Pro-tumor M2 Macrophages Research Articles

Nano-Ayurvedic Medicine Approaches Using Ginkgo biloba-Phytochemicals Functionalized Gold Nanoparticles Against Breast Cancer.

Breast cancer is a significant global health issue, contributing to 15% of cancer-related deaths. Our laboratory has pioneered a novel approach, combining Ayurvedic principles with green nanotechnology, to develop a scientifically rigorous medical modality referred to as Nano-Ayurvedic Medicine, recently approved by the US Patents and Trademarks Office. Here in we report a new Nano-Ayurvedic medicine agent derived from gold nanoparticles encapsulated with phytochemicals from Ginkgo biloba plant (GB-AuNPs). We have developed biocompatible gold nanoparticles using electron-rich phytochemicals from Ginkgo biloba as reducing agent cocktail. Ginkgo biloba phytochemical-encapsulated gold nanoparticles (GB-AuNPs) were fully characterized, and their anticancer activity, including immunomodulatory profiles, were evaluated against breast (MDAMB-231) cancer cell lines. Characterization revealed spherical morphology for GB-AuNPs and possessed optimum in vitro stability through high zeta potential of -34 mV for optimum in vivo stability. The core size of GB-AuNPs of 19 nm allows for penetration into tumor cells through both EPR effects as well as through the receptor-mediated endocytosis. The Antitumor efficacy of this nano-ayurvedic medicine agent revealed strong antitumor effects of GB-AuNPs towards MDAMB-231. Our investigations reveal that GB-AuNPs enhance anti-tumor cytokines (IL-12, TNF-α, IFN-γ) and reduce pro-tumor cytokines (IL-10, IL-6), promoting the conversion of protumor M2 macrophages into M1-like macrophage antitumor phenotype. Cellular studies show that GB-AuNPs offer superior anti-tumor efficacy and a better safety profile against breast tumors compared to cisplatin. Our investigations have demonstrated that the nano-ayurvedic medicine agent, GB-AuNPs, treats cancers through an immunomodulatory mechanism facilitated by elevated levels of anti-tumor cytokines (TNF-α, IFN-γ and IL-12) with concomitant downregulation of pro-tumor cytokines expression (IL-6 and IL-10). The green nanotechnology approach for the development of nano-ayurvedic medicine agent (GB-AuNPs), as described in this paper, presents new and attractive opportunities for treating human cancers and other debilitating diseases and disorders.

The tumor immune microenvironment in resected treatment-naive pancreatic cancer patients with long-term survival

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Presently, only a fraction of patients undergo successful surgical resection, the most effective treatment. Enhancing treatment strategies necessitates a deep comprehension of the factors underlying extended survival after surgical resection in patients. MethodsThis study aims to identify the important factors of PDAC patients’ long-term survival with metatranscriptomics and multiplex immunofluorescence (IF) staining analyses. Specifically, differences in tumor immune microenvironment (TIME) were investigated between treatment-naïve PDAC short-term survivors (STS, overall survival <6 months) and long-term survivors (LTS, overall survival >5 years). ResultsAs a result, we detected 589 over-expressed genes, including HOXB9, CDA, and HOXB8, and 507 under-expressed genes, including AMY2B, SCARA5, and SLC2A2 in LTS. Most of the Reactome overbiological pathways enriched in our data were over-expressed in LTS, such as RHO GTPase Effectors and Cell Cycle Checkpoints. Eleven microbiomes significantly differed between LTS and STS, including Sphingopyxis and Capnocytophaga. Importantly, we demonstrate that the TIME profile with an increased abundance of memory B cells and the reduction of M0 and pro-tumoral M2 macrophages are associated with a good prognosis in PDAC. ConclusionsIn this study, we delved into the TIME with metatranscriptomics and IF staining analyses to understand the prerequisite of prolonged survival in PDAC patients. In LTS, several biological pathways were overexpressed, and specific microbiomes were identified. Furthermore, apparent differences in driven immune factors were found that provide valuable insights into developing new treatment strategies.

Abstract LB346: Dynamic changes in the tumor microenvironment from initial diagnosis through sequential therapies in patients with large B cell lymphoma undergoing axicabtagene ciloleucel (axi-cel) treatment

