M2-like Tumor-associated Macrophages Research Articles

Size-switchable and dual-targeting nanomedicine for cancer chemoimmunotherapy by potentiating deep tumor penetration and antitumor immunity

The effectiveness of commonly adopted therapeutic regimen is low for treating triple-negative breast cancer (TNBC), especially for inoperable patients at advanced stage. Herein, we reported a nano-based recipe for orthotopic and metastatic TNBC management by potentiating the deep penetration of antitumor agents in solid tumors and remodeling the tumor immune microenvironment through combinational chemoimmunotherapy. Specifically, doxorubicin (DOX) was loaded in DSPE-PEG2000-modified poly(amidoamine) (PAMAM) dendrimer to obtain small-sized DOX@P nanoparticles, which were further inserted into the phospholipid monolayer of imiquimod (R837)-encapsulated reconstituted high-density lipoprotein (rHDL) to acquire R@rHDP. Finally, M2 macrophage-targeting peptide (M2pep) was modified on the surface of R@rHDP to obtain R@rHDP-M2pep nanoparticles with special targeting ability towards M2-like tumor-associated macrophages (TAMs). Upon intravenous injection, R@rHDP-M2pep actively anchored TNBC sites owing to the natural tumor targeting potential of rHDL. Once reaching tumor sites, small-sized DOX@P was released from large-sized R@rHDP-M2pep due to the breakdown of imine bond in acidic tumor microenvironment (TME), further carrying DOX into the deep tumor core to exert chemotherapeutic effect. On the other hand, the remaining R@rH-M2pep continued to target M2-like TAMs to deliver R837 for effective polarization of TAM phenotype from protumoral M2-like into antitumoral M1-like type. Consequently, the immunosuppressive TME was remodeled to facilitate improved immunotherapeutic performance. Hence, the designed R@rHDP-M2pep nanoparticles displayed potent antitumor efficacy against TNBC, which not only remarkably suppressed orthotopic tumor growth, but also prevented the lethal lung metastasis of TNBC, largely prolonging the survival time of mice suffered from TNBC. Altogether, the proposed strategy exhibited promising research value in advancing the therapeutic protocols for TNBC.

Correlation of tumor-associated macrophage density and proportion of M2 subtypes with the pathological stage of colorectal cancer.

Colorectal cancer (CRC) is a prevalent global malignancy with complex prognostic factors. Tumor-associated macrophages (TAMs) have shown paradoxical associations with CRC survival, particularly concerning the M2 subset. We aimed to establish a simplified protocol for quantifying M2-like TAMs and explore their correlation with clinicopathological factors. A cross-sectional study included histopathological assessment of paraffin-embedded tissue blocks obtained from 43 CRC patients. Using CD68 and CD163 immunohistochemistry, we quantified TAMs in tumor stroma and front, focusing on M2 proportion. Demographic, histopathological, and clinical parameters were collected. TAM density was significantly higher at the tumor front, with the M2 proportion three times greater in both zones. The tumor front had a higher M2 proportion, which correlated significantly with advanced tumor stage (P = 0.04), pathological nodal involvement (P = 0.04), and lymphovascular invasion (LVI, P = 0.01). However, no significant association was found between the M2 proportion in the tumor stroma and clinicopathological factors. Our study introduces a simplified protocol for quantifying M2-like TAMs in CRC tissue samples. We demonstrated a significant correlation between an increased M2 proportion at the tumor front and advanced tumor stage, nodal involvement, and LVI. This suggests that M2-like TAMs might serve as potential indicators of disease progression in CRC, warranting further investigation and potential clinical application.

