, the targeting of CSF-1R using NPs has not been explored yet.

2.1. Nanomedicines to target tumor associated macrophages Tumor Associated Macrophages (TAMs) are a key component of the tumor microenvironment. They may represent up to 50% of the tumor mass and their prominent role in the evasion from immune surveillance has been established [13,14]. TAM infiltration in tumor tissues has been shown to support tumor growth, angiogenesis, invasion and metastasis, and their high density in tumors is correlated with tumor progression and resistance to therapies (Fig. 1) [11]. These findings point out TAMs as promising targets for novel antitumoral therapeutic strategies. These strategies can be divided into three main groups: i) inhibition of TAM recruitment to the tumor, ii) direct killing of TAMs, iii) re-education of TAM from their M2-like protumoral phenotype into a M1-like antitumoral phenotype [14–16]. Thus, the development of new nanomedicines to target and impact on TAM stands as a promising opportunity to switch the tumor-promoting immune suppressive microenvironment, characteristic of tumors rich in macrophages, to one that kills tumor cells, is anti-angiogenic and promotes adaptive immune responses (Fig. 2). Several nanotechnological approaches with this purpose have been reported.

2.1.2. Kill TAM in the tumor A few anticancer nanomedicines have demonstrated ability to kill TAM. Interestingly, more than 20 years ago, nanotechnology approaches were developed to deliver bisphosphonates, such as clodronate or zoledronate, to tumors resulting in depletion of TAM, better antitumoral effect, impaired angiogenesis and decreased metastasis (Fig. 2) [22,23]. The intratumoral injection of alendronate conjugated with glucomannan into sarcoma-bearing mice, was used to target the mannose receptors in TAM, achieving their effective depletion [24]. The development of folate-decorated liposomes loaded with zoledronic acid was also applied to target TAM in tumors, however, this composition did not result in a reduction of tumor growth when applied to tumor-bearing mice: KB (human nasopharyngeal) and C-26 (mouse colon adenocarcinoma) [25,26]. Despite of this, nowadays, the use of clodronate-liposomes is still a common approach used by biomedical researchers to deplete macrophages in a non-located manner, thus room to improve the specific delivery of bisphosphonates towards TAM in specific diseased-tissues (i.e. solid tumor), by means of new nanocarriers, is still available. Several nanocarriers, including liposomes and PLGA nanoparticles, have been decorated with mannose for the specific targeting of the mannose receptor (MR or CD206), highly expressed in TAM (M2-like macrophages) [27,28]. In order to favour the uptake of the mannosylated-nanomedicines by TAM in the tumor, and prevent their uptake by macrophages in other locations (i.e. MPS see Section 3), Zhu et al. engineered mannosylated-PLGA NPs shielded with pH-sensitive-PEG moieties for the delivery of doxorubicin (DOX) [28,29] or siRNA into TAM [30] (Fig. 2). Several peptides, proteins and aptamers were also investigated to target TAM specifically. For example, PEGylated liposomes conjugated with the peptide LyP-1 and loaded with DOX were shown to reach TAM in metastatic lymph nodes, causing the inhibition

2.1.1. Inhibition of TAM recruitment to the tumor Monocytes/macrophages are recruited from the blood and infiltrate the tumor as an immune reaction to a damage event (see Section 2.2). Thus, it is feasible to impair the generation of TAM by targeting and impacting the monocytes, either in the blood, in the bone marrow or in the lymphoid organs. In 2011 siRNA-loaded lipid NPs were proposed as a way to reduce the expression of the chemokine receptor CCR2, which is required for the recruitment of monocytes to the tumor. The intravenous injection of these NPs led to their accumulation in the spleen and bone marrow, where they delivered the associated siRNA into the Ly6Chigh monocytes (TAM precursors) resulting in reduced tumor growth in two different xenograft tumor models [17]. A similar approach has been recently presented by Ban et al. through the targeting of the CCL5-CCR5 axis. They developed bone marrow-targeted biodegradable mesoporous silicon nanoparticles (MSVs) loaded with liposomal CCL5-siRNA and decorated on their surface with a thioaptamer for E-selectin, expressed on the bone marrow endothelium (Fig. 2). The CCL5-siRNA-loaded-MSVs intravenously injected into 4T1 bearing mice resulted in the re-programming of immunosuppressive myeloid cells in the bone marrow (evaluated as defective expression of CCR5), and this result was translated into a significant reduction of tumor growth and 104

