Selective targeting of tumor associated macrophages in different tumor models.
ABSTRACT: Tumor progression largely depends on the presence of alternatively polarized (M2) tumor-associated macrophages (TAMs), whereas the classical M1-polarized macrophages can promote anti-tumorigenic immune responses. Thus, selective inhibition of M2-TAMs is a desirable anti-cancer approach in highly resistant tumor entities such as hepatocellular carcinoma (HCC) or breast cancer. We here examined whether a peptide that selectively binds to and is internalized by in vitro-differentiated murine M2 macrophages as compared to M1 macrophages, termed M2pep, could be used to selectively target TAMs in HCC and breast carcinoma. We confirmed selectivity of M2pep for in vitro M2 polarized macrophages. Upon incubation of suspended mixed 4T1 tumor cells with M2pep, high amounts of the TAMs were found to be associated with M2pep, whereas in mixed tumor cell suspensions from two HCC mouse models, M2pep showed only low-degree binding to TAMs. M2pep also showed low-degree targeting of liver macrophages. This indicates that the TAMs in different tumor entities show different targeting of M2pep and that M2pep is a very promising approach to develop selective M2-TAM-targeting in tumor entities containing M2-TAMs with significant amounts of the so far elusive M2pep receptor(s).
Project description:The tumor microenvironment in the majority of cancers is known to favor polarization of tumor-associated macrophages (TAMs) to alternatively activated M2 phenotype, promoting disease progression and reducing patient survival. Effective therapy targeting this M2 macrophage population is thus a promising adjuvant to approved cancer therapies. One of the challenges in targeting M2-like TAMs is a lack of high affinity targeting ligand with good selectivity over anti-tumor M1-like TAMs. We have previously identified an M2 macrophage-targeting peptide (M2pep) that binds preferentially to murine M2 macrophages and M2-like TAMs. A fusion peptide of M2pep with pro-apoptotic peptide KLA (M2pepKLA) was further used to reduce TAM population in vivo but high concentrations and frequent dosing were required due to low binding affinity of M2pep for M2 macrophage. The goal of this study was to develop more potent TAM depletion constructs by increasing the valency of both the M2pep targeting and KLA drug domains. Divalent and tetravalent displays of M2pep ([M2pep]2-Biotin and [M2pep]4-Biotin) were synthesized and evaluated for improvement in binding avidity to the murine macrophages. High avidity and selective binding of [M2pep]2-Biotin to M2 macrophages were achieved with at least 10-fold lower concentration than required for monovalent M2pep activity. Increasing M2pep valency to four, however, resulted in a reduction in both binding activity and selectivity. Surprisingly, both divalent and tetravalent M2pep, without conjugation of any cytotoxic drug cargo, exhibited M2 macrophage-selective toxicity not observed in monovalent M2pep treatment. We next synthesized divalent M2pep with monovalent and divalent KLA ([M2pep]2-[KLA] and [M2pep]2-[KLA]2) to evaluate its enhanced potency compared to M2pepKLA. While both constructs were significantly more toxic than M2pepKLA to primary, bone marrow-derived M2 macrophage, desired selectivity was retained only with [M2pep]2-[KLA]. Finally, we evaluated all multivalent M2pep and M2pepKLA analogs using a syngeneic CT-26 tumor cell suspension. In this setting, [M2pep]4-Biotin and [M2pep]2-[KLA]2 exhibited selective toxicity to both M2-like TAMs and malignant cells but not to M1-like TAMs. Therefore, these constructs are promising anti-cancer constructs with dual-modality mechanisms: malignant cell killing and TAM-based immunomodulation.
Project description:<h4>Purpose</h4>Tumor-associated macrophages (TAMs) with immune-suppressive M2-like phenotype constitute a significant part of tumor and support its growth, thus making an attractive therapeutic target for cancer therapy. To improve the delivery of drugs that control the survival and/or functions of TAMs, we developed nanoparticulate drug carriers with high affinity for TAMs.<h4>Methods</h4>Poly(lactic-co-glycolic acid) nanoparticles were coated with M2pep, a peptide ligand selectively binding to M2-polarized macrophages, via a simple surface modification method based on tannic acid-iron complex. The interactions of M2pep-coated nanoparticles with macrophages of different phenotypes were tested in vitro and in vivo. PLX3397, an inhibitor of the colony stimulating factor-1 (CSF-1)/CSF-1 receptor (CSF-1R) pathway and macrophage survival, was delivered to B16F10 tumors via M2pep-modified PLGA nanoparticles.<h4>Results</h4>In bone marrow-derived macrophages polarized to M2 phenotype, M2pep-coated nanoparticles showed greater cellular uptake than those without M2pep. Consistently, M2pep-coated nanoparticles showed relatively high localization of CD206<sup>+</sup> macrophages in B16F10 tumors. PLX3397 encapsulated in M2pep-coated nanoparticles attenuated tumor growth better than the free drug counterpart.<h4>Conclusion</h4>These results support that M2pep-coating can help nanoparticles to interact with M2-like TAMs and facilitate the delivery of drugs that control the tumor-supportive functions of TAMs.