Codonopsis pilosula-derived glycopeptide dCP1 promotes the polarization of tumor-associated macrophage from M2-like to M1 phenotype

The majority of the immune cell population in the tumor microenvironment (TME) consists of tumor-associated macrophages (TAM), which are the main players in coordinating tumor-associated inflammation. TAM has a high plasticity and is divided into two main phenotypes, pro-inflammatory M1 type and anti-inflammatory M2 type, with tumor-suppressive and tumor-promoting functions, respectively. Considering the beneficial effects of M1 macrophages for anti-tumor and the high plasticity of macrophages, the conversion of M2 TAM to M1 TAM is feasible and positive for tumor treatment. This study sought to evaluate whether the glycopeptide derived from simulated digested Codonopsis pilosula extracts could regulate the polarization of M2-like TAM toward the M1 phenotype and the potential regulatory mechanisms. The results showed that after glycopeptide dCP1 treatment, the mRNA relative expression levels of some M2 phenotype marker genes in M2-like TAM in simulated TME were reduced, and the relative expression levels of M1 phenotype marker genes and inflammatory factor genes were increased. Analysis of RNA-Seq of M2-like TAM after glycopeptide dCP1 intervention showed that the gene sets such as glycolysis, which is associated with macrophage polarization in the M1 phenotype, were significantly up-regulated, whereas those of gene sets such as IL-6-JAK-STAT3 pathway, which is associated with polarization in the M2 phenotype, were significantly down-regulated. Moreover, PCA analysis and Pearson's correlation also indicated that M2-like TAM polarized toward the M1 phenotype at the transcriptional level after treatment with the glycopeptide dCP1. Lipid metabolomics was used to further explore the efficacy of the glycopeptide dCP1 in regulating the polarization of M2-like TAM to the M1 phenotype. It was found that the lipid metabolite profiles in dCP1-treated M2-like TAM showed M1 phenotype macrophage lipid metabolism profiles compared with blank M2-like TAM. Analysis of the key differential lipid metabolites revealed that the interconversion between phosphatidylcholine (PC) and diacylglycerol (DG) metabolites may be the central reaction of the glycopeptide dCP1 in regulating the conversion of M2-like TAM to the M1 phenotype. The above results suggest that the glycopeptide dCP1 has the efficacy to regulate the polarization of M2-like TAM to M1 phenotype in simulated TME.

Reprogramming Tumor-Associated Macrophage Using Nanocarriers: New Perspectives to Halt Cancer Progression.

Cancer remains a significant challenge for public healthcare systems worldwide. Within the realm of cancer treatment, considerable attention is focused on understanding the tumor microenvironment (TME)-the complex network of non-cancerous elements surrounding the tumor. Among the cells in TME, tumor-associated macrophages (TAMs) play a central role, traditionally categorized as pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. Within the TME, M2-like TAMs can create a protective environment conducive to tumor growth and progression. These TAMs secrete a range of factors and molecules that facilitate tumor angiogenesis, increased vascular permeability, chemoresistance, and metastasis. In response to this challenge, efforts are underway to develop adjuvant therapy options aimed at reprogramming TAMs from the M2 to the anti-tumor M1 phenotype. Such reprogramming holds promise for suppressing tumor growth, alleviating chemoresistance, and impeding metastasis. Nanotechnology has enabled the development of nanoformulations that may soon offer healthcare providers the tools to achieve targeted drug delivery, controlled drug release within the TME for TAM reprogramming and reduce drug-related adverse events. In this review, we have synthesized the latest data on TAM polarization in response to TME factors, highlighted the pathological effects of TAMs, and provided insights into existing nanotechnologies aimed at TAM reprogramming and depletion.

Reprogramming tumor-associated macrophages and inhibiting tumor neovascularization by targeting MANF-HSF1-HSP70-1 pathway: An effective treatment for hepatocellular carcinoma

