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Origins of Brain Tumor Macrophages
Cancer Cell
Previews Origins of Brain Tumor Macrophages Michele De Palma1,* 1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2016.11.015
The ontogeny of brain-tumor-associated macrophages is poorly understood. New findings indicate that both resident microglia and blood-derived monocytes generate the pool of macrophages that infiltrate brain tumors of either primary or metastatic origin. Cell-tracing studies in mice have shown that several types of tissue-resident macrophages originate from yolk-sac progenitors early during embryonic development and persist throughout adulthood by selfrenewing in situ. Tissue-resident macrophages can also be replenished from circulating monocytes, which function as macrophage precursors when tissue homeostasis is perturbed, such as during inflammation, organ injury, and regeneration, and in multiple pathological processes (Varol et al., 2015). In cancer, tumor-associated macrophages (TAMs) largely derive from bone marrow hematopoietic stem cells (HSCs) through monocyte intermediates, apparently with minor, if any, contribution from locally derived, tissue-resident macrophages (Lahmar et al., 2016). Brain macrophages, also called microglia, belong to the category of earlyseeded, long-lived, and self-renewing macrophages (Varol et al., 2015). However, some brain pathologies can cause the breakdown of the selectively permeable interface that separates the blood circulation from the nervous tissues, known as blood-brain barrier (BBB), enabling monocyte infiltration into the diseased brain. Therefore, both brainresident microglia and monocyte-derived macrophages may potentially contribute to the TAM pool in brain malignancies (Lahmar et al., 2016). In Cell Reports, Bowman et al. (2016) analyze macrophage ontogeny in mouse models of brain cancer using cell-tracing strategies that enable the discrimination of microgliaderived TAMs from monocyte-derived TAMs. They identify brain TAMs originating from either cellular compartment, but also document profound in situ programming of these cells upon tumor colonization.
Previous studies interrogated brain TAM origins using cell-tracing techniques based on transplanting genetically marked HSCs into mice following whole-body irradiation to facilitate HSC engraftment, a procedure also called myeloablation. Under these conditions, HSC-derived monocytes contributed substantially to the pool of brain TAMs (De Palma et al., 2005; Lahmar et al., 2016). However, tissue irradiation may alter macrophage homeostasis and bias it toward increased turnover from HSCderived monocytes. In order to circumvent potentially confounding factors, Bowman et al. (2016) employed two cellmarking approaches not requiring irradiation or other forms of host conditioning. According to one strategy, the HSCderived monocytes, but not the resident microglia, were permanently marked; in a complementary setting, only the microglia were marked instead. Both experimental setups revealed mixed contributions of microglia and monocytes to the TAMs of intracranial gliomas, which included transplant as well as genetically engineered mouse models. Therefore, brain TAMs are an amalgam of locally and peripherally derived cells, irrespective of the tumor model and independent of brain irradiation or systemic myeloablation (Bowman et al., 2016). Do the two TAM subsets maintain memory of their ancestry and portray distinguishing features, possibly suggestive of different functions, after their recruitment to the tumor microenvironment? To address these questions, Bowman et al. (2016) analyzed the TAM’s transcriptomes by RNA sequencing. They found that the brain TAMs, regardless of their origins, were clearly distinct from normal microglia or blood monocytes. This result is consistent with the notion that macro-
832 Cancer Cell 30, December 12, 2016 ª 2016 Elsevier Inc.
phages undergo significant in situ programming, or ‘‘education,’’ in the tumor microenvironment, a process that involves the concerted action of various tumor-derived factors that impinge on the inherent plasticity of macrophage-lineage cells (Ginhoux et al., 2016). However, Bowman et al. (2016) also noted significant differences between the two subsets. For example, the monocyte-derived TAMs expressed higher levels of gene transcripts involved in antigen presentation and T cell costimulation, compared to the microglia-derived TAMs. However, genes with known immunosuppressive functions, such as interleukin-10 (IL10), CCL17, and CCL22, were also upregulated. In human glioblastomas, macrophages produce more IL10 than microglia (Gabrusiewicz et al., 2016), consistent with the findings of Bowman et al. (2016). Moreover, human glioblastomas contain infiltrates of regulatory T cells, which are immunosuppressive and can be recruited to the tumor microenvironment through macrophagederived CCL17 and CCL22 (Hussain et al., 2006). While these results illustrate complex immunomodulatory profiles of brain TAMs, overall they suggest that monocyte-derived TAMs may acquire tolerogenic properties in brain tumors, consistent with findings in other cancer types (Lahmar et al., 2016). Interestingly, a few markers could readily distinguish the two TAM subsets. Among these, integrin ITGA4 (also known as CD49d) was specifically expressed in the monocyte-derived TAMs. Of note, expression of ITGA4 was also restricted to monocyte-derived TAMs in mouse models of breast-cancer-derived brain metastasis, indicating that lineage fidelity supersedes idiosyncratic influences of the tumor type on ITGA4 expression.
Cancer Cell
Previews Moreover, human glioblastomas contained ITGA4-positive and -negative TAMs that likely mirrored the monocyteand microglia-derived subsets identified in mice. Indeed, the expression of ITGA4 strongly correlated with a ‘‘monocytederived TAM’’ gene signature in human glioblastomas (Bowman et al., 2016). Although the two brain TAM subsets displayed transcriptional profiles indicative of in situ programming, they also maintained epigenetic memory of their ancestry. Indeed, transcription factor landscapes inferred from chromatin accessibility analyses by ATAC-seq highlighted enriched activity, specifically in the monocyte-derived TAMs, of transcription factors involved in monocyte-to-macrophage differentiation, including RUNX and CEBP, and macrophage activation, including STAT3 and IRF4. On the other hand, the microglia-derived TAMs displayed increased MEF2 activity, which is known to control microglia identity. Furthermore, the genomic occupancy of selected transcription factors, such as PU.1, differed in the target genes of normal microglia and blood monocytes, and, remarkably, such differences were conserved in their TAM progenies (Bowman et al., 2016). Overall, the molecular profiles of monocyte- and microgliaderived TAMs support a multidimensional mode of macrophage activation regulated by both tissue-specific (microenvironmental) signals and lineage-restricted (developmental) epigenetic mechanisms (Ginhoux et al., 2016). The findings of Bowman et al. (2016) indicate that resident macrophages, namely microglia, contribute significantly to the TAM compartment in brain malignancies of the mouse. Work in other mouse cancer models has suggested that locally derived TAMs are progressively diluted by monocyte-derived TAMs dur-
ing tumor progression and growth (Lahmar et al., 2016). However, kinetics analyses of locally versus peripherally derived macrophages in progressing mouse brain tumors may be complicated by the constraints imposed by the relatively small volume that experimental tumors can attain in the mouse brain, compared to the human condition. The gene signatures specific to either brain TAM subset should, therefore, be interrogated in TAMs isolated from human surgical specimens, which often involve advanced and bulky brain tumors. Glioblastoma multiforme, the most frequent and malignant primary brain tumor, is markedly resistant to available therapies. Because TAMs contribute to suppressing antitumor immune responses in various cancer types, their selective targeting may offer opportunities to alleviate immunosuppression in glioblastoma, especially combined with other immunotherapies. Monoclonal antibodies that block CSF1R signaling efficiently eliminate TAMs in mice and patients with cancer, at least in part, by disrupting the monocyte-to-macrophage differentiation program (Ries et al., 2015). While the findings of Bowman et al. (2016) imply that anti-CSF1R antibodies could help impede glioblastoma colonization by monocytederived TAMs, the effects of anti-CSF1R antibodies on microglia-derived TAMs are more difficult to predict. As opposed to small-molecule inhibitors, anti-CSF1R antibodies do not cross an intact BBB and do not affect the brain-resident microglia (Ries et al., 2015), so a potential advantage of this approach is that the effects on microglia and macrophages should be restricted to the tumor. The identification of monocyte-derived TAMs in mouse and human glioblastoma also encourages the application of gene and cell therapy strategies based on targeting
biological therapeutics to tumors via engineered HSC-derived monocytes (De Palma et al., 2008). The outstanding clinical success of HSC-based gene therapy of neurodegenerative diseases (Biffi, 2016) provides a strong rationale for implementing similar strategies in glioblastoma patients, who currently lack curative treatment options. ACKNOWLEDGMENTS The author received research grants from Hoffmann-La Roche, including support for evaluating CSF1R inhibitors in mouse cancer models. REFERENCES Biffi, A. (2016). Hum. Mol. Genet. 25, R65–R75. Bowman, R.L., Klemm, F., Akkari, L., Pyonteck, S.M., Sevenich, L., Quail, D.F., Dhara, S., Simpson, K., Gardner, E.E., Iacobuzio-Donahue, C.A., et al. (2016). Cell Rep. 17, 2445–2459. De Palma, M., Venneri, M.A., Galli, R., Sergi Sergi, L., Politi, L.S., Sampaolesi, M., and Naldini, L. (2005). Cancer Cell 8, 211–226. De Palma, M., Mazzieri, R., Politi, L.S., Pucci, F., Zonari, E., Sitia, G., Mazzoleni, S., Moi, D., Venneri, M.A., Indraccolo, S., et al. (2008). Cancer Cell 14, 299–311. Gabrusiewicz, K., Rodriguez, B., Wei, J., Hashimoto, Y., Healy, L.M., Maiti, S.N., Thomas, G., Zhou, S., Wang, Q., Elakkad, A., et al. (2016). JCI Insight 1, e85841. Ginhoux, F., Schultze, J.L., Murray, P.J., Ochando, J., and Biswas, S.K. (2016). Nat. Immunol. 17, 34–40. Hussain, S.F., Yang, D., Suki, D., Aldape, K., Grimm, E., and Heimberger, A.B. (2006). Neurooncol. 8, 261–279. Lahmar, Q., Keirsse, J., Laoui, D., Movahedi, K., Van Overmeire, E., and Van Ginderachter, J.A. (2016). Biochim. Biophys. Acta 1865, 23–34. Ries, C.H., Hoves, S., Cannarile, M.A., and €ttinger, D. (2015). Curr. Opin. Pharmacol. 23, Ru 45–51. Varol, C., Mildner, A., and Jung, S. (2015). Annu. Rev. Immunol. 33, 643–675.
Cancer Cell 30, December 12, 2016 833
Previews Origins of Brain Tumor Macrophages Michele De Palma1,* 1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, E ´ cole Polytechnique Fe´de´rale de Lausanne (EPFL), 1015 Lausanne, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2016.11.015
The ontogeny of brain-tumor-associated macrophages is poorly understood. New findings indicate that both resident microglia and blood-derived monocytes generate the pool of macrophages that infiltrate brain tumors of either primary or metastatic origin. Cell-tracing studies in mice have shown that several types of tissue-resident macrophages originate from yolk-sac progenitors early during embryonic development and persist throughout adulthood by selfrenewing in situ. Tissue-resident macrophages can also be replenished from circulating monocytes, which function as macrophage precursors when tissue homeostasis is perturbed, such as during inflammation, organ injury, and regeneration, and in multiple pathological processes (Varol et al., 2015). In cancer, tumor-associated macrophages (TAMs) largely derive from bone marrow hematopoietic stem cells (HSCs) through monocyte intermediates, apparently with minor, if any, contribution from locally derived, tissue-resident macrophages (Lahmar et al., 2016). Brain macrophages, also called microglia, belong to the category of earlyseeded, long-lived, and self-renewing macrophages (Varol et al., 2015). However, some brain pathologies can cause the breakdown of the selectively permeable interface that separates the blood circulation from the nervous tissues, known as blood-brain barrier (BBB), enabling monocyte infiltration into the diseased brain. Therefore, both brainresident microglia and monocyte-derived macrophages may potentially contribute to the TAM pool in brain malignancies (Lahmar et al., 2016). In Cell Reports, Bowman et al. (2016) analyze macrophage ontogeny in mouse models of brain cancer using cell-tracing strategies that enable the discrimination of microgliaderived TAMs from monocyte-derived TAMs. They identify brain TAMs originating from either cellular compartment, but also document profound in situ programming of these cells upon tumor colonization.
Previous studies interrogated brain TAM origins using cell-tracing techniques based on transplanting genetically marked HSCs into mice following whole-body irradiation to facilitate HSC engraftment, a procedure also called myeloablation. Under these conditions, HSC-derived monocytes contributed substantially to the pool of brain TAMs (De Palma et al., 2005; Lahmar et al., 2016). However, tissue irradiation may alter macrophage homeostasis and bias it toward increased turnover from HSCderived monocytes. In order to circumvent potentially confounding factors, Bowman et al. (2016) employed two cellmarking approaches not requiring irradiation or other forms of host conditioning. According to one strategy, the HSCderived monocytes, but not the resident microglia, were permanently marked; in a complementary setting, only the microglia were marked instead. Both experimental setups revealed mixed contributions of microglia and monocytes to the TAMs of intracranial gliomas, which included transplant as well as genetically engineered mouse models. Therefore, brain TAMs are an amalgam of locally and peripherally derived cells, irrespective of the tumor model and independent of brain irradiation or systemic myeloablation (Bowman et al., 2016). Do the two TAM subsets maintain memory of their ancestry and portray distinguishing features, possibly suggestive of different functions, after their recruitment to the tumor microenvironment? To address these questions, Bowman et al. (2016) analyzed the TAM’s transcriptomes by RNA sequencing. They found that the brain TAMs, regardless of their origins, were clearly distinct from normal microglia or blood monocytes. This result is consistent with the notion that macro-
832 Cancer Cell 30, December 12, 2016 ª 2016 Elsevier Inc.
phages undergo significant in situ programming, or ‘‘education,’’ in the tumor microenvironment, a process that involves the concerted action of various tumor-derived factors that impinge on the inherent plasticity of macrophage-lineage cells (Ginhoux et al., 2016). However, Bowman et al. (2016) also noted significant differences between the two subsets. For example, the monocyte-derived TAMs expressed higher levels of gene transcripts involved in antigen presentation and T cell costimulation, compared to the microglia-derived TAMs. However, genes with known immunosuppressive functions, such as interleukin-10 (IL10), CCL17, and CCL22, were also upregulated. In human glioblastomas, macrophages produce more IL10 than microglia (Gabrusiewicz et al., 2016), consistent with the findings of Bowman et al. (2016). Moreover, human glioblastomas contain infiltrates of regulatory T cells, which are immunosuppressive and can be recruited to the tumor microenvironment through macrophagederived CCL17 and CCL22 (Hussain et al., 2006). While these results illustrate complex immunomodulatory profiles of brain TAMs, overall they suggest that monocyte-derived TAMs may acquire tolerogenic properties in brain tumors, consistent with findings in other cancer types (Lahmar et al., 2016). Interestingly, a few markers could readily distinguish the two TAM subsets. Among these, integrin ITGA4 (also known as CD49d) was specifically expressed in the monocyte-derived TAMs. Of note, expression of ITGA4 was also restricted to monocyte-derived TAMs in mouse models of breast-cancer-derived brain metastasis, indicating that lineage fidelity supersedes idiosyncratic influences of the tumor type on ITGA4 expression.
Cancer Cell
Previews Moreover, human glioblastomas contained ITGA4-positive and -negative TAMs that likely mirrored the monocyteand microglia-derived subsets identified in mice. Indeed, the expression of ITGA4 strongly correlated with a ‘‘monocytederived TAM’’ gene signature in human glioblastomas (Bowman et al., 2016). Although the two brain TAM subsets displayed transcriptional profiles indicative of in situ programming, they also maintained epigenetic memory of their ancestry. Indeed, transcription factor landscapes inferred from chromatin accessibility analyses by ATAC-seq highlighted enriched activity, specifically in the monocyte-derived TAMs, of transcription factors involved in monocyte-to-macrophage differentiation, including RUNX and CEBP, and macrophage activation, including STAT3 and IRF4. On the other hand, the microglia-derived TAMs displayed increased MEF2 activity, which is known to control microglia identity. Furthermore, the genomic occupancy of selected transcription factors, such as PU.1, differed in the target genes of normal microglia and blood monocytes, and, remarkably, such differences were conserved in their TAM progenies (Bowman et al., 2016). Overall, the molecular profiles of monocyte- and microgliaderived TAMs support a multidimensional mode of macrophage activation regulated by both tissue-specific (microenvironmental) signals and lineage-restricted (developmental) epigenetic mechanisms (Ginhoux et al., 2016). The findings of Bowman et al. (2016) indicate that resident macrophages, namely microglia, contribute significantly to the TAM compartment in brain malignancies of the mouse. Work in other mouse cancer models has suggested that locally derived TAMs are progressively diluted by monocyte-derived TAMs dur-
ing tumor progression and growth (Lahmar et al., 2016). However, kinetics analyses of locally versus peripherally derived macrophages in progressing mouse brain tumors may be complicated by the constraints imposed by the relatively small volume that experimental tumors can attain in the mouse brain, compared to the human condition. The gene signatures specific to either brain TAM subset should, therefore, be interrogated in TAMs isolated from human surgical specimens, which often involve advanced and bulky brain tumors. Glioblastoma multiforme, the most frequent and malignant primary brain tumor, is markedly resistant to available therapies. Because TAMs contribute to suppressing antitumor immune responses in various cancer types, their selective targeting may offer opportunities to alleviate immunosuppression in glioblastoma, especially combined with other immunotherapies. Monoclonal antibodies that block CSF1R signaling efficiently eliminate TAMs in mice and patients with cancer, at least in part, by disrupting the monocyte-to-macrophage differentiation program (Ries et al., 2015). While the findings of Bowman et al. (2016) imply that anti-CSF1R antibodies could help impede glioblastoma colonization by monocytederived TAMs, the effects of anti-CSF1R antibodies on microglia-derived TAMs are more difficult to predict. As opposed to small-molecule inhibitors, anti-CSF1R antibodies do not cross an intact BBB and do not affect the brain-resident microglia (Ries et al., 2015), so a potential advantage of this approach is that the effects on microglia and macrophages should be restricted to the tumor. The identification of monocyte-derived TAMs in mouse and human glioblastoma also encourages the application of gene and cell therapy strategies based on targeting
biological therapeutics to tumors via engineered HSC-derived monocytes (De Palma et al., 2008). The outstanding clinical success of HSC-based gene therapy of neurodegenerative diseases (Biffi, 2016) provides a strong rationale for implementing similar strategies in glioblastoma patients, who currently lack curative treatment options. ACKNOWLEDGMENTS The author received research grants from Hoffmann-La Roche, including support for evaluating CSF1R inhibitors in mouse cancer models. REFERENCES Biffi, A. (2016). Hum. Mol. Genet. 25, R65–R75. Bowman, R.L., Klemm, F., Akkari, L., Pyonteck, S.M., Sevenich, L., Quail, D.F., Dhara, S., Simpson, K., Gardner, E.E., Iacobuzio-Donahue, C.A., et al. (2016). Cell Rep. 17, 2445–2459. De Palma, M., Venneri, M.A., Galli, R., Sergi Sergi, L., Politi, L.S., Sampaolesi, M., and Naldini, L. (2005). Cancer Cell 8, 211–226. De Palma, M., Mazzieri, R., Politi, L.S., Pucci, F., Zonari, E., Sitia, G., Mazzoleni, S., Moi, D., Venneri, M.A., Indraccolo, S., et al. (2008). Cancer Cell 14, 299–311. Gabrusiewicz, K., Rodriguez, B., Wei, J., Hashimoto, Y., Healy, L.M., Maiti, S.N., Thomas, G., Zhou, S., Wang, Q., Elakkad, A., et al. (2016). JCI Insight 1, e85841. Ginhoux, F., Schultze, J.L., Murray, P.J., Ochando, J., and Biswas, S.K. (2016). Nat. Immunol. 17, 34–40. Hussain, S.F., Yang, D., Suki, D., Aldape, K., Grimm, E., and Heimberger, A.B. (2006). Neurooncol. 8, 261–279. Lahmar, Q., Keirsse, J., Laoui, D., Movahedi, K., Van Overmeire, E., and Van Ginderachter, J.A. (2016). Biochim. Biophys. Acta 1865, 23–34. Ries, C.H., Hoves, S., Cannarile, M.A., and €ttinger, D. (2015). Curr. Opin. Pharmacol. 23, Ru 45–51. Varol, C., Mildner, A., and Jung, S. (2015). Annu. Rev. Immunol. 33, 643–675.
Cancer Cell 30, December 12, 2016 833