Functional Genetic Screens Reveal Key Pathways Instructing the Molecular Phenotypes of Tumor-Associated Macrophages.

<div>Abstract<p>Tumor-associated macrophages (TAM) display remarkable functional heterogeneity, yet the molecular mechanisms driving their diverse phenotypes remain elusive. Using CRISPR screens in primary macrophages, we identified tumor-derived factors, including lactic acid, prostaglandin E2, and GM-CSF, as key modulators of TAM polarization. These factors interacted cooperatively and antagonistically to shape distinct TAM phenotypes that were highly conserved across human cancers. Mechanistically, lactic acid and PGE2 jointly induced angiogenic gene programs while suppressing GM-CSF–driven MHC-II expression at the chromatin level, creating mutually exclusive distributions of proangiogenic and MHC-II<sup>+</sup> TAMs, which were differentially localized to specific spatial niches in the tumor microenvironment. Furthermore, we showed that shifting TAMs to an interferon-responsive phenotype, triggered by <i>Adar</i> inactivation, significantly promoted the infiltration of effector CD8<sup>+</sup> T cells through specific receptor–ligand interactions. These findings uncover a conserved mechanism of TAM polarization and offer insights into therapeutic strategies for TAM reprogramming to potentiate cancer immunotherapy.</p></div>

High-Density Gene Expression Analysis of Tumor-Associated Macrophages from Mouse Mammary Tumors

SY27-02: Heterogeneity of tumor-associated macrophages: Implications for antiangiogenic therapy

BackgroundHigh frequencies of Tumor Associated Macrophages (TAMs) are related to poor patient prognosis. The Tumor Microenvironment (TME) is characterised by resource scarcity, toxic metabolic by-products, and low pH, together creating...

Tumour associated macrophages (TAMs) are thought to be biased towards an M2 phenotype, suppressing anti-tumour immune responses and thus therapies to eradicate macrophages have been proposed. However, current knowledge on TAMs in human cancer is based on a limited number of markers; their comprehensive M1 (anti-tumour) or M2 (pro-tumour) signatures, as well as their role in the anti-tumour immune response, have not yet been determined. Here, for the first time, we have generated transcriptomic data from TAMs isolated from human lung cancer, in comparison with non-tumour lung macrophages from the same patients. We found out that TAMs in lung cancer show a broad pro-tumour M2 signature, but that this signature is not associated with a weaker anti-immune response. However, it is the activation of an M1 signature in TAMs that determines whether TAMs can promote or not a strong anti-tumour immune response. We describe a novel set of M1high TAMs that simultaneously activate M1 and M2 signatures. M1high TAMs strongly activate antigen-presenting genes and express high levels of T-cell chemoattractants (CXCL9, 10 and 11), associating with a higher infiltration of anti-tumour CD8+ T-cells and a better clinical outcome. AIM2 and IL1beta levels are increased in M1high TAMs, indicating a direct involvement in pyroptosis and clearance of cancer cells. We provide a unique transcriptomic resource of TAMs in lung cancer and propose that TAMs can be used to improve immunotherapy due to a novel and pivotal role for M1high TAMs in orchestrating the immune adaptive anti-tumour responses.

