Immunosuppressive M2 Phenotype Research Articles

Unveiling the Role of Endoplasmic Reticulum Stress Pathways in Canine Demodicosis.

Canine demodicosis is a prevalent skin disease caused by overpopulation of a commensal species of Demodex mite, yet its precise cause remains unknown. Research suggests that T-cell exhaustion, increased immunosuppressive cytokines, induction of regulatory T cells and increased expression of immune checkpoint inhibitors may contribute to its pathogenesis. This study aimed to gain a deeper understanding of the molecular changes occurring in canine demodicosis using mass spectrometry and pathway enrichment analysis. The results indicate that endoplasmic reticulum stress promotes canine demodicosis through regulation of three linked signalling pathways: eIF2, mTOR, and eIF4 and p70S6K. These pathways are involved in the modulation of Toll-like receptors, most notably TLR2, and have been shown to play a role in the pathogenesis of skin diseases in both dogs and humans. Moreover, these pathways are also implicated in the promotion of immunosuppressive M2 phenotype macrophages. Immunohistochemical analysis, utilising common markers of dendritic cells and macrophages, verified the presence of M2 macrophages in canine demodicosis. The proteomic analysis also identified immunological disease, organismal injury and abnormalities and inflammatory response as the most significant underlying diseases and disorders associated with canine demodicosis. This study demonstrates that Demodex mites, through ER stress, unfolded protein response and M2 macrophages contribute to an immunosuppressive microenvironment, thereby assisting in their proliferation.

Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype.

Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.

Expression of PD-1 mitigates phagocytic activities TAM in osteosarcoma

The high expression of programmed death 1 (PD-1) is a hallmark of T cell exhaustion, consequently inhibiting the anti-tumor immunity, tumor-associated macrophages (TAMs) aggravate Osteosarcoma (OS) progression. However, PD-1 expression on TAMs in OS metastasis remains unclear. Here, we used scRNA-Seq of 15500 individual cells from human OS lung metastatic lesion, identified thirteen major cell clusters. Our data revealed that tumor-infiltrating lymphocytes (TILs) OS lung metastatic accompanied by accumulation of exhausted T cells and regulatory T cells (Tregs). CD3+ T cells from human OS lung metastatic exhibited lower proliferation than in primary tissue. Importantly, TAMs mainly comprise immunosuppressive M2 phenotype in OS metastasis. Mechanistically, we found that PD-1 of TAMs inhibits the phagocytic potency, further promoting the progression of OS metastasis. Therefore, the study provides a strong technical support for OS immunotherapy based on PD-1 inhibitors.

NIR-Actuated Targeted Janus Nanomotors Remodel Immunosuppressive Tumor Microenvironment for Augmented Cancer Immunotherapy.

Tumor-associated macrophages (TAMs) always display immunosuppressive M2 phenotype in the tumor microenvironment to facilitate tumor growth, invasion, and metastasis. Ibrutinib (IBR), a novel irreversible Bruton's tyrosine kinase (BTK) inhibitor, has been employed to repolarize the BTK-overexpressed TAMs from M2 to M1 phenotype to remodel the immunosuppressive tumor microenvironment. However, the poor solubility of IBR extremely hinders its bioavailability, which results in low tumor accumulation and TAMs uptake in vivo. Herein, NIR laser-actuated Janus nanomotors are proposed for the effective and deep delivery of IBR to TAMs in solid tumor for targeted immunotherapy. Under NIR irradiation, the Janus nanomotors exhibit efficient photothermal conversion to produce powerful propulsion via self-thermophoresis with a speed of 12.15µm s-1 . Combined with the salic acid targeting and IBR loading, the nanomotors significantly boost their binding and uptake efficacy by M2-like macrophages during the active motion, which highly facilitate the reprogramming of M2 to M1 macrophages in vitro. Furtherly, the autonomous motion also validly improves in vivo accumulation and penetration depth in tumors to alter the M1/M2 polarization balance and activate T cells. Overall, the synthesized IC@MSA JNMs would provide a promising strategy for the efficient delivery of immunological agents toward targeted cancer immunotherapy.