In advanced hepatocellular carcinoma (HCC) tissues, M2-like tumor-associated macrophages (TAMs) are in the majority and promotes HCC progression. Contrary to the pro-tumor effect of M2-like TAMs, M1-like TAMs account for a small proportion and have anti-tumor effects. Since TAMs can switch from one type to another, reprogramming TAMs may be an important treatment for HCC therapy. However, the mechanisms of phenotypic switch and reprogramming TAMs are still obscure. In this study, we analyzed differential genes in normal macrophages and TAMs, and found that loss of MANF in TAMs accompanied by high levels of downstream genes negatively regulated by MANF. MANF reprogrammed TAMs into M1 phenotype. Meanwhile, loss of MANF promoted HCC progression in HCC patients and mice HCC model, especially tumor neovascularization. Additionally, macrophages with MANF supplement suppressed HCC progression in mice, suggesting MANF supplement in macrophage was an effective treatment for HCC. Mechanistically, MANF enhanced the HSF1-HSP70-1 interaction, restricted HSF1 in the cytoplasm of macrophages, and decreased both mRNA and protein levels of HSP70-1, which in turn led to reprogramming TAMs, and suppressing neovascularization of HCC. Our study contributes to the exploration the mechanism of TAMs reprogramming, which may provide insights for future therapeutic exploitation of HCC neovascularization.

Comprehensive Molecular Analyses of an M2-Like Tumor-Associated Macrophage for Predicting the Prognosis and Immunotherapy in Breast Cancer.

The involvement of M2-like tumor-associated macrophages (TAMs) in the advancement and treatment of cancer has been widely documented. This study aimed to develop a new signature associated with M2-like TAMs to predict the prognosis and treatment response in individuals diagnosed with breast cancer (BC). Weighted gene co-expression network analysis (WGCNA) was used to identity for M2-like TAM-related modular genes. The M2-like TAM-related modular subtype was identified using unsupervised clustering. WGCNA identified 722 M2-like TAM genes, 204 of which were associated with recurrence-free survival (RFS). Patients in cluster 1 exhibited upregulated cancer-related pathways, a higher proportion of triple-negative breast cancer (TNBC) subtypes, lower expression of immune checkpoints, and worse prognosis. Cluster 2 was characterized by upregulated immune-related pathways, a higher proportion of luminal A subtypes, and higher expression of immune checkpoints. A prognostic signature was created and confirmed using an independent dataset. A well-built nomogram can accurately forecast the survival outcomes for every individual. Furthermore, patients classified as low-risk exhibited a more favorable outlook, elevated tumor microenvironment (TME) score, and superior reaction to immunotherapy. In conclusion, we discovered 2 different types of M2-like TAMs and developed a prognostic signature revealing the diversity of M2-like TAMs in BC and their correlation with immune status and prognosis. This feature can predict the prognosis and immunotherapeutic effects of BC and offer novel concepts and approaches for tailoring BC treatment.

Optical controlled and nuclear targeted CECR2 competitor to downregulate CSF-1 for metastatic breast cancer immunotherapy

The crosstalk between breast cancer cells and tumor associated macrophages (TAMs) greatly contributes to tumor progression and immunosuppression. In this work, cat eye syndrome chromosome region candidate 2 (CECR2) is identified to overexpress in breast cancer patients, which can recognize v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) and activate nuclear factor κB (NF-κB) to release colony stimulating factor-1 (CSF-1). Pharmacological inhibition of CECR2 by the bromodomain competitor (Bromosporine, Bro) can downregulate CSF-1 to inhibit M2 type TAMs. To amplify the immunotherapeutic effect, a chimeric peptide-based and optical controlled CECR2 competitor (designated as N-PB) is constructed to enhance the nuclear targeted delivery of Bro and initiate an immunogenic cell death (ICD). In vivo results indicate a favorable breast cancer targeting ability and primary tumor suppression effect of N-PB under optical irradiation. Importantly, N-PB downregulates CSF-1 by competitive inhibition of CECR2 and NF-κB(RelA) interactions, thus inhibiting immunosuppressive M2-like TAMs while improving the antitumorigenic M1-like phenotype. Ultimately, the systemic anti-tumor immunity is activated to suppress the metastatic breast cancer in an optical controlled manner. This study provides a promising therapeutic target and reliable strategy for metastatic breast cancer treatment by interrupting immunosuppressive crosstalk between tumor cells and macrophages.

IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME). The symbiotic interactions between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAM) in the TME are critical for tumor progression. Here, we identified that IFI35, a transcriptional regulatory factor, plays both cell-intrinsic and cell-extrinsic roles in maintaining GSCs and the immunosuppressive TME. IFI35 induced non-canonical NF-kB signaling through proteasomal processing of p105 to the DNA-binding transcription factor p50, which heterodimerizes with RELB (RELB/p50), and activated cell chemotaxis in a cell-autonomous manner. Further, IFI35 induced recruitment and maintenance of M2-like TAMs in TME in a paracrine manner. Targeting IFI35 effectively suppressed in vivo tumor growth and prolonged survival of orthotopic xenograft-bearing mice. Collectively, these findings reveal the tumor-promoting functions of IFI35 and suggest that targeting IFI35 or its downstream effectors may provide effective approaches to improve GBM treatment.

Discovery of a Novel CSF-1R Inhibitor with Highly Improved Pharmacokinetic Profiles and Superior Efficacy in Colorectal Cancer Immunotherapy.

Blocking CSF-1/CSF-1R pathway has emerged as a promising strategy to remodel tumor immune microenvironment (TME) by reprogramming tumor-associated macrophages (TAMs). In this work, a novel CSF-1R inhibitor C19 with a highly improved pharmacokinetic profile and in vivo anticolorectal cancer (CRC) efficiency was successfully discovered. C19 could effectively reprogram M2-like TAMs to M1 phenotype and reshape the TME by inducing the recruitment of CD8+ T cells into tumors and reducing the infiltration of immunosuppressive Tregs/MDSCs. Deeper mechanistic studies revealed that C19 facilitated the infiltration of CD8+ T cells by enhancing the secretion of chemokine CXCL9, thus significantly potentiating the anti-CRC efficiency of PD-1 blockade. More importantly, C19 combined with PD-1 mAb could induce durable antitumor immune memory, effectively overcoming the recurrence of CRC. Taken together, our findings suggest that C19 is a promising therapeutic option for sensitizing CRC to anti-PD-1 therapy.

Prenatal inflammation exposure accelerates lung cancer tumorigenesis in offspring mouse: possible links to IRE1α/XBP1-mediated M2-like polarization of TAMs and PD-L1 up-expression

BackgroundPrenatal inflammation exposure (PIE) can increase the disease susceptibility in offspring such as lung cancer. Our purpose was to investigate the mechanisms of PIE on lung cancer.MethodsPrenatal BALB/c mice were exposed to lipopolysaccharide (LPS), and then, their offspring were intraperitoneally instilled with urethane to establish the two-stage lung cancer carcinogenesis model. At the 48 weeks of age, the offspring mice were killed and lung tissues were collected for HE, immunohistochemistry, immunofluorescence, and Luminex MAGPIX®-based assays. CD11b + F4/80 + tumor-associated macrophages (TAMs) were sorted out from lung tumor tissues by cell sorting technique. Flow cytometry was employed to evaluate the extent of M2-like polarization of TAMs and PD-L1 expression.ResultsThe offspring of PIE mice revealed more lung lesion changes, including atypical hyperplasia and intrapulmonary metastases. The number of lung nodules, lung organ index, and PCNA, MMP-9 and Vimentin positive cells in lung tissue of PIE group were higher than those of Control group. The increases of mRNA encoding M2 macrophage markers and cytokines in offspring of prenatal LPS-treated mice confirmed the induced effect of PIE on macrophage polarization. Additionally, PIE treatment increased the percentage of CD163 + CD206 + cells in the sorted TAMs. Importantly, endoplasmic reticulum (ER) stress-markers like GRP78/BIP and CHOP, p-IRE1α and XBP1s, and PD-L1 were up-regulated in TAMs from PIE group. Besides, we also observed that IRE1α inhibitor (KIRA6) reversed the M2-like TAMs polarization and metastasis induced by PIE.ConclusionsIRE1α/XBP1-mediated M2-like TAMs polarization releases the pro-tumorigenic cytokines and PD-L1 expression, which may be the regulatory mechanism of accelerating lung cancer in offspring of mice undergoing PIE.

Surgical tumor-derived nanoplatform targets tumor-associated macrophage for personalized postsurgical cancer immunotherapy.