Project description:Tumor-associated macrophages (TAMs) display a spectrum of phenotypes ranging from pro-tumoral/anti-inflammatory "M2-like" to anti-tumoral/pro-inflammatory "M1-like" subtypes and, consequently, high intratumoral M2-to-M1 ratios are typically indicative of poor disease prognosis. Cancer immunotherapies that selectively modulate M2-like TAMs, enabling reversal of the M2-to-M1 ratio, represent a promising anti-cancer intervention but are difficult to implement due to the lack of effective targeting systems. In this study, we report the development of high avidity, M2 macrophage-selective targeted drug delivery platforms based on M2 macrophage-targeting peptides (M2pep) grafted onto poly(N-(2-hydroxypropyl) methacrylamide). Furthermore, these M2pep-grafted polymers also exhibit improved serum stability along with M2 macrophage-selective toxicity.
Project description:Most current cancer therapies focus on killing malignant cells, but these cells are often genetically unstable and can become resistant to chemotherapy. Tumor-associated macrophages (TAMs) facilitate disease progression by promoting angiogenesis and tumor cell growth, as well as by suppressing the adaptive immune response. TAMs are therefore potential targets for adjuvant anticancer therapies. However, resident macrophages are critical to host defense, and preferential ablation of TAMs remains challenging. Macrophage activation is broadly categorized as classically activated, or M1, and alternatively activated, or M2, and TAMs in the tumor microenvironment have been shown to adopt the anti-inflammatory, M2-like phenotype. To date, there are no methods for specific molecular targeting of TAMs. In this work, we report the discovery of a unique peptide sequence, M2pep, identified using a subtractive phage biopanning strategy against whole cells. The peptide preferentially binds to murine M2 cells, including TAMs, with low affinity for other leukocytes. Confocal imaging demonstrates the accumulation of M2pep in TAMs in vivo after tail vein injection. Finally, tail vein injection of an M2pep fusion peptide with a proapoptotic peptide delays mortality and selectively reduces the M2-like TAM population. This work therefore describes a molecularly targeted construct for murine TAMs and provides proof of concept of this approach as an anticancer treatment. In addition, M2pep is a useful tool for murine M2 macrophage identification and for modulating M2 macrophages in other murine models of disease involving M2 cells.
Project description:Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep(RY) outperformed M2pep in both tumor localization and selective accumulation in M2-like TAMs. In conclusion, we report cyclic M2pep(RY) as our lead M2pep analog with improved serum stability and M2 macrophage-binding activity. Its enhanced utility as an in vivo M2-like-TAM-targeting agent was demonstrated in two tumor models, and is expected to be applicable for other tumor models or in models of M2 macrophage-related diseases.
Project description:Traditional Chinese medicine (TCM) has been used as a significant cancer treatment method for many years in China. It has been demonstrated that TCM could assist in inhibiting the growth of tumors and prolonging the survival rates of cancer patients. Although the mechanism of TCM are still not clear, accumulating evidence has shown that they may be related to the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) play a significant role in TME and are polarized to two phenotypes, M1 (classically activated) and M2 (alternatively activated) TAMs. The two different phenotypes of TAMs play converse roles in the TME and M2-polarized tumor-associated macrophages (M2-TAMs) always lead to poor prognosis in cancer patients compared to M1-polarized tumor-associated macrophages (M1-TAMs). In this review, the potential correlation between TCM and TAMs (especially the M2 phenotype) in tumor progression and promising TCM strategies targeting TAMs in cancer are discussed.
Project description:<b>Background:</b> Among the many immunosuppressive cells in the tumor microenvironment, tumor-associated-macrophages (TAMs) are well known to contribute to tumor development. TAMs can be conditioned (polarized) to transition between classical M1-like macrophages, or alternatively to M2-like macrophages. Both are regulated by signaling molecules in the microenvironment. M1-like TAMs can secrete classic inflammatory cytokines that kill tumors by promoting tumor cell necrosis and immune cell infiltration into the tumor microenvironment. In contrast, M2-like TAMs exhibit powerful tumor-promoting functions, including degradation of tumor extracellular matrix, destruction of basement membrane, promotion of angiogenesis, and recruitment of immunosuppressor cells, all of which further promote tumor progression and distal metastasis. Therefore, remodeling the tumor microenvironment by reversing the TAM phenotype will be favorable for tumor therapy, especially immunotherapy. <b>Methods:</b> PLGA nanoparticles encapsulating baicalin and melanoma antigen Hgp peptide fragment 25-33 were fabricated using the ultrasonic double-emulsion technique. The nanoparticles were further loaded with CpG fragments and used conjugated M2pep and ?-pep peptides on their surfaces to produce novel nano-complexes. The capability to target M2-like TAMs and anti-tumor immunotherapy effects of nano-complexes were evaluated by flow cytometry and confocal microscopy <i>in vitro</i>. We also investigated the survival and histopathology of murine melanoma models administrated with different nanocomplexes. Improvements in the tumor microenvironment for immune attack of melanoma-bearing mice were also assessed. <b>Results:</b> The nano-complexes were effectively ingested by M2-like TAMs <i>in vitro</i> and <i>in vivo</i>, and the acidic lysosomal environment triggered the disintegration of polydopamine from the nanoparticle surface, which resulted in the release of the payloads. The released CpG played an important role in transforming the M2-like TAMs into the M1-like phenotype that further secreted inflammatory cytokines. The reversal of TAM released cytokines and gradually suppressed tumor angiogenesis, permitting the remodeling of the tumor microenvironment. Moreover, the activated TAMs also presented antigen to T cells, which further stimulated the antitumor immune response that inhibited tumor metastasis. Activated T cells released cytokines, which stimulated NK cell infiltration and directly resulted in killing tumor cells. The baicalin released by M1-like TAMs also killed tumor cells. <b>Conclusion:</b> The nano-complexes facilitated baicalin, antigen, and immunostimulant delivery to M2-like TAMs, which polarized and reversed the M2-like TAM phenotype and remodeled the tumor microenvironment to allow killing of tumor cells.
