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. 2020 Sep 15;26(18):4970-4982.
doi: 10.1158/1078-0432.CCR-19-3890. Epub 2020 Jun 25.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer

Sophie J Cowman  1 , Daniel G Fuja  1 , Xian-De Liu  2 , Rebecca S Slack Tidwell  2 , Neelima Kandula  1 , Deepika Sirohi  1 , Archana M Agarwal  1 , Lyska L Emerson  3 , Sheryl R Tripp  4 , Jeffrey S Mohlman  1 , Miekan Stonhill  1 , Guillermina Garcia  5 , Christopher J Conley  3 , Adam A Olalde  5 , Timothy Sargis  5 , Adela Ramirez-Torres  5 , Jose A Karam  2 , Christopher G Wood  2 , Kanishka Sircar  2 , Pheroze Tamboli  2 , Kenneth Boucher  3 , Benjamin Maughan  3 , Benjamin T Spike  3 , Thai H Ho  6 , Neeraj Agarwal  3 , Eric Jonasch  2 , Mei Yee Koh  7
Affiliations

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer

Sophie J Cowman et al. Clin Cancer Res. .

Erratum in

  • Correction: Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.
    Cowman SJ, Fuja DG, Liu XD, Tidwell RSS, Kandula N, Sirohi D, Agarwal AM, Emerson LL, Tripp SR, Mohlman JS, Stonhill M, Garcia G, Conley CJ, Olalde AA, Sargis T, Ramirez-Torres A, Karam JA, Wood CG, Sircar K, Tamboli P, Boucher K, Maughan B, Spike BT, Ho TH, Agarwal N, Jonasch E, Koh MY. Cowman SJ, et al. Clin Cancer Res. 2021 Jun 1;27(11):3265. doi: 10.1158/1078-0432.CCR-21-1235. Clin Cancer Res. 2021. PMID: 34074655 No abstract available.

Abstract

Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC.

Experimental design: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC.

Results: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs.

Conclusions: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.

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Conflict of interest statement

Conflict of interest: MYK is founder and has equity in Kuda Therapeutics Inc. All other authors declare no conflict of interest.

Figures

Figure 1:
Figure 1:. High HIF-1α expression in tumor stroma is associated with worse outcome, whereas high tumor HAF or HIF-2α expression is associated with better outcome.
Kaplan Meier plots showing associations of HIF-1α (stroma), (A), HIF-2α, (B), and HAF, (C) with overall survival. D) Table showing multivariable overall survival analysis by patient and tumor characteristics. NI=Not Included.
Figure 2:
Figure 2:. Levels of HAF and the HIFs during ccRCC progression.
A) Representative images of large sections of paired uninvolved and T stage 1 tumor sections stained for HAF, HIF-2α and HIF-1α. Scale bars = 100μM. B-D) Quantitation of staining percent nuclear positivity of large tissue sections from T stage 1 tumors containing both tumor and uninvolved tissue (B), multi-grade tumor microarrays (C), and large tissue sections (D), based on an arbitrary threshold for HAF, HIF-2α and HIF-1α as determined by Aperio digital imaging software. Each dot on the plots indicates a single patient with patient numbers indicated in brackets. Average values ± SEM are shown with p-values indicated for (B), students’ paired T-tests, or (C,D) Mann Whitney U- tests. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. NS: not significant.
Figure 3:
Figure 3:. HIF-1α is primarily expressed in tumor-associated macrophages (TAMs).
A) Percent of immune cells as a percentage of total cells in tumors as determined by pathologists’ analysis of hematoxylin and eosin (H&E) stained tissue sections. B,C) Percent of immune cells that stained positive for HIF-2α (B) or HIF-1α (C) as determined by pathologists’ analysis of HIF-1/2α stained sections with reference to H&E stained sections. Each dot on the plots indicates a single patient with patient numbers indicated in brackets. D-E) IHC showing HIF-1α (D) and HIF-2α (E) localization in matched high grade ccRCC. Scale bars = 50μM. F) T-SNE plots showing clustering of indicated cell types from single cell sequencing of ccRCC tissue from 3 patients. V = vasculature; Tu = tumor cells; T = T-cells, NK = NK cells, P = plasma cells, B = B-cells; M = macrophages. G) Heatmap showing average transcript expression levels for HIF-1α (HIF1A), HIF2-α (EPAS1) and a panel of HIF regulated genes for the indicated cell type clusters. H) Heatmap showing average expression levels of transcripts associated with M1 or M2 polarization in the three macrophage clusters.
Figure 4:
Figure 4:. Characterization of HIF-1α expressing TAMs.
A) RNA in-situ hybridization (RNA-ISH) showing proximity of HIF1A and CD163 in TAMs, and HIF target, GLUT1 (SLC2A1), with ccRCC tumor cell -specific transcript, NDUFA4L2. Scale bars = 10μM. B) Quantitation of average number of HIF1A or GLUT1 mRNA molecules within a 5μM radius of NDUFA4L2 and CD163 molecules. At least 200 NDUFA4L2 and CD163 molecules were assessed across all patients. *** p < 0.0001. C-G) Double staining of HIF-1α with indicated markers. Scale bar = 100μM. H) Top 10 upregulated genes in HIF1A+ macrophages from differential gene expression analysis of HIF1A+ TAMs versus HIF1A− TAMs I) The most positively enriched pathways identified from gene set enrichment analysis on upregulated genes (using a 10% FDR cut-off) in HIF1A+ TAMs.
Figure 5:
Figure 5:. HIF-1α is upregulated in hypoxic M2-polarized macrophages and is associated with resistance to antiangiogenic therapy.
A) Western blots showing effect of exposure of PMA-differentiated THP-1 macrophages to M1 or M2-polarizing cytokines in normoxia or hypoxia. B-C) Taqman-qRT-PCR showing effects of hypoxia on HIF1A normalized to B2M in M1 or M2 polarized macrophages ± SEM. D-G) Western blots showing impact of indicated durations of hypoxia on a panel of RCC cells (D), with quantitation in E-G). H-I) HIF-1α (H) and HIF-2α (I) levels in ccRCC tissue and association with response to angiogenic therapy. *p < 0.05 using unpaired t-tests for log-transformed values. (J) Revised role of HAF and the HIFs in ccRCC: HIF-1α positive TAMs increase with ccRCC progression in the context of HIF-1α loss in tumor cells and is an independent predictor of poor outcome. By contrast, HAF and HIF-2α levels decrease with tumor stage and predict for better outcome.
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References

    1. Lara PN, Jonasch E. Kidney cancer : principles and practice. Heidelberg: Springer-Verlag; 2012. 309 p.
    1. Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet 1994;7(1):85–90 doi 10.1038/ng0594-85. - DOI - PubMed
    1. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995;92(12):5510–4. - PMC - PubMed
    1. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer 2011;11(6):393–410 doi http://www.nature.com/nrc/journal/v11/n6/suppinfo/nrc3064_S1.html. - PubMed
    1. Noman MZ, Desantis G, Janji B, Hasmim M, Karray S, Dessen P, et al. PD-L1 is a novel direct target of HIF-1alpha, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med 2014;211(5):781–90 doi 10.1084/jem.20131916. - DOI - PMC - PubMed

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