Directly activating CD8+ T cells within the tumor through antigen-presenting cells (APCs) hold promise for tumor elimination. However, M2-like tumor-associated macrophages (TAMs), the most abundant APCs in tumors, hinder CD8+ T cell activation due to inefficient antigen cross-presentation. Here, we demonstrated a personalized nanotherapeutic platform using surgical tumor-derived galactose ligand-modified cancer cell membrane (CM)-coated cysteine protease inhibitor (E64)-loaded mesoporous silica nanoparticles for postsurgical cancer immunotherapy. The platform targeted M2-like TAMs and released E64 within lysosomes, which reshaped antigen cross-presentation and directly activated CD8+ T cells, thus suppressing B16-OVA melanoma growth. Furthermore, this platform, in combination with anti-PD-L1 antibodies, enhanced the therapeutic efficacy and substantially inhibited 4T1 tumor growth. CMs obtained from surgically resected tumors were used to construct a personalized nanotherapeutic platform, which, in synergy with immune checkpoint blockade (ICB), effectively inhibited postsurgical tumor recurrence in 4T1 tumor. Our work offered a robust, safe strategy for cancer immunotherapy and prevention of postsurgical tumor recurrence.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Abstract 6818: Obesity-mediated perineural invasion promotes PDAC tumorigenesis

Abstract Obesity is a modifiable host factor that has been reported to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by ~60% and the mortality by two-fold, however, the exact mechanisms by which obesity enhances PDAC aggressiveness is incompletely understood. Given that nearly 74% of U.S. adults are overweight or obese (US Center for Disease Control), resolving the complex association between obesity and PDAC may unveil new targets for prevention and/or intervention to decrease incidence and improve outcomes. The prevailing notion is that obesity-induced tumor in situ factors (i.e., oxidative, hypoxic, nutrient, and biochemical) play crucial roles in enhancing cancer cell fitness and survival. However, these concepts have yet to lead to the identification of any actionable target in patients, with obesity-related cancer in general and PDAC in particular. Prior studies and our preliminary data show the presence of a distinctive tumor immune profile in PDAC models under conditions of obesity, which encompasses immune suppressive myeloid and type 2 immune cells, namely M2-like tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), that inhibit effector CD8+ T cell expansion and function enabling PDAC progression. However, the genesis of this ‘type 2 immune cell’ dominant tumor-immune microenvironment (TIME) of obesity-driven PDAC remains to be fully understood. This led us to take a holistic approach utilizing multiple genetically engineered mouse models (GEMMs), pharmacological inhibitors, transcriptomic analysis (RNA-seq, spatial transcriptomic, and scRNA-seq), fresh PDAC patient samples, and imaging technologies (confocal microscopy) to understand the molecular mechanisms. Our novel mechanistic study using some of the above-mentioned tools shows that obesity-driven PDAC has significantly higher perineural invasion (PNI) that enhances PDAC progression. We found that sympathetic nerve terminals (of the PNI) via neurotransmitter release control the expression of integral cytokines that cause a dramatic remodeling of the TIME. Specifically, the PNI-mediated neuro-adrenergic signaling activates β2-adrenergic receptor (β2-AR) on PDAC cells, leading to increased tumor cell production/proliferation and secretion of the ‘alarmins’ IL-33 and IL-1β. Importantly, we showed that mitigation of adrenergic signaling using two separate approaches: (i) pharmacological antagonists of β2-ARs (β-blockers), and (ii) the use of a thermoneutral model leads to substantial abrogation of obesity-mediated tumor progression. These, and other key data raise the provocative question that reducing adrenergic signaling-dependent IL-33 expression and release favor a pro-inflammatory TIME and significantly increase patient survival in obesity-driven PDAC. Citation Format: Shyamananda Singh Mayengbam, Aftab Alam, Sharon Senchanthisai, Irmak Bektas, Prasenjit Dey. Obesity-mediated perineural invasion promotes PDAC tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6818.

