Metabolic reprogramming of efferocytosis in the tumour microenvironment: From apoptotic-cell clearance to therapeutic targeting.

Tumor associated macrophages (TAMs) are one of the most common types of stromal cells in solid tumors. They are closely related to the immunosuppressive status of tumor microenvironment and potentiate the malignant progress of tumors. Studies have shown that metabolism in tumor associated macrophages has been reprogrammed and involved in the regulation of their own polarization and corresponding functions and phenotypes. Metabolic reprogramming refers to the alteration of key enzymes activity, substrate and its associated metabolites' concentration in a certain metabolic pathway, which accounts for the disorder of original metabolic states. In this paper, we mainly concentrated on the lipid metabolic reprogramming of TAMs, including triglycerides, fatty acids and their derivatives, cholesterol, phospholipids, and their regulations on tumor progression. However, the metabolism of tumor and tumor microenvironment cells is highly heterogeneous. It is worthy of further exploration on the similarities and differences of lipid metabolism reprogramming between stromal cells and tumor cells, and the mechanism of how reprogramming modulates cell activity. It will be a new strategy for immunotherapy of tumor with metabolic intervention to accurately target the lipid metabolism reprogramming of TAMs, so as to promote the polarization of TAMs to M1 like macrophages, when synthetically considering the diverse types of tumors and different stages of development.

Macrophage-mediated Bystander Effect Triggered by Tumor Cell Apoptosis

Author response: Comprehensive characterization of tumor microenvironment in colorectal cancer via molecular analysis

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

Background The tumor microenvironment (TME) plays integral roles in prostate cancer progression and therapeutic resistance. The development of effective TME-targeted therapies is limited by current technologies which are insufficient to replicate or analyze this complex environment. To address these challenges, we have developed a microfluidic cell culture platform known as STACKS, which permits co-culture of up to 6 patient-derived cell populations as well as compartmentalized, multiplexed analysis of gene expression, cell signaling, and matrix remodeling. We have focused on investigation of tumor-associated macrophages (TAMs), which are traditionally classified as M2 (tumor-supportive) or M1 (tumor-destructive) and are high value therapeutic targets with roles in prostate cancer growth, metastasis, survival, and therapeutic resistance. Methods Cell line (THP-1) and patient-derived monocytes were differentiated into macrophages within the STACKs device and polarized to either a M1 or M2 state or left unpolarized. Unpolarized macrophages were cultured with androgen dependent (LNCaP) and independent (DU145,C4-2B) prostate tumor lines and cancer-associated fibroblasts (CAFs) derived from patient biopsies. Cells were cultured on 2D surfaces as well as within 3D matrix environments. Individual cell populations were isolated and analyzed for RNA, protein, and secretory factor expression. Results We report that M2 (CCL18, MRC1) as well as M1 (CXCL10, CXCL11) associated genes were more highly upregulated in THP-1s cultured with C4-2Bs than with LNCaPs. Select M1 and M2 genes (CXCL10 and CCL18) were also more highly expressed in THP-1s cultured with CAFs than with DU145s. While IL-10 expression was higher in THP-1s in co-culture with LNCaPs than with C4-2Bs on a 2D surface, the opposite was true when THP-1s were cultured within a 3D collagen I matrix. Conclusions Within a microscale co-culture environment, we have demonstrated that macrophage gene expression is influenced by androgen dependent and independent tumor cells, stromal cells, and the structural microenvironment. Expression of certain genes, such as IL-10, was strongly dependent on the integration of multiple TME signals, such as paracrine tumor factors and cell-matrix interactions. Additionally, while macrophages are traditionally thought to polarize either towards a M1 or a M2 state, there was concomitant elevation of both M1 and M2 genes with C4-2B and CAF co-culture. These findings highlight the complexity of macrophage polarization within the TME and represent potential therapeutic targets. We will continue to build on this data through 2D and 3D multi-culture of macrophages with tumor, stromal, and immune cells to target pathways involved in TAM polarization, tumor promotion, and therapeutic resistance. Citation Format: David Kosoff, Jiaquan Yu, Jennifer L. Schehr, David J. Beebe, Joshua M. Lang. Microscale engineering of the tumor microenvironment for therapeutic targeting of tumor-associated macrophages in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4922. doi:10.1158/1538-7445.AM2017-4922