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immunomodulatory protein histidine-rich glycoprotein (HRG), antiCD47 antibodies, PI3Kγ or BTK inhibitors, and also a modified form of vitamin-D-binding protein (EF-022) for the antitumoral activation of TAM [14]. Despite these promising results, a more effective and long-lasting functional re-education of TAM is still needed, and a few nanotechnological approaches have been explored with this purpose [23]. Considering the targeting of the mannose receptor in TAM, Ortega et al. developed mannose-decorated polymeric nanocapsules, loaded with IκBα siRNA to restore the classical NF-κB activity in TAMs, and showed their good targeting and re-educative properties in vitro [42]. In another instance, mannan-conjugated MnO2 and low molecular weight Hyaluronic Acid (HA), intended to induce pro-inflammatory signals in macrophages, were developed by Song et al. [43] (Fig. 2). In a 4T1 breast cancer model, these Mannan-HA-MnO2-NPs were able to reach TAM, increase production of O2 and tumor oxygenation, regulate the pH and down-regulate HIF-1α and VEGF in the tumor microenvironment. Furthermore, Man-HA-MnO2-NPs demonstrated to be interesting nanosystems for T1- and T2-MRI performance for tumor imaging and detection, and their antitumor activity was enhanced in combination with Doxorubicin. Another approach to target and to re-educate TAM, consisted in the development of galactosylated cationic dextran nanocomplexes intended to target the galactose-type lectin receptor and to deliver anti-IL-10 and anti-IL-10R oligodeoxinucleotide in TAM [44]. These Gal-IL10-NCs were made of pH-sensitive PEG-histidine-modified alginate meant to hide the nanocarriers from macrophage uptake till they reach the acidic microenvironment of the tumor, and then be released allowing the specific binding of TAM in the tumor. The intravenous administration of these nanocomplexes in an allograft hepatoma murine model resulted in the suppression of TAM protumor functions and stimulation of macrophage anti-tumor activities [44]. To inhibit STAT3 in TAM, several nanotechnology approaches involving the use of PLG-PEG NPs and liposomes have been explored. For example, PLGA-PEG NPs loaded with sunitinib [45], hydrazinocurcumin or imidazole-loaded legumain-targeted liposomes [46,47] have shown a good capability for remodelling the tumor microenvironment. In other cases, engineered PEG-PLA and DOTAP-NPs containing IL-12 plasmids showed ability to inhibit angiogenesis and reduce tumor growth in subcutaneous and peritoneal CT26 colon cancer murine models [48]. A different approach has involved the use of liposomal polymeric gels of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers capable of co-delivering small hydrophobic TGF-β inhibitors and water-soluble IL-2, demonstrating satisfactory antitumoral efficacy in melanoma bearing mice [49]. With a similar rational, lipid/protamine/hyaluronic acid nanoparticles were also developed to deliver TGF-β siRNA into tumors [50]. Conde et al. developed PEGylated gold nanoparticles (15 nm) conjugated to TAMs-targeting peptides (M2pep) and loaded with VEGF siRNA (Fig. 2). This formulation showed the ability to target murine lung TAMs and lung cancer cells, demonstrating a good ability to reeducate the tumor microenvironment, stimulating a host immune response that was critical for long-lived tumor eradication [51]. One of the most striking observations regarding the re-education of TAM induced by NPs was recently reported by Zanganeh et al. The authors showed that Ferumoxytol®, dextran-coated iron oxide NPs FDAapproved for the treatment of iron deficiency, present antitumoral effects towards early mammary cancers and lung and liver metastasis. Ferumoxytol® accumulates in TAM in tumors and induces their reeducation towards M1-like antitumoral macrophages, increased production of TNF-α and generation of reactive oxygen species through iron oxide Fenton reactions. This treatment inhibited the growth of subcutaneous adenocarcinomas in mice and was able to prevent the development of liver metastasis and tumor re-challenge [52]. Finally, a combined therapy consisting of PLGA NPs loaded with the photothermally-active dye indocyanine green and a TLR7 agonist were intravenously administered in mice and followed by localized near-

of lymphatic tumor metastasis [31]. Another approach consisted in the development of RNA aptamers with affinity for the murine or human IL4 receptor a (IL-4Ra/CD124) which resulted in preferential binding to M-MDSC in the spleen, and to M-MDSC and TAM in murine tumors [32]. Hence, several molecules, receptors overexpressed in the surface of TAM, have been identified for the targeting of TAM, which can be applied to the decoration of drug delivery nanocarriers; however, lack of selectivity is still the major challenge to be addressed, as these molecules/receptors are also present in other immune cells (i.e. IL-4R in T or B cells, CD206 in subpopulations of endothelial cells) or in macrophages in other locations (i.e. MPS, see Section 3.1). In addition to the targeting molecules, the selection of the ‘pharmacological’ load of nanomedicines to kill TAMs is not trivial. In 2013, Trabectedin (Yondelis ®), registered for soft tissue sarcoma and ovarian cancer, was the first marketed drug showing a selective cytotoxic activity towards Ly6Chigh monocytes in circulation and in the spleen, as well as a reduction of TAM in the tumor, and this effect was found to be key for the antitumoral efficacy of this drug [33]. The distinct ability of other chemotherapeutics drugs to kill TAMs and their effect on the tumor microenvironment has been studied [11]. For example, while Doxorubicin has been demonstrated to deplete myeloid derived suppressor cells (MDSCs), induce immunogenic cell death and activate anti-tumor immune responses [34]. Platinum agents showed protumor skewing effects [35]. The capability to abolish the immunosuppressive microenvironment has been also observed for Docetaxel or Gemcitabine, among others. And the loading of cytotoxic drugs into nanocarriers to reach immunosuppressive cells has been also explored [23]. For example, Sasso et al. have developed gemcitabine lipid nanocapsules with ability to target monocytic MDSCs and to unleash antitumor responses (i.e. activation of T cells) in lymphoma and melanomabearing mice. Another emerging approach of interest for the killing of TAM is the application of photodynamic or photothermal therapy using engineered nanostructures. Ben-Nun et al. developed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification and kill TAM upon light stimulation, leading to significant tumor shrinkage in a breast cancer murine model [36]. Two additional reports, aimed to target the mannose receptor, consisted in the development of mannose-conjugated chlorin-nanoconjugates which were intraperitoneally injected in a murine colon cancer model [37], and the phthalocyanine dye conjugated to a monoclonal anti-CD206 antibody (IRD-aCD206) which demonstrated good antitumoral activity and prevention of lung metastasis when applied intravenously in 4T1 breast cancer tumors resistant to Sorafenib [38]. These results provide new evidences for the importance of TAM depletion in tumors resistant to current antitumoral therapies. 2.1.3. Re-educate TAM in the tumor The re-education of TAM consists in the re-programming of macrophages with M2-like protumoral properties to M1-like macrophages with active defensive activity, including anti-tumor functions (i.e. direct killing of tumor cells and eliciting of vascular damage and tissue destruction). Several pharmacological molecules with the ability to reswitch the polarization of macrophages from M2-like immunosuppressive macrophages to M1-like cytotoxic effectors have been identified in the last years [14]. Among them, IFN-γ is a classic inducer of macrophage M1 polarization and killing of cancer cells. In order to avoid the problems related to systemic macrophage activation, IFN-γ has been administered intraperitoneally in women with ovarian cancer resulting in enhanced clinical responses [39]. Other clinically approved approaches involve the use of an agonist antibody CD40 to patients with advanced pancreatic cancer or the administration of BLZ945 (a highly selective small molecule inhibitor of CSF-1R) to patients with glioblastoma multiforme [23,40,41]. Both strategies resulted in the functional re-polarization of TAM and a significant enhancement of the patient’s survival. Additional investigations comprise the use of 105

