production-characterization-human-macrophages-from-pluripotent-stem

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

University of Edinburgh

Oncogenic targets acting in both tumor cells and tumor stromal cells may offer special therapeutic appeal. Interrogation of the Oncomine database revealed that 52 of 53 human breast carcinomas showed substantial upregulation of WNT family ligand WNT7B. Immunolabeling of human mammary carcinoma showed that WNT7B immunoreactivity was associated with both tumor cells and with tumor-associated macrophages. In the MMTV-PymT mouse model of mammary carcinoma, we found tumor progression relied upon WNT7B produced by myeloid cells in the microenvironment. Wnt7b deletion in myeloid cells reduced the mass and volume of tumors due to a failure in the angiogenic switch. In the tumor overall, there was no change in expression of Wnt/β-catenin pathway target genes, but in vascular endothelial cells (VEC), expression of these genes was reduced, suggesting that VECs respond to Wnt/β-catenin signaling. Mechanistic investigations revealed that failure of the angiogenic switch could be attributed to reduced Vegfa mRNA and protein expression in VECs, a source of VEGFA mRNA in the tumor that was limiting in the absence of myeloid WNT7B. We also noted a dramatic reduction in lung metastasis associated with decreased macrophage-mediated tumor cell invasion. Together, these results illustrated the critical role of myeloid WNT7B in tumor progression, acting at the levels of angiogenesis, invasion, and metastasis. We suggest that therapeutic suppression of WNT7B signaling might be advantageous due to targeting multiple aspects of tumor progression.

Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer.

Pulmonary metastasis of breast cancer cells is promoted by a distinct population of macrophages, metastasis-associated macrophages (MAMs), which originate from inflammatory monocytes (IMs) recruited by the CC-chemokine ligand 2 (CCL2). We demonstrate here that, through activation of the CCL2 receptor CCR2, the recruited MAMs secrete another chemokine ligand CCL3. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces the number of lung metastasis foci, as well as the number of MAMs accumulated in tumor-challenged lung in mice. Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM-cancer cell interactions. These findings collectively indicate that the CCL2-triggered chemokine cascade in macrophages promotes metastatic seeding of breast cancer cells thereby amplifying the pathology already extant in the system. These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets.

CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages.

Classical inflammatory monocytes and their derivative macrophages promote tumor metastasis whereas CD8(+) T and NK cells restrict tumor growth. In a recent paper published in Science, Hanna and colleagues demonstrate that another monocyte population, nonclassical patrolling monocytes, is enriched in the microvasculature of tumor-challenged lung and reduces tumor metastasis by recruiting NK cells.

Cancer immunosurveillance: role of patrolling monocytes.

The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies.

Isolation of Mouse and Human Tumor-Associated Macrophages.

Tumor-associated macrophages (TAMs) contribute to breast cancer progression and dissemination; TAM-targeting strategies aimed at their reprogramming show promising preclinical results. In a new report Guerriero and colleagues demonstrate that a novel HDAC Class IIa inhibitor, TMP195, can reprogram monocytes and macrophages in the tumor into cells able to sustain a robust CD8 T cell-mediated anti-tumoral immune response.

Repolarizing macrophages improves breast cancer therapy.

Metastasis-associated macrophages (MAMs) play pivotal roles in breast cancer metastasis by promoting extravasation and survival of metastasizing cancer cells. In a metastatic breast cancer mouse model, we previously reported that circulating classical monocytes (C-MOs) preferentially migrated into the tumor-challenged lung where they differentiated into MAMs. However, the fate and characteristics of C-MOs in the metastatic site has not been defined. In this study, we identified that adoptively transferred C-MOs (F4/80CD11bLy6C) differentiated into a distinct myeloid cell population that is characterized as F4/80CD11bLy6C and gives rise to MAMs (F4/80CD11bLy6C) within 18 h after migration into the metastatic lung. In mouse models of breast cancer, the CD11bLy6C MAM precursor cells (MAMPCs) were commonly found in the metastatic lung, and their accumulation was increased during metastatic tumor growth. The morphology and gene expression profile of MAMPCs were distinct from C-MOs and had greater similarity to MAMs. For example MAMPCs expressed mature macrophage markers such as CD14, CD36, CD64, and CD206 at comparable levels with MAMs, suggesting that MAMPCs have committed to a macrophage lineage in the tumor microenvironment. MAMPCs also expressed higher levels of , and  than C-MOs to a comparable level to MAMs. Expression of these MAM-associated genes in MAMPCs was reduced by genetic deletion of colony-stimulating factor 1 receptor (CSF1R). On the other hand, transient CSF1R blockade did not reduce the number of MAMPCs in the metastatic site, suggesting that CSF1 signaling is active in MAMPCs but is not required for their accumulation. Functionally MAMPCs suppressed the cytotoxicity of activated CD8 T cells  in part through superoxide production. Overall, our results indicate that immediately following migration into the metastatic tumors C-MOs differentiate into immunosuppressive cells that have characteristics of monocytic myeloid-derived suppressor cell phenotype and might be targeted to enhance efficacy of immunotherapy for metastatic breast cancer.

Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer.

Inhibition of immune checkpoint pathways in CD8 T cell is a promising therapeutic strategy for the treatment of solid tumors that has shown significant anti-tumor effects and is now approved by the FDA to treat patients with melanoma and lung cancer. However the response to this therapy is limited to a certain fraction of patients and tumor types, for reasons still unknown. To ensure success of this treatment, CD8 T cells, the main target of the checkpoint inhibitors, should exert full cytotoxicity against tumor cells. However recent studies show that tumor-associated macrophages (TAM) can impede this process by different mechanisms. In this mini-review we will summarize recent studies showing the effect of TAM targeting on immune checkpoint inhibitors efficacy. We will also discuss on the limitations of the current strategies as well on the future scientific challenges for the progress of the tumor immunology field.

Targeting Tumor-Associated Macrophages as a Potential Strategy to Enhance the Response to Immune Checkpoint Inhibitors.

We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The CAG-driven ZsGreen expression cassette was inserted into the  locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the  locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for  tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and  tracking of therapeutic cells.

Tumour-infiltrating immune cells regulate tumour development and progression either negatively or positively. For example, cytotoxic lymphocytes (CTL) such as CD8 T and natural killer (NK) cells can recognize and eliminate cancer cells, and thereby restrict the tumour growth and metastasis, if they exert full cytotoxicity. In contrast, tumour-infiltrating myeloid cells such as tumour-associated macrophages (TAM) promote the expansion and dissemination of cancer cells depending on their functional states. Given the tumour-killing ability of CTL, the augmentation of CTL-induced antitumour immune reactions has been considered as an attractive therapeutic modality for lethal solid tumours and several promising strategies have emerged, which include immune checkpoint inhibitors, cancer vaccines and adoptive CTL transfer. These immunotherapies are now tested in clinical trials and have shown significant antitumour effects in patients with lymphoma and some solid tumours such as melanoma and lung cancer. Despite these encouraging results, these therapies are not efficient in a certain fraction of patients and tumour types with tumour cell-intrinsic mechanisms such as impaired antigen presentation and/or tumour cell-extrinsic mechanisms including the accumulation of immunosuppressive cells. Several animal studies suggest that tumour-infiltrating myeloid cells, especially TAM, are one of the key targets to improve the efficacy of immunotherapies as these cells can suppress the functions of CD8 T and NK cells. In this review, we will summarize recent animal studies regarding the involvement of TAM in the immune checkpoint, cancer vaccination and adoptive CTL transfer therapies, and discuss the therapeutic potential of TAM targeting to improve the immunotherapies.

Macrophage targeting: opening new possibilities for cancer immunotherapy.

Infiltration of macrophages in solid tumours is associated with poor prognosis and correlates with chemotherapy resistance in most cancers. In mouse models of cancer, macrophages promote cancer initiation and malignant progression by stimulating angiogenesis, increasing tumour cell migration, invasion and intravasation and suppressing antitumour immunity. At metastatic sites, macrophages promote tumour cell extravasation, survival and subsequent growth. Each of these pro-tumoural activities is promoted by a subpopulation of macrophages that express canonical markers but have unique transcriptional profiles, which makes tumour-associated macrophages (TAMs) good targets for anticancer therapy in humans through either their ablation or their re-differentiation away from pro-tumoural towards antitumoural states. In this Review, we evaluate the state of the art of TAM-targeting strategies, focusing on the limitations and potential side effects of the different therapies such as toxicity, rebound effects and compensatory mechanisms. We provide an extensive overview of the different types of therapy used in the clinic and their limitations in light of known macrophage biology and propose new strategies for targeting TAMs.

