Mechanisms Driving Macrophage Diversity and Specialization in Distinct Tumor Microenvironments and Parallelisms with Other Tissues

Macrophages are extremely versatile cells that adopt a distinct phenotype in response to a changing microenvironment. Consequently, macrophages are involved in diverse functions, ranging from organogenesis and tissue homeostasis to recognition and destruction of invading pathogens. In cancer, tumor-associated macrophages (TAM) often contribute to tumor progression by increasing cancer cell migration and invasiveness, stimulating angiogenesis, and suppressing anti-tumor immunity. Accumulating evidence suggests that these different functions could be exerted by specialized TAM subpopulations. Here, we discuss the potential underlying mechanisms regulating TAM specialization and elaborate on TAM heterogeneity in terms of their ontogeny, activation state, and intra-tumoral localization. In addition, parallels are drawn between TAM and macrophages in other tissues. Together, a better understanding of TAM diversity could provide a rationale for novel strategies aimed at targeting the most potent tumor-supporting macrophages

The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity

Various steady state and inflamed tissues have been shown to contain a heterogeneous DC population consisting of developmentally distinct subsets, including cDC1s, cDC2s and monocyte-derived DCs, displaying differential functional specializations. The identification of functionally distinct tumour-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrate that multiple mouse tumours as well as human tumours harbour ontogenically discrete TADC subsets. Monocyte-derived TADCs are prominent in tumour antigen uptake, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADCs have lymph node migratory potential, whereby cDC1s efficiently activate CD8(+) Tcells and cDC2s induce Th17 cells. Mice vaccinated with cDC2s displayed a reduced tumour growth accompanied by a reprogramming of pro-tumoural TAMs and a reduction of MDSCs, while cDC1 vaccination strongly induces anti-tumour CTLs. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors

Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy

Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM

AhR transcriptional activity in serum of Inuits across Greenlandic districts

<p>Abstract</p> <p>Background</p> <p>Human exposure to lipophilic persistent organic pollutants (POPs) including polychlorinated dibenzo-<it>p</it>-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and organochlorine pesticide is ubiquitous. The individual is exposed to a complex mixture of POPs being life-long beginning during critical developmental windows. Exposure to POPs elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR). The aim of this study was to compare the actual level of integrated AhR transcriptional activity in the lipophilic serum fraction containing the actual POP mixture among Inuits from different districts in Greenland, and to evaluate whether the AhR transactivity is correlated to the bio-accumulated POPs and/or lifestyle factors.</p> <p>Methods</p> <p>The study included 357 serum samples from the Greenlandic districts: Nuuk and Sisimiut (South West Coast), Qaanaaq (North Coast) and Tasiilaq (East Coast). The bio-accumulated serum POPs were extracted by ethanol: hexane and clean-up on Florisil columns. Effects of the serum extract on the AhR transactivity was determined using the Hepa 1.12cR mouse hepatoma cell line carrying an AhR-luciferase reporter gene, and the data was evaluated for possible association to the serum levels of 14 PCB congeners, 10 organochlorine pesticide residues and/or lifestyle factors.</p> <p>Results</p> <p>In total 85% of the Inuit samples elicited agonistic AhR transactivity in a district dependent pattern. The median level of the AhR-TCDD equivalent (AhR-TEQ) of the separate genders was similar in the different districts. For the combined data the order of the median AhR-TEQ was Tasiilaq > Nuuk ≥ Sisimiut > Qaanaaq possibly being related to the different composition of POPs. In overall, the AhR transactivity was inversely correlated to the levels of sum POPs, age and/or intake of marine food.</p> <p>Conclusion</p> <p>i) We observed that the proportion of dioxin like (DL) compounds in the POP mixture was the dominating factor affecting the level of serum AhR transcriptional activity even at very high level of non DL-PCBs; ii) The inverse association between the integrated serum AhR transactivity and sum of POPs might be explained by the higher level of compounds antagonizing the AhR function probably due to selective POP bioaccumulation in the food chain.</p

Exposure determinants of cadmium in European mothers and their children

© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother–child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24–52 years of age), and 1689 children (5–12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011–2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1–28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18–86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1–13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9–12 years had 7% (95% CI 1–13%) higher UCd (p=0.04) than children 5–8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.Financially supported by the 7th EU framework programe(DGResearch – No. 244237-COPHES),LIFE+ 2009(DG Environment – LIFE09ENV/BE000410-DEMOCOPHES),with addi- tional co-funding from DEMOCOPHES partners

Mechanisms driving macrophage diversity and specialization in distinct tumor microenvironments and parallelisms with other tissues.