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positive outcome of this work encourages for the investment of future efforts in this direction. Another interesting ligand to reach monocytes in circulation could be β-glucan to target the pattern recognition receptor Dectin-1 receptor [64]. In addition to the targeting possibilities, the use of nanotechnology offers the potential to load the Live Cell-mediated Drug Delivery Systems (LCDDS) with different pharmacological molecules and to maintain these drugs safely confined, until they reach the tumor site. Once the LCDDS reaches the appropriate location for its therapeutic action (i.e. the center of the tumor) the drug must be released. For this, an option has been the implementation of nanotechnology approaches that trigger the release of the drug upon physical or chemical stimulation. On the other hand, passive release of the drug at the targeted tissue may be achieved through the selection of the appropriate components of the nanocarrier, which must have a good biodegradation profile when the LCDDS reaches the target. For example, well-known biodegradable polymers with satisfactory clinical profile, such as Polylactic acid (PLA), are available and their biodegradation profiles have been properly studied [65,66]. Alternatively, to achieve the active release of the drug, more advanced nanotechnological strategies are needed, to open up the nanocarrier and to trigger the release of the drug at a chosen time. For this, external stimuli, such as temperature, light, magnetic field or ultrasound approaches can be exploited [54]. He at al. have associated, through disulphide linkages, L-asparaginase, a drug for acute lymphoblastic leukaemia, to a cell penetrating peptide (CCP). This complex was encapsulated into red blood cells (RBC), and when it reaches the cytoplasm the reduction of disulfide bonds by glutathione results in the disassociation of the drug from the peptide, drug release and therapeutic action [67]. In essence, the loading of monocytes/macrophages with nanomedicines, before they reach the tumor, is still in early stages of investigation and arduous to apply in the clinic. On the other hand, this strategy already takes advantage of the physiological properties of the ‘carrier’ cells, thus minimizing problems of toxicity, and of their innate ability to penetrate the tumor microenvironment even reaching the most difficult hypoxic regions in the center of the tumor.

infrared light application to the tumor, resulting in tumor ablation by heating and stimulation of immune antitumoral responses [53]. The antitumoral effect of these PLGA-ICG-TLR7 NPs was significantly enhanced in combination with checkpoint blockade inhibitors. Overall, major challenges for the targeting and re-education of TAM are: first, the specificity of ligands to reach the protumoral M2-like macrophages in tumors and not other populations of macrophages (i.e. MPS-macrophages, see Section 3.1); second, the resilient nature of the immunosuppressive microenvironment, as it often takes time for the immune system to become “activated/stimulated/re-educated” and to acquire the capacity to mount a sufficient anti-tumor response which must be long-lasting for the definitive elimination of cancer. For this, the engineering of new nanomedicines provides new opportunities by: i) improving the selectivity of TAM-targeting, through the synergic effect of the ligand-receptor recognition with the preferential appetite of macrophages for particles in the nanometre size, ii) combining into an unique therapeutic nanostructure several pharmacological molecules with ability to kill TAM, thus reducing their amount in the tumor and making it more ‘manageable’, or combinations of molecules to re-educate TAM in a long-lasting manner, taking advantage of the controlled release properties of the nanocarriers. The effective development of such nanomedicines could lead to a new breakthrough in the field of cancer immunotherapy. 2.2. Monocytes/macrophages loaded with nanomedicines: new live cellmediated drug delivery systems for cancer treatment A new generation of drug delivery strategies, so-called Live Cellmediated Drug Delivery Systems (LCDDS), consists in the use of the patient host’s cells (i.e. monocytes, macrophages, erythrocytes or stem cells) [54], either as a whole or by employing selected key components of these cells (i.e. external cellular membranes) [55–58], as ‘Trojan Horses’ loaded with drugs. Major advantages of live cell properties, of interest for their application as ‘Trojan Horses’, include: long circulation time in blood, flexible morphology, ratio of surface ligands and physiological metabolism [54]. In addition, the transient lifespan of live cells (i.e. leukocytes) could be exploited as an appropriate time for the delivery, therapeutic action and physiological elimination of the ‘carrier’. The latest investigations, related to the cellular dynamics of the tumor microenvironment, indicate that TAMs are primarily derived from circulating monocytes, which are recruited from the peripheral blood in response to a broad range of molecular signals (i.e. CCL2/MCP1 released from tumor cells) [59]. Of interest, though most of the chemotherapeutic drugs used in clinic can only reach the vascular area, monocytes/macrophages present a high ability to penetrate the tumor tissue in depth, even reaching the hypoxic regions [14]. This thrilling ability of myeloid cells to infiltrate diseased tissues and to penetrate challenging biological barriers, even when they are loaded with a therapeutic cargo, has started to be investigated. In addition, it has been demonstrated, mainly in vitro, that the phagocytic nature of monocytes/macrophages is beneficial for their easy loading with drug-containing-nanostructures [60,61]. For example, Qin et al. have studied the ex vivo loading of THP-1 monocytes with cRGD-modified liposomes containing an anti-depressive macromolecular drug (trefoil factor 3). The intravenous injection of these ‘loaded cells’ in relevant murine models of disease resulted in a satisfactory performance to cross the blood brain barrier (BBB) and the improvement of symptoms [62]. A more challenging approach comprises the in vivo targeting and loading of monocytes in the circulation with nanostructures, which must then be able to reach the bulk of the tumor. For this, Smith et al. have developed RGD (Arg-Gly-Asp) peptide labelled single-walled carbon nanotubes (SWCNTs) which were specifically engulfed by Ly6Chigh monocytes in blood circulation, and subsequently delivered into the tumor, showing significant tumor penetration capabilities [63]. Although these RGD-SWCNTs were not loaded with any drug, the

