Tumor-Derived Retinoic Acid Regulates Intratumoral Monocyte Differentiation to Promote Immune Suppression.

The immunosuppressive tumor microenvironment (TME) is a major barrier to immunotherapy. Within solid tumors, why monocytes preferentially differentiate into immunosuppressive tumor-associated macrophages (TAMs) rather than immunostimulatory dendritic cells (DCs) remains unclear. Using multiple murine sarcoma models, we find that the TME induces tumor cells to produce retinoic acid (RA), which polarizes intratumoral monocyte differentiation toward TAMs and away from DCs via suppression of DC-promoting transcription factor Irf4. Genetic inhibition of RA production in tumor cells or pharmacologic inhibition of RA signaling within TME increases stimulatory monocyte-derived cells, enhances T cell-dependent anti-tumor immunity, and synergizes with immune checkpoint blockade. Furthermore, an RA-responsive gene signature in human monocytes correlates with an immunosuppressive TME in multiple human tumors. RA has been considered as an anti-cancer agent, whereas our work demonstrates its tumorigenic capability via myeloid-mediated immune suppression and provides proof of concept for targeting this pathway for tumor immunotherapy.

Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages.

Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor SPI-C is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80(+)VCAM1(+) bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor BACH1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Furthermore, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insights into iron homeostasis.

Batf3 maintains autoactivation of Irf8 for commitment of a CD8α(+) conventional DC clonogenic progenitor.

The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.

Neutrophils promote tumor resistance to radiation therapy.

Nearly two-thirds of cancer patients are treated with radiation therapy (RT), often with the intent to achieve complete and permanent tumor regression (local control). RT is the primary treatment modality used to achieve local control for many malignancies, including locally advanced cervical cancer, head and neck cancer, and lung cancer. The addition of concurrent platinum-based radiosensitizing chemotherapy improves local control and patient survival. Enhanced outcomes with concurrent chemoradiotherapy may result from increased direct killing of tumor cells and effects on nontumor cell populations. Many patients treated with concurrent chemoradiotherapy exhibit a decline in neutrophil count, but the effects of neutrophils on radiation therapy are controversial. To investigate the clinical significance of neutrophils in the response to RT, we examined patient outcomes and circulating neutrophil counts in cervical cancer patients treated with definitive chemoradiation. Although pretreatment neutrophil count did not correlate with outcome, lower absolute neutrophil count after starting concurrent chemoradiotherapy was associated with higher rates of local control, metastasis-free survival, and overall survival. To define the role of neutrophils in tumor response to RT, we used genetic and pharmacological approaches to deplete neutrophils in an autochthonous mouse model of soft tissue sarcoma. Neutrophil depletion prior to image-guided focal irradiation improved tumor response to RT. Our results indicate that neutrophils promote resistance to radiation therapy. The efficacy of chemoradiotherapy may depend on the impact of treatment on peripheral neutrophil count, which has the potential to serve as an inexpensive and widely available biomarker.

Transcription factor Zeb2 regulates commitment to plasmacytoid dendritic cell and monocyte fate.

Dendritic cells (DCs) and monocytes develop from a series of bone-marrow-resident progenitors in which lineage potential is regulated by distinct transcription factors. Zeb2 is an E-box-binding protein associated with epithelial-mesenchymal transition and is widely expressed among hematopoietic lineages. Previously, we observed that Zeb2 expression is differentially regulated in progenitors committed to classical DC (cDC) subsets in vivo. Using systems for inducible gene deletion, we uncover a requirement for Zeb2 in the development of Ly-6Chi monocytes but not neutrophils, and we show a corresponding requirement for Zeb2 in expression of the M-CSF receptor in the bone marrow. In addition, we confirm a requirement for Zeb2 in development of plasmacytoid DCs but find that Zeb2 is not required for cDC2 development. Instead, Zeb2 may act to repress cDC1 progenitor specification in the context of inflammatory signals.

Origin, development, and homeostasis of tissue-resident macrophages.

Macrophages are versatile cells of the hematopoietic system that display remarkable functional diversity encompassing innate immune responses, tissue development, and tissue homeostasis. Macrophages are present in almost all tissues of the body and display distinct location-specific phenotypes and gene expression profiles. Recent studies also demonstrate distinct origins of tissue-resident macrophages. This emerging picture of ontological, functional, and phenotypic heterogeneity within tissue macrophages has altered our understanding of these cells, which play important roles in many human diseases. In this review, we discuss the different origins of tissue macrophages, the transcription factors regulating their development, and the mechanisms underlying their homeostasis at steady state.

Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin.

Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, we genetically labeled and ablated Pax3(+) and Pax7(+) cells. Pax3(+)Pax7(-) cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7(+) cells. Subsequently, Pax7(+) cells give rise to and are required for fetal myogenesis. Thus, Pax3(+) and Pax7(+) cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements we conditionally inactivated or activated beta-catenin, an important regulator of myogenesis, in Pax3- or Pax7-derived cells. beta-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, beta-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for beta-catenin.

A conditional mouse model of synovial sarcoma: insights into a myogenic origin.

Synovial sarcoma is an aggressive soft-tissue malignancy marked by a unique t(X;18) translocation leading to expression of a chimeric SYT-SSX fusion protein. We report here a mouse model of synovial sarcoma based on conditional expression of the human SYT-SSX2. Using this model, we have identified myoblasts as a potential source of synovial sarcoma. Remarkably, within the skeletal muscle lineage, while expression of the oncoprotein in immature myoblasts leads to induction of synovial sarcoma with 100% penetrance, its expression in more differentiated cells induces myopathy without tumor induction. We also show that early widespread expression of the fusion protein disrupts normal embryogenesis, causing lethality.

RALDH1 Inhibition Shows Immunotherapeutic Efficacy in Hepatocellular Carcinoma.

Globally, hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related death. We previously identified an immune evasion pathway whereby tumor cells produce retinoic acid (RA) to promote differentiation of intratumoral monocytes into protumor macrophages. Retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, and RALDH3 are the three isozymes that catalyze RA biosynthesis. In this study, we have identified RALDH1 as the key driver of RA production in HCC and demonstrated the efficacy of RALDH1-selective inhibitors (Raldh1-INH) in suppressing RA production by HCC cells. Raldh1-INH restrained tumor growth in multiple mouse models of HCC by reducing the number and tumor-supporting functions of intratumoral macrophages as well as increasing T-cell infiltration and activation within tumors. Raldh1-INH also displayed favorable pharmacokinetic, pharmacodynamic, and toxicity profiles in mice thereby establishing them as promising new drug candidates for HCC immunotherapy.

Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes antitumor immunity in solid cancers, but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional coactivator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend on oncogene-mediated ECM composition and remodeling.

How meningeal macrophages control medulloblastoma induction.

In this issue of Developmental Cell, Pokrajac et al. report that Wnt signaling in endothelial cells maintain CXCL4 expression in meningeal macrophages, which suppresses induction of Sonic hedgehog medulloblastoma by antagonizing the pro-tumor effects of CXCL12. This work highlights how homeostatic tissue macrophages can regulate early stages of tumor induction.

Mapping and modeling human colorectal carcinoma interactions with the tumor microenvironment.

The initiation and progression of cancer are intricately linked to the tumor microenvironment (TME). Understanding the function of specific cancer-TME interactions poses a major challenge due in part to the complexity of the in vivo microenvironment. Here we predict cancer-TME interactions from single cell transcriptomic maps of both human colorectal cancers (CRCs) and mouse CRC models, ask how these interactions are altered in human tumor organoid (tumoroid) cultures, and functionally recapitulate human myeloid-carcinoma interactions in vitro. Tumoroid cultures suppress gene expression programs involved in inflammation and immune cell migration, providing a reductive platform for re-establishing carcinoma-immune cell interactions in vitro. Introduction of human monocyte-derived macrophages into tumoroid cultures instructs macrophages to acquire immunosuppressive and pro-tumorigenic gene expression programs similar to those observed in vivo. This includes hallmark induction of SPP1, encoding Osteopontin, an extracellular CD44 ligand with established oncogenic effects. Taken together, these findings offer a framework for understanding CRC-TME interactions and provide a reductionist tool for modeling specific aspects of these interactions.

Dll1-Mediated Notch Signaling Drives Tumor Cell Cross-talk with Cancer-Associated Fibroblasts to Promote Radioresistance in Breast Cancer.

SIGNIFICANCE: Dll1+ breast cancer cells activate Notch signaling in cancer-associated fibroblasts that increases Wnt ligand secretion and leads to ß-catenin-driven radioresistance and metastasis, opening new therapeutic avenues for breast cancer.

Of mice and men: opportunities to use genetically engineered mouse models of synovial sarcoma for preclinical cancer therapeutic evaluation.

