Tumor-associated Macrophages Populations Research Articles

HUNK Regulation of Interleukin 4 in Triple Negative Breast Cancer

Triple‐negative breast cancer (TNBC) is a type of breast cancer that does not express hormone receptors (estrogen receptor or progesterone receptor) or human epidermal growth factor receptor 2. Therefore, contrary to other types of breast cancer, TNBC is not sensitive to endocrine therapy or HER2 targeted inhibitors. Some new FDA approve as treatments for TNBC include immunotherapy targets such as immune checkpoint inhibitor (ICI). Unfortunately, inadequate anti‐tumor T‐cell effector function and high abundances of tumor‐associated macrophages (TAMs) has limited the efficacy of ICI therapy. TAMs constituted one of the most abundant immune cell population in mammary tumors. Within the tumor TAMS can be polarized to classicallyactivatedM1‐like and alternativelyactiveM2‐likephenotypes.M2‐likemacrophagescontribute to cancer progression by inducing tumor angiogenic responses, promoting tumor growth and metastasis. In cancer the polarization of macrophages toward a M2 phenotype is directed by cancer‐cell derived factors such as pleiotropic cytokines like interleukin‐4 (IL‐4). New knowledge suggests that IL‐4 expression in cancer cells is regulated by the signal transducer and activator of transcription 3 (STAT3) transcription factor. However, it is still undescribed what signaling pathways are responsible for mediating IL‐4 in breast cancer cells. Intriguingly, we observed that Hormonally Up‐Regulated Neu‐Associated Kinase (HUNK), is responsible for IL‐4 production in the 4T1 mammary tumor cell line, which is considered a metastatic TNBC model. We hypothesis that HUNK regulation of IL‐4 drives TAM’s polarization in triple negative breast cancer cells. We engineered4T1 cells expressing HUNK and HUNK knockdown by shRNA. Our current data shown that4T1 cells expressing HUNK, have elevated levels of STAT3 phosphorylation, IL‐4 production and secretion compared to 4T1 cells where HUNK has been downregulated by shRNA. Furthermore, the loss of IL‐4 secretion in 4T1 HUNK knockdown cells corresponds to a reduced ability of conditioned medium from HUNK knockdown cells to induce alternative activation of macrophages. We also observed that HUNK has a significant effect on M2‐likeTAMspresence in the tumor microenvironment, where tumors derived from HUNK knockdown 4T1 cells have reduced TAMs compared to control tumors. Therefore, our results proposed the identification of a HUNK signaling pathway that is responsible for pro‐metastatic TAM function in TNBC. Our study will evaluate HUNK as a therapeutic target for TNBC metastasis by modulating the TAM population within the tumor microenvironment.

Tumor-associated macrophages related signature in glioma

Background: Glioma is the most common malignant primary tumor with a poor prognosis. Infiltration of tumor-associated macrophages (TAMs) is a hallmark of glioma. However, the regulatory mechanism of TAMs and the prognostic value of related signature in glioma remain unclear.Methods: TAMs were analyzed by EPIC, MCPCOUNTER and XCELL methods in multiple cohorts, including the TCGA merged GBMLGG, CGGA mRNAseq-325, and CGGA mRNAseq-693. Weighted correlation network analysis (WGCNA) were performed to identify candidate hub genes that might be related to TAMs. The prognostic genes were selected by Univariate Cox regression, Kaplan-Meier analysis and the least absolute shrinkage and selection operator (LASSO) multivariate Cox regression algorithm, and were used to construct a high efficacy prediction model.Results: Compared with LGG, TAMs of GBM in the TCGA merged GBMLGG, CGGA mRNAseq-693, and CGGA mRNAseq-325 cohorts were increased, and high TAMs levels predicted poorer overall survival for gliomas. The prediction model constructed by nine prognostic genes was highly efficient. The TAMs related risk-score was an independent risk factor for glioma. Moreover, high risk score was correlated with an increased population of TAMs in glioma, as well as the high immune scores, stromal scores and ESTIMATE scores.Conclusions: Increased TAMs might be an immune evasion mechanism of glioma. In addition, our findings suggested that TAMs-related signature was a valuable prognostic biomarker in glioma and provided therapeutic targets for glioma.

Aqueous Extract of Cimicifuga dahurica Reprogramming Macrophage Polarization by Activating TLR4-NF-κB Signaling Pathway.

