Early monocyte response following local ablation in hepatocellular carcinoma

Abstract

Local ablative therapies are established treatment modalities in the treatment of early- and intermediate-stage hepatocellular carcinoma (HCC). Systemic effects of local ablation on circulating immune cells may contribute to patients' response. Depending on their activation, myeloid cells are able to trigger HCC progression as well as to support anti-tumor immunity. Certain priming of monocytes may already occur while still in the circulation. By using flow cytometry, we analyzed peripheral blood monocyte cell populations from a prospective clinical trial cohort of 21 HCC patients following interstitial brachytherapy (IBT) or radiofrequency ablation (RFA) and investigated alterations in the composition of monocyte subpopulations and monocytic myeloid-derived suppressor cells (mMDSCs) as well as receptors involved in orchestrating monocyte function. We discovered that mMDSC levels increased following both IBT and RFA in virtually all patients. Furthermore, we identified varying alterations in the level of monocyte subpopulations following radiation compared to RFA. (A) Liquid biopsy liquid biopsy of circulating monocytes in the future may provide information on the inflammatory response towards local ablation as part of an orchestrated immune response.

Keywords: brachytherapy; flow cytometry; hepatocellular carcinoma; local ablation; monocytes; radiofrequency ablation.

Figure 1

Gating strategies for myeloid cell populations. Representative dot plots from one HCC patient. (A) Panel 1: Monocytes were defined as HLA-DR+CD14+ cells. Monocyte subsets (classical, intermediate, non-classical; all presented as % frequency of monocytes) were further defined by their expression of CD14 and CD16: (i) classical monocytes (CD14++CD16−), (ii) intermediate monocyte (CD14++CD16+), and (iii) non-classical monocytes (CD14+CD16++). Monocytic MDSCs (mMDSCs) were defined as HLA-DR-CD11b+CD14+ cells (% of live cells). Cells were fixed prior to staining. (B) Panel 2: CD11b+CD64+ monocytes were further analyzed for CD86, CD163, and CD200R marker expression. Cells were not fixed prior to staining. Following staining, cells of both panels were fixed with 4% PFA and subsequently analyzed. FVD, Fixable Viability Dye-eFluor^®780.

Figure 2

Macrophage polarization within HCC biopsies. A representative sample shows the appearance of macrophages in tumor tissue obtained before local ablation. H&E staining as well as CD68 (pan macrophage), CD86 (M1), and CD163 (M2) IHC allowed the identification of large areas with M2-type macrophages (CD163+) whereas M1-type macrophages (CD86+) were almost completely absent.

Figure 3

Leukocyte changes following different local ablation treatments. Pre and post analysis of (A) LMR, (B) NMR, (C) NLR, (D) monocytes, (E) lymphocytes, and (F) neutrophils. Differential blood values from 12 IBT- and 7 RFA-treated patients were analyzed. Each dot represents an individual patient. Numbers next to dots represent patient IDs and ensure assignment of pre and post values. Data were analyzed using paired t-test (IBT: B, C, E, and F; RFA: A–C, E, F) or Wilcoxon-test (IBT: A, D; RFA: D). Intraindividual differences were analyzed using unpaired t-test (A, B, D–F) or Mann–Whitney U-test (C). p-values < 0.05 indicate statistical significance.

Figure 4

Therapy-specific alterations in monocyte cell populations. Percentage of myeloid cells as measured by flow cytometry. Pre and post analysis of 12 IBT- and 9 RFA-treated patients. (A) Classical monocytes (CD14++CD16−), (B) intermediate monocytes (CD14++CD16+), (C) non-classical monocytes (CD14+CD16++), (D) mMDSC, (E) CD86+, (F) CD163+, and (G) CD200R+ monocytes (shown as percentage of monocytes). Each dot represents an individual patient. Numbers next to dots represent patient IDs and ensure assignment of pre and post values. Data were analyzed using paired t-test (IBT: A, F; RFA: A, C–F) or Wilcoxon test (IBT: B–E, G; RFA: B, G). Intraindividual differences were analyzed using unpaired t-test (A, B, D, E) or Mann–Whitney U-test (C, F, G). p-values < 0.05 indicate statistical significance.