The presence of immunosuppressive molecules and cells in the tumor microenvironment (TME) can lead to T cell dysfunction. CD8-positive cytotoxic T cells (CTLs) are ineffective in killing tumor cells primarily due to upregulated expression of inhibitory checkpoint molecules and decreased production of effective cytokines. In addition, immune suppressive cell types such as regulatory T cells (Tregs) and tumor associated macrophages (TAMs) are recruited to the TME, further establishing a suppressive immune environment. In this study, we evaluated expression profiles of key immunosuppressive molecules and cell types by applying RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, and dual ISH-IHC staining. First, we evaluated CD8-positive cell infiltration in TME of archived human tissues from non-small cell lung cancer and ovarian cancer. Selected tissues with either high or low CD8-positive cell number (CD8-high or CD8-low) were evaluated for (1) the presence of Tregs (FOXP3+CD4+) and TAMs (CD163+, including IL10 and CCL22), (2) the expression of immune checkpoint molecules including PD1, PD-L1, TIM3, and LAG3, (3) the expression of immune suppressive molecules IDO1 and TGFβ, and (4) IFNγ expression in CD8-positive subsets. Examination of immune inhibitory molecules expressed in single cells in the tumor and stromal microenvironment revealed that in general, CD8-high tissues expressed higher level of immune checkpoint molecules, often co-expressed in the same individual cells in the same TME, while the expression of IDO1 and TGFβ was independent of CD8-positive cell inflammation. Tregs and TAMs often co-existed with IFNγ-positive CTLs in the same TME. Unexpectedly, all investigated inhibitory molecules were expressed in both tumor cells and stromal/immune cells in some tumors. Beyond PD-L1, immune checkpoint molecules PD1, TIM3, and LAG3 were frequently expressed in tumor cells but at lower level than in immune cells. Expression of IDO1 and TGFβ was observed in many cell types, including tumor cells, with various expression levels in each tumor cell. The single-cell tumor expression profiles suggest a potential tumor-intrinsic mechanism of expression for these inhibitory molecules. This exploratory study highlights the potential of RNAscope® ISH to better understand the cellular and molecular suppressive mechanisms associated with T cell dysfunction and exhaustion in the TME. The robust RNAscope® ISH platform is well suited for evaluating critical secreted factors and other key molecules in a highly sensitive and cell type-specific manner. As multiple therapeutic approaches to augment the CTL function are being developed, the presented method may facilitate the identification and development of key biomarkers to stratify patients based on their specific tumor and immune cell states. Citation Format: Annelies Laeremans, Na Li, Jeff Kim, Xiao-Jun Ma, Emily Park. Evaluation of potential factors contributing to the exhaustion of T lymphocytes in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1022.

Bacterial minicells to the rescue: cyto-Immunotherapy for the treatment of late stage cancers with minimal to no toxicity.

Reinforcing Suppression Using Regulators: A New Link between STAT3, IL-23, and Tregs in Tumor Immunosuppression

Breast cancer is the most common invasive malignancy and a leading cause of cancer-associated deaths in women worldwide. Metastasis is the major cause of morbidity and mortality in women afflicted with breast cancer. In the metastatic process, intravasation is an important step and largely dependent on interactions between tumor cells and stromal cells in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), an integral component of the TME, have been found to associate and co-migrate with tumor cells during intravasation. Invasive TAMs that co-migrate with tumor cells in response to epidermal growth factor display a unique transcriptome in comparison to the general TAM population found in the TME. Furthermore, intercellular signaling between tumor cells and TAMs generates a positive feedback loop that promotes tumor cell invasion and intravasation. Chemokines play a major role in cell migration and are an important component of the TME. In particular, chemokine (C-X-C motif) receptor 2 (CXCR2) has been shown to be important in tumor growth and angiogenesis. MIF and IL8, two ligands of CXCR2, correlate with poor outcome in breast cancer. Our objective is to elucidate the role of macrophage CXCR2 in tumor cell intravasation. We have used flow cytometry to characterize the basal expression of CXCR2 in a murine macrophage cell line, BAC-1.2F5 (BAC). We have also analyzed mRNA expression of CXCR2 in BAC and primary TAMs isolated from a murine model of breast cancer, the polyoma middle T oncoprotein (PyMT), by qRT-PCR. To test the functional significance of CXCR2 in intravasation, we have examined the effects of an inhibitor of CXCR2 on the ability of tumor cells to migrate through the endothelium using an in vitro transendothelial migration assay (iTEM). In this assay, MDA-MB-231 tumor cells, a human breast cancer cell line, are co-cultured with BAC cells and subsequently placed in an environment that is in contact with the basal surface of endothelial cells. We have observed that BAC cells produce CXCR2 mRNA and express surface CXCR2. We have also found elevated expression of CXCR2 in invasive TAMs as compared to the general TAM population. When MDA-MB-231 cells are co-cultured with BAC cells in the iTEM assay in the presence of SB265610, an inhibitor of CXCR2, we have found a significant reduction (p<10-4) in iTEM. Together, these results suggest that cellular signaling through macrophage CXCR2 is critical to tumor cell intravasation. Citation Format: Serena Chiang, Zhenni Zhou, Jeffrey E. Segall. Examining mechanisms by which macrophages enhance intravasation in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4019. doi:10.1158/1538-7445.AM2015-4019

