Cryoablation remodels the immune microenvironment in hepatocellular carcinoma: From mechanistic insights to clinical translation in combination immunotherapy

Table 6 Proposed minimal marker set and endpoints for assessing macrophage reprogramming in preclinical and translational studies

Category	Key markers / endpoints	Rationale and interpretation	Suggested supporting reference
Phenotypic and characteristic markers	Pro-inflammatory/anti-tumor (M1-like) phenotype: Surface: CD86, MHC-II (human leukocyte antigen-DR isotype); Cytokine: IL-12high, TNF-αhigh	Indicates antigen-presenting capacity and Th1-type immune activation. Increased ratio of these markers to M2 markers suggests successful reprogramming	CD86/MHC-II: Noy et al[63] and De Palma et al[64]; IL-12/TNF-α: Mills et al[59] and Wynn et al[60]
	Immunosuppressive/pro-tumor (M2-like) phenotype: Surface: CD206 (MRC1), CD163; enzyme: Arg1; immune checkpoint: PD-L1	Associated with T-cell suppression, tissue repair, and angiogenesis. Reduction post-treatment indicates attenuation of immunosuppressive tumor microenvironment	CD206/Arg1: Mills et al[59] and Wynn et al[60]; PD-L1: Tan et al[54]
	Phagocytic and “eat-me” signals: Phagocytosis receptor: FcγR; pro-phagocytic signal: CALR exposure on tumor cells	Essential for antibody-dependent cellular phagocytosis and immunogenic cell death. Upregulation enhances tumor cell clearance	CALR/phagocytosis: Fucikova et al[32] and Zhou et al[34]
Functional and prognostic endpoints	In vitro co-culture suppression assay: Inhibition of T-cell proliferation or interferon-gamma production	Direct functional readout of macrophage-mediated immunosuppression. Decreased suppression indicates functional reprogramming	Mandt et al[27], Shewarega et al[37], and Wang et al[50]
	Spatial context (multiplex immunohistochemistry/immunofluorescence): Co-localization with CD8+ T cells (permissive vs excluded)	Defines the physical interaction between macrophages and effector cells, critical for predicting immunotherapy response	Shewarega et al[37] and Santana et al[121]
	Correlation with treatment outcome: Inverse correlation with CD8+ T-cell infiltration: Direct correlation with tumor growth or survival in vivo	Validates the clinical relevance of the macrophage phenotype. Successful reprogramming should correlate with improved T-cell infiltration and survival	Mauda-Havakuk et al[20], Tan et al[54], and Gu et al[83]

It proposes a framework for standardizing the assessment of macrophage polarization states beyond the simplistic M1/M2 dichotomy, focusing on functional relevance in the context of cryoablation and immunotherapy combinations. These markers and endpoints are recommended for use in preclinical studies (murine models) and translational research (analysis of human biopsy samples pre- and post-treatment) to evaluate the efficacy of combination strategies in reprogramming the immunosuppressive macrophage compartment. A shift in the balance or ratio of pro-inflammatory to immunosuppressive markers is often more informative than absolute changes in a single marker. Spatial analysis (e.g., via multiplex imaging) is crucial for understanding the functional impact within the tissue architecture. Arg1: Arginase-1; CALR: Calreticulin; IL: Interleukin; MHC: Major histocompatibility complex; PD-L1: Programmed death-ligand 1; TNF-α: Tumor necrosis factor-alpha.