PurposeCimicifuga dahurica (C. dahurica), which has been used in traditional oriental medicine for a long period, was reported to exert extensive antitumor activity, but the effect and molecular biological mechanism of C. dahurica on multiple myeloma (MM) has not been elaborated. Tumor-associated macrophages (TAMs) exhibit a sustained polarization between tumor killing M1 subtype and tumor supporting M2 subtype. And a lower ratio of M1/M2 is associated with tumor angiogenesis, proliferation and invasion. We explored the inhibitory effect of the aqueous extract of the root of C. dahurica (CRAE) on tumor growth by reprogramming macrophage polarization in the tumor microenvironment.MethodsMice bearing SP2/0 multiple myeloma were treated with CRAE. Western blotting (WB), immunohistochemistry (IHC) and immunofluorescence staining were utilized to assess tumor growth and TAM populations. Macrophages were depleted by injection of clodronate liposomes to determine and measure the role of CRAE as an anti-tumor agent by targeting macrophages. To simulate tumor microenvironment, MM cells H929 and TAMs were co-cultured using the transwell co-culture system. By using CRAE as an immunoregulator in M2-like macrophages, we analyzed CRAE-treated macrophage-associated surface markers and cytokines by flow cytometry and WB.ResultsThe results indicated that CRAE treatment could reduce tumor burden of MM mice and a high degree of M1-like macrophages infiltration was detected in tumor tissues. In vitro co-culture system, CRAE significantly promoted the polarization of M2 to M1 phenotype, which led to the increase in apoptosis of myeloma cells. It was found that the M1 polarization induced by CRAE depended on the TLR4-MyD88-TAK1-NF-κB signal transduction.ConclusionThis study elucidated the anticancer mechanism of the aqueous extract of C. dahurica (CRAE) through reprogramming macrophage polarization and highlighted that CRAE could act as a potential novel option for cancer immunotherapy.

Immunostimulation of tumor microenvironment by targeting tumor-associated macrophages with hypoxia-responsive nanocomplex for enhanced anti-tumor therapy

Tumor-associated macrophages (TAMs), which dampen the therapeutic efficacy of cancer immunotherapy, are the key players in the immunosuppressive tumor microenvironment (TME). Therefore, reprogramming TAMs into tumoricidal M1 macrophages possesses considerable potential as a novel immunotherapy. However, the low bioavailability of polarization agents and limited accumulation of TAMs restrict their anti-tumor efficacy. In this study, we developed a polymer-based hypoxia-responsive nanocomplex to target TAMs in hypoxia for enhanced cancer immunotherapy. We synthesized a hypoxia-cleavable polymer poly(ethylene glycol)-azo-poly(l-lysine) (PEG-azo-PLL) and formulated a nanocomplex by simple mixing PEG-azo-PLL and poly(I:C). By mimicking in vitro hypoxia conditions, PEG-azo-PLL/poly(I:C) complexes could transform the physicochemical properties to enhance the delivery efficiency of poly(I:C) to tumor hypoxia, where M2-like TAMs are accumulated. Furthermore, PEG-azo-PLL/poly(I:C) could successfully reduce the population of M2-like TAMs in hypoxic tumors and promoted infiltration of CD8+ T cells in vivo, resulting in the favorable conversion of immunosuppressive TME. Finally, PEG-azo-PLL/poly(I:C) could elicit a significant in vivo anti-tumor effect in B16F10-bearing mice in addition to a prolonged survival time, demonstrating that the hypoxia-responsive nanocomplex PEG-azo-PLL/poly(I:C) is a promising approach for TAM reprogramming immunotherapy for solid tumors.

“THE EFFECT OF GROWTH DIFFERENTIATION FACTOR 11 (GDF11) ON THE RESPONSE OF TUMOR-ASSOCIATED MACROPHAGES IN HEPATOCELLULAR CARCINOMA DERIVED CELLS”