Abstract Axicabtagene ciloleucel (axi-cel), marketed as YESCARTA®, is an approved autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory large B cell lymphoma patients following one prior therapeutic intervention. The pivotal ZUMA-1 trial (NCT02348216), a Phase 1/2 multicenter study, reported an impressive 83% objective response rate and a robust 58% complete response rate at 5 years (Locke et al., Lancet Oncol. 2019). Subsequently, ZUMA-7 (NCT03391466), a Phase 3 multicenter trial focusing on patients with one prior systemic therapy, demonstrated comparable outcomes with an 83% objective response rate and a remarkable 65% complete response rate at 2 years (Locke et al., Lancet Oncol. 2022). Previous studies have highlighted the crucial connection between the tumor microenvironment (TME) and CAR T-cell therapy outcomes. Notably, the pre-treatment tumor immune contexture has been proposed as a major determinant of clinical outcomes in ZUMA-1 and ZUMA-7 patients (Scholler et al., Nature Medicine 2022; Locke et al., Nature Medicine, in press; ASH2023 oral presentation #226). This study aims to provide novel insights into the dynamic changes in the TME from initial diagnosis through sequential therapeutic interventions in large B-cell lymphoma patients treated with axi-cel. Utilizing multiplex immunohistochemistry (Brightplex®), we analyzed 165 commercial tumor biopsies (at diagnosis), 100 ZUMA-7 samples (40 at diagnosis, 60 after 1 prior therapeutic line), and 35 ZUMA-1 samples (20 pre- and 15 post-CAR T infusion). Three Brightplex® assays were developed to assess T-cell (CD3 CD8 FOXP3 TIM3 PD1 LAG3 GZMB) and macrophages infiltration (CD68 CD64 CD163 CD204 CD206 PDL1), along with single staining for the NK cell marker, NKP46. Our findings revealed significant changes and evolution from diagnosis through post-axi-cel infusion: a significant decrease in lymphocyte levels (cytotoxic, helper, and regulatory, with p-values of 0.042, 1.3e-05, 0.024, respectively), a notable decrease in M1 macrophages proportion (p-value=5.8e-06), and a substantial increase in M2 macrophages proportion (p-value=4.5e-10) across different lines of standard care therapy. Importantly, high baseline proportions of protumor M2 macrophages may serve as predictors of disease relapse post-axi-cel treatment, while low baseline M2 proportions could be associated with a complete response. This comprehensive characterization of the immune contexture provides crucial insights to better highlight TME modification depending on the line of therapy, and further correlative analysis of cell subsets will be presented, contributing to a deeper understanding of treatment impact on the TME in large B cell lymphoma. Citation Format: Michael Mattie, Regis Perbost, Aurelie Auguste, Sarah Turcan, Jenny J. Kim, Christina To, Jenny Nater, Simone Filosto, Rhine R. Shen, Jérôme Galon. Dynamic changes in the tumor microenvironment from initial diagnosis through sequential therapies in patients with large B cell lymphoma undergoing axicabtagene ciloleucel (axi-cel) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB346.

Metformin boosts antitumor immunity and improves prognosis in upfront resected pancreatic cancer: an observational study.

Beyond demographic and immune factors, metabolic considerations, particularly metformin's recognized impact in oncology, warrant exploration in treating pancreatic cancer. This study aimed to investigate the influence of metformin on patient survival and its potential correlation with distinct immune profiles in pancreatic ductal adenocarcinoma (PDAC) tumors. We included 82 upfront resected and 66 gemcitabine-based neoadjuvant chemoradiotherapy (nCRT)-treated patients from the PREOPANC randomized controlled trial (RCT). Transcriptomic NanoString immunoprofiling was performed for a subset of 96 available resected specimens. Disparities in survival outcomes and immune profiles were apparent between metformin and non-metformin users in upfront resected patients but lacking in nCRT-treated patients. Compared to non-metformin users, upfront resected metformin users showed a higher median overall survival (OS) of 29 vs 14 months and a better 5-year OS rate of 19% vs 5%. Furthermore, metformin use was a favorable prognostic factor for OS in the upfront surgery group (HR = 0.56; 95% CI = 0.32 to 0.99). Transcriptomic data revealed that metformin users significantly underexpressed genes related to pro-tumoral immunity, including monocyte to M2 macrophage polarization and activation. Furthermore, the relative abundance of anti-inflammatory CD163+ MRC1+ M2 macrophages in non-metformin users and immune-activating CD1A+ CD1C+ dendritic cells in metformin users was heightened (P < .001). This study unveils immune profile changes resulting from metformin use in upfront resected pancreatic cancer patients, possibly contributing to prolonged survival outcomes. Specifically, metformin use may decrease the abundance and activity of pro-tumoral M2 macrophages and increase the recruitment and function of tumor-resolving DCs, favoring antitumor immunity.[PREOPANC trial EudraCT: 2012-003181-40].

Abstract 6098: Spatial single-cell immune microenvironment analysis reveals the transition from innate to adaptive immune response during early lung adenocarcinoma carcinogenesis