Macrophages are extremely versatile cells that adopt a distinct phenotype in response to a changing microenvironment. Consequently, macrophages are involved in diverse functions, ranging from organogenesis and tissue homeostasis to recognition and destruction of invading pathogens. In cancer, tumor-associated macrophages (TAM) often contribute to tumor progression by increasing cancer cell migration and invasiveness, stimulating angiogenesis, and suppressing anti-tumor immunity. Accumulating evidence suggests that these different functions could be exerted by specialized TAM subpopulations. Here, we discuss the potential underlying mechanisms regulating TAM specialization and elaborate on TAM heterogeneity in terms of their ontogeny, activation state and intratumoral localization. In addition, parallels are drawn between TAM and macrophages in other tissues. Together, a better understanding of TAM diversity could provide a rationale for novel strategies aimed at targeting the most potent tumor-supporting macrophages

Functional relationship between tumor-associated macrophages and macrophage colony stimulating factor as contributors to cancer progression

The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the - sometimes conflicting - effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM

The timing of surgery after neoadjuvant radiotherapy influences tumor dissemination in a preclinical model

peer reviewedNeoadjuvant radiotherapy (neoRT) used in cancer treatments aims at improving local tumor control and patient overall survival. The neoRT schedule and the timing of the surgical treatment (ST) are empirically based and influenced by the clinician’s experience. The current study examines how the sequencing of neoRT and ST affects metastatic dissemination. In a breast carcinoma model, tumors were exposed to different neoRT schedules (2x5Gy or 5x2Gy) followed by surgery at day 4 or 11 post- RT. The impact on the tumor microenvironment and lung metastases was evaluated through immunohistochemical and flow cytometry analyses. After 2x5Gy, early ST (at day 4 post-RT) led to increased size and number of lung metastases as compared to ST performed at day 11. Inversely, after 5x2Gy neoRT, early ST protected the mice against lung metastases. This intriguing relationship between tumor aggressiveness and ST timing could not be explained by differences in classical parameters studied such as hypoxia, vessel density and matrix remodeling. The study of tumor-related inflammation and immunity reveals an increased circulating NK cell percentage following neoRT as compared to non irradiated mice. Then, radiation treatment and surgery were applied to tumor-bearing NOD/SCID mice. In the absence of NK cells, neoRT appears to increase lung metastatic dissemination as compared to non irradiated tumor-bearing mice. Altogether our data demonstrate that the neoRT schedule and the ST timing affect metastasis formation in a pre-clinical model and points out the potential role of NK cells. These findings highlight the importance to cautiously tailor the optimal window for ST following RT

Genetic depletion of the dual specificity protein phosphatase DUSP3 promotes LLC Lung tumour metastasis

DUSP3, also called Vaccinia-H1 Related (VHR) is a small dual specificity phosphatase dephosphorylating both tyrosine and serine/threonine phosphorylated residues. DUSP3 plays an important role in cell cycle regulation and is up-regulated in several human cancers. The physiological role of this phosphatase is, however, poorly understood. We have recently generated a DUSP3 knockout mouse by homologous recombination. The obtained mice have no spontaneous phenotype or pathology. However, DUSP3 deficiency prevented neo-vascularization of subcutaneously transplanted Matrigel plugs and Lung Lewis Carcinoma (LLC) tumours, suggesting an involvement of DUSP3 in tumour angiogenesis. Considering the importance of angiogenesis in metastatic formation, our study aimed to investigate the role of DUSP3 in metastatic dissemination. To do so, we used the LLC experimental metastasis model that shortcuts the intravasation/extravasation processes by injecting intravenously the LLC and the B16 (metastatic melanoma cell line) cells. Surprisingly, LLC, but not B16, lung metastasis developed twice faster in DUSP3-KO than WT mice. The enhanced LLC metastatic growth in DUSP3-/- mice was transferable to WT mice via DUSP3-/- bone marrow adoptive transfer, suggesting an involvement of the hematopoietic compartment in the observed phenotype. This was confirmed by a higher infiltration of neutrophils and macrophages in the lungs of DUSP3-KO compared to WT mice after LLC injection. This infiltration was correlated with higher expression of the chemokine receptor CCR2 in LLC-bearing DUSP3-KO lungs macrophages. Interestingly, LLC, but not B16 cells, were found to secrete high level of CCL2/MCP1, the CCR ligand chemokine. In line with this observation, we found that DUSP3-/- bone marrow derived-macrophages have a higher migration potential in response to LLC, but not B16, -conditionned medium. Altogether, our results suggest that DUSP3 plays an important role in metastatic dissemination/growth by a mechanism involving the control of CCR2-CCL2 chemoattraction axis in macrophages