BACKGROUND: Synovial sarcoma is a soft tissue malignancy with a predilection for adolescents and young adults. Despite recent improvements in the understanding of its character and etiology, few therapeutic advances have been made. The mortality rate is high among the young population it affects. The low incidence of most subtypes of sarcoma, such as synovial sarcoma, makes disease-specific trials difficult to organize. The biological differences between sarcoma subtypes make inclusion of multiple types in general trials unsatisfactory as well. METHODS: A review of the literature regarding targetable pathways in synovial sarcoma was undertaken. A strategy has been devised to utilize available technologies in order to prioritize drug trial planning. RESULTS: Cell culture and xenograft research with synovial sarcoma cell lines have identified some critical pathways that may be targetable. Promising therapeutic strategies include newer cytotoxic chemotherapies, antiangiogenic agents, anti-IGF1R pathway agents, anti-Bcl-2/proapoptotic agents, and histone deacetylase complex inhibitors. CONCLUSIONS: We propose to prioritize potential therapeutic strategies via preclinical testing of agents in a genetic mouse model of synovial sarcoma. Preclinical optimization of treatment regimens can guide the development of more focused patient trials.

Isolation of myeloid cells from mouse brain tumors for single-cell RNA-seq analysis.

Current single-cell RNA sequencing (scRNA-seq) protocols are limited by the number of cells that can be simultaneously sequenced, restricting the ability to resolve heterogeneity of rare cell types. We describe here a protocol for rapid isolation of myeloid cells from tumor-harboring mouse cerebellum without cell sorting to minimize cell damage for scRNA-seq. This protocol includes the procedures for further enrichment of myeloid cells using CD11b+ magnetic beads, followed by the generation of scRNA library and sequencing analysis. For complete details on the use and execution of this protocol, please refer to Dang et al. (2021).

Macrophages in SHH subgroup medulloblastoma display dynamic heterogeneity that varies with treatment modality.

Tumor-associated macrophages (TAMs) play an important role in tumor immunity and comprise of subsets that have distinct phenotype, function, and ontology. Transcriptomic analyses of human medulloblastoma, the most common malignant pediatric brain cancer, showed that medulloblastomas (MBs) with activated sonic hedgehog signaling (SHH-MB) have significantly more TAMs than other MB subtypes. Therefore, we examined MB-associated TAMs by single-cell RNA sequencing of autochthonous murine SHH-MB at steady state and under two distinct treatment modalities: molecular-targeted inhibitor and radiation. Our analyses reveal significant TAM heterogeneity, identify markers of ontologically distinct TAM subsets, and show the impact of brain microenvironment on the differentiation of tumor-infiltrating monocytes. TAM composition undergoes dramatic changes with treatment and differs significantly between molecular-targeted and radiation therapy. We identify an immunosuppressive monocyte-derived TAM subset that emerges with radiation therapy and demonstrate its role in regulating T cell and neutrophil infiltration in MB.

Thymic stromal lymphopoietin induces adipose loss through sebum hypersecretion.

Emerging studies indicate that the immune system can regulate systemic metabolism. Here, we show that thymic stromal lymphopoietin (TSLP) stimulates T cells to induce selective white adipose loss, which protects against obesity, improves glucose metabolism, and mitigates nonalcoholic steatohepatitis. Unexpectedly, adipose loss was not caused by alterations in food intake, absorption, or energy expenditure. Rather, it was induced by the excessive loss of lipids through the skin as sebum. TSLP and T cells regulated sebum release and sebum-associated antimicrobial peptide expression in the steady state. In human skin, TSLP expression correlated directly with sebum-associated gene expression. Thus, we establish a paradigm in which adipose loss can be achieved by means of sebum hypersecretion and uncover a role for adaptive immunity in skin barrier function through sebum secretion.

Intratumoral Monocyte Transfer to Examine Monocyte Differentiation in the Tumor Microenvironment.

The regulation of monocyte differentiation in the tumor microenvironment is of significant interest to tumor immunologists. Monocytes injected into the circulation may not track into tumors in sufficient numbers, making intratumoral injections a preferred experimental approach. Monocyte enrichment with antibody-based positive selection may activate downstream signaling, while cell sorters expose monocytes to mechanical stress. Here, we describe an approach of intratumoral monocyte transfer that circumvents these limitations by using negative selection and fluorescent reporter mice. For complete details on the use and execution of this protocol, please refer to Devalaraja et al. (2020).

TGFß and Hippo Pathways Cooperate to Enhance Sarcomagenesis and Metastasis through the Hyaluronan-Mediated Motility Receptor (HMMR).

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFß signaling. Therefore, we investigated crosstalk between YAP1 and TGFß resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFß and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFß-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFß cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.