Hepatocellular carcinoma (HCC) is the most common and aggressive form of liver cancer. Until 2020, worldwide, it has been ranked 5 th and 3 rd in terms of incidence and mortality, respectively. HCC has various etiologies, but tumors formed by high cholesterol intake induce an aggressive phenotype and high tumor-associated macrophages (TAM) infiltration. TAM acquires an alternative polarity (M2) with immunosuppressive and pro-tumoral activities. HCC patients with high TAM recruitment have a poor prognosis with a low free-survival rate. The design of therapies represents a challenge. The growth differentiation factor 11 (GDF11) has been proposed as a promising molecule for treating HCC, favors a reduction in proliferation, invasion, and lipid metabolism, and therefore reduces the aggressive phenotype. In our research group, we have found that GDF11 also has various effects on cells belonging to the tumor microenvironment, particularly in TAM. To evaluate TAM polarity and their intercommunication with HCC-derived cells during treatment with GDF11. THP-1-macrophages cell line was used; lipopolysaccharides were used for the M1 polarity acquisition and IL-4/13 or conditioned medium (CM) derived from Huh-7 cells for M2 polarity acquisition, flow cytometry (FCM) was used. GDF11 (50 ng/mL) was applied every 24 h up to 3 days. We used anti-Smad2 and 3 antibodies in Western blot (WB). Cell viability and proliferation studies were performed using crystal violet. The quantification of cholesterol was performed using o-Phthaldialdehyde and free radicals’ detection was performed using dihydroethidium. Data were presented as mean ± standard error of the mean (SEM). The analysis of variance (ANOVA) test was used to compare the mean values ​​between groups. Each result has at least 3 independent experiments. Statistical significance was indicated with an asterisk (*P<0.05). GDF11 treatments induced Smad proteins activation by specific phosphorylation, indicating that THP-1 macrophages respond to treatment in times from 5 to 60 min, but maintaining their response up to 72 h. This could suggest that the difference in the subsequent response may be involved in the binding or activation of additional adapter proteins of this signaling pathway. Furthermore, it was observed that GDF11 does not affect the cell viability, proliferation, and morphology studied in their different polarities. Macrophage re-education or re-polarization assays were carried out by adding GDF11 for an additional 72h. The data obtained show that TAM populations lost CD206 marker, going from 90% to 40% of the population, suggesting a loss of this pro-tumor polarity and a re-education towards anti-tumor macrophages responses. However, experiments show a dual role of GDF11 in macrophages due to the increase of specific markers in non-activated cells, indicating that it has a different effect depending on the activation state. Since GDF11 compromises cholesterol metabolism, which is observed in a decrease in total cholesterol levels in non-activated macrophages, which is observed in other studies using HCC cell lines. Also, it was possible to corroborate that GDF11 is behaving like a statin used as a positive control in macrophages and Huh-7 cells. Finally, GDF11 increases ROS levels, specifically superoxide anion, characteristic of phagocytic M1 macrophages. To evaluate response on HCC cells, wound-healing assays indicated that macrophage secretion is a key factor for cell displacement rather than viability and proliferation. In the present study, we found that GDF11 has immunomodulatory effects in TAM, decreasing pro-tumoral markers and lipid content that could be important to design HCC therapies. The authors declare that there is no conflict of interest.

Leptin Augments Antitumor Immunity in Obesity by Repolarizing Tumor-Associated Macrophages.

Although obesity can promote cancer, it may also increase immunotherapy efficacy in what has been termed the obesity-immunotherapy paradox. Mechanisms of this effect are unclear, although obesity alters key inflammatory cytokines and can promote an inflammatory state that may modify tumor-infiltrating lymphocytes and tumor-associated macrophage populations. To identify mechanisms by which obesity affects antitumor immunity, we examined changes in cell populations and the role of the proinflammatory adipokine leptin in immunotherapy. Single-cell RNAseq demonstrated that obesity decreased tumor-infiltrating lymphocyte frequencies, and flow cytometry confirmed altered macrophage phenotypes with lower expression of inducible NO synthase and MHC class II in tumors of obese animals. When treated with anti-programmed cell death protein 1 (PD-1) Abs, however, obese mice had a greater absolute decrease in tumor burden than lean mice and a repolarization of the macrophages to inflammatory M1-like phenotypes. Mechanistically, leptin is a proinflammatory adipokine that is induced in obesity and may mediate enhanced antitumor immunity in obesity. To directly test the effect of leptin on tumor growth and antitumor immunity, we treated lean mice with leptin and observed tumors over time. Treatment with leptin, acute or chronic, was sufficient to enhance antitumor efficacy similar to anti-PD-1 checkpoint therapy. Further, leptin and anti-PD-1 cotreatment may enhance antitumor effects consistent with an increase in M1-like tumor-associated macrophage frequency compared with non-leptin-treated mice. These data demonstrate that obesity has dual effects in cancer through promotion of tumor growth while simultaneously enhancing antitumor immunity through leptin-mediated macrophage reprogramming.