M2pep-Modified Cyclodextrin-siRNA Nanoparticles Modulate the Immunosuppressive Tumor Microenvironment for Prostate Cancer Therapy.

Prostate cancer (PCa) is the most prevalent cause of cancer deaths in men. Conventional strategies, such as surgery, radiation, or chemotherapy, face challenges including poor prognosis and resistance. Therefore, the development of new improved strategies is vital to enhance patient outcomes. Recently, immunotherapy has shown potential in the treatment of a range of cancers, including PCa. Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment (TME) and reprogramming of TAMs is associated with remodeling the TME. The colony-stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) signaling pathway is closely related to the polarization of TAMs. The downregulation of CSF-1R, using small interfering RNA (siRNA), has been shown to achieve the reprogramming of TAMs, from the immunosuppressive M2 phenotype to the immunostimulatory M1 one. To maximize specific cellular delivery an M2 macrophage-targeting peptide, M2pep, was formulated with an amphiphilic cationic β-Cyclodextrin (CD) incorporating CSF-1R siRNA. The resulting nanoparticles (NPs) increased M2 macrophage targeting both in vitro and in vivo, promoting the release of M1 factors and simultaneously downregulating the levels of M2 factors through TAM reprogramming. The subsequent remodeling of the TME resulted in a reduction in tumor growth in a subcutaneous PCa mouse model mainly mediated through the recruitment of cytotoxic T cells. In summary, this M2pep-targeted CD-based delivery system demonstrated significant antitumor efficacy, thus presenting an alternative immunotherapeutic strategy for PCa treatment.

Flagella of Tumor-Targeting Bacteria Trigger Local Hemorrhage to Reprogram Tumor-Associated Macrophages for Improved Antitumor Therapy.

Tumor-associated macrophages (TAMs) exhibit an immunosuppressive M2 phenotype and lead to failure of antitumor therapy. Infiltrated erythrocytes during hemorrhage are recognized as a promising strategy for polarizing TAMs. However, novel materials that precisely induce tumor hemorrhage without affecting normal coagulation still face challenges. Here, tumor-targeting bacteria (flhDC VNP) are genetically constructed to realize precise tumor hemorrhage. FlhDC VNP colonizes the tumor and overexpresses flagella during proliferation. The flagella promote the expression of tumor necrosis factor α, which induces local tumor hemorrhage. Infiltrated erythrocytes during the hemorrhage temporarily polarize macrophages to the M1 subtype. In the presence of artesunate, this short-lived polarization is transformed into a sustained polarization because artesunate and heme form a complex that continuously produces reactive oxygen species. Therefore, the flagella of active tumor-targeting bacteria may open up new strategies for reprogramming TAMs and improving antitumor therapy.

Sambucus adnata Polysaccharide SPS-1 Inhibits Growth of Transplanted Lung Cancer via Polarizing Tumor Associated Macrophage From M2 into M1

Macrophages are the primary immune cells in the tumor microenvironment, where they exhibit the immunosuppressive M2 phenotype to support tumor development. Reshaping M2 tumor-associated macrophages (TAMs) into the antitumor M1 phenotype is a promising approach in tumor immunotherapy. We had previously demonstrated that a high-purity homogeneous Sambucus adnata wall polysaccharide SPS-1 isolated from S. adnata exerted a good immunoregulatory effect. In the present study, we explored whether SPS-1 can remodel TAMs and inhibit the development of lung cancer. In the tumor mouse model transplanted with Lewis lung cancer, SPS-1 promoted the apoptosis of tumor cells and inhibited angiogenesis in tumor tissues by remodeling M2 macrophages in the tumor microenvironment into M1 macrophages, which exerted effective antitumor effects. Therefore, SPS-1 may be used as a novel immunostimulator in tumor immunotherapy, and our study also provides a novel pathway and material for reshaping immune defense to prevent and treat tumors.