Abstract Introduction: Lung cancer remains the leading cause of cancer death worldwide. Although prevention holds the promise to reduce lung cancer mortality, the results from randomized trials have been disappointing, likely due to our rudimentary knowledge of early lung cancer carcinogenesis. Cancer initiation and progression involve complex processes dependent on the dynamic interplay of individual cells within the tumor microenvironment (TME). Our understanding of the multicellular composition, interaction, spatial distribution, and functional dynamics during the early evolution from precancer to invasive LUAD is limited. Methods: We performed spatial single cell immune microenvironment analysis of 10 normal, 40 AAH, 22 AIS, 18 MIA, and 34 IAC by Imaging Mass Cytometry (IMC) and analyzed high-dimensional images of 1,618 regions-of-interest (ROIs) using a 34-plex marker. Using machine learning approaches, we classified 4,828,879 cells into 14 major cell types, which were subjected to further analysis for multicellular composition, interaction, spatial distribution, and functional dynamics in LUAD precursors of different stages. Results: From AAH to AIS, MIA, and IAC, we observed a gradual escalation in the density (number of cells/mm2) of all immune subsets. Proportionally, adaptive immune cells exhibited a progressive increase, whereas innate immune cells demonstrated a concurrent decrease with the neoplastic progression from normal lung to AAH, AIS, MIA, and ADC. Notably, macrophages emerged as the predominant immune cells within the immune microenvironment of these LUAD precursors. Upon closer examination of subtypes, protumor M2 macrophages showed a gradual increase from AAH to AIS, MIA, and IAC, whereas anti-tumor M1 macrophages increased from normal to AIH, AIS and subsequently decreased in AIS, MIA, and IAC stages. These alterations resulted in a progressive increase in M2/M1 ratios from AAH to IAC stages. Furthermore, M2 macrophages exhibited higher heterotypic interactions with epithelial cells than M1 across LUAD precursors of different stages, especially in the later stages. Co-expression analysis revealed 5 modules that distinguishes each histological stage. Tim-3 module was significantly enriched in AAH lesions, while NK cell-related features were enriched at AIS stage, B cell-related features enriched at MIA stage and Ki-67-related adaptive immune features enriched in IAC stage. Conclusion: The spatial single cell immune microenvironment analysis revealed a transition from innate to adaptive immunity during initiation and early progression of LUAD progression and innate immunity may play critical roles during early LUAD development. Therefore, reprograming innate immunity such as repolarization of M2/M1 may provide avenues to intercept LUAD precursors to prevent invasive LUAD. Citation Format: Bo Zhu, Pingjun Chen, Muhammad Aminu, Junya Fujimoto, Lingzhi Hong, Andre L. Moreira, Jian-Rong Li, Yanhua Tian, Luisa M. Solis Soto, Parra Cuentas Edwin Roger, Ou Shi, Hong Chen, Frank R. Rojas, Eduardo Vilar, Anirban Maitra, Ken Chen, Nicolas Navin, Cara L. Haymacker, Vamsidhar Velcheti, Daniel H. Sterman, P Andrew Futreal, Don L. Gibbons, Ignacio I. Wistuba, John V. Heymach, Chao Cheng, Harvey I. Pass, Jia Wu, Jianjun Zhang. Spatial single-cell immune microenvironment analysis reveals the transition from innate to adaptive immune response during early lung adenocarcinoma carcinogenesis [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 6098.

Abstract 1122: Inhibition of HDAC6 enhanced radiation therapy induced antitumor response in melanoma

Abstract Patients with advanced melanoma are often treated with radiation therapy (RT) in combination with immunotherapies. Immunogenic tumor cell death induced by radiation is known to activate innate and adaptive immunity. Macrophage activity in the irradiated tumor microenvironment (TME) has been implicated to play a critical role in post-RT innate immune responses. However, tumor-associated macrophages (TAMs) due to their phenotypic plasticity convert from initial antitumor M1 phenotype to protumor M2 macrophages resulting in relapse and metastasis of any surviving tumor cells. Therefore, strategies regulating tumor macrophages post-radiation can enhance the therapeutic effectiveness of radiation therapy. Here, we report targeting HDAC6 function with a novel selective inhibitor (SP-2-225) as a potential therapeutic candidate for combination with RT. When administered intraperitoneally at a dose of 25mg/kg, SP-2-225 resulted in significant tumor suppression in the immunocompetent SM1 murine melanoma model. Phenotyping of tumor immune infiltrate indicated a profound decrease in M2 macrophages resulting in an M1/M2 macrophage ratio and an increase in CD8 effector memory T-cells. Further, in-vitro co-culture studies using trans-well assays indicated that irradiated tumor cells attract M2 macrophages, and treatment with SP-2-225 abrogated the M2 macrophage migration but did not alter M1 macrophage migration. In addition, 24 hours post-RT, intratumor administration of SP-2-225 treated M1 macrophages as adoptive cell therapy resulted in diminished tumor growth. The combination of conditioned M1 macrophages with an irradiated tumor microenvironment resulted in an increased M1/M2 ratio and infiltration of effector memory and central memory CD8 T-cells underscoring the influence of transplanted macrophages on the local antitumor immunity. Therefore, our findings demonstrate the ability of HDAC6 inhibitors to regulate macrophage function and phenotype as a cell therapy for use in combination with radiation therapy. Citation Format: Xintang Li, Manasa Suresh, Hawa Coulibaly, David Quiceno-Torres, Nithya Gajendran, Karen Tan, Satish Noonepalle, Scott Grindrod, Anatoly Dritschilo, Alejandro Villagra. Inhibition of HDAC6 enhanced radiation therapy induced antitumor response 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 1122.