Imaging of Glioblastoma Tumor-Associated Myeloid Cells Using Nanobodies Targeting Signal Regulatory Protein Alpha

Glioblastoma (GBM) is the most common malignant primary brain tumor. Glioblastomas contain a large non-cancerous stromal compartment including various populations of tumor-associated macrophages and other myeloid cells, of which the presence was documented to correlate with malignancy and reduced survival. Via single-cell RNA sequencing of human GBM samples, only very low expression of PD-1, PD-L1 or PD-L2 could be detected, whereas the tumor micro-environment featured a marked expression of signal regulatory protein alpha (SIRPα), an inhibitory receptor present on myeloid cells, as well as its widely distributed counter-receptor CD47. CITE-Seq revealed that both SIRPα RNA and protein are prominently expressed on various populations of myeloid cells in GBM tumors, including both microglia- and monocyte-derived tumor-associated macrophages (TAMs). Similar findings were obtained in the mouse orthotopic GL261 GBM model, indicating that SIRPα is a potential target on GBM TAMs in mouse and human. A set of nanobodies, single-domain antibody fragments derived from camelid heavy chain-only antibodies, was generated against recombinant SIRPα and characterized in terms of affinity for the recombinant antigen and binding specificity on cells. Three selected nanobodies binding to mouse SIRPα were radiolabeled with 99mTc, injected in GL261 tumor-bearing mice and their biodistribution was evaluated using SPECT/CT imaging and radioactivity detection in dissected organs. Among these, Nb15 showed clear accumulation in peripheral organs such as spleen and liver, as well as a clear tumor uptake in comparison to a control non-targeting nanobody. A bivalent construct of Nb15 exhibited an increased accumulation in highly vascularized organs that express the target, such as spleen and liver, as compared to the monovalent format. However, penetration into the GL261 brain tumor fell back to levels detected with a non-targeting control nanobody. These results highlight the tumor penetration advantages of the small monovalent nanobody format and provide a qualitative proof-of-concept for using SIRPα-targeting nanobodies to noninvasively image myeloid cells in intracranial GBM tumors with high signal-to-noise ratios, even without blood-brain barrier permeabilization.

Inhibition of FGFR Reactivates IFNγ Signaling in Tumor Cells to Enhance the Combined Antitumor Activity of Lenvatinib with Anti-PD-1 Antibodies.

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb. SIGNIFICANCE: FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

P-060: Subclone-specific microenvironmental impact and drug response in refractory multiple myeloma revealed by single cell transcriptomics

<h3>Background</h3> Relapsed/refractory multiple myeloma (RRMM) is characterized by a high inter- and intratumor heterogeneity and a complex interplay of myeloma cells with the bone marrow microenvironment (BME). Accordingly, there is an urgent need to dissect subclone structure, transcriptional heterogeneity and cellular interactions to unravel the molecular mechanisms underlying drug resistance in RRMM. <h3>Methods</h3> Single cell RNA sequencing (scRNA-seq) of ~210,000 cells from bone marrow aspirates sorted into CD138+ and CD138– fractions was conducted for 20 heavily pretreated RRMM patients. RRMM subclones were called from the scRNA-seq data based on a copy number aberration (CNA) analysis that was confirmed by interphase fluorescence in situ hybridization and whole genome sequencing. From the scRNA-seq data the composition and abundance of immune cell types in the BME was determined. Interactions of myeloma cells with the BME were characterized by an analysis of the correlated expression of ligand-receptor pairs. Selected findings from the scRNA-seq analysis were validated by flow cytometry. <h3>Results</h3> Subclones with distinct chromosomal aberrations were reliably identified at the single cell level based on CNAs inferred from scRNA-seq data. The analysis revealed a subclonal 1q-gain (+1q) in 10/20 samples, for which a gene expression signature of recurrently upregulated genes was derived. These +1q subclones frequently expanded during treatment. Furthermore, RRMM cells shaped an immune suppressive BME by upregulation of inflammatory cytokines and close interplay with the myeloid compartment. RRMM cells appear to reprogram the BME by upregulation of inflammatory cytokines and ligands of inhibitory receptors expressed on T and NK-cells. Specifically, the immune cell compartment of RRMM patients displayed an accumulation of PD1+ γδ T-cells and myeloid populations compared to healthy donors or early disease. In addition, we observed a depletion of hematopoietic progenitors in patients with high expression of genes associated with inflammatory signaling in the BME. Upon treatment with immunomodulatory drugs, reprogrammed plasmacytoid dendritic cells expanded. Focusing on patients with +1q, we found an enrichment of a rare M2-like tumor associated macrophage population while GZMB+ NK effector cells were depleted in these patients, indicating that +1q has a distinct effect on the BME in RRMM. <h3>Conclusions</h3> Our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming. The insight gained in our study on the evolution of RRMM heterogeneity and its bone marrow milieu will support the development of novel treatment approaches and potentially guide clinical decision making.