Targeting macrophages: therapeutic approaches in cancer.

In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates.

Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.

AhR controls redox homeostasis and shapes the tumor microenvironment in BRCA1-associated breast cancer.

Red blood cells mature within the erythroblastic island (EI) niche that consists of specialized macrophages surrounded by differentiating erythroblasts. Here we establish an in vitro system to model the human EI niche using macrophages that are derived from human induced pluripotent stem cells (iPSCs), and are also genetically programmed to an EI-like phenotype by inducible activation of the transcription factor, KLF1. These EI-like macrophages increase the production of mature, enucleated erythroid cells from umbilical cord blood derived CD34 haematopoietic progenitor cells and iPSCs; this enhanced production is partially retained even when the contact between progenitor cells and macrophages is inhibited, suggesting that KLF1-induced secreted proteins may be involved in this enhancement. Lastly, we find that the addition of three secreted factors, ANGPTL7, IL-33 and SERPINB2, significantly enhances the production of mature enucleated red blood cells. Our study thus contributes to the ultimate goal of replacing blood transfusion with a manufactured product.

Genetic programming of macrophages generates an in vitro model for the human erythroid island niche.

The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.

Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets.

Tumor-associated macrophages (TAMs) are becoming a promising target for cancer immunotherapy. Significant efforts have been made to study the detrimental role of TAMs both in vivo and in vitro. However, it remains challenging to isolate these macrophages to study their function in human cancers and there is the need to seek alternatives to address these limitations. In this review, we will focus on the three most relevant approaches to obtain in vitro fully differentiated macrophages i.e. peripheral blood, immortalized cell lines such as THP-1 or human induced pluripotent stem cells. We will also provide protocols for the polarization of human macrophages to a TAM-like cells in vitro.

Methods for macrophage differentiation and in vitro generation of human tumor associated-like macrophages.

Cassetta and Pollard introduce tumor-associated macrophages and discuss their origin, diversity, function and plasticity.

Article	Year
Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer. Cancer research\| PubMed ID: 24638982	2014
CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages. The Journal of experimental medicine\| PubMed ID: 26056232	2015
Cancer immunosurveillance: role of patrolling monocytes. Cell research\| PubMed ID: 26634605	2016
Isolation of Mouse and Human Tumor-Associated Macrophages. Advances in experimental medicine and biology\| PubMed ID: 27325269	2016
Repolarizing macrophages improves breast cancer therapy. Cell research\| PubMed ID: 28429765	2017
Monocytes Differentiate to Immune Suppressive Precursors of Metastasis-Associated Macrophages in Mouse Models of Metastatic Breast Cancer. Frontiers in immunology\| PubMed ID: 29387063	2017
Targeting Tumor-Associated Macrophages as a Potential Strategy to Enhance the Response to Immune Checkpoint Inhibitors. Frontiers in cell and developmental biology\| PubMed ID: 29670880	2018
A human iPSC line capable of differentiating into functional macrophages expressing ZsGreen: a tool for the study and tracking of therapeutic cells. Philosophical transactions of the Royal Society of London. Series B, Biological sciences\| PubMed ID: 29786554	2018
Macrophage targeting: opening new possibilities for cancer immunotherapy. Immunology\| PubMed ID: 29963704	2018
Targeting macrophages: therapeutic approaches in cancer. Nature reviews. Drug discovery\| PubMed ID: 30361552	2018
Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates. Cancer immunology, immunotherapy : CII\| PubMed ID: 30684003	2019
AhR controls redox homeostasis and shapes the tumor microenvironment in BRCA1-associated breast cancer. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30733286	2019
Genetic programming of macrophages generates an in vitro model for the human erythroid island niche. Nature communications\| PubMed ID: 30787325	2019
Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets. Cancer cell\| PubMed ID: 30930117	2019
Methods for macrophage differentiation and in vitro generation of human tumor associated-like macrophages. Methods in enzymology\| PubMed ID: 32000892	2020
Tumor-associated macrophages. Current biology : CB\| PubMed ID: 32208142	2020

Luca Cassetta

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