BackgroundGastric cancer with peritoneal metastasis (PM-GC), recognized as one of the deadliest cancers. However, whether and how the tumor cell-extrinsic tumor microenvironment (TME) is involved in the therapeutic failure remains unknown. Thus, this study systematically assessed the immunosuppressive tumor microenvironment in ascites from patients with PM-GC, and its contribution to dissemination and immune evasion of ascites-disseminated tumor cells (aDTCs).MethodsSixty-three ascites and 43 peripheral blood (PB) samples from 51 patients with PM-GC were included in this study. aDTCs in ascites and circulating tumor cells (CTCs) in paired PB were immunophenotypically profiled. Using single-cell RNA transcriptional sequencing (scRNA-seq), crosstalk between aDTCs and the TME features of ascites was inspected. Further studies on the mechanism underlying aDTCs-immune cells crosstalk were performed on in vitro cultured aDTCs.ResultsImmune cells in ascites interact with aDTCs, prompting their immune evasion. Specifically, we found that the tumor-associated macrophages (TAMs) in ascites underwent a continuum lineage transition from cathepsinhigh (CTShigh) to complement 1qhigh (C1Qhigh) TAM. CTShigh TAM initially attracted the metastatic tumor cells to ascites, thereafter, transitioning terminally to C1Qhigh TAM to trigger overproliferation and immune escape of aDTCs. Mechanistically, we demonstrated that C1Qhigh TAMs significantly enhanced the expression of PD-L1 and NECTIN2 on aDTCs, which was driven by the activation of the C1q-mediated complement pathway.ConclusionsFor the first time, we identified an immunosuppressive macrophage transition from CTShigh to C1Qhigh TAM in ascites from patients with PM-GC. This may contribute to developing potential TAM-targeted immunotherapies for PM-GC.Graphical Schematic of the immune TME of ascites and the crosstalk with aDTCs in patients with PM-GC. In ascites with TAM-dominant TME, the ascitic TAMs undergo CTS-to-C1Q transition to support multiple phases of aDTC dissemination, including aDTC homing, proliferation, immune escape, and therapeutic resistance. While in ascites with T-cell-dominant TME, enriched T cells do not imply “immune-hot” TME. Infiltrated CD8+ T cells are GZMK+ precursor-exhausted cells that have lost their capacity to kill tumor cells. (Abbreviations: aDTC ascites-disseminated tumor cells, CTS cathepsin, TAM Tumor-associated macrophages, TME tumor microenvironment)Supplementary InformationThe online version contains supplementary material available at 10.1007/s10120-024-01486-6.

The recreation of the tumor microenvironment remains a significant challenge in the development of experimental cancer models. The present study constitutes an investigation into the interconnection between tumor, endothelial and stromal cells in heterotypic breast cancer spheroids. The generation of models was achieved through the utilization of MCF7, MDA-MB-231, and SK-BR-3 tumor cell lines, in conjunction with endothelial TIME-RFP cells and either cancer-associated (BrC4f) or normal (BN120f) fibroblasts, within ultra-low attachment plates. It was established that stromal cells, most notably fibroblasts, were conducive to the aggregation of tumor cells into spheroids and the formation of pseudovessels in close proximity to fibroblast bands. In contrast to the more aggressive tumor models MDA-MB-231 and SK-BR-3, microenvironment cells do not influence the migration ability of MCF7 tumor cells. Heterotypic spheroids incorporating CAFs demonstrated a more aggressive and immunosuppressive phenotype. Multiplex immunoassay analysis of cytokines, followed by STRING cluster analysis, was used to identify key processes including angiogenesis, invasion, stem cell maintenance, and immunosuppression. Furthermore, a cluster of cytokines (LIF, SDF-1, HGF, SCGFb) was identified as potentially involved in the regulation of PD-L1 expression by tumor cells. This finding reveals a potential mechanism of immune evasion and suggests new avenues for therapeutic investigation.