2.3. Nanoparticles for imaging monocytes/macrophages: application for diagnosis and prognosis of cancer A central role for monocytes and macrophages has been described not only in cancer, but also in other pathologies, including cardiovascular disease (CVD) [68], diabetes [69], tuberculosis [70] and rheumatoid arthritis [71]. Therefore, the specific targeting and visualization of monocytes and macrophages represents a unique opportunity to monitor and analyse the progression of these diseases. A wide variety of diagnostic imaging modalities have been combined with nanoparticle contrast agents and targeting probes for molecular imaging approaches, such as MRI, PET, x-ray computed tomography (CT) and single-photon emission computed tomography (SPECT) [72,73]. Nowadays, optical imaging represents a valid and simple alternative to conventional clinical imaging techniques for basic research in small animals, where the need of deeper tissue penetration is reduced (Fig. 3) [74]. Moreover, the development of new fluorescent nanomaterials (i.e. quantum dots) allows now for improved performance of preclinical optical imaging methods [75]. Macrophage-targeting imaging is being increasingly used for the investigation of cancer [15,33,76–80]. The imaging of macrophage infiltration in solid tumors provides relevant prognostic information, allowing the identification of neoplastic margins and in some cases providing an indication for the efficacy of anti-tumor therapies [76]. For example, MRI enables the visualization of TAM in a variety of tumors, including breast cancer [81]. Superparamagnetic iron oxide NPs (SPIO) are useful contrast agents for MRI, and they can be appropriately engineered, with appropriate particle size and surface properties, to be phagocytosed by macrophages in different tissues. While 80–150 nm 106

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Fig. 3. Two-photon intravital imaging can be used to follow in real-time the ability of nanomedicines labelled with fluorescent molecules to penetrate the tumor microenvironment. This technique can also be applied to study the amount and/or localization of TAM in the tumor. In this picture, in a fibrosarcoma murine tumor model, collagen can be observed in blue by the second harmonic generation signal, CX3CR1-GFP monocytes/ macrophages are visualized in green and HA-Cy5 labelled nanocapsules in red. Courtesy of Eduardo Fernández Megía, University of Santiago de Compostela (HA-Cy5 polymer) and Diego Morone, Istituto Clínico Humanitas (two-photon image). Fig. 4. Major challenges and possible solutions for the efficacy of nanomedicine in cancer. (A) The mononuclear phagocyte system (MPS), mainly resident macrophages in the competing organs (liver, lung and spleen), has been designated as the major responsible of the undesirable clearance of nanomedicines from the blood circulation. Solutions to address this issue encompass: (i) the therapeutic manipulation of the MPSmacrophages; or (ii) the strict engineering of nanomedicines with features to avoid the macrophage-uptake in these organs, but allowing their uptake by the target cells. (B) Once the nanomedicines reach the tumor, the abnormal blood and lymphatic vessels, the excessive deposition of extracellular matrix (i.e. collagen, hyaluronan or fibronectin) and the immunosuppressive tumor microenvironment prevent the penetration and efficacy of cancer nanomedicines in solid tumors, which in theory can be resolved by: (iii) use of tumor penetration peptides (i.e. LyP-1), (iv) enzymatic depletion of the extracellular matrix (i.e. collagenase or hyaluronidase), (v) tumor vessel normalization (i.e. by application of VEGF or HIF inhibitors) and (vi) TAM elimination or re-education into antitumoral macrophages, switching the tumor-promoting immune suppressive microenvironment to one that kills tumor cells, is anti-angiogenic, promotes adaptive immune responses and provides a normalized tissular structure which allows the better penetration of nanomedicines.