Dual role of ANGPTL8 in promoting tumor cell proliferation and immune escape during hepatocarcinogenesis

The interplay between hepatocellular carcinoma (HCC) cells and the tumor microenvironment is essential for hepatocarcinogenesis, but their contributions to HCC development are incompletely understood. We assessed the role of ANGPTL8, a protein secreted by HCC cells, in hepatocarcinogenesis and the mechanisms through which ANGPTL8 mediates crosstalk between HCC cells and tumor-associated macrophages. Immunohistochemical, Western blotting, RNA-Seq, and flow cytometry analyses of ANGPTL8 were performed. A series of in vitro and in vivo experiments were conducted to reveal the role of ANGPTL8 in the progression of HCC. ANGPTL8 expression was positively correlated with tumor malignancy in HCC, and high ANGPTL8 expression was associated with poor overall survival (OS) and disease-free survival (DFS). ANGPTL8 promoted HCC cell proliferation in vitro and in vivo, and ANGPTL8 KO inhibited the development of HCC in both DEN-induced and DEN-plus-CCL4-induced mouse HCC tumors. Mechanistically, the ANGPTL8–LILRB2/PIRB interaction promoted polarization of macrophages to the immunosuppressive M2 phenotype in macrophages and recruited immunosuppressive T cells. In hepatocytes, ANGPTL8-mediated stimulation of LILRB2/PIRB regulated the ROS/ERK pathway and upregulated autophagy, leading to the proliferation of HCC cells. Our data support the notion that ANGPTL8 has a dual role in promoting tumor cell proliferation and immune escape during hepatocarcinogenesis.

O094 Urinary-derived immune cell profiling to predict bladder cancer progression and treatment response

Abstract Introduction A quarter of newly diagnosed bladder cancers are muscle-invasive (MIBC) (45% 5-year disease free survival). 30% patients with apparent non-muscle-invasive cancer (NMIBC) have MIBC on histology following cystectomy (RARC). Tumour associated macrophages (TAMs) polarise into an immunogenic M1 phenotype or immunosuppressive M2 phenotype. This study assesses accuracy and validity of quantifying urinary macrophages for earlier recognition of treatment resistant bladder cancer. Methods Urine-derived cells and tumour and adjacent normal bladder derived cells were collected from 18 RARC patients between May-October 2022. Flow cytometry performed for immune cell profiling (MACSQuant). Results The urinary immune cell profile closely reflected the tumour immune profile. Females had higher CD206+CD163+ M2 phenotype cells in urine and in tumour (p<0.05). In adjacent normal bladder and in males we identified higher CD3+CD8+ T cell expression in tumour tissue (p<0.05) despite non-significant difference in distribution of male and female disease stage at final histology. NMIBC demonstrated an immunogenic urinary and tumour profile with more M1 macrophages (CD68+CD80+) (p<0.05) vs. MIBC. The 3 males upstaged from NMIBC to MIBC on final histology had lower representation of CD68+CD80+ M1 phenotype cells in urine and tumour (p<0.05). Conclusion The TAM immune cell profile is reflected accurately in urine sampled at RARC: a higher proportion of M2 phenotypic macrophages and lower proportion of M1 macrophages in the tumour and urine is predictive of more aggressive disease. This study highlights the potential application of urinary derived TAM analysis for disease outcome prediction and early consideration of adjuvant immune checkpoint therapy.

Abstract No. 5 ▪ FEATURED ABSTRACT Adjuvant Macrophage Repolarization to M1 Phenotype Augments Post-Ablation Local Tumor Control and Improves Overall Survival in a Murine Model of Bladder Tumors

To investigate the impact of adjuvant macrophage repolarization to inflammatory M1 phenotype on local tumor control and overall survival following irreversible electroporation (IRE) of murine bladder tumors. Mouse bladder cancer cell line (MB49) was used to induce subcutaneous tumors in C57BL/6 (n = 8/group) or NOD-SCID (n = 10/group) mice. Animals were randomized to receive (i) IRE, (ii) supramolecular nanotherapeutic for M1 repolarization (SNT), (iii) IRE+SNT or (iv) sham control. Mice assigned to IRE treatment received electric pulses (875 or 1500V/cm for C57BL/6 mice and 875V/cm for NOD-SCID mice, 90 pulses, 100 us duration) with tweezer electrodes. SNT treatment was administered by intravenous injection (40 mg/kg, 1,3- and 5-days post treatment). Mice were sacrificed at Day 1 or 6 for flow cytometry following IRE or were kept under observation in a survival study. Flow cytometry for macrophage markers (M1: CD80, M2: CD206) and T Cell markers (CD8, CD4 and FoxP3) from tumor samples was performed. Tumor samples underwent immunohistochemistry for cell death, macrophages (F4/80) or T cell markers. Local tumor volume and overall survival were evaluated and compared between cohorts and mouse strains to understand cross-interactions with adaptive immunity. Ablation of subcutaneous tumor with IRE stimulated robust macrophage infiltration that uniformly exhibited an immunosuppressive M2 phenotype at Day 3 and 5. SNT effectively repolarized macrophages at the site of ablation to M1 phenotype, with corresponding change in gene expression of NOS2 and IL-6. There was significantly higher population of CD80+ cells and reduced CD206+ cells at Day 1. Adjuvant SNT did not alter CD4+ and CD8+ cells but greatly reduced FoxP3+ population in vivo. IRE+SNT treated mice had greater reduction in local tumor growth, reduced lung metastases and high overall survival. These findings were reduced in NOD-SCID mice, indicating contribution of adaptive immunity under conditions of M1 repolarization. Repolarization of macrophages attracted to the site of ablation to a M1 phenotype promotes local tumor control, reducing the development of metastatic disease and prolonging survival in a mouse model of bladder cancer.

Moringa oleifera leaf polysaccharides exert anti-lung cancer effects upon targeting TLR4 to reverse the tumor-associated macrophage phenotype and promote T-cell infiltration.

Tumor-associated macrophages (TAMs) participate in tumorigenesis, growth, invasion as well as metastasis by facilitating an immunosuppressive tumor microenvironment. Reversing the pro-tumoral M2 phenotype of TAMs has become a hot spot in advancing cancer immunotherapy. In the current study, the content of Moringa oleifera leaf polysaccharides (MOLP) was determined and characterized, along with the anti-cancer mechanism of MOLP studied in a Lewis lung cancer (LLC) tumor-bearing mouse model and bone marrow-derived macrophages. The monosaccharide composition and gel permeation chromatography analyses show that MOLP are mainly composed of galactose, glucose, and arabinose, with approximately 17.35 kDa average molecular weight (Mw). In vivo studies demonstrate that MOLP convert TAMs from the immunosuppressive M2 phenotype to the antitumor M1 phenotype, thus inducing CXCL9 and CXCL10 expression and increasing T-cell infiltration in the tumor. Furthermore, macrophage depletion and T cell suppression demonstrated that the tumor suppressive effect of MOLP was reliant on reprogramming macrophage polarization and T cell infiltration. In vitro studies revealed that MOLP could induce the phenotypic switch from M2 macrophages to M1 by targeting TLR4. The current study highlights that MOLP are promising anticancer plant-derived polysaccharides with potential in modulating the immune microenvironment and have a bright application prospect in the immunotherapy of lung cancer.