Targeting TREM2 on tumor-associated macrophages enhances immunotherapy.

Converting checkpoint inhibitor (CPI)-resistant individuals to being responsive requires identifying suppressive mechanisms. We identify TREM2+ tumor-associated macrophages (TAMs) as being correlated with exhausted CD8+ tumor-infiltrating lymphocytes (TILs) in mouse syngeneic tumor models and human solid tumors of multiple histological types. Fc domain-enhanced anti-TREM2 monoclonal antibody (mAb) therapy promotes anti-tumor immunity by elimination and modulation of TAM populations, which leads to enhanced CD8+ TIL infiltration and effector function. TREM2+ TAMs are most enriched in individuals with ovarian cancer, where TREM2 expression corresponds to disease grade accompanied by worse recurrence-free survival. In an aggressive orthotopic ovarian cancer model, anti-TREM2 mAb therapy drives potent anti-tumor immunity. These results highlight TREM2 as a highly attractive target for immunotherapy modulation in individuals who are refractory to CPI therapy and likely have a TAM-rich tumor microenvironment.

Cross-tissue single-cell landscape of human monocytes and macrophages in health and disease

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1+CD274(PD-L1)+IDO1+ macrophages, which accumulated in the tumor periphery in a Tcell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory Tcell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states.

OTME-6. Deep sequencing reveals heterogeneity of brain metastasis-associated macrophages and microglia and uncovers their cell type-specific functions within the tumor microenvironment

Macrophages represent a highly plastic cell type,indispensable for tissue and organ homeostasis, as well as innate immunity. Basic and translational research attributed tumor-promoting functions to macrophages, and their presence is often associated to poor patient prognosis and therapy resistance. While brain-resident macrophages, the so-called microglia (MG), represent the major immune cell type in the parenchyma under normal conditions, primary and metastatic brain tumors induce the recruitment of different immune cell types from the periphery, including monocyte-derived macrophages (MDM). Controversy remained about the redundancy of disease-associated molecular signatures and functions. The identification of markers that reliably distinguish brain-resident from blood-borne tumor-associated macrophages (TAMs) allowed the interrogation of molecular traits of different TAM populations in mouse and human brain tumors.Using RNA-Seq, we demonstrated that TAMs rapidly acquire disease-associated transcriptional programs upon initial tumor infiltration, while gene expression remained stable during different stages of BrM progression. Across different BrM models, disease-associated transcriptional changes revealed lineage-specific, non-redundant functions of TAM populations, which was further reflected by cell type-specific occupation of different niches within the BrM microenvironment. Furthermore, we observed dose- and cell type-specific immune modulatory effects of whole brain radiotherapy on myeloid cells in BrM leading to a transient loss of disease-associated transcriptional programs predominately in blood-borne myeloid populations. This effect can at least in part be attributed to a replenishment of the recruited macrophage pool. This observation was further supported by scRNA-Seq analyses revealing higher heterogeneity of TAM-MDM compared to TAM-MG under treatment-naïve conditions and in response to radiotherapy.Together, our results point towards the phenotypic plasticity of TAMs, especially MDMs, and the contribution of each compartment in instigating cancer-associated inflammation or the establishment of an immuno-suppressive TME. While TAM-MG exert functions related to pro-inflammatory responses, TAM-MDM are rather involved in tissue repair and regulation of adaptive immune cell functions.