SPIO are rapidly engulfed by macrophages of the mononuclear phagocyte system (MPS), such as spleen and liver, in contrast, ultra-small SPIO (USPIO) with a smaller diameter (< 50 nm), suffer a lower retention in MPS-macrophages, remain more time in blood circulation and accumulate in inflamed tissues and tumor sites, reaching tissue macrophages and TAMs [82,83]. Of note, Ferumoxytol® (described in detail in the previous section) is an USPIO which has been successfully used to quantify macrophage infiltration by MRI in tumors. In addition, Ferumoxytol® has been used to predict penetration efficacy of delivery systems and it could be applied to predict patients’ outcome in breast cancer and to monitor TAM-targeted therapies in clinical practice [84,85]. Recently, TAM-targeting dextran NPs, a fluorescent and crosslinked version of Ferumoxytol®, have been developed to visualize TAM infiltration in a 3D organ imaging model of lung carcinoma [86]. Lymphotropic superparamagnetic NPs, which are internalized by macrophages, have been used in combination with high-resolution MRI for the non-invasive detection of small lymph node metastasis in prostate cancer [87]. Another approach, consisting in the generation, by radiolabelling, of small dextran NPs (known to accumulate in macrophages) with 89Zr, has been applied to quantify TAM infiltration in a syngenic colon carcinoma mouse model [88]. In addition, 89Zr radiolabelled reconstituted high-density lipoprotein (rHDL) has been used in PET imaging for the non-invasive monitoring of TAM in a mouse model of breast cancer [89]. In another study, fluorescent molecular tomography (FMT) was combined with MRI to visualize macrophages in sarcoma bearing mice [90]. In this manuscript, TAMs were selectively labelled with surface-tuned magneto-fluorescent NPs allowing their tracking within the tumor microenvironment both by MRI and FMT. Another approach for the identification of pro-angiogenic TAM in tumors was reported by Movahedi et al. They generated 99mTc-labeled macrophage mannose receptor targeting nanobodies (single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies) with excellent properties for the molecular imaging by SPECT of macrophages overexpressing the mannose receptor in the tumor stroma [91]. As a whole, these imaging approaches, in some cases with pertinent modifications, are applicable to study the macrophage infiltration in solid tumors, and more importantly, to evaluate their specific localization (i.e. periphery or center), phenotype and functions. This comprehensive study of macrophages in tumors, which can be achieved using new nanotechnological imaging-approaches, has the potential to lead for a better evaluation of the cancer disease and for a better prediction of response to antitumoral therapies. These diagnostic and prognostic investigations gain increasing relevance with the rising use of cancer immunotherapeutics (i.e. checkpoint inhibitors) which are applied now in the clinic to fight against the immunosuppressive cells

of the tumor microenvironment, among them TAM [92,93]. 3. Challenges for the efficacy of cancer nanomedicine in patients and pre-clinical tumor models 3.1. Undesirable clearance of nanomedicines by the mononuclear phagocytic system The Mononuclear Phagocytic System (MPS) is composed of a family of cells, mainly macrophages, which are characterized for their high phagocytic activity, increased expression of antigen presentation molecules and secretion of cytokines [94,95]. Upon systemic administration, nanomedicines smaller than 5 nm are commonly excreted by the renal system (kidney), while larger NPs may be identified by the immune system, specifically the MPS (mainly macrophages in liver, lungs and spleen), as foreign substances which need to be sequestered, degraded and eliminated (Fig. 4) [10,23,96–99]. Although tissue resident macrophages seem to be the major responsible for NP clearance, a few investigations have also showed the ability of cells circulating in blood, such as monocytes, neutrophils or red blood cells (RBCs) to sequester NPs. The interaction of NPs with RBCs has been explored using in vitro, in vivo and mathematical models [100,101], with evidence that beyond the nature of the circulating cells, NPs dispersion rate, hydrodynamic interactions, flow speed and size of capillaries, are important factors influencing the clearance of NPs. In addition to the capacity of monocytes or neutrophils to phagocyte NPs, other mechanisms could be implicated. For example, neutrophils can release DNA and proteins (neutrophil extracellular traps) to trap pathogens at infection sites, which by a similar mechanism may trap NPs [102,103]. Another issue to be considered is the rate of the circulating cells in specific disease conditions (i.e. neutropenia in patients treated with chemotherapy). An 107

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undesirable uptake of nanomedicines by MPS-macrophages, mainly in the liver [99]. Physicochemical properties of nanoparticles, such as size, shape, surface charge and even flexibility have been demonstrated to influence macrophage uptake, and have been implemented to avoid liver uptake [99,105]. The coating of NPs with specific molecules to avoid the uptake of macrophages is by large the most explored strategy (mainly PEG, discovered in the 90′s) [112]. However, the PEGylation of therapeutics is not a trivial task, which still requires investigation. Some PEGylated-therapeutics have presented problems, such as inhibition of cellular uptake and endosomal escape into cancer cells, and even more striking, the appearance of an unexpected immunogenic response known as the ‘accelerated blood clearance’ (ABC) phenomenon, where a second dose of PEGylated-therapeutics is rapidly cleared when given several days after the first dose [113]. Mechanistic studies have demonstrated that the first injection of PEG-NPs induces the generation of anti-PEG IgM, which upon the second injection results in the immediate and specific recognition of NPs by the macrophages, capturing all the NPs in the liver. More recent investigations, have suggested strategies to solve these problems. For example, the conjugation of neutral PEG ligands with the appropriate size has resulted highly efficient to extend the time of NPs, and also other drugs, in blood circulation [113–115]. Other ‘coating’ strategies have been used to avoid the uptake of NPs by macrophages, such as: zwitterionic polymers, ‘don’t eat me proteins’ CD47 [116] or even the covering with components of cellular membranes derived from leukocytes or erythrocytes [55–58]. Indeed, some of these strategies have demonstrated an almost ‘perfect’ capacity to avoid macrophages uptake and to increase the time of nanomedicines in blood circulation, however, some of these stealth NP have also shown a reduced uptake by the target cells (usually cancer cells) thus resulting in a diminished therapeutic effect. Another approach, which has been hardly explored, consists in the therapeutic manipulation of the MPS prior to the administration of nanomedicines to avoid their clearance and consequently enhancing their delivery to the targeted diseased tissue [117,118]. This strategy is conceived to prevent the uptake of nanoparticles by MPS-macrophages for a short-term period of time, providing a therapeutic window for the administration of the nanomedicines and increasing their effectiveness. Some investigations, applied with this rational, have used an extremely aggressive approach, which consists in the ‘killing’ of liver macrophages using liposomes loaded with clodronate or dichloromethylene-biphosphonate. This depletion of Kupffer cells in mice resulted in diminished nanomedicine clearance, increased time of circulation in blood and increased accumulation in the tumor (i.e. pancreatic or breast xenograft tumor models) [117]. However, this indiscriminate killing of macrophages in the liver, and other organs, may reduce the hepatic function and the integrity of the immune system for the whole life. More recent studies have tried to induce a mild and transient loss of function of MPS-macrophages prior to the administration of nanomedicines. For example, gadolinium chloride (GdCl3) [119,120], phosphatidylcholine:cholesterol liposomes or other cationic liposomes have been successfully applied to reduce MPS-clearance of nanomedicines [121,122]. Altogether, liver and lung macrophages, with a minor role for the spleen macrophages, have been identified as major responsible for the undesirable uptake and clearance of intravenously injected nanomedicines (Fig. 4). Importantly, some researchers have started to study in detail the major factors influencing these interactions, such as: i) anatomical and physiological properties of the ‘competing organs’, ii) cellular localization and phenotype and iii) physicochemical and surface properties of the NPs. In addition, a few strategies to manipulate these factors have been presented. However, more studies are needed to understand the biodistribution profile of nanomedicines in different patients or pre-clinical animal models. For example, it has been shown that local and systemic immune response to cancer increases particle clearance by liver and spleen, due to an increase in M2-like macrophages in these organs [123]. Similarly, it was observed in both, human patients and mice with recurrent epithelial ovarian cancer, that the