Microglial Activation Modulated by P2X4R in Ischemia and Repercussions in Alzheimer’s Disease

There are over 80 million people currently living who have had a stroke. The ischemic injury in the brain starts a cascade of events that lead to neuronal death, inducing neurodegeneration which could lead to Alzheimer’s disease (AD). Cerebrovascular diseases have been suggested to contribute to AD neuropathological changes, including brain atrophy and accumulation of abnormal proteins such as amyloid beta (Aβ). In patients older than 60 years, the incidence of dementia a year after stroke was significantly increased. Nevertheless, the molecular links between stroke and dementia are not clearly understood but could be related to neuroinflammation. Considering that activated microglia has a central role, there are brain-resident innate immune cells and are about 10–15% of glial cells in the adult brain. Their phagocytic activity is essential for synaptic homeostasis in different areas, such as the hippocampus. These cells polarize into phenotypes or subtypes: the pro-inflammatory M1 phenotype, or the immunosuppressive M2 phenotype. Phenotype M1 is induced by classical activation, where microglia secrete a high level of pro- inflammatory factors which can cause damage to the surrounding neuronal cells. Otherwise, M2 phenotype is the major effector cell with the potential to counteract pro-inflammatory reactions and promote repair genes expression. Moreover, after the classical activation, an anti-inflammatory and a repair phase are initiated to achieve tissue homeostasis. Recently it has been described the concepts of homeostatic and reactive microglia and they had been related to major AD risk, linking to a multifunctional microglial response to Aβ plaques and pathophysiology markers related, such as intracellular increased calcium. The upregulation and increased activity of purinergic receptors activated by ADP/ATP, specially P2X4R, which has a high permeability to calcium and is mainly expressed in microglial cells, is observed in diseases related to neuroinflammation, such as neuropathic pain and stroke. Thus, P2X4R is associated with microglial activation. P2X4R activation drives microglia motility via the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. Also, these receptors are involved in inflammatory-mediated prostaglandin E2 (PGE2) production and induce a secretion and increase the expression of BDNF and TNF-α which could be a link between pathologies related to aging and neuroinflammation.

Tumor microcalcification-mediated relay drug delivery for photodynamic immunotherapy of breast cancer

Spatiotemporal targeting of tumor-associated macrophages (TAMs) and tumor cells is emerging as a promising strategy for tumor therapy. Tumor microcalcifications that specifically bind to bisphosphonates are potentially used to design efficient relay drug delivery nanosystems to achieve spatiotemporal drug modulation. Here, we developed manganese dioxide (MnO2)-embedded and LyP-1 peptide-labeled liposomal nanoparticles (NPs) for photodynamic immunotherapy of breast cancer; zoledronic acid (Zol) was encapsulated in the hydrophilic cavity of liposomes, and a hydrophobic photosensitizer (IR780) was embedded in the phospholipid bilayer of liposomes. These Lipo Zol/IR NPs generated O2 bubbles through MnO2 in response to H2O2 in the tumor microenvironment, leading to the degradation of the liposomal membrane, which triggered the release of Zol and provided O2 for photodynamic therapy. The released Zol attached to microcalcifications and was selectively phagocytosed by TAMs, leading to the induction of death or repolarization of TAMs from the immunosuppressive M2 phenotype to the immunostimulatory M1 phenotype. The remaining liposomal fragments embedded with IR780 then preferentially targeted tumor cells through LyP-1 peptide and produced abundant reactive oxygen species (ROS) under near infrared (NIR) laser irradiation, resulting in the death of tumor cells and mild immune activation. All in vitro and in vivo studies demonstrated the effective photodynamic and immunoregulatory performance of Lipo Zol/IR NPs. Statement of significanceSpatiotemporal targeting of tumor-associated macrophages (TAMs) and tumor cells remains a challenge in tumor photodynamic immunotherapy for promoting synergy and reducing side effects. Here, we developed tumor microcalcification-mediated relay drug delivery nanoliposomes for breast cancer therapy. H2O2 in the tumor microenvironment (TME) triggers the breakage of nanoliposomes, thereby causing the separation of zoledronic acid (Zol) and the photosensitizer IR780 and allowing them to perform their respective functions. Microcalcifications enable Zol to target TAMs, resulting in immunomodulation. LyP-1 guides IR780 to target tumor cells for PDT with adequate O2 supply. These nanoliposomes enable precise spatiotemporal targeting of different types of cells in the TME and promote the synergy between immunotherapy and PDT while ensuring the effectiveness of both methods.