Abstract 113: Lyve-1 expressing macrophages direct the expansion of pericytes within the perivascular niche to support angiogenesis in cancer

Abstract Despite the many pro-tumoral functions that have been described for the various stromal populations in cancer, it is becoming apparent that these cells are not working alone, but instead in concert with one another as part of a wider network of cross-communication to facilitate disease progression. Identifying non-redundant signaling pathways within the stromal network is desirable, as therapeutically targeting these signals could result in an unraveling of the stromal support network upon which the tumor cells rely. Tumor associated macrophages (TAMs) have been implicated in a variety of pro-tumoral processes and are polarized by signals in the tumor microenvironment (TME) to adopt a range of functionally distinct subsets to perform these tasks. Single-cell RNA-sequencing of the F4/80+ TAMs from the MMTV-PyMT murine model of mammary adenocarcinoma identified one subset selectively expressing Lyve-1 and adopting a CD206hiMHCIIlo phenotype. This population was identified to spatially reside in the niche proximal to blood vasculature within the tumor. We demonstrate that selective depletion of the Lyve-1+ TAM population using liposome-based approaches controlled tumor growth and was associated with a loss of perivascular alpha smooth muscle actin (αSMA)-expressing stromal cells. The αSMA+ stromal cells were characterized ex vivo and expressed a surface phenotype associated with pericytes (CD90+CD34-NG2+), highlighting a possible role for Lyve-1+ TAMs in maintaining the population. To exclude a role of Lyve-1+ TAMs in recruiting a pericyte-progenitor cell to the tumor, we selectively photo-labelled the TME using MMTV-PyMT mice ubiquitously expressing the photo-convertible green/red Kaede protein. Labelling the TME Kaede-red through exposure to ultraviolet light, demonstrated that pericytes and CD90+ mesenchymal cells more broadly, were not recruited from a peripheral site but instead suggested these populations must rely on local proliferation to maintain their prevalence in the TME. Indeed, we demonstrate through Edu incorporation, that loss of Lyve-1+ TAMs from the perivascular niche renders pericytes senescent. Using computational approaches, we characterized the signaling cross-talk in the perivascular niche between Lyve-1+ TAMs, pericytes and endothelial cells and identified a specific interaction involving platelet derived growth factor-CC (PDGF-CC) expression by Lyve-1+ TAMs and PDGF-receptor alpha (PDGFRα) on the pericytes. Using blocking antibodies to PDGF-CC in tumor bearing mice we demonstrate that blocking this axis of stromal crosstalk can render pericytes senescent. These data highlight that local expansion of pericytes in cancer is not an autonomous event, nor regulated by the endothelium alone, but tightly regulated by the perivascular Lyve-1+ TAM population, which dictates the success of angiogenesis in cancer. Citation Format: James W. Opzoomer, Joanne E. Anstee, Isaac Dean, Emily J. Hill, Ihssane Bouybayoune, Jonathan Caron, Tamara Muliaditan, Peter Gordon, Rosamond Nuamah, Sarah E. Pinder, Tony Ng, Francesco Dazzi, Shahram Kordasti, David R. Withers, Toby Lawrence, James N. Arnold. Lyve-1 expressing macrophages direct the expansion of pericytes within the perivascular niche to support angiogenesis in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 113.

Overexpression of Interferon-Inducible Protein 16 Promotes Progression of Human Pancreatic Adenocarcinoma Through Interleukin-1β-Induced Tumor-Associated Macrophage Infiltration in the Tumor Microenvironment.

Activation of inflammasomes has been reported in human pancreatic adenocarcinoma (PAAD); however, the expression pattern and functional role of inflammasome-related proteins in PAAD have yet to be identified. In this study, we systemically examined the expression and role of different inflammasome proteins by retrieving human expression data. Several genes were found to be differentially expressed; however, only interferon-inducible protein 16 (IFI16) expression was found to be adversely correlated with the overall survival of PAAD patients. Overexpression of IFI16 significantly promoted tumor growth, increased tumor size and weight in the experimental PAAD model of mice, and specifically increased the population of tumor-associated macrophages (TAMs) in the tumor microenvironment. Depletion of TAMs by injection of liposome clodronate attenuated the IFI16 overexpression-induced tumor growth in PAAD. In vitro treatment of conditioned medium from IFI16-overexpressing PAAD cells induced maturation, proliferation, and migration of bone marrow-derived monocytes, suggesting that IFI16 overexpression resulted in cytokine secretion that favored the TAM population. Further analysis suggested that IFI16 overexpression activated inflammasomes, thereby increasing the release of IL-1β. Neutralization of IL-1β attenuated TAM maturation, proliferation, and migration induced by the conditioned medium from IFI16-overexpressing PAAD cells. Additionally, knockdown of IFI16 could significantly potentiate gemcitabine treatment in PAAD, which may be associated with the reduced infiltration of TAMs in the tumor microenvironment. The findings of our study shed light on the role of IFI16 as a potential therapeutic target for PAAD.

A phase 1, multicenter, open-label, dose-escalation, safety, pharmacodynamic, pharmacokinetic study of Q702 with a cohort expansion at the RP2D in patients with advanced solid tumors.

TPS2673 Background: Immune checkpoint inhibitors directly targeting T cell activation have been successfully used in the treatment of various malignancies, nevertheless, the durable ORRs are low for certain indications. The low ORRs have been attributed to the immune suppressive tumor microenvironment (TME), composed of innate immune suppressive components such as tumor associated macrophages (TAM) and myeloid-derived suppression cells (MDSC). The potential contributions of innate immune modulation to anti-tumor immunity, suggest the need for the novel strategies to elicit a more efficient/robust immune response against the targeted malignant cells. Axl, Mer and CSF1R receptor tyrosine kinases play vital roles in promoting an immune suppressive TME by affecting TAM and MDSC populations and by decreasing antigen presentation on tumor cells. Q702 is a novel Axl/Mer/CSF1R inhibitor, able to modulate the TAM and MDSC population leading to CD8+ T cell activation and to increase antigen presentation of the tumor cells in syngeneic animal models. Q702, as a monotherapy, shows significant tumor growth inhibition in multiple syngeneic tumor models, and demonstrates synergistic effects with anti-PD-1 treatment particularly in high myeloid containing tumor models. Interestingly, intermittent administration of Q702 monotherapy demonstrates a more favorable immune cell population changes, possibly through preventing immune exhaustion secondary to negative feedback with continuous activation. These results suggest that Q702 monotherapy or in combination with existing therapies have a good potential to become a novel treatment strategy for patients with advanced solid tumors. Methods: “A Phase 1, Multicenter, Open-label, Dose-Escalation, Safety, Pharmacodynamic, Pharmacokinetic Study of Q702 with a Cohort Expansion at the RP2D in Patients with Advanced Solid Tumors. (NCT04648254)” is open and recruiting patients at 4 US investigative sites. Patients with histologically or cytologically confirmed advanced or metastatic solid tumors, that have progressed following SOC or for which there is no SOC which confers clinical benefit are being enrolled in this study. The study follows a standard dose escalation. The study will enroll up to 78 patients. The primary endpoint is to establish safety, PK profile and define the recommended phase 2 dose. The secondary and exploratory endpoints include establishing pharmacokinetic/pharmacodynamic relationship, potential biomarkers and preliminary anti-tumor activity. Clinical trial information: NCT04648254.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Abstract PR-005: Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment

Abstract Introduction/Purpose: Radiation therapy drives pro-inflammatory changes to the tumor microenvironment (TME). We have shown that this may be countered by the induction of immunosuppressive changes that drive therapy resistance. Here, we test the hypothesis that radiation therapy induces immunosuppressive myeloid cells in the TME in an adenosine-dependent fashion. Methods: Using the 4T1 orthotopic breast cancer mouse model, we tested the immunophenotypic changes to the TME by spectral flow cytometry and bulk RNASeq of tumor infiltrating leukocytes (CD45+, TIL) 72-hours after tumor-directed radiation therapy (8 Gy x 1) using the small animal radiation research platform (SARRP, Xstrahl; 220 kV). We cultured human peripheral blood mononuclear cells (PBMCs) with 0.1, 1, and 10uM of adenosine receptor agonist, 5’-N-ethylcarboxamindoadenosine (NECA), in the presence of CD3/CD28 beads. Cytokine quantification of cell-free supernatant was performed 48-hours later using the Meso Scale Discovery platform. Results: Radiation increases the abundance of CD45+CD11b+ myeloid cells (77.4% vs. 84.4%) and decreased CD45+TCRb+ lymphocytes (6.3% vs. 3.6%) in the TME, compared to untreated controls. The reduction of lymphocytes was driven by CD4+ T-cells (5.2% vs 2.7%), including a significant reduction Tregs (1.4% vs 0.63%), with no change in abundance of CD8+ T-cells (0.53% vs 0.43%). The increase in myeloid cells was predominantly influenced by an induction of granulocytic and monocytic myeloid-derived suppressor cells (PMN-MDSCs: 22% vs 34.5%; M-MSCSs: 3.0% vs 10.0%). Radiation caused a decrease in the tumor-associated macrophage population (TAM: 50% vs 31.1%) in the setting of a phenotypic shift to from pro-inflammatory M1-like to immunosuppressive M2-like TAM phenotype: M2-like TAM/M1-like TAM ratio 2.0 vs 13.3. Radiation increased the expression of adenosine 2A receptor (A2AR, log2 fold change 1.13) and A2BR (log2 fold change 0.92) by TILs, but not CD73 that metabolizes AMP to adenosine. The addition of NECA to human PBMCs resulted in significant reduction in effector T-cell cytokines (IL-2, IFN- γ, granzyme B) at all NECA concentrations tested and a dose-dependent decrease in GM-CSF, IL-12p40 and IL-23. There was a significant reduction of IL-1 β, IL-8, IL-12p70 and MCP-1 only at the highest dose (10uM) tested. IL-6 was increased at the intermediate dose (1uM) of NECA with an overall reduction with high-dose NECA. High-dose NECA alone significantly increased both IFN- β and TGF- β. Conclusion: Radiation induces an immunosuppressive TME, increasing the abundance of PMN-MDSCs, M-MDSCs and a shifting the TAM population towards an immunosuppressive phenotype. Radiation increases the expression of adenosine receptors on TILs and treatment of human PBMCs with adenosine receptor agonist resulted in loss of effector T-cell function and suppressive changes to cytokine milieu suggesting a mechanistic link between radiation and adenosine signaling. Citation Format: Shruti Bansal, Mathew Chaimowitz, Casey R. Ager, Aleksandar A. Obradovic, Charles G. Drake, Catherine S. Spina. Adenosine signaling and radiation-induced immunosuppressive changes to the tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PR-005.