attempt to look into this has been described by Jones et al. They have observed a higher number of circulating neutrophils in BALB/c compared to C57BL/6 mice, however these variations were not correlated with differences in NP clearance [104]. Macrophages in the liver, traditionally denominated Kupffer cells, are the major responsible for the clearance of nanomedicines, being able to accumulate and sequester between 30 and 99% of the NPs from the bloodstream [105–109]. Tsoi et al. have recently studied in detail the mechanisms of hard-nanomaterial (i.e. quantum dots, gold and silica NPs) clearance by the liver. They found that NPs-velocity in the bloodstream is reduced 1000-fold as they enter and traverse the liver, and that cells located in the hepatic sinusoid near the vascular inlet are the main responsible of take up NPs. At 12 h post-injection 84.8% of Kupffer cells, 81.5% of B cells, 14% of endothelial cells, and a smaller percentage of T cells showed quantum dots (QDs) uptake. QDs were not detected in hepatocytes [98]. In the same study, the comparison of NPs with different diameters (10–90 nm), showed a higher accumulation of smaller NPs in the liver sinusoid due to the blood flow dynamics and organ microarchitecture rather than to the properties of the NPs. In another study, Talamini et al. found a significant influence of NP-shape in the biodistribution of gold nanoparticles (GNPs). Spherical and starlike-GNPs intravenously administered in specific pathogen-free animals resulted in the same percentage of accumulation, but a different localization in the liver; and only the star-like GNPs were accumulated in the lung [110]. Additional difficulties, to understand NPs-biodistribution, appear when the complexity of the therapeutic nanostructure is increased. Kreyling et al. have recently studied the degradation profile of polymer-coated-GNPs after their intravenous injection in rats. Their detailed in vitro and in vivo studies demonstrated the partial removal of the polymer shell in the liver and its ubiquitous observation in many locations around the body, while the gold-core of the NPs was only observed in the liver for the whole duration of the experiments (24 h) [111]. These investigations highlight the difficulties associated with the study of nanomedicines vs small drugs, and the importance of evaluating the biodistribution profile of each of the main components of the nanomedicines (i.e. polymer and pharmacological molecule) before its application in the clinic. The clearance of NPs by spleen macrophages has also been studied by Tsoi et al. Their in vivo experiments revealed that splenic macrophages take up ten times less quantum dots (QDs) per cell basis, and only 25.4% of splenic macrophages were QD-positive, compared to 84.8% of the Kupffer cells. To test the influence of macrophage phenotype on NP-uptake and clearance, they performed in vitro analysis comparing primary splenic and hepatic macrophages which confirmed the different uptake of QDs [98]. However, the liver-spleen clearance difference was significantly pronounced in vivo, thus pointing again to the bloodstream dynamics and organ microarchitecture. Of note, the liver receives 20% of cardiac output via hepatic artery and portal vein, whereas the spleen receives only 1% via splenic artery. These results suggest that both macrophage phenotype and anatomical and physiological differences are responsible for the higher clearance of NPs in the liver as compared to the spleen. The accumulation of NPs in lung macrophages has been recently studied in detail by Wibroe et al. Their investigations have shown that intravenous injection of different nanopharmaceuticals, including PEGylated and non-PEGylated NPs, induce adverse cardiopulmonary reactions, which are majorly due to the interaction of the NPs with lung macrophages, rather than the interaction with the complement system (which was the major hypothesis). No significant advantage was observed for PEGylated-NPs. However, the modification of particle geometry from a spherical shape to a rod- or disk-shape morphology or the NP-coating with erythrocytes were able to delay the particle recognition by lung macrophages within the first few minutes of injection, and this was enough to overcome the cardiopulmonary distress associated with nanomedicine administration [106]. Numerous nanotechnology strategies have been devised to avoid the 108