Project description:Typically, tumor-associated macrophages (TAMs), an abundant population of leukocytes in lung cancer, are affected by tumor microenvironment (TME) and shift towards either a pro-tumor (M2-like) or an anti-tumor phenotype (M1-like). M2-polarized macrophages, are one of the primary tumor-infiltrating immune cells and were reported to be associated with the promotion of cancer cell growth, invasion, metastasis, and angiogenesis. TAMs are considered a potential target for adjuvant anticancer therapies, and recent therapeutic approaches targeting the M2 polarization of TAMs have shown encouraging results. The present review discusses recent developments in the role of TAMs in cancer, in particular TAMs functions, clinical implication and prospective therapeutic strategies in lung cancer.
Project description:Tumor-associated macrophages (TAMs) polarize to M1 and M2 subtypes exerting anti-tumoral and pro-tumoral effects, respectively. To date, little is known about TAMs, their subtypes, and their roles in non-functional pituitary adenomas (NFPAs). We performed flow cytometry on single cell suspensions from 16 NFPAs, revealing that CD11b<sup>+</sup> myeloid cells comprise an average of 7.3% of cells in NFPAs (range = 0.5%-27.1%), with qPCR revealing most CD11b<sup>+</sup> cells to be monocyte-derived TAMs rather than native microglia. The most CD11b-enriched NFPAs (10-27% CD11b<sup>+</sup>) were the most expansile (size>3.5 cm or MIB1>3%). Increasing CD11b<sup>+</sup> fraction was associated with decreased M2 TAMs and increased M1 TAMs. All NFPAs with cavernous sinus invasion had M2/M1 gene expression ratios above one, while 80% of NFPAs without cavernous sinus invasion had M2/M1<1 (<i>P</i> = 0.02). Cultured M2 macrophages promoted greater invasion (<i>P</i> < 10<sup>-5</sup>) and proliferation (<i>P</i> = 0.03) of primary NFPA cultures than M1 macrophages in a manner inhibited by siRNA targeting S100A9 and EZH2, respectively. Primary NFPA cultures were of two types: some recruited more monocytes in an MCP-1-dependent manner and polarized these to M2 TAMs, while others recruited fewer monocytes and polarized them to M1 TAMS in a GM-CSF-dependent manner. These findings suggest that TAM recruitment and polarization into the pro-tumoral M2 subtype drives NFPA proliferation and invasion. Robust M2 TAM infiltrate may occur during an NFPA growth phase before self-regulating into a slower growth phase with fewer overall TAMs and M1 polarization. Analyses like these could generate immunomodulatory therapies for NFPAs.
Project description:Tumor-associated macrophages (TAMs), the most abundant infiltrating immune cells in tumor microenvironment, have distinct functions in hepatocellular carcinoma (HCC) progression. CD68? TAMs represent multiple polarized immune cells mainly containing CD86? antitumoral M1 macrophages and CD206? protumoral M2 macrophages. TAMs expression and density were assessed by immunohistochemical staining of CD68, CD86, and CD206 in tissue microarrays from 253 HCC patients. Clinicopathologic features and prognostic value of these markers were evaluated. We found that CD68? TAMs were not associated with clinicopathologic characteristics and prognosis in HCC. Low presence of CD86? TAMs and high presence of CD206? TAMs were markedly correlated with aggressive tumor phenotypes, such as multiple tumor number and advanced tumor-node-metastasis (TNM) stage; and were associated with poor overall survival (OS) (p = 0.027 and p = 0.024, respectively) and increased time to recurrence (TTR) (p = 0.037 and p = 0.031, respectively). In addition, combined analysis of CD86 and CD206 provided a better indicator for OS (p = 0.011) and TTR (p = 0.024) in HCC than individual analysis of CD86 and CD206. Moreover, CD86?/CD206? TAMs predictive model also had significant prognosis value in ?-fetoprotein (AFP)-negative patients (OS: p = 0.002, TTR: p = 0.005). Thus, these results suggest that combined analysis of immune biomarkers CD86 and CD206 could be a promising HCC prognostic biomarker.