Chemotherapy Induces Tumor-Associated Macrophages that Aid Adaptive Immune Responses in Ovarian Cancer.

Neoadjuvant chemotherapy (NACT) may stimulate anticancer adaptive immune responses in high-grade serous ovarian cancer (HGSOC), but little is known about effects on innate immunity. Using omental biopsies from HGSOC, and omental tumors from orthotopic mouse HGSOC models that replicate the human tumor microenvironment, we studied the impact of platinum-based NACT on tumor-associated macrophages (TAM). We found that chemotherapy reduces markers associated with alternative macrophage activation while increasing expression of proinflammatory pathways, with evidence of inflammasome activation. Further evidence of a shift in TAM functions came from macrophage depletion via CSF1R inhibitors (CSF1Ri) in the mouse models. Although macrophage depletion in established disease had no impact on tumor weight or survival, CSF1Ri treatment after chemotherapy significantly decreased disease-free and overall survival. This decrease in survival was accompanied by significant inhibition of adaptive immune response pathways in the tumors. We conclude that chemotherapy skews the TAM population in HSGOC toward an antitumor phenotype that may aid adaptive immune responses, and therapies that enhance or sustain this during remission may delay relapse.

Characterization of the inflammatory microenvironment and hepatic macrophage subsets in experimental hepatocellular carcinoma models.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)- and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

M2 macrophage-targeted iron oxide nanoparticles for magnetic resonance image-guided magnetic hyperthermia therapy

Tumor-associated macrophages (TAMs) play an important role in tumor development and progression. In particular, M2 TAMs can promote tumor growth by facilitating tumor progression and malignant behaviors. Selectively targeted elimination of M2 TAMs to inhibit tumor progression is of great significance for cancer treatment. Iron oxide nanoparticles based magnetic hyperthermia therapy (MHT) is a classical approach to destroy tumor tissue with deep penetration depth. In this study, we developed a typical M2 macrophage-targeted peptide (M2pep) functionalized superparamagnetic iron oxide nanoparticle (SPIO) for magnetic resonance imaging (MRI)-guided MHT in an orthotopic breast cancer mouse model. The obtained multifunctional SPIO-M2pep with a hydrodynamic diameter of 20 nm showed efficient targeting capability, high transverse relaxivity (149 mM−1 s−1) and satisfactory magnetic hyperthermia performance in vitro. In vivo studies demonstrated that the SPIO-M2pep based MRI can monitor the distribution of nanoparticles in tumor and indicate the suitable timing for MHT. The M2 macrophage-targeted MHT significantly reduced the tumor volume and the population of pro-tumoral M2 TAMs in tumor. In addition, the SPIO-M2pep based MHT can remodel the tumor immune microenvironment (TIME). The multifunctional SPIO-M2pep with M2 macrophage-targeting ability, high magnetic hyperthermia efficiency, MR imaging capability and effective role in remodeling the TIME hold great potential to improve clinical cancer therapy outcomes.