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nanomedicines, as a strategy to improve their penetration into solid tumors [143]. Unfortunately, this strategy led to an enhanced tumor penetration of 10 nm NPs, but the same effect was not observed for 100 nm NPs, a size that is close to the average pore size of the normalized blood vessels (125 nm) [144]. Another study comparing NPs with different sizes and tumors with different properties (i.e. microvessel density, vascular permeability, lymphatics, stromal content and tumor-associated immune cells) confirmed the specific NP accumulation trends, showing a major accumulation of larger NPs closer to the blood vessels and smaller NPs throughout the whole tumor tissue [145]. In addition, these investigations showed a higher NP-tumor accumulation in orthotopic vs flank lung cancer models, whereas NP-tumor accumulation was constant in orthotopic vs flank ovarian cancer models. These examples illustrate the variability of the so-called enhanced permeation and retention (EPR) effect, which drives the passive accumulation of NPs (reviewed elsewhere) [146,147] between tumors with different anatomical locations. An additional proof of knowledge related to the forces driving the penetration of NPs into solid tumors has been recently presented by Matsumoto et al., who have observed that, the ‘vascular bursts’ followed by stochastic ‘eruptions’ of fluid from the tumor blood vessels into the tumor interstitial space is a dynamic event which enhances the permeability of tumor blood vessels and improves the delivery of NPs [148]. To address the barrier imposed by the extracellular matrix (ECM) for the penetration of nanomedicines, but also antibodies or other drugs, several therapeutic strategies have been studied. For example, Losartan has been used to inhibit collagen-1 synthesis in the tumor resulting in a significant improved penetration of nanomedicines [149,150]. Another approach, consisting in the removal of hyaluronan (HA) by a PEGylated recombinant hyaluronidase (PEDPH20) has been used to improve the penetration of liposomal doxorubicin (Doxil®) in different tumor models [151,152]. In another study, Kohli et al. have developed liposomes with Methylumbelliferone (MU), an oral inhibitor of HA synthesis. The administration of these liposomes, followed by the intravenous injection of Doxil® in orthotopic 4T1 tumors, resulted in a higher therapeutic efficacy and extended overall survival compared to Doxil® alone, while the efficacy of free doxorubicin was not improved by the elimination of HA [153]. These results demonstrate that the elimination of ECM gains major importance for the implementation of therapies with a nanometric size. Interestingly, it has been discovered that some peptides, initially developed for the targeting of cells in the tumor, improve the ability of their coupled payload, ranging from small molecule drugs to NPs, to penetrate into solid tumors (i.e. iRGD or LyP-1) [154]. Mechanistic studies have demonstrated that these, so-called tumor-penetration peptides, activate an endocytic transport, C-end-rule pathway, related to but distinct to macropinocytosis, which mediates the extravasation transport through the extravascular tumor tissue of payloads. Remarkably, it was observed that the payload does not have to be coupled to the peptide. Further studies suggest that these peptides present a higher ability to improve the antitumoral efficacy of their payload in tumors overexpressing the receptor neuropilin-1 (NRP1) [155]. This receptor, initially identified as regulator of the nervous system, has been also shown to recognize VEFG, and recent studies have shown the key role of this receptor in the migration ability of macrophages towards the tumor hypoxic areas [156]. Furthermore, the Lyp-1 peptide has been found to present a primary accumulation in TAM, even reaching these cells in hypoxic areas of tumors [157,158], making it an attractive ligand for the targeting of TAM. Despite this evidence, further studies are required to improve our understanding of the interaction between tumor-penetrating peptides and macrophages inside stromal tumors. This knowledge could also allow for a better selection of tumors which can benefit the most from these therapies. As a whole, the major challenges for the penetration of nanomedicines or other drugs into the bulk of the tumor can be resolved by tumor vessel normalization and depletion of the excessive extracellular matrix

increased plasmatic levels of CCL2 and CCL5, lead to a higher clearance of PEGylated liposomal doxorubicin [124]. Thus, could hepato-pulmonary diseases, liver or lung abnormalities or macrophage-phenotype alterations in these organs be responsible of the difference biodistribution of nanomedicines? Could the therapeutic manipulation of macrophages in these organs, combined to the engineering of NPs with precise surface properties, allow us to give personalized nanomedicines with ability to avoid or to reach specific macrophages in a particular location? 3.2. Inefficient ability of nanomedicines to penetrate the tumor microenvironment: abnormal vasculature and extracellular matrix In the case that nanomedicines are able to efficiently reach the tumor, still additional difficulties are encountered for the efficient transport of their therapeutic load into the center of solid tumors and to reach the desired cellular target (i.e. cancer cells or tumor associated macrophages). The abnormal tumor tissular structure, highly influenced by the tumor immunosuppressive microenvironment, presents a series of challenges for the delivery of nanomedicines which can be summarized as abnormal vasculature, low oxygen concentration (hypoxia) and the important barrier of extracellular matrix (ECM) (Fig. 4). This nearly impenetrable barrier, which prevents the penetration of drugs, notably nanomedicines or antibodies, is mainly present in highly stromal tumors, such as pancreatic cancer, and it consists mainly of fibroblasts, endothelial cells and immune cells, embedded into a net of ECM formed by hyaluronan, collagen and fibronectin [125–127]. In addition, the abnormal and malformed vasculature of these tumors fail to deliver enough oxygen and nutrients to the growing tumor mass resulting in the appearance of hypoxic avascular regions. These nearly impenetrable cancer niches hinder the delivery of therapeutic agents and benefit the resistance of hypoxic tolerant tumor cells to chemotherapy, radiotherapy and photodynamic therapy which depend upon the availability of oxygen molecules to generate reactive oxygen species (ROS) and promote apoptosis of cancer cells [128]. Several therapeutic strategies have been presented to address these challenges. Since enhanced angiogenesis promotes tumor progression, several anti-angiogenic drugs, mainly vascular endothelial growth factor inhibitors (anti-VEGF treatments), have been used to reduce the blood supply and nutrients to the tumor [129,130]. In parallel, the resistance to the lower oxygen supply to the center of the tumor has been correlated with the presence of hypoxia-inducible factors (HIFs), which promote the invasion of tumor cells and the immune suppressive activity of MDSCs and TAMs [131–133], thus several HIF inhibitors have been investigated [134]. In a recent study, Frumovitz et al. have combined Topotecan (HIF inhibitor), Bevacizumab (anti-VEGF antibody) and Paclitaxel for the treatment of small cell neuroendocrine cervix carcinoma [135]. Accordingly, several nanomedicines have been developed to regulate angiogenesis and hypoxia [136–138]. Biodegradable PLGA NPs (300–350 nm) containing quantum dots, superparamagnetic FeO3 nanocrystals and doxorubicin plus VEGF shRNA on their surface have been demonstrated to reach the cytoplasm of the cancer cells via FA receptor-mediated pathway, resulting in the killing of the cancer cells and VEGF suppression. In addition, these luminescent/magnetic hybrid NPs were used as a dual-modality imaging nanoprobe for enhanced MR and tumor fluorescence imaging [139]. In a different study, lipid/calcium phosphate NPs containing gemcitabine and VEGF siRNA were applied intravenously for the treatment of subcutaneous and orthotopic xenograft models of human non-small-cell lung cancer (NSLCL). The results showed a greater antitumoral efficacy for the drug combinations when compared to the single therapies [140]. Interestingly, it has been observed that silver NPs can inhibit HIF-1, resulting in the inhibition of tumor progression [141]. Similarly, folate-decorated cationic liposomes containing HIF-1-siRNA have been used in murine melanoma models [142]. Another important study has reported on the use of anti-VEGF therapy prior to the administration of 109