Abstract 162: Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer

Abstract Background: Immune checkpoint blockade (ICB), which unleashes potent CD8+ T cell-based anti-tumor responses, has emerged as a promising therapeutic option for several cancer types. However, response rates stay limited in gastric cancer (GC) patients, indicating the necessity of combinational strategies to overcome other immunosuppressive factors. Fatty acid synthesis (FAS) is an indispensable metabolic pathway proven to support cancer cell survival. The rate-limiting enzyme of FAS, FASN, is upregulated in several cancers and could contribute to cancer immune escape. Though some investigations propose that specific gene alterations can cause FASN upregulation under the GC setting, the immunoregulatory role and therapeutic value of FASN in the tumor microenvironment (TME) of GC have not been fully elucidated. Methods: FASN expression was measured by Western Blotting and flow cytometry. Bioinformatic analysis was conducted via an online website (www.camoip.net). The syngeneic GC mouse model was established by subcutaneous inoculation of MFC cells in 615-line mice, treated by vehicles, FASN inhibitor (FASNi), anti-PD-1 antibodies, or FASNi+anti-PD-1 antibodies. TAM and CD8+ T cell depletion was implemented by anti-CSF1R and anti-CD8α antibodies. Bone marrow-derived macrophages (BMDMs) were extracted from the femurs of 615-line mice, induced by M-CSF and stimulated by LPS or IL-4. The proportions and functional status of immune cells were identified by flow cytometry. Results: FASN was upregulated in GC tissues and GC human cell lines. Bioinformatic analyses demonstrated that FASN expression was related to poor infiltration of cytotoxic lymphocytes, as well as the downregulation of pathways related to anti-cancer immune responses. FASNi inhibited tumor growth in a syngeneic GC mouse model, with elevated PD-1 and IFN-γ expression in CD8+ T cells and increased NK cell infiltration, suggesting the enhancement in anti-cancer cytotoxicity. Meanwhile, FASNi induced a shift from M2-like to M1-like in tumor-associated macrophages (TAM). We detected that M2-like TAM and CD8+ T cells harbored higher FASN expression than other immune cells in GC mouse tissues, and tumor-suppressing effects of FASN inhibition could be reversed by TAM and CD8+ T cell depletion, which corroborated the key role of TAM and CD8+ T cells in shaping FASN-mediated tumor regulation. FASNi could also repolarize BMDMs from M1-like to M2-like in vitro. Finally, FASNi exhibited synergistic tumor control with anti-PD-1 therapy in GC and melanoma mouse models. Conclusion: This work sheds light on the impact of FASN on TME modulation of GC. We proved that FASNi could repolarize M2-like TAM to M1 phenotypes and promote cytotoxic lymphocyte-mediated anti-tumor immunity. Our results propose FASN inhibition as a potential therapeutic strategy to sensitize ICB therapy in GC. Citation Format: Hanbing Wang, Tao Shi, Yue Wang, Yuting Luo, Yunfeng Pan, Xiaoyu Zhou, Yue Zhang, Baorui Liu, Jia Wei. Fatty acid synthesis blockade synergizes with immune checkpoint blockade by repolarizing macrophages and enhancing anti-tumor cytotoxicity in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 162.

Abstract 5247: Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma

Abstract The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5247.

Hypoxia-induced TREM1 promotes mesenchymal-like states of glioma stem cells via alternatively activating tumor-associated macrophages

The mesenchymal subtype of glioblastoma (GBM) cells characterized by aggressive invasion and therapeutic resistance is thought to be dependent on cell-intrinsic alteration and extrinsic cellular crosstalk. Tumor-associated macrophages (TAMs) are pivotal in tumor progression, chemo-resistance, angiogenesis, and stemness maintenance. However, the impact of TAMs on the shifts in glioma stem cells (GSCs) states remains largely uncovered. Herein, we showed that the triggering receptor expressed on myeloid cells-1 (TREM1) preferentially expressed by M2-like TAMs and induced GSCs into mesenchymal-like states by modulating the secretion of TGFβ2, which activated the TGFβR/SMAD2/3 signaling in GSCs. Furthermore, we demonstrated that TREM1 was transcriptionally regulated by HIF1a under the hypoxic environment and thus promoted an immunosuppressive type of TAMs via activating the TLR2/AKT/mTOR/c-MYC axis. Collectively, this study reveals that cellular communication between TAMs and GSCs through the TREM1-mediated TGFβ2/TGFβR axis is involved in the mesenchymal-like transitions of GSCs. Our study provides valuable insights into the regulatory mechanisms between the tumor immune microenvironment and the malignant characteristics of GBM, which can lead to potential novel strategies targeting TAMs for tumor control.