Cyclin D1b induces changes in the macrophage phenotype resulting in promotion of tumor metastasis.

In breast cancer, tumor-associated macrophages with activated phenotypes promote tumor invasion and metastasis. The more aggressive mesenchymal-like breast cancer cells have a selective advantage, skewing macrophages toward the more immunosuppressive subtype. However, the mechanism underlying this shift is poorly understood. Cyclin D1b is a highly oncogenic variant of cyclin D1. Our previous study showed that non-metastatic epithelial-like breast cancer cells were highly metastatic in vivo when cyclin D1b was overexpressed. The present study determined whether cyclin D1b contributed to the interaction between breast cancer cells and macrophages. The results showed that cyclin D1b promoted the invasion of breast cancer cells in vitro. Specifically, through overexpression of cyclin D1b, breast cancer cells regulated the differentiation of macrophages into a more immunosuppressive M2 phenotype. Notably, tumor cells overexpressing cyclin D1b activated macrophages and induced migration of breast cancer cells. Further investigations indicated that SDF-1 mediated macrophage activation through breast cancer cells overexpressing cyclin D1b. These results revealed a previously unknown link between aggressive breast cancer cells and Tumor-associated macrophages, and highlighted the importance of cyclin D1b activity in the breast cancer microenvironment.

Tumor-derived exosomal circFARSA mediates M2 macrophage polarization via the PTEN/PI3K/AKT pathway to promote non-small cell lung cancer metastasis

BackgroundExosomes in the tumor microenvironment (TME) facilitate tumor progression by enabling inter-cellular communication. Tumor cell-derived exosomes can polarize tumor-associated macrophages (TAMs) to an immunosuppressive M2 phenotype. The aim of this study was to determine the role of exosomal circFARSA in non-small cell lung cancer (NSCLC) and elucidate the underlying mechanisms. MethodsIn situ circFARSA expression in NSCLC tissues was analyzed using qRT-PCR. The in vitro migration of NSCLC cells was evaluated using a transwell assay or through indirect co-culture with M2 macrophages, as appropriate. Immunoprecipitation (IP), western blotting, RNA binding protein immunoprecipitation (RIP), and RNA pull down assays were conducted for mechanistic studies. ResultsCircFARSA was significantly upregulated in NSCLC tissues, and the ectopic overexpression of circFARSA enhanced NSCLC cell metastasis. Furthermore, NSCLC cell-derived exosomal circFARSA polarized the macrophages to a M2 phenotype. The NSCLC cells co-cultured with macrophages transfected with circFARSA or pre-treated with exosomal circFARSA showed enhanced EMT and metastasis. Mechanistically, exosomal circFARSA induced M2 polarization via PTEN ubiquitination and degradation, which further activated the PI3K/AKT signaling pathway. In addition, eIF4A3 promoted circRNA biogenesis and cyclization by binding to its flanking sequences. ConclusionExosomal circFARSA plays a crucial role in cross-talk between macrophages and NSCLC cells through the PTEN/PI3K/AKT signaling pathway, and is a promising diagnostic/prognostic biomarker for NSCLC.

Oesophageal squamous cell carcinoma-associated IL-33 rewires macrophage polarization towards M2 via activating ornithine decarboxylase.