Abstract 4583: Development of selective HDAC6 inhibitors to improve cancer immunotherapy

Abstract Histone Deacetylases (HDACs) are enzymes that modify histones and non-histone proteins. Our group has previously demonstrated that HDAC6 inhibitors (HDAC6is) modulate multiple immune pathways, including those that suppress the antitumoral M2 phenotype, leading to improved antitumor immunity. Despite the potential of HDAC6is, current inhibitors have poor selectivity and off-target effects and only work at micromolar concentrations. In this work, we designed a screening pipeline of HDAC6i, considering selectivity, potency, capacity to prevent the protumoral phenotype of macrophages, and low cytotoxicity on macrophages and other immune cells. This approach has not been tested before, as the standard screening method for antitumoral HDACis has focused on its cytotoxic effect on cancer cells. We initially designed in silico 980 compound derivatives from previously reported HDAC6i, such as SS208, Nexturastat A, and Suprastat. Twenty-four compounds with a high capacity to bind HDAC6 and chemical stability were screened by their ability to inhibit HDAC6 in cell-free conditions. Sixteen compounds demonstrated high potency and were further evaluated in murine macrophages by their cytotoxic effect using CellTox. The capacity to impede tubulin deacetylation was assessed by western blot and deacetylase activity by HDAC-Glo. The top six candidates were then evaluated by their selectivity compared to other HDAC members and by their effect on repressing the expression of protumoral M2 macrophages Arginase 1 and Fizz1, and activation of antitumoral markers TNFa and iNOS by western blot and qRT-PCR. The best three candidates were then evaluated at nanomolar concentrations to explore their immunomodulatory potential of M1 against M2 by performing antigen presentation (SIINFEKL presentation) and phagocytic capacity using microscopy. Two of these compounds, SM-06-09 and SM-05-947, will be further tested in syngeneic murine melanoma models for their ability in tumor regression and antitumor immunity. This study has yielded important insights into developing new and improved HDAC6is with minimal cytotoxicity, and we believe our work will help advance cancer research and ultimately lead to better patient outcomes. Citation Format: Nithya Gajendran, Manasa Suresh, David Quiceno, Xintang Li, Marie Durr, Karen Tan, Satish Kumar Reddy Noonepalle, Alejandro Villagra. Development of selective HDAC6 inhibitors to improve cancer immunotherapy [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 4583.

Macrophages as Targets in Hepatocellular Carcinoma Therapy.

Hepatocellular carcinoma (HCC) is a malignant tumor with a complex and diverse immunosuppressive microenvironment. Tumor-associated macrophages (TAM) are an essential component of the tumor immune microenvironment. TAMs typically exist in two primary states: anti-tumor M1 macrophages and protumor M2 macrophages. Remarkably, TAMs possess high plasticity, enabling them to switch between different subtypes or alter their biological functions in response to the tumor microenvironment. Based on research into the biological role of TAMs in the occurrence and development of malignant tumors, including HCC, TAMs are emerging as promising targets for novel tumor treatment strategies. In this review, we provide a detailed introduction to the origin and subtypes of TAMs, elucidate their interactions with other cells in the complex tumor microenvironment of HCC, and describe the biological roles, characteristics, and mechanisms of TAMs in the progression of HCC. Furthermore, we furnish an overview of the latest therapeutic strategies targeting TAMs.

Pre- and Post-Treatment Immune Contexture Correlates with Long Term Response in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel (axi-cel)