Mutational, immune microenvironment, and clinicopathological profiles of diffuse large B-cell lymphoma and follicular lymphoma with BCL6 rearrangement.

BCL6-rearrangement (BCL6-R) is associated with a favorable prognosis of follicular lymphoma (FL), but the mechanism is unknown. We analyzed the clinicopathological, immune microenvironment (immune checkpoint, immuno-oncology markers), and mutational profiles of 10 BCL6-R-positive FL, and 19 BCL6-R-positive diffuse large B-cell lymphoma (DLBCL) cases (both BCL2-R and MYC-R negative). A custom-made panel included 168 genes related to aggressive B-cell lymphomas and FL. FL cases were nodal, histological grade 3A in 70%, low Ki67; and had a favorable overall and progression-free survival. DLBCL cases were extranodal in 60%, IPI high in 63%, non-GCB in 60%, EBER-negative; and had a progression-free survival comparable to that of DLBCL NOS. The microenvironment had variable infiltration of M2-like tumor-associated macrophages (TAMs) that were CD163, CSF1R, LAIR1, PD-L1, and CD85A (LILRB3) positive; but had low IL10 and PTX3 expression. In comparison to FL, DLBCL had higher TAMs, IL10, and PTX3 expression. Both lymphoma subtypes shared a common mutational profile with mutations in relevant pathogenic genes such as KMT2D, OSBPL10, CREBBP, and HLA-B (related to chromatin remodeling, metabolism, epigenetic modification, and antigen presentation). FL cases were characterized by a higher frequency of mutations of ARID1B, ATM, CD36, RHOA, PLOD2, and PRPRD (p < 0.05). DLBCL cases were characterized by mutations of BTG2, and PIM1; and mutations of HIST1H1E and MFHAS1 to disease progression (p < 0.05). Interestingly, mutations of genes usually associated with poor prognosis, such as NOTCH1/2 and CDKN2A, were infrequent in both lymphoma subtypes. Some high-confidence variant calls were likely oncogenic, loss-of-function. MYD88 L265P gain-of-function was found in 32% of DLBCL. In conclusion, both BCL6-R-positive FL and BCL6-R-positive DLBCL had a common mutational profile; but also, differences. DLBCL cases had a higher density of microenvironment markers.

Hybrid Cellular Nanovesicles Block PD-L1 Signal and Repolarize M2 Macrophages for Cancer Immunotherapy.

The PD1/PD-L1 immune checkpoint blocking is a promising therapy, while immunosuppressive tumor microenvironment (TME) and poor tumor penetration of therapeutic antibodies limit its efficacy. Repolarization of tumor-associated macrophages (TAMs) offers a potential method to ameliorate immunosuppression of TME and further boost T cell antitumor immunity. Herein, hybrid cell membrane biomimetic nanovesicles (hNVs) are developed by fusing M1 macrophage-derived nanovesicles (M1-NVs) and PD1-overexpressed tumor cell-derived nanovesicles (PD1-NVs) to improve cancer immunotherapy. The M1-NVs promote the transformation of M2-like TAMs to M1-like phenotype and further increase the release of pro-inflammatory cytokines, resulting in improved immunosuppressive TME. Concurrently, the PD1-NVs block PD1/PD-L1 pathway, which boosts cancer immunotherapy when combined with M1-NVs. In a breast cancer mouse model, the hNVs efficiently accumulate at the tumor site after intravenous injection and significantly inhibit the tumor growth. Mechanically, the M1 macrophages and CD8+ T lymphocytes in TME increase by twofold after the treatment, indicating effective immune activation. These results suggest the hNVs as a promising strategy to integrate TME improvement with PD1/PD-L1 blockade for cancer immunotherapy.