BackgroundThe tumour microenvironment primarily constitutes macrophages in the form of an immunosuppressive M2 phenotype, which promotes tumour growth. Thus, the development of methodologies to rewire M2‐like tumour‐associated macrophages (TAMs) into the M1 phenotype, which inhibits tumour growth, might be a critical advancement in cancer immunotherapy research.MethodsThe expressions of IL‐33 and indicators related to macrophage polarization in oesophageal squamous cell carcinoma (ESCC) tissues and peripheral blood mononuclear cell (PBMC)–derived macrophages were determined. Inhibition of ornithine decarboxylase (ODC) with small interfering RNA was used to analyse the phenotype of macrophage polarization and polyamine secretory signals. CCK‐8, wound‐healing and Transwell assays were used to detect the proliferation and migration of ECA109 cells in vitro. The tumour xenograft assay in nude mice was used to examine the role of IL‐33 in ESCC development in vivo.ResultsThis study showed the substantially elevated IL‐33 expression in ESCC tissues compared with the normal tissues. Additionally, enhanced infiltration of M2‐like macrophages into the ESCC tumour tissue was also observed. We observed a strong correlation between the IL‐33 levels and the infiltration of M2‐like macrophages in ESCC tumours locally. Mechanistically, IL‐33 induces M2‐like macrophage polarization by activating ODC, a key enzyme that catalyses the synthesis of polyamines. Inhibition of ODC suppressed M2‐like macrophage polarization. Finally, in vivo, we confirmed that IL‐33 promotes tumour progression.ConclusionsThis study revealed an oncogenic role of IL‐33 by actively inducing M2‐like macrophage differentiation; thus, contributing to the formation of an immunosuppressive ESCC tumour microenvironment. Thus, IL‐33 could act as a novel target for cancer immunotherapies.

Abstract IA26: Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549

Abstract The PI3 kinases (PI3K) belong to a family of signal-transducing enzymes that mediate key cellular functions in cancer and immunity. The PI3K-gamma (γ) isoform is highly expressed in myeloid cells/macrophages and plays an important role in M2 macrophage polarization (Kaneda, Nature 2016). PI3K-γ knockout mice also demonstrated the role for PI3K-γ in tumor growth and immune tolerance (Gunderson, Cancer Discovery 2015; Kaneda, 2016). IPI-549 is an oral, potent, and selective inhibitor of PI3K-γ that shows single-agent antitumor activity in multiple murine tumor models (Kaneda, 2016) and enhanced antitumor activity and improved survival when combined with immune-checkpoint blockade (ICB) (De Henau, Nature 2016). This antitumor activity is dependent on the presence of both immune-suppressive tumor-associated CD11b+ myeloid cells and CD8+ cytotoxic T cells. IPI-549 can reduce the suppression of T-cell activity by both murine and human myeloid-derived suppressor cells in vitro and in vivo. IPI 549 treatment of tumor-bearing mice leads to a shift in tumor-associated myeloid cells from an immunosuppressive M2 phenotype to a proinflammatory M1 phenotype and an increased frequency of circulating tumor-specific T cells and tumor-infiltrating CD8+ T cells. In addition, preclinical tumor model systems show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells (De Henau, Nature 2016). Treatment of ICB-resistant 4T1 and B16GMCSF tumor-bearing mice with IPI-549 and anti-PD1 for 14 days led to a significant antitumor activity and an increased CD8+/T-reg cell ratio, confirming that selective pharmacologic targeting PI3K-γ by IPI-549 restores sensitivity to ICB. We are conducting a Ph 1/1b study IPI-549-01 (ClinicalTrials.gov NCT02637531) evaluating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunomodulatory activity of IPI-549 to determine its recommended phase 2 dose and preliminary clinical activity, as both a monotherapy and in combination with nivolumab, in patients with advanced solid tumors. Citation Format: Jeffery L. Kutok. Reprogramming tumor-associated macrophages by targeting PI3K-gamma with IPI-549 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA26.

In Vivo siRNA Delivery to Immunosuppressive Liver Macrophages by α-Mannosyl-Functionalized Cationic Nanohydrogel Particles