Background: Axi-cel is a US and EU-approved autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy for pts with relapsed/refractory large B cell lymphoma after 1 prior therapy as a result of ZUMA-7 (NCT. In ZUMA-1 (NCT02348216), the pivotal study which initially led to the approval of axi-cel in the 3L+ setting, the objective response rate was 83% (58% complete response rate; Locke et al. Lancet Oncol. 2019) and at 5 years follow up an OS rate of 42.6%, 5 years durable response and 5 years disease specific survival at 51% ( Neelapu et al. Blood 2023). T cell-related biology (Immunosign 21; Immunoscore®IC) measured pretreatment in the tumor microenvironment (TME) was associated with response to axi-cel ( Scholler et al., 2022). Increased density of activated PD-1+LAG-3+/−TIM-3−CD8+ T cells, measurable pretreatment, facilitates clinical response in pts post-axi-cel. This expanded analysis characterized the impact of axi-cel treatment on the TME immune contexture and examined associations between immune cell subsets and relapse. Methods: Baseline and post-infusion tumor biopsy samples were analyzed by multiplex immunohistochemistry ( Brightplex®). Four panels were developed and applied to assess T cell infiltration (CD3 CD8 FOXP3 TIM3 PD1 LAG3 TOX), regulatory T cell subtyping (CD3 CD8 GATA3 TBET RORg BCL6), T cell activation &amp; exhaustion (CD3 CD8 TIM3 LAG3 PD1 GZMB KI67), macrophage (CD68 CD64 CD163 CD204 CD206 PDL1) and MDSC (CD3 CD11B CD68 CD14 CD15 LOX1 S100A9) subsets. Transcriptomic analysis was performed using nCounter® Pan Cancer panel. The association between T cell subtypes and macrophages cell subset density, and probability to relapse was evaluated. T test values were based on Brightplex analysis. Results: 34 tumor biopsies (16 at baseline, 18 post-infusion) from 26 pts were analyzed including 11 relapsed (6 CR/5 PR) and 15 durable response (15 CR). The baseline and post-infusion TME comprised all major macrophages, MDSC and T cell subsets, with diverse distribution across samples analyzed. Low proinflammatory M1 macrophage density was observed at baseline and post-infusion across patients. In relapsed patients, baseline tumors were enriched with a significantly higher proportion of protumoral M2 macrophage (p&amp;lt;0.0001). A shift of M1-M2 polarization was observed between baseline and post-infusion (Baseline 33%M1M2 / 65%M2, post infusion 76%M1M2 / 21%M2, p&amp;lt;0,0001) across the cohort. In contrast, in relapsed patients, M-MDSC cell density was significantly increased post treatment (p=0.0420). In addition, Innate lymphocyte cells subtype 2 (ILC2) cell density, previously described to be positively correlated with improved overall survival in CRC ( Huang et al. 2021 Cancers), was significantly decreased in relapsed patients post infusion (p=0.019).Post-infusion, durable response was associated with a significant increase in pan-Macrophages (p=0.0360) and cytotoxic T cell subset densities: CD8 naïve T cells (p=0.016); Activated Cytotoxic T cell (GZMB+)(p=0.0203); PD1+TIM3+LAG3- cytotoxic T cell (p=0.0117); CD8 regulatory T cell (FOXP3+)(p=0.012). Regarding T helper lineage, ongoing response was associated with a significant increase of: CD4 naïve T cells (p=0.021); T helper Th2 (p=0.020); cytotoxic T lymphocyte TC2 and TC1/TC2 cell densities (respectively p=0.0052 and 0.011).Additional characterization of the immune contexture and correlative analysis of cell subsets will be presented. Conclusion: These results suggest that baseline proportions of protumor M2 macrophages may predict disease relapse after axi-cel treatment. Following infusion, relapse is also associated with spatial enrichment of M-MDSC, whereas durable response is related to increases in T cell subpopulation densities inclusive of cytotoxic T cells, T helper, TC1/TC2 ratios and innate T-subpopulations. These findings suggest that axi-cel treatment drastically impacts tumor immune mobilization, infiltration and contexture, which correlates with long term response.

Emodin reduces surgical wounding-accelerated tumor growth and metastasis via macrophage suppression in a murine triple-negative breast cancer model.

It has been suspected that tumor resection surgery itself may accelerate breast cancer (BC) lung metastasis in some patients. Emodin, a natural anthraquinone found in the roots and rhizomes of various plants, exhibits anticancer activity. We examined the perioperative use of emodin in our established surgery wounding murine BC model. Emodin reduced primary BC tumor growth and metastasis in the lungs in both sham and surgical wounded mice, consistent with a reduction in proliferation and enhanced apoptosis (primary tumor and lungs). Further, emodin reduced systemic inflammation, most notably the number of monocytes in the peripheral blood and reduced pro-tumoral M2 macrophages in the primary tumor and the lungs. Consistently, we show that emodin reduces gene expression of select macrophage markers and associated cytokines in the primary tumor and lungs of wounded mice. Overall, we demonstrate that emodin is beneficial in mitigating surgical wounding accelerated lung metastasis in a model of triple-negative BC, which appears to be mediated, at least in part, by its actions on macrophages. These data support the development of emodin as a safe, low-cost, and effective agent to be used perioperatively to alleviate the surgery triggered inflammatory response and consequential metastasis of BC to the lungs.

Enhanced antitumour immunity following neoadjuvant chemoradiotherapy mediates a favourable prognosis in women with resected pancreatic cancer

BackgroundThis study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or resection preceded by gemcitabine-based neoadjuvant chemoradiotherapy...

Macrophage Profiling in Head and Neck Cancer to Improve Patient Prognosis and Assessment of Cancer Cell-Macrophage Interactions Using Three-Dimensional Coculture Models.

Tumor-associated macrophages are key components of the tumor microenvironment and play important roles in the progression of head and neck cancer, leading to the development of effective strategies targeting immune cells in tumors. Our study demonstrated the prognostic potential of a new scoring system (Macroscore) based on the combination of the ratio and the sum of the high and low densities of M1 (CD80+) and M2 (CD163+) macrophages in a series of head and neck cancer patients, including a training population (n = 54) and a validation population (n = 19). Interestingly, the Macroscore outperformed TNM criteria and p16 status, showing a significant association with poor patient prognosis, and demonstrated significant predictive value for overall survival. Additionally, 3D coculture spheroids were established to analyze the crosstalk between cancer cells and monocytes/macrophages. Our data revealed that cancer cells can induce monocyte differentiation into protumoral M2 macrophages, creating an immunosuppressive microenvironment. This coculture also induced the production of immunosuppressive cytokines, such as IL10 and IL8, known to promote M2 polarization. Finally, we validated the ability of the macrophage subpopulations to induce apoptosis (M1) or support proliferation (M2) of cancer cells. Overall, our research highlights the potential of the Macroscore as a valuable prognostic biomarker to enhance the clinical management of patients and underscores the relevance of a spheroid model in gaining a better understanding of the mechanisms underlying cancer cell-macrophage interactions.