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pharmacological molecules into a unique nanomedicine, may allow to treat cancer in a ‘tumor personalized manner’, with the view of enabling greater progress in the treatment of tumors and ultimately lead to improved outcomes for cancer patients.

present in highly stromal tumors (Fig. 4). Importantly, as described in the first section of this review (Section 2.1), these tumors present a hostile immunosuppressive tumor microenvironment, of which TAMs can constitute up to 50% of the cellular population, that contributes to the maintenance and growth of the abnormal tumor tissular structure. It has been recently demonstrated that TAMs instruct the deposition, cross-linking, and linearization of collagen fibers during tumor development [159]. Nevertheless, good news has been recently presented by Tian et al., who reported that the reprogramming of immunosuppressive cells through immune checkpoint blockade treatments results in the tumor vessel normalization and reduction of hypoxia [160]. Thus, we, and other [138] conceive that the progressive normalization of the tumor vasculature and hypoxia in highly stromal tumors can be theoretically achieved through the elimination of immunosuppressive TAM and/or their re-education into anti-tumoral macrophages, and this strategy has the potential to improve the penetration of nanomedicines in highly stromal tumors, thus enhancing their efficacy for the treatment of cancer.

Declaration of interest The authors declare no conflicts of interest. The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Acknowledgments This work was supported by the Worldwide Cancer Research, UK AND IG grants from the Italian Association for Cancer Research (AIRC) (P. A.). F.T.A. is recipient of a Marie Skłodowska-Curie Individual European Fellowship (H2020-MSCA-IF-2014-EF-ST) from the European Commission for the project NANOTAM, No. 658592. A.M. is recipient of a Marie Skłodowska-Curie Individual European Fellowship (H2020MSCA-IF-2015-EF-ST) from the European Commission for the project MONONANOCHEM, No. 706557.

4. Conclusions and future perspectives In most cancer patients, chronic inflammation and immune suppression are dominant effects in the tumor microenvironment which hamper the efficacy of current antitumoral treatments. New therapeutic nanostructures (TNs), allowing the combination of ‘targeting’ molecules to reach TAM with ‘pharmacological’ molecules to kill or re-educate TAM in a unique nanomedicine, have the potential to abolish these immunosuppressive functions and unleash anti-tumor immune responses in the tumor microenvironment. The inclusion of fluorescent molecules, nanoparticle contrast agents or radiolabelled compounds into TNs, allows their application to visualize macrophages, certainly leading for a better evaluation of the cancer disease and a better prediction of response to antitumoral therapies. Newer ‘Myeloid Trojan Horses’ (monocytes/macrophages-loaded with TNs), with high infiltration capabilities, are starting to be explored, and they could also offer a new plethora of therapeutic opportunities with different rational; for example, to design ‘pre-programed’ myeloid cells with intrinsic antitumor properties which could be activated when they reach the bulk of the tumor. Of note, macrophages in ‘competing organs’ (liver, lung and spleen) are the major responsible of the undesirable uptake of nanomedicines in the bloodstream. Thus, traditionally, nanomedical researchers have endeavoured sophisticated nanotechnological strategies (i.e. surface modification of NPs) to avoid the uptake by MPS-macrophages, with the principal inconvenient that most of these coating-approaches also avoid the NP-uptake by their intended target cells. An emerging strategy, designed by immunological researchers, to surpass the MPSclearance considers the therapeutic manipulation of MPS-macrophages prior to the injection of the nanomedicines, with the potential to provide an extraordinary solution for the intravenous administration of nanomedicines which have already been developed in the last years, but just failed in their antitumoral efficacy because of MPS-clearance. In a similar manner, the challenging penetration of nanomedicines and other drugs (i.e. antibodies) into stromal tumors, may be addressed through the application of ‘nanotechnological strategies’ (i.e. penetration peptides) or by the implementation of ‘immunological treatments’ intended to achieve the tumor vessel normalization, the depletion of the excessive extracellular matrix and the re-education of the immunosuppressive cells in the tumor microenvironment with TAM-targeted nanomedicines. Although further investigations are required to understand the full details of nanomedicine-macrophage interactions, and subsequent phenomenon’s reviewed here, we witness a new generation of therapeutic nanostructures (TNs), which will take advantage of the latest discoveries made by nanotechnologists and immunologists in the field of cancer. New TNs, loaded with appropriate ligand and

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