Introduction: Tumor-associated macrophages (TAMs) constitute a significant population of immune cells in the tumor microenvironment (TME), mainly exhibiting an anti-inflammatory M2-like phenotype. They secrete angiogenic growth factors that support tumor growth with an immunosuppressive TME and contribute to developing resistance against immunotherapies. Therefore, there is an increasing effort to switch TAMs from protumoral M2-like to antitumoral and pro-inflammatory M1-like phenotype. Aging is a critical risk factor, with global cancer incidences increasing from 45 to 75 years of age and declining thereafter. The reason behind this decline beyond 75 years of age is unknown but needs further investigation. With the underrepresentation of aged population in preclinical and clinical studies, the role of aging in cancer development and the TME are understudied. Considering the multidimensional role of macrophages in the TME, this study determines the effects of age-associated changes in macrophage function and phenotype on tumor development. Objective: To study the changes in macrophage biology with aging and understand the implications of aging macrophages on the TME in murine cancer models. Methods: Wildtype mice of ages 6-8 WKS, 3-6 MO, 10-12 MO, and 20-24 MO were implanted with tumor cells (SM1 melanoma, 4T1 breast cancer) to study tumor growth kinetics. Immune cell profiling of TME, including TAMs, was performed using flow cytometry, single-cell RNAseq, and secretome analysis. Bone marrow-derived macrophages isolated from tumor-bearing and healthy mice of the above four age groups were polarized to M1 and M2 phenotypes to study age- and cancer-associated changes in macrophage function, phenotype, and metabolic signature. The changes in macrophage cytokine and chemokine secretion levels with aging were determined by multiplex ELISA assays. Results: Tumor growth in older mice was significantly slower than in younger mice. A distinct change in immune cell populations within the TME and reduced secretion profile of TAMs with aging was observed from single-cell analyses. Additionally, the single-cell secretome analysis reflected an inefficient monocyte-to-macrophage transition in tumors of older mice. The defect in macrophage polarization to M1- and M2-like phenotypes with aging complemented with the sub-optimal function (phagocytosis, antigen presentation, and migration) and metabolic signatures of aged macrophages. Conclusions: The phenotypic, functional, and metabolic changes in macrophages with aging significantly affect tumor growth in murine cancer models. Our study further emphasizes the important role of macrophages in the TME and the need for better representation of aged populations in preclinical and clinical studies. Citation Format: Manasa Suresh, Marie Durr, Xintang Li, Bryan Weselman, Francisco Tapia Belmonte, Nithya Gajendran, David Quiceno-Torres, Sonia Sebaoui, Adhithi Rajesh, Matias Hepp, Alexis Salas-Burgos, Satish Noonepalle, Alejandro Villagra. Changes in macrophage phenotype and function with aging affects tumor growth and tumor microenvironment in murine cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6139.

Tumor-Associated Macrophages in the Cutaneous SCC Microenvironment Are Heterogeneously Activated

In lung cancer, tumor‐associated macrophages (TAMs), especially M2‐like TAMs, represent the main tumor progression components in the tumor microenvironment (TME). Therefore, M2‐like TAMs may serve as a therapeutic target. The purpose of this study was to investigate the effect of M2‐like TAM depletion in the TME on tumor growth and chemotherapy response in lung cancer. The levels of secreted monocyte chemoattractant protein (MCP‐1) and prostaglandin E2 (PGE2) in the supernatants of lung cancer cell lines A549 and LLC were evaluated via ELISA. Cell migration assays were performed to assess the recruitment ability of macrophage cell lines THP‐1 and J774‐1 cells. Differentiation of macrophages was assessed via flow cytometry. Immunohistochemical staining was performed to visualize M2‐like TAMs in transplanted lung cancer in mouse. We used the COX‐2 inhibitor nimesulide to inhibit the secretion of MCP‐1 and PGE2, which promotes macrophage migration and M2‐like differentiation. Nimesulide treatment decreased the secretion of MCP‐1 and PGE2 from lung cancer cells. Nimesulide treatment suppressed the migration of macrophages by blocking MCP‐1. Lung cancer supernatant induced the differentiation of macrophages toward the M2‐like phenotype, and nimesulide treatment inhibited M2‐like differentiation by blocking MCP‐1 and PGE2. In the lung cancer mouse model, treatment with nimesulide depleted M2‐like TAMs in the TME and enhanced the tumor inhibitory effect of cisplatin. Our results indicated that blocking the secretion of MCP‐1 and PGE2 from tumor cells depleted M2‐like TAMs in the TME and the combination therapy with cisplatin considerably suppressed tumor growth in the LLC mouse model.