Sophisticated genetically engineered macrophages, CAR-Macs, in hitting the bull's eye for solid cancer immunotherapy approaches.

Studies have begun to employ macrophages engineered with chimeric antigen receptor (CAR-Macs) against solid tumors since they can enter solid tumor tissue and interact with approximately all cellular components in the tumor microenvironment. The chimeric antigen receptor (CAR) has emerged as an appealing strategy for improving immune cells' ability to detect cancer. Tumor-associated macrophages (TAMs) generated with CAR designs exhibit appropriate potency based on their capacity to enter solid tumors and communicate through the inhibitory tumor microenvironment. CAR-Macs technology is a new therapeutic method for attacking cancer cells by switching pro-tumoral M2 macrophages to anti-tumoral M1 macrophages, enhancing macrophage phagocytosis, or increasing antigen presentation activity. CAR-Macs may have a prevailing impact on surrounding immune cells, indicating that they retain anti-tumor activity in the presence of human M2 macrophages, demonstrating their use in CAR technology. Understanding the biology of TAM and targeting novel domains for the advanced CAR-Macrophage platform, it will be feasible to add a new dimension to immunotherapy techniques used exclusively in solid malignancies. This review describes how CAR-Macs technologies modulate CAR-Macrophage production, potential target biomarkers on these platforms, their role in immunotherapeutic approaches, and tumor microenvironment.

Precision Killing of M2 Macrophages with Phage-Displayed Peptide-Photosensitizer Conjugates.

Among the immunosuppressive cells recruited to the tumor microenvironment, macrophages are particularly abundant and involved in angiogenesis, metastasis, and resistance to current cancer therapies. A strategy that simultaneously targets tumor cells and macrophages, particularly pro-tumoral M2 macrophages, would have significant clinical impact for various types of solid malignancies. By the use of phage display technology, we have recently developed a synthetic peptide, named NW, which binds to M1 and M2 macrophages with high affinity. Additional affinity selection on M2 macrophages identified only dominant peptides whose binding motifs are similar to that of the NW peptide. To reduce the frequency of selecting such dominating peptides, the peptide library was affinity selected on M2 macrophages blocked with NW peptide. This approach resulted in the selection of peptides that bind to M2, but not M1 macrophages. To explore the therapeutic potential of the selected peptides, the M13 phage-displayed peptides were conjugated to the photosensitizer IR700, which has been used for cancer photoimmunotherapy. The phage displaying a dominant peptide (SPILWLNAPPWA) killed both M1 and M2 macrophages, while those displaying the M2-specific peptides killed M2 macrophages only upon near-infrared light exposure. A significant fraction of the M2 macrophages were also killed with the untargeted M13 phage-IR700 conjugates. Hence, M2 macrophages can also be selectively targeted by the wild type M13 phage, which displayed a significant tropism to these cells. The benefits of this photoimmunotherapy include an automatic self-targeting ability of the wild type M13 phage, and the option of genetic manipulation of the phage genome to include tumor targeting peptides, allowing the killing of both M2 macrophages and cancer cells.

The current understanding of the immune landscape relative to radiotherapy across tumor types.

Radiotherapy is part of the standard of care treatment for a great majority of cancer patients. As a result of radiation, both tumor cells and the environment around them are affected directly by radiation, which mainly primes but also might limit the immune response. Multiple immune factors play a role in cancer progression and response to radiotherapy, including the immune tumor microenvironment and systemic immunity referred to as the immune landscape. A heterogeneous tumor microenvironment and the varying patient characteristics complicate the dynamic relationship between radiotherapy and this immune landscape. In this review, we will present the current overview of the immunological landscape in relation to radiotherapy in order to provide insight and encourage research to further improve cancer treatment. An investigation into the impact of radiation therapy on the immune landscape showed in several cancers a common pattern of immunological responses after radiation. Radiation leads to an upsurge in infiltrating T lymphocytes and the expression of programmed death ligand 1 (PD-L1) which can hint at a benefit for the patient when combined with immunotherapy. In spite of this, lymphopenia in the tumor microenvironment of 'cold' tumors or caused by radiation is considered to be an important obstacle to the patient's survival. In several cancers, a rise in the immunosuppressive populations is seen after radiation, mainly pro-tumoral M2 macrophages and myeloid-derived suppressor cells (MDSCs). As a final point, we will highlight how the radiation parameters themselves can influence the immune system and, therefore, be exploited to the advantage of the patient.

Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions

Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, the effect of chalcones 1-18 against the metabolic viability of cervical (HeLa) and prostate (PC-3 and LNCaP) tumor cell lines was tested, to compare the activity against solid and liquid tumor cells. Their effect was also evaluated on the Jurkat cell line. Chalcone 16 showed the highest inhibitory effect on the metabolic viability of the tested tumor cells and was selected for further studies. Recent antitumor therapies include compounds with the ability to influence immune cells on the tumor microenvironment, with immunotherapy being one actual goal in cancer treatment. Therefore, the effect of chalcone 16 on the expression of mTOR, HIF-1α, IL-1β, TNF-α, IL-10, and TGF-β, after THP-1 macrophage stimulation (none, LPS or IL-4), was evaluated. Chalcone 16 significantly increased the expression of mTORC1, IL-1β, TNF-α, and IL-10 of IL-4 stimulated macrophages (that induces an M2 phenotype). HIF-1α and TGF-β were not significantly affected. Chalcone 16 also decreased nitric oxide production by the RAW 264.7 murine macrophage cell line, this effect probably being due to an inhibition of iNOS expression. These results suggest that chalcone 16 may influence macrophage polarization, inducing the pro-tumoral M2 macrophages (IL-4 stimulated) to adopt a profile closer to the antitumor M1 profile.

Carbon dots with two-photon fluorescence imaging for efficient synergistic trimodal therapy

Applying the fluorescent carbon dots as smart materials in anticancer therapy is of great interest. However, carbon dots for multimodal synergistic anticancer therapy, especially for the triple modality, is rarely reported. Herein, we successfully synthesized OCDs by citric acid and (1R,2S)-2-amino-1,2-diphenylethan-1-ol, which show aggregation-induced emission property and two-photon fluorescence imaging. Meanwhile, OCDs are ideal photosensitizers for photothermal therapy under 808 nm and Type I photodynamic therapy with white light. Hydroxyl radicals, generated by Type I photodynamic therapy based on OCDs can transform protumoral M2 macrophages into antitumoral M1 macrophages, which exhibited immunotherapy ability. The synergism trimodal of OCDs results in potent anticancer efficacy, showing great potential in cancer therapy.

Monocytes educated by cancer-associated fibroblasts secrete exosomal miR-181a to activate AKT signaling in breast cancer cells

BackgroundCancer-associated fibroblasts (CAFs), one of the major components of the tumor stroma, contribute to an immunosuppressive tumor microenvironment (TME) through the induction and functional polarization of protumoral macrophages. We have herein investigated the contribution of CAFs to monocyte recruitment and macrophage polarization. We also sought to identify a possible paracrine mechanism by which CAF-educated monocytes affect breast cancer (BC) cell progression.MethodsMonocytes were educated by primary CAFs and normal fibroblast (NF); the phenotypic alterations of CAF- or NF-educated monocytes were measured by flow cytometry. Exosomes isolated from the cultured conditioned media of the educated monocytes were characterized. An in vivo experiment using a subcutaneous transplantation tumor model in athymic nude mice was conducted to uncover the effect of exosomes derived from CAF- or NF-educated monocytes on breast tumor growth. Gain- and loss-of-function experiments were performed to explore the role of miR-181a in BC progression with the involvement of the AKT signaling pathway. Western blotting, enzyme-linked immunosorbent assay, RT-qPCR, flow cytometry staining, migration assay, immunohistochemical staining, and bioinformatics analysis were performed to reveal the underlying mechanisms.ResultsWe illustrated that primary CAFs recruited monocytes and established pro-tumoral M2 macrophages. CAF may also differentiate human monocyte THP-1 cells into anti-inflammatory M2 macrophages. Besides, we revealed that CAFs increased reactive oxygen species (ROS) generation in THP-1 monocytes, as differentiating into M2 macrophages requires a level of ROS for proper polarization. Importantly, T-cell proliferation was suppressed by CAF-educated monocytes and their exosomes, resulting in an immunosuppressive TME. Interestingly, CAF-activated, polarized monocytes lost their tumoricidal abilities, and their derived exosomes promoted BC cell proliferation and migration. In turn, CAF-educated monocyte exosomes exhibited a significant promoting effect on BC tumorigenicity in vivo. Of clinical significance, we observed that up-regulation of circulating miR-181a in BC was positively correlated with tumor aggressiveness and found a high level of this miRNA in CAF-educated monocytes and their exosomes. We further clarified that the pro-oncogenic effect of CAF-educated monocytes may depend in part on the exosomal transfer of miR-181a through modulating the PTEN/Akt signaling axis in BC cells.ConclusionsOur findings established a connection between tumor stromal communication and tumor progression and demonstrated an inductive function for CAF-educated monocytes in BC cell progression. We also proposed a supporting model in which exosomal transfer of miR-181a from CAF-educated monocytes activates AKT signaling by regulating PTEN in BC cells.

RF11 | PSAT272 Suppressive Tumor Immune Microenvironment is Associated with Aggressive Differentiated Thyroid Cancer

Abstract Background The incidence of differentiated thyroid cancer (DTC) has tripled over the last 30 years in the US. While most patients respond to conventional treatment, some have an aggressive course with a dismal 5-year survival of 15.3%. There is a need to identify novel markers in addition to clinico-pathologic criteria to better prognosticate DTC patients. In addition, exploration of immunotherapies in advanced DTC underlies the critical need to investigate the immune interface of DTC to identify potential therapeutic targets and those likely to respond to immunotherapies. Aims We aimed to identify tumor-infiltrating immune cells and checkpoints [programmed cell death protein 1 (PD-1) and its ligand (PD-L1)] associated with DTC and investigate their association with metastatic and advanced DTC. Methods We performed multiplex immunofluorescence on de-paraffinized thyroid tissue collected at initial thyroidectomy from 17 adults with DTC. We identified cell-surface molecules to characterize the tumor immune microenvironment for leukocytes (CD45+), T cells (CD3+), B cells (CD19+), natural killer cells (CD56+), T regulatory cells (CD3+FOXP3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), macrophages (CD68+), M2 macrophages (CD68+CD163+), and immune checkpoints PD-1 and PD-L1. Tonsil tissue was used as positive control. Immunofluorescence staining was quantified by cell counting HALO software. We compared the mean percentage expression of immune markers between tumor and adjacent thyroid tissue from the same patient by paired t-test, and in exploratory analysis between intra-tumoral tissue in patients with versus without distant metastases by student's t-test; p value&amp;lt;0.05 was used for significance. Results Overall, in 17 DTC patient samples, most leukocyte infiltrate occurred along the leading edge of the tumor and in the peritumoral thyroid tissue. Immune checkpoints PD-1 and PD-L1 were highly expressed in the tumor as compared to adjacent thyroid tissue (p&amp;lt;0.05). A higher trend for M2 macrophages was observed in the tumor but was not statistically significant. Intra-tumoral expressions of T regulatory cells, macrophages and PD-L1+ cells were more in samples for DTC patients with metastases (n=3) as compared to those without (n=14). Conclusions Immune profiling demonstrated significant differences between tumor and adjacent thyroid tissue, particularly for PD-1 and PD-L1, indicating their role in DTC occurrence. Intra-tumor infiltration of T regulatory cells, macrophages and PD-L1+ cells was more in metastatic DTC indicating their potential role in aggressive behavior of DTC. These data suggest that suppressive tumor immune microenvironment portends more aggressive behavior of DTC, and that specific immune suppressive markers may be potential therapeutic targets. We are continuing to expand our sample size to have adequate power for comparing groups with various clinico-pathologic severities and analyzing the role of pro-tumoral M2 macrophages in DTC prognosis. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Saturday, June 11, 2022 1:36 p.m. - 1:41 p.m.

Paclitaxel-loaded ginsenoside Rg3 liposomes for drug-resistant cancer therapy by dual targeting of the tumor microenvironment and cancer cells

IntroductionInherent or acquired resistance to paclitaxel (PTX) is a pivotal challenge for chemotherapy treatment of multidrug-resistant (MDR) breast cancer. Although various targeted drug-delivery systems, including nanoparticles and liposomes, are effective for MDR cancer treatment, their efficacy is restricted by immunosuppressive tumor microenvironment (TME). MethodsGinsenosides Rg3 was used to formulate unique Rg3-based liposomes loaded with PTX to establish Rg3-PTX-LPs, which were prepared by the thin-film hydration method. The stability of the Rg3-PTX-LPs was evaluated by particle size analysis through dynamic light scattering. The active targeting effect of Rg3-based liposomes was examined in an MCF-7/T xenograft model by an in a vivo imaging system. To evaluate the antitumor activity and mechanism of Rg3-PTX-LP, MTT, apoptosis assays, TAM regulation, and TME remodeling were performed in MCF-7/T cells in vitro and in vivo. ResultsRg3-PTX-LPs could specifically distribute to MCF7/T cancer cells and TME simultaneously, mainly through the recognition of GLUT-1. The drug resistance reversing capability and in vivo antitumor effect of Rg3-PTX-LPs were significantly improved compared with conventional cholesterol liposomes. The TME remodeling mechanisms of Rg3-PTX-LPs included inhibiting IL-6/STAT3/p-STAT3 pathway activation to repolarize protumor M2 macrophages to antitumor M1 phenotype, suppressing myeloid-derived suppressor cells (MDSCs), decreasing tumor-associated fibroblasts (TAFs) and collagen fibers in TME, and promoting apoptosis of tumor cells. Hence, through the dual effects of targeting tumor cells and TME remodeling, Rg3-PTX-LPs achieved a high tumor inhibition rate of 90.3%. ConclusionOur multifunctional Rg3-based liposome developed in the present study offered a promising strategy for rescuing the drug resistance tumor treatment.