Crosstalk between Macrophages and Endothelial Cells in the Tumor Microenvironment

Despite objective responses to poly(ADP-ribose) polymerase (PARP) inhibition and improvements in progression-free survival (PFS) compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high-dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in breast cancer susceptibility (BRCA)-associated TNBC. Macrophages are an innate immune cell that play a critical role in host defense and maintaining tissue homeostasis, however their infiltration into tumors has been associated with disease progression and resistance to therapy. Tumor associated macrophages (TAMs) represent a significant proportion of solid tumors, including breast cancer. TAMs play a major role in tumorigenesis as they can enhance tumor cell growth, angiogenesis and metastasis. In addition, TAMs can inhibit anti-tumor responses of T cells. Our recent work has shown that removal or conversion of TAMs to an anti-tumor phenotype enhances chemo- and immuno-therapy establishing TAMs as targets for anti-cancer therapy. Through multi-omics profiling, we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming, driven by the sterol regulatory element-binding protein 1 (SREBF1, SREBP1) pathway. Combining PARP inhibitor therapy with colony-stimulating factor 1 receptor (CSF1R)-blocking antibodies significantly enhanced innate and adaptive antitumor immunity and extended survival in mice with BRCA-deficient tumors in vivo, and this was mediated by CD8+ T cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate that combined PARP inhibition and macrophage-targeting therapy induces a durable reprogramming of the tumor microenvironment (TME), thus constituting a promising therapeutic strategy for TNBC. Therefore, targeting TAMs offers great potential to enhance both chemo- and immuno-therapy. Deep analysis of TAMs in solid tumors has revealed the complexity of TAMs and revealed major gaps in our knowledge of the functional and phenotypic characterization of TAM subsets associated with cancer, before and after treatment. Here we will discuss the complexity of TAMs in solid tumors including characterizing TAM subsets, location, and crosstalk with neighboring cells, as well as novel TAM-modulating strategies and combinations that are likely to enhance current therapies and overcome chemo- and immuno-therapy resistance. Citation Format: Jennifer Guerriero. Immunosuppressive macrophages and PARP inhibitor resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr F2-3.

Despite objective responses to poly(ADP-ribose) polymerase (PARP) inhibition and improvements in progression-free survival (PFS) compared to standard chemotherapy in patients with BRCA-associated triple-negative breast cancer (TNBC), benefits are transitory. Using high-dimensional single-cell profiling of human TNBC, here we demonstrate that macrophages are the predominant infiltrating immune cell type in breast cancer susceptibility (BRCA)-associated TNBC. Macrophages are an innate immune cell that play a critical role in host defense and maintaining tissue homeostasis, however their infiltration into tumors has been associated with disease progression and resistance to therapy. Tumor associated macrophages (TAMs) represent a significant proportion of solid tumors, including breast cancer. TAMs play a major role in tumorigenesis as they can enhance tumor cell growth, angiogenesis and metastasis. In addition, TAMs can inhibit anti-tumor responses of T cells. Our recent work has shown that removal or conversion of TAMs to an anti-tumor phenotype enhances chemo- and immuno-therapy establishing TAMs as targets for anti-cancer therapy. Through multi-omics profiling, we show that PARP inhibitors enhance both anti- and pro-tumor features of macrophages through glucose and lipid metabolic reprogramming, driven by the sterol regulatory element-binding protein 1 (SREBF1, SREBP1) pathway. Combining PARP inhibitor therapy with colony-stimulating factor 1 receptor (CSF1R)-blocking antibodies significantly enhanced innate and adaptive antitumor immunity and extended survival in mice with BRCA-deficient tumors in vivo, and this was mediated by CD8+ T cells. Collectively, our results uncover macrophage-mediated immune suppression as a liability of PARP inhibitor treatment and demonstrate that combined PARP inhibition and macrophage-targeting therapy induces a durable reprogramming of the tumor microenvironment (TME), thus constituting a promising therapeutic strategy for TNBC. Therefore, targeting TAMs offers great potential to enhance both chemo- and immuno-therapy. Deep analysis of TAMs in solid tumors has revealed the complexity of TAMs and revealed major gaps in our knowledge of the functional and phenotypic characterization of TAM subsets associated with cancer, before and after treatment. Here we will discuss the complexity of TAMs in solid tumors including characterizing TAM subsets, location, and crosstalk with neighboring cells, as well as novel TAM-modulating strategies and combinations that are likely to enhance current therapies and overcome chemo- and immuno-therapy resistance. Citation Format: Jennifer L. Guerriero. The complexities of tumor associated macrophages and the key to effective targeting for anti-cancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY17-01.

Cutaneous diffuse large B-cell lymphoma induce a macrophage immunosuppressive phenotype through IL-10 secretion