Isolation and separation of murine tumor-associated macrophages (TAMs) subpopulations from orthotopic 4T1 breast tumors.

Tumor-associated macrophages (TAMs) are highly heterogenous regarding their intratumoral localization, surface marker expression, and molecular properties. This protocol describes the complete procedure for isolation and digestion of murine breast cancer samples and fluorescence-activated cell sorting (FACS) of TAMs from murine orthotopic 4T1 breast tumors. This includes steps to separate PoEMs (podoplanin-expressing macrophages) and non-PoEMs (podoplanin-negative macrophages). Our FACS separation approach could also be used for other tumor types with TAM infiltration. For complete details on the use and execution of this protocol, please refer to Bieniasz-Krzywiec et al. (2019).

DNA methylation repels binding of hypoxia-inducible transcription factors to maintain tumor immunotolerance.

BACKGROUND: Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. RESULTS: We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. CONCLUSIONS: Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

Podoplanin-Expressing Macrophages Promote Lymphangiogenesis and Lymphoinvasion in Breast Cancer.

Among mammary tumor-infiltrating immune cells, the highest expression of podoplanin (PDPN) is found in a subset of tumor-associated macrophages (TAMs). We hereby demonstrate that PDPN is involved in the attachment of this TAM subset to lymphatic endothelial cells (LECs). Mechanistically, the binding of PDPN to LEC-derived galectin 8 (GAL8) in a glycosylation-dependent manner promotes the activation of pro-migratory integrin ß1. When proximal to lymphatics, PDPN-expressing macrophages (PoEMs) stimulate local matrix remodeling and promote vessel growth and lymphoinvasion. Anti-integrin ß1 blockade, macrophage-specific Pdpn knockout, or GAL8 inhibition impairs TAM adhesion to LECs, restraining lymphangiogenesis and reducing lymphatic cancer spread. In breast cancer patients, association of PoEMs with tumor lymphatic vessels correlates with incidences of lymph node and distant organ metastasis.

Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism.

Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

MIF-Mediated Hemodilution Promotes Pathogenic Anemia in Experimental African Trypanosomosis.

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells (RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF (macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis.