- Short report
- Open Access
Tumor-associated macrophage expression of interferon regulatory Factor-8 (IRF8) is a predictor of progression and patient survival in renal cell carcinoma
© The Author(s). 2019
- Received: 11 February 2019
- Accepted: 4 June 2019
- Published: 20 June 2019
Tumor-associated macrophages have been well-characterized in solid malignancies, including renal cell carcinoma and generally correlate with poor prognosis. However, the molecular mechanisms which govern intratumoral macrophage behavior and patient outcome are unclear. Here, we investigated whether alterations in macrophage expression of the transcriptional regulator for myeloid commitment and function, interferon regulatory factor-8 (IRF8), could predict survival of clear cell renal cell carcinoma patients. Transcriptional analysis of publicly available data revealed elevated IRF8 expression was associated with prolonged disease-free survival. Evaluation of protein expression within histologic sections of primary clear cell renal cell carcinoma patient samples showed intensity of IRF8 by CD68+ macrophages correlated inversely with stage. Survival outcomes of patients with primary or metastatic disease could be stratified on the basis of IRF8 levels by macrophages. Patients with high levels of IRF8 expression within metastatic sites had prolonged overall survival (log-rank P < 0.01, HR = 0.44, 95% C.I.: 0.23–0.84) compared to patients with low levels of IRF8 expression. When patient cohorts were further separated based on macrophage infiltration within metastatic lesions, patients with a macrophagelo IRF8hi profile had a more than 10 year increase in median overall survival compared to patients with a macrophagelo IRF8lo profile (log-rank, P < 0.001). In summary, we report that macrophage expression of IRF8 is inversely correlated with tumor mass and directly related to survival outcome. These findings support the utilization of IRF8 expression by macrophages to predict patient outcome, which may have important implications for guiding treatment decisions for renal cell carcinoma patients with metastatic disease.
- Interferon regulatory factor-8
- Renal cell carcinoma
- Tumor progression
The clinical behavior of metastatic renal cell carcinoma (RCC) can vary significantly from patient-to-patient, which complicates the tracking of disease progression and designing treatment paradigms. Biomarkers that correlate with progression are likely to improve management by providing indicators of tumor behavior, as well as potential targets for therapeutic intervention. To date, most identified biomarkers reflect tumor-intrinsic properties within the heterogeneous tumor microenvironment (TME) that, depending on which region is evaluated, may or may not express the biomarker . Stromal components that interact directly with the TME may present a more reliable representation of the aggressiveness of malignant disease. Indeed, recent findings have demonstrated that the stromal elements of the TME play a significant role in progression, response to therapy, and even prognosis at time of diagnosis .
In diverse solid tumor types, including RCC, tumor-associated macrophages (TAMs) reside within the TME in large numbers and generally correlate with poorer outcomes . Conversely, infiltration of macrophages characterized by an antitumor phenotype has been correlated with improved survival . The complexity of TAM-based prognosis is supported by recent mass cytometry findings that have characterized 17 subtypes of TAMs within clear cell (cc) RCC . In that study, the increased infiltration of 2 macrophage subsets in concert with a reduced accumulation of a third subtype predicted progression-free survival . Thus, the relationship between infiltration and prognosis may reflect the balance between immunosuppressive versus immune-activating subpopulations of intratumoral macrophages. Compared to bulk infiltration, evaluation of TAM behavior may therefore provide a more reliable method to predict outcome. The capacity of TAMs to transition from one functional state to another depends upon the cytokine milieu , which may influence their functional contributions. These changes are generally thought to be governed by distinct transcription factors that act as master regulators of cellular identity and function.
Interferon regulatory factor-8 (IRF8) is a myeloid-dependent transcription factor that is indispensable for myeloid commitment and adaptive immunity through its ability to control: 1) the development of monocytes/macrophages and dendritic cells into productive antigen-presenting cells (APCs) ; 2) the production of pro-inflammatory cytokines, such as IL-12, from APCs  which promotes the differentiation of CD4+ Th1 and CD8+ cytotoxic T cells and activation of NK cells, that in turn secrete IFN-γ, an important component of antitumor immunity . Expression of IRF8 in the human myeloid compartment is essential for the development of adaptive immunity, whereby patients with mutations in IRF8 harbor significant deficiencies in circulating monocytes or dendritic cells . Moreover, recent work from our laboratory has identified IRF8 as a previously unrecognized negative regulator of myeloid-derived suppressor cells (MDSCs), which are known to potently inhibit innate and adaptive immunity . In addition to its effects on lineage commitment, in vitro functional studies have demonstrated that IRF8-deficiency within human and murine macrophages abrogates IL-12p40 production in response to IFN-γ [7, 10].
While IRF8 acts as a regulator of lineage commitment and macrophage responses to pathogens [10, 11], less is known regarding the importance of IRF8 as a transcriptional marker of TAM behavior and disease progression in human malignancies. Recent studies performed in our laboratory demonstrate that IRF8 expression in macrophages is important for an anti-metastatic program in preclinical models of mammary cancer and melanoma . We extended these studies here to address whether the level of IRF8 in nephrectomy and metastatic tissues from ccRCC patients could be correlated with disease progression. These studies evaluated IRF8 expression by TAMs and provide the first evidence that protein expression of this transcription factor is decreased in advanced stage patient specimens and can be used to predict long-term survival in a subset of ccRCC patients.
TCGA data analysis
The results reported here, in part, are based upon data generated by The Cancer Genome Atlas (TCGA) Research Network: https://www.cancer.gov/tcga. ccRCC patient data including clinical and gene expression profiles were obtained from TCGA and cBioPortal. Only patients with complete clinical data were used in analysis. TAMs (defined by M0 macrophages, P value ≤0.1) were determined following deconvolution of TCGA data by CIBERSORT analysis . IRF8 and TAM hi vs low cutoffs were determined using the tertile method, wherein values > 66% expression were considered high and values < 33% expression were considered low; values contained within 33–66% were censored. Correlations with outcome were performed using the survival package in R studio (https://cran.r-project.org/web/packages/survival/index.html).
Tissue microarray and immunohistochemistry
This retrospective study was performed using tissues that required informed consent for donation. Patient materials selected for tissue microarray (TMA) construction were from those with histologically confirmed RCC and no known prior oncologic treatments. H&E sections were reviewed by pathologists who selected representative areas of RCC. Attention was made to exclude areas of extensive necrosis and hemorrhage within tumors. TMAs were constructed from 0.6 mm tissue cores from formalin-fixed, paraffin-embedded blocks and arrayed into a new recipient paraffin block. A previous study showed that 3 to 4 cores from each sample gave optimal statistical results ; therefore TMAs were constructed using 3 cores from each sample.
4-μm sections were cut, placed on charged slides, and dried at 60 °C for 1 hour. Slides were cooled to room temperature, deparaffinized in xylene, and rehydrated using graded alcohols. Antigen retrieval was performed using citrate buffer (BioCare Medical, catalog # CB910) for 60 min in a steamer and cooled for 20 min. Peroxidase was quenched with aqueous 0.3% H2O2 for 10 min. Slides were loaded on an autostainer and serum-free protein block (DAKO, X0909) was applied for 5 min. The primary antibody ICSBP (Santa Cruz, sc-6058) was applied at 1/400 (goat IgG) for 1 hour, followed by Bio-2-Gt (Vector Labs, PK-6105) for 30 min. Elite ABC (Vector Laboratories, PK-6100) was applied for 30 min followed by DAB chromogen (Dako, K3468) for 5 min. Serum-free protein block was applied again for 5 min. The second primary antibody, CD68 (Dako, M0814), was applied for 1 h at 1/1321 (mouse IgG1) followed by DakoLink for 30 min and DakoEnhancer for 30 min. Fast Red (Agilent, K5355) was applied for 10 min. CD3 staining was performed at 1/100 (Dako, A0452), followed by biotinylated anti-rabbit (Vector Laboratories, BA-1000), then Elite ABC, and finally DAB. Slides were counterstained with Hematoxylin, dehydrated, cleared, and cover-slipped.
Studies were performed in accordance with de-identified IRB-approved protocols. The sections were evaluated by two pathologists for specificity of staining. The percentage of cells expressing the marker (reported from 0 to 100 in deciles), and the intensity of expression (reported on a scale from 0 to 3) within CD68+ macrophages were quantified in a blinded manner by a board certified GU pathologist (G.A.). The intensity of IRF8 and CD68 staining were scored from 1 to 3 and the quantity of their expression was scored in percent positive cells of all stromal cells in the section. While the TMA consisted of additional renal tumor histologies, only ccRCC specimens were analyzed.
The Biomedical Data Science Department provided all demographic and clinical data associated with the TMAs in a de-identified manner.
Selection of IRF8 and TAM cutoffs and statistical analysis
Cutoff values for IRF8 intensity and CD68+ TAM infiltration were determined using median values from primary samples. Statistical analyses were performed using GraphPad version 7.0. Results were compared using an unpaired Kruskal-Wallis or Mann-Whitney test, as indicated. Patients without staging information were removed from analysis. Progression-free and overall survival information was available for all patients who had specimens within the TMAs and estimated via the Kaplan-Meier method. Comparisons between groups were performed using the log-rank test. P-values less than 0.05 were deemed significant.
Transcriptional analysis of IRF8 expression and macrophage infiltration
IRF8 expression within primary RCC specimens
Characteristics of clear cell RCC patients utilized in analysis of primary tumor expression of IRF8
Age at diagnosis, yr (mean ± SD)
58.6 ± 12.8
Pathological stage, n
We next addressed whether the intensity of IRF8 expression by TAMs was associated with stage or outcome. We observed increased IRF8 intensity (score) by CD68+ TAMs in stage I patient tumors (Fig. 2b). We found no association between IRF8 intensity and tumor grade (Additional file 1: Figure S2D). Comparisons of IRF8 scores with survival data showed a trend towards improved progression-free survival with high levels of IRF8 expression that did not reach significance (P = 0.0596, Fig. 2c). Elevated IRF8 protein in primary disease was associated with an increase in overall survival (P = 0.0481, Fig. 2d). We found no difference in overall survival when comparing outcomes of stage I patients with low versus high IRF8 expression (P = 0.2637, Additional file 1: Figure S3).
IRF8 expression within tumor metastases and correlations with patient survival
Characteristics of clear cell RCC patients utilized in analysis of metastatic expression of IRF8
Age at diagnosis, yr (mean ± SD)
55.6 ± 9.0
RCC is one of the most heavily immune infiltrated human tumor types  with lymphocytes and macrophages representing approximately 80% of intratumoral immune cells . Interestingly, while ccRCC also has the highest level of T cell infiltration and cytolytic activity compared with more than 18 human tumor types [5, 21] and is responsive to multiple classes of immunotherapy [18, 21], T cell infiltration is not a prognostic indicator of survival . Rather, foundational studies have shown a high proliferative capacity of intratumoral T cells is associated with survival , signifying that behavior rather than quantity of the infiltrate is a more reliable prognostic indicator.
Here, we evaluated the expression of a key transcription factor of monocyte/macrophage development and function, IRF8 [7, 9, 11] in TAM-based prognosis of patients with ccRCC. The characterization of TAMs in RCC has been performed multiple times with groups using as many as 30 markers to distinguish 17 different subpopulations of TAMs within human RCC . However, there has been little investigation of TAM expression of master transcriptional regulators, including IRF8, in human disease and whether such regulators correlate with disease status and survival.
We found that the intensity of IRF8 staining on TAMs within primary ccRCC was correlated with pathologic T-stage. Evidence that TAMs from larger primary RCC patient tumors produce more immunosuppressive cytokines on a per cell basis  than TAMs from smaller tumors further supports the hypothesis that the function, not quantity, of TAMs can influence disease aggressiveness. Our key findings that IRF8 expression by TAMs within both primary and metastatic disease was a better prognostic indicator of survival than TAM infiltration suggests that the behavior of TAMs can be represented by the status of master or major transcriptional regulators. Functionally, TAMs can also directly or indirectly influence infiltration and differentiation states of other key leukocyte subsets, including effector T cells that can promote local anti-tumor responses .
A potential limitation of this study, however, is that the analysis was based on conventional pathologic microscopic evaluation, compared to digital methods. However, our reasoning reflected the observation that macrophages within ccRCC displayed diverse morphologic shapes, potentially making it problematic for accurate digital assessment.
In summary, our study supports the notion that TAM-based prognosis carries greater clinical merit if the expression of transcription factors, which provide important insights into functionality, is assessed as part of a broader cellular signature.
The authors would like to thank the Roswell Park Comprehensive Cancer Center Pathology Shared Resource Network for assistance coordinating IHC evaluation of patient specimens.
Research reported here was supported by R01CA172105 from the National Cancer Institute/NIH (to SIA) and the National Center for Advancing Translational Sciences of the NIH under award number UL1TR001412 to the University at Buffalo. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work was also supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Cancer Institute’s Flow and Image Cytometry as well as Biostatistics Shared Resources. Research funding was also provided by the Sklarow Foundation, Elsa Kreiner Memorial Fund, Fraternal Order of Eagles, and RPCI Friends of Urology (T. Schwaab and J. Muhitch).
Conception and design: JBM, NH, TS, SIA. Development of methods: JBM, NH, GA, WB, CDM, TS, SIA. Acquisition of data: NH, GA, WB. Analysis and interpretation of data: JBM, NH, GA, AM, TS, SIA. Writing, review, and/or revision of manuscript: JBM, NH, GA, AM, WB, CDM, TS, SIA. Administrative, technical, or material support: GA, AM, WB. Study supervision: JBM, TS, SIA. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Patient samples were obtained with informed consent at Roswell Park Comprehensive Cancer Center. Studies were performed under Roswell Park Comprehensive Cancer Center Institutional Review Board approved protocols.
Consent for publication
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Parker AS, Kosari F, Lohse CM, Houston Thompson R, Kwon ED, Murphy L, et al. High expression levels of survivin protein independently predict a poor outcome for patients who undergo surgery for clear cell renal cell carcinoma. Cancer. 2006;107(1):37–45.View ArticleGoogle Scholar
- Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang TH, et al. The immune landscape of. Cancer Immun. 2018;48(4):812–30 e14.Google Scholar
- Dannenmann SR, Thielicke J, Stockli M, Matter C, von Boehmer L, Cecconi V, et al. Tumor-associated macrophages subvert T-cell function and correlate with reduced survival in clear cell renal cell carcinoma. Oncoimmunology. 2013;2(3):e23562.View ArticleGoogle Scholar
- Edin S, Wikberg ML, Dahlin AM, Rutegard J, Oberg A, Oldenborg PA, et al. The distribution of macrophages with a M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PLoS One. 2012;7(10):e47045.View ArticleGoogle Scholar
- Chevrier S, Levine JH, Zanotelli VRT, Silina K, Schulz D, Bacac M, et al. An immune atlas of clear cell renal cell carcinoma. Cell. 2017;169(4):736–49 e18.View ArticleGoogle Scholar
- Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122(3):787–95.View ArticleGoogle Scholar
- Salem S, Langlais D, Lefebvre F, Bourque G, Bigley V, Haniffa M, et al. Functional characterization of the human dendritic cell immunodeficiency associated with the IRF8(K108E) mutation. Blood. 2014;124(12):1894–904.View ArticleGoogle Scholar
- Masumi A, Tamaoki S, Wang IM, Ozato K, Komuro K. IRF-8/ICSBP and IRF-1 cooperatively stimulate mouse IL-12 promoter activity in macrophages. FEBS Lett. 2002;531(2):348–53.View ArticleGoogle Scholar
- Waight JD, Netherby C, Hensen ML, Miller A, Hu Q, Liu S, et al. Myeloid-derived suppressor cell development is regulated by a STAT/IRF-8 axis. J Clin Invest. 2013;123(10):4464–78.View ArticleGoogle Scholar
- Raetz M, Kibardin A, Sturge CR, Pifer R, Li H, Burstein E, et al. Cooperation of TLR12 and TLR11 in the IRF8-dependent IL-12 response to toxoplasma gondii profilin. J Immunol. 2013;191(9):4818–27.View ArticleGoogle Scholar
- Langlais D, Barreiro LB, Gros P. The macrophage IRF8/IRF1 regulome is required for protection against infections and is associated with chronic inflammation. J Exp Med. 2016;213(4):585–603.View ArticleGoogle Scholar
- Twum DY, Colligan SH, Hoffend NC, Katsuta E, Cortes Gomez E, Hensen ML, et al. IFN regulatory factor-8 expression in macrophages governs an antimetastatic program. JCI Insight. 2019;4(3):e124267.Google Scholar
- Newman AM, Liu CL, Green MR, Gentles AJ, Feng W, Xu Y, et al. Robust enumeration of cell subsets from tissue expression profiles. Nat Methods. 2015;12(5):453–7.View ArticleGoogle Scholar
- Rubin MA, Dunn R, Strawderman M, Pienta KJ. Tissue microarray sampling strategy for prostate cancer biomarker analysis. Am J Surg Pathol. 2002;26(3):312–9.View ArticleGoogle Scholar
- Singh AK, Winslow TB, Kermany MH, Goritz V, Heit L, Miller A, et al. A pilot study of stereotactic body radiation therapy combined with Cytoreductive nephrectomy for metastatic renal cell carcinoma. Clin Cancer Res. 2017;23(17):5055–65.View ArticleGoogle Scholar
- Yang D, Thangaraju M, Browning DD, Dong Z, Korchin B, Lev DC, et al. IFN regulatory factor 8 mediates apoptosis in nonhemopoietic tumor cells via regulation of Fas expression. J Immunol. 2007;179(7):4775–82.View ArticleGoogle Scholar
- Kim HL, Seligson D, Liu X, Janzen N, Bui MH, Yu H, et al. Using tumor markers to predict the survival of patients with metastatic renal cell carcinoma. J Urol. 2005;173(5):1496–501.View ArticleGoogle Scholar
- Atkins M, Regan M, McDermott D, Mier J, Stanbridge E, Youmans A, et al. Carbonic anhydrase IX expression predicts outcome of interleukin 2 therapy for renal cancer. Clin Cancer Res. 2005;11(10):3714–21.View ArticleGoogle Scholar
- Choueiri TK, Motzer RJ. Systemic therapy for metastatic renal-cell carcinoma. N Engl J Med. 2017;376(4):354–66.View ArticleGoogle Scholar
- Rooney MS, Shukla SA, Wu CJ, Getz G, Hacohen N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015;160(1–2):48–61.View ArticleGoogle Scholar
- Senbabaoglu Y, Gejman RS, Winer AG, Liu M, Van Allen EM, de Velasco G, et al. Tumor immune microenvironment characterization in clear cell renal cell carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures. Genome Biol. 2016;17(1):231.View ArticleGoogle Scholar
- Bindea G, Mlecnik B, Tosolini M, Kirilovsky A, Waldner M, Obenauf AC, et al. Spatiotemporal dynamics of intratumoral immune cells reveal the immune landscape in human cancer. Immunity. 2013;39(4):782–95.View ArticleGoogle Scholar
- Nakano O, Sato M, Naito Y, Suzuki K, Orikasa S, Aizawa M, et al. Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity. Cancer Res. 2001;61(13):5132–6.PubMedPubMed CentralGoogle Scholar
- Daurkin I, Eruslanov E, Stoffs T, Perrin GQ, Algood C, Gilbert SM, et al. Tumor-associated macrophages mediate immunosuppression in the renal cancer microenvironment by activating the 15-lipoxygenase-2 pathway. Cancer Res. 2011;71(20):6400–9.View ArticleGoogle Scholar