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

Table 3 Summary of selected clinical studies on cryoablation combined with immunotherapy for liver cancers

Design	Patient population (line of therapy)	Intervention details	Key efficacy and survival outcomes (assessment criteria)	Key baseline confounders (tumor burden, PVTT, liver function)	Key immunological findings	Ref.
Case report	Advanced HCC, post-multiline therapy (post-resection, post-lenvatinib failure)	Bevacizumab + immune checkpoint inhibitors + intratumoral cryoablation (of a single metastasis)	mRECIST, sustained CR lasting > 24 months	Tumor burden: High (multifocal intrahepatic metastases + lymph node involvement); PVTT: Yes (noted in primary tumor); liver function: NR (HBV-positive, well-controlled on therapy)	Tumor mutational burden increased from 3 Muts/Mb to 18.67 Muts/Mb post-cryoablation (biopsy of a separate, non-ablated lesion)	Li et al[91], 2021
Case report	Advanced, multifocal HCC, post-sorafenib and nivolumab (human immunodeficiency virus/HBV co-infection)	Cryoablation (partial, of 2 sites) + nivolumab - liver transplant	The mRECIST, durable CR; bridged to successful liver transplantation > 4 years post-treatment	Tumor burden: High (multifocal, bilobar, infiltrative progression); PVTT: NR; liver function: Cirrhosis (Child-Pugh score NR, HBV-DNA undetectable)	Potent “abscopal effect”: Cryoablation of partial lesions triggered a systemic immune