Abstract PR-004: Targeting EPHB2/ABL1 restores anti-tumor immunity in a preclinical model of ependymoma

Abstract Purpose: Ependymoma (EPN) is a common type of brain tumor in children and is often resistant to available cytotoxic therapies. Molecular profiling studies have led to a better understanding of unique EPN subtypes and revealed a critical role of EPHB2. However, the immune system's role in tumor progression and treatment response remains poorly understood. New treatments for EPN are desperately needed and should be developed in a molecular subtype-specific fashion. Experimental Design: We developed a syngeneic mouse model using subtype-specific EPHB2-driven genetically engineered EPN tumor cells. Druggable targets were identified by matching transcriptomic signatures with the target spectrum of FDA-approved drugs. After identifying dasatinib as a potentially effective agent, we measured the changes in the immune microenvironment during EPN growth and after dasatinib treatment. Results: Transcriptomic profiling of EPHB2-driven EPN and analysis of a human EPN dataset revealed multiple protein kinases as potential druggable targets. We found that the multikinase inhibitor dasatinib potently inhibited the growth of EPN both in vitro and in vivo, mainly through blocking EPHB2 and ABL1 signaling. We found an increased frequency of immunosuppressive M2-like tumor-associated macrophages (TAMs), which proportionally increased with tumor size during tumor progression. However, treatment with dasatinib reprogrammed the EPN immune microenvironment by polarizing the TAMs toward an anti-tumor M1-like phenotype and increasing CD8 T cell activation. Furthermore, dasatinib treatment induced complete regression of established EPN tumors in 78% of the animals and protected survivors against tumor recurrence. Depletion of CD8 T cells compromised the durability of EPN responses and reduced overall survival. Conclusions: These data indicate that dasatinib may be an effective therapy for EPHB2-driven molecular subgroup of EPNs by activating the anti-tumor immune response and support further investigations of dasatinib in clinical trials. Citation Format: Taylor P. Uccello, Jun Ren, Zohreh Amoozgar, Yuhui Zhao, Pin-Ji Lei, William W. Ho, Sylvie Roberge, Peigen Huang, Dan G. Duda, Lei Xu, Rakesh K. Jain. Targeting EPHB2/ABL1 restores anti-tumor immunity in a preclinical model of ependymoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-004.

Branched glycopolymer prodrug-derived nanoassembly combined with a STING agonist activates an immuno-supportive status to boost anti-PD-L1 antibody therapy

Despite the great potential of anti-PD-L1 antibodies for immunotherapy, their low response rate due to an immunosuppressive tumor microenvironment has hampered their application. To address this issue, we constructed a cell membrane-coated nanosystem (mB4S) to reverse an immunosuppressive microenvironment to an immuno-supportive one for strengthening the anti-tumor effect. In this system, Epirubicin (EPI) as an immunogenic cell death (ICD) inducer was coupled to a branched glycopolymer via hydrazone bonds and diABZI as a stimulator of interferon genes (STING) agonist was encapsulated into mB4S. After internalization of mB4S, EPI was acidic-responsively released to induce ICD, which was characterized by an increased level of calreticulin (CRT) exposure and enhanced ATP secretion. Meanwhile, diABZI effectively activated the STING pathway. Treatment with mB4S in combination with an anti-PD-L1 antibody elicited potent immune responses by increasing the ratio of matured dendritic cells (DCs) and CD8+ T cells, promoting cytokines secretion, up-regulating M1-like tumor-associated macrophages (TAMs) and down-regulating immunosuppressive myeloid-derived suppressor cells (MDSCs). Therefore, this nanosystem for co-delivery of an ICD inducer and a STING agonist achieved promotion of DCs maturation and CD8+ T cells infiltration, creating an immuno-supportive microenvironment, thus potentiating the therapy effect of the anti-PD-L1 antibody in both 4T1 breast and CT26 colon tumor mice.