Tumor-associated macrophages (TAMs) may be noticed in gastric carcinomas (GC), but their clinicopathological significance has not been yet explored. From a histological review of 400 cases of tubular/papillary adenocarcinomas, 24 cases of stage I–II gastric adenocarcinomas with intraglandular and stromal TAMs were identified. Their clinicopathological features were compared with 72 pT-matched as well as stage-matched control cases of adenocarcinomas without TAMs. TAMs present in GC cases were present either in glands or in neoplastic stroma, showing an immunoreactivity for CD68 and CD80; sometimes, they were organized in mature granulomas with occasional giant cells. Therefore, the stained TAMs were reminiscent of a specific polarized macrophage M1 phenotype; however, in any case of our cohort, no M2 phenotype macrophages were documented by CD 163 and CD 204 immunostainings. Statistically, no significant differences in age, gender, tumor location, size, and lymphovascular and perineural invasion between the case group with TAMs and pT- as well as stage-matched controls were reported; furthermore, the case group showed lower frequency of lymph node metastasis (p = 0.02). In addition, a significantly different clinical course and overall survival rate were also observed in gastric adenocarcinomas with M1 TAMs (p = 0.02) in comparison to controls. These results suggest that tumor-associated M1 macrophages are related to a quite indolent growth and a better prognosis of patients with this peculiar variant of gastric adenocarcinomas.

A new inflammatory role for p53 in human macrophages

High-Throughput Characterization and New Insight into the Role of Tumor Associated Macrophages (TAMs) in Multiple Myeloma (MM)

Obesity has been established as a leading risk factor for many cancers and can drive tumor progression and metastasis. Paradoxically, obesity has in some cases been associated with better survival and improved response to immune checkpoint blockade therapies. The role of the immune system in the obesity-cancer connection and how obesity affects immunotherapy responses, however, have been unclear. Here we show that obesity enhanced PD-1 expression on macrophages to reduce phagocytosis and antigen presentation to T cells that correlated with reduced T cell expansion and function. This obesity associated immune dysfunction, however, primed for enhanced anti-tumor response to PD-1 blockade. Single cell RNAseq showed obesity remodels myeloid and T cell populations but does not impact non-immune cell populations such as fibroblasts and epithelial cells. Specifically, obesity increased the overall number of tumor-associated macrophages (TAM) while effector T cells were decreased in abundance. Interestingly, the frequency of macrophages expressing PD-1 increased, while the remaining T cells maintained similar or reduced PD-1 expression and appeared less activated. Further, T cell depletion from the tumor microenvironment, enhanced macrophage PD-1 expression and worsened PD1-associated TAM dysfunction in an obesity enhanced manor. Obesity associated cytokines and adipokines including IFN-ã, leptin and insulin induced PD-1 expression on macrophages. This expression is associated with mTOR and c-myc activation signaling pathways; as such inhibiting these signaling pathways blocked PD1 expression. Bulk RNA sequencing analysis revealed PD-1+ TAMs had an altered gene profile compared to PD-1− TAMs. PD1 expressing macrophages exhibited increased mitochondrial respiration and expression of oxidative phosphorylation, increased lipid uptake and increased cell cycling related genes. Conversely PD1- TAMs exhibited increased phagocytosis and antigen presentation. PD-1/PDL1 interaction directly regulated TAMs, as recombinant PDL-1 reduced glycolysis and phagocytosis in purified macrophages, and these effects could be reversed with blocking PD-1 antibody. Conversely, PD-1−/− TAMs had reduced lipid uptake but high rates of glycolysis, phagocytosis, and expression of MHC-II. Myeloid-specific PD-1 deficiency correlated with slower tumor growth and decreased LAG3 and increased CD69 on CD8 T cells. In addition, myeloid specific PD1 deficiency enhanced TAM antigen presentation through OVA specific CD8 T cell activation. These findings identify PD-1 as a metabolic regulator in TAM dysfunction and reveal a unique PD-1 mediated and macrophage-specific mechanism for immune tumor surveillance and checkpoint blockade. This may contribute to improved immunotherapy response in TAM-enriched tumors and obesity. Citation Format: Jackie Bader, Jeffrey Rathmell. PD-1 directly suppresses macrophage metabolism to restrict anti-tumor immunity in an obesity-cancer connection [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 1369.

Simple SummaryWe recently proved that in human colorectal cancer, the presence of small or large tumor-associated macrophages (TAMs) is associated with different outcomes. To translate this biological data into a robust clinical marker means to identify in a single slide all TAMs, hundreds of cells, and then evaluate the area of each of them, a task unfeasible in the routine pathology workout. With the aim to develop a deep-learning pipeline to tackle this challenge, we selected, trained and tested three different approaches. The deep-learning pipeline based on the DeepLab-v3 architecture and semantic segmentation technique warrants the separation of TAMs from the background and the identification of single TAMs: this will easily allow the evaluation of their area.Quantitative analysis of Tumor Microenvironment (TME) provides prognostic and predictive information in several human cancers but, with few exceptions, it is not performed in daily clinical practice since it is extremely time-consuming. We recently showed that the morphology of Tumor Associated Macrophages (TAMs) correlates with outcome in patients with Colo-Rectal Liver Metastases (CLM). However, as for other TME components, recognizing and characterizing hundreds of TAMs in a single histopathological slide is unfeasible. To fasten this process, we explored a deep-learning based solution. We tested three Convolutional Neural Networks (CNNs), namely UNet, SegNet and DeepLab-v3, with three different segmentation strategies, semantic segmentation, pixel penalties and instance segmentation. The different experiments are compared according to the Intersection over Union (IoU), a metric describing the similarity between what CNN predicts as TAM and the ground truth, and the Symmetric Best Dice (SBD), which indicates the ability of CNN to separate different TAMs. UNet and SegNet showed intrinsic limitations in discriminating single TAMs (highest SBD ), whereas DeepLab-v3 accurately recognized TAMs from the background (IoU ) and separated different TAMs (SBD ). This deep-learning pipeline to recognize TAMs in digital slides will allow the characterization of TAM-related metrics in the daily clinical practice, allowing the implementation of prognostic tools.

Infiltration of macrophages into tumors is a hallmark of cancer progression, and re-educating tumor-associated macrophages (TAMs) toward an antitumor status is a promising immunotherapy strategy. However, the mechanisms through which cancer cells affect macrophage education are unclear, limiting the therapeutic potential of this approach. Here we conducted an unbiased genome-wide CRISPR screen of primary macrophages. Our study confirms the function of known regulators in TAM responses and reveals new insights into the behavior of these cells. We identify olfactory and vomeronasal receptors, or chemosensors, as important drivers of a tumor-supportive macrophage phenotype across multiple cancers. In vivo deletion of selected chemosensors in TAMs resulted in cancer regression and increased infiltration of tumor-reactive CD8+ T cells. In human prostate cancer tissues, palmitic acid bound to olfactory receptor 51E2 (OR51E2) expressed by TAMs, enhancing their protumor phenotype. Spatial lipidomics analysis further confirmed the presence of palmitic acid in close proximity to TAMs in prostate cancer, supporting the function of this lipid mediator in the tumor microenvironment. Overall, these data implicate chemosensors in macrophage sensing of the lipid-enriched milieu and highlight these receptors as possible therapeutic targets for enhancing antitumor immunity.

The tumor microenvironment (TME) is an essential contributor to the development and progression of malignancy. Within the TME, tumor associated macrophages (TAMs) mediate angiogenesis, metastasis, and immunosuppression, which inhibits infiltration of tumor-specific cytotoxic CD8+ T cells. In previous work, we demonstrated that the synthetic triterpenoid CDDO-methyl ester (CDDO-Me) converts breast TAMs from a tumor-promoting to a tumor-inhibiting activation state in vitro. We show now that CDDO-Me remodels the breast TME, redirecting TAM activation and T cell tumor infiltration in vivo. We demonstrate that CDDO-Me significantly attenuates IL-10 and VEGF expression but stimulates TNF production, and reduces surface expression of CD206 and CD115, markers of immunosuppressive TAMs. CDDO-Me treatment redirects the TAM transcriptional profile, inducing signaling pathways associated with immune stimulation, and inhibits TAM tumor infiltration, consistent with decreased expression of CCL2. In CDDO-Me-treated mice, both the absolute number and proportion of splenic CD4+ T cells were reduced, while the proportion of CD8+ T cells was significantly increased in both tumors and spleen. Moreover, mice fed CDDO-Me demonstrated significant reductions in numbers of CD4+ Foxp3+ regulatory T cells within tumors. These results demonstrate for the first time that CDDO-Me relieves immunosuppression in the breast TME and unleashes host adaptive anti-tumor immunity.