Macrophages are the front soldiers of the innate immune system and are vital for immune defense, tumor surveillance, and tissue homeostasis. In chronic diseases, including cancer and liver fibrosis, macrophages can be forced into an immunosuppressive and profibrotic M2 phenotype. M2-type macrophages overexpress the mannose receptor CD206. Targeting these cells via CD206 and macrophage repolarization towards an immune stimulating and antifibrotic M1 phenotype through RNA interference represents an appealing therapeutic approach. We designed nanohydrogel particles equipped with mannose residues on the surface (ManNP) that delivered siRNA more efficiently to M2 polarized macrophages compared to their untargeted counterparts (NonNP) in vitro. The ManNP were then assessed for their in vivo targeting potential in mice with experimental liver fibrosis that is characterized by increased profibrotic (and immunosuppressive) M2-type macrophages. Double-labelled siRNA-loaded ManNP carrying two different near infrared labels for siRNA and ManNP showed good biocompatibility and robust uptake in fibrotic livers as assessed by in vivo near infrared imaging. siRNA–ManNP were highly colocalized with CD206+ M2-type macrophages on a cellular level, while untargeted NP (NonNP) showed little colocalization and were non-specifically taken up by other liver cells. ManNP did not induce hepatic inflammation or kidney dysfunction, as demonstrated by serological analysis. In conclusion, α-mannosyl-functionalized ManNP direct NP towards M2-type macrophages in diseased livers and prevent unspecific uptake in non-target cells. ManNP are promising vehicles for siRNA and other drugs for immunomodulatory treatment of liver fibrosis and liver cancer.

Significance of Immunosuppressive Cells as a Target for Immunotherapies in Melanoma and Non-Melanoma Skin Cancers.

Tumor-associated macrophages (TAMs) have been detected in most skin cancers. TAMs produce various chemokines and angiogenic factors that promote tumor development, along with other immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated neutrophils. TAMs generated from monocytes develop into functional, fully activated macrophages, and TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment. Since TAMs express PD1 to maintain the immunosuppressive M2 phenotype by PD1/PD-L1 signaling from tumor cells, and the blockade of PD1/PD-L1 signaling by anti-PD1 antibodies (Abs) activate and re-polarize TAMs into immunoreactive M1 phenotypes, TAMs represent a potential target for anti-PD1 Abs. The main population of TAMs comprises CD163+ M2 macrophages, and CD163+ TAMs release soluble (s)CD163 and several proinflammatory chemokines (CXCL5, CXCL10, CCL19, etc.) as a result of TAM activation to induce an immunosuppressive tumor microenvironment together with other immunosuppressive cells. Since direct blockade of PD1/PD-L1 signaling between tumor cells and tumor-infiltrating T cells (both effector T cells and Tregs) is mandatory for inducing an anti-immune response by anti-PD1 Abs, anti-PD1 Abs need to reach the tumor microenvironment to induce anti-immune responses in the tumor-bearing host. Taken together, TAM-related factors could offer a biomarker for anti-PD1 Ab-based immunotherapy. Understanding the crosstalk between TAMs and immunosuppressive cells is important for optimizing PD1 Ab-based immunotherapy.

Targeted delivery of chlorogenic acid by mannosylated liposomes to effectively promote the polarization of TAMs for the treatment of glioblastoma

Tumor-associated macrophages (TAMs) generally display an immunosuppressive M2 phenotype and promote tumor progression and metastasis, suggesting their potential value as a target in cancer immunotherapy. Chlorogenic acid (CHA) has been identified as a potent immunomodulator that promotes the polarization of TAMs from an M2 to an M1 phenotype. However, rapid clearance in vivo and low tumor accumulation have compromised the immunotherapeutic efficacy of CHA in clinical trials. In this study, mannosylated liposomes are developed for targeted delivery of CHA to TAMs. The immunoregulatory effects of CHA, along with the overall antitumor efficacy of CHA-encapsulated mannosylated liposomes, are investigated through in vitro and in vivo experiments. The prepared CHA-encapsulated mannosylated liposomes exhibit an ideal particle size, favorable stability, and preferential accumulation in tumors via the mannose receptor-mediated TAMs-targeting effects. Further, CHA-encapsulated mannosylated liposomes inhibit G422 glioma tumor growth by efficiently promoting the polarization of the pro-tumorigenic M2 phenotype to the anti-tumorigenic M1 phenotype. Overall, these findings indicate that CHA-encapsulated mannosylated liposomes have great potential to enhance the immunotherapeutic efficacy of CHA by inducing a shift from the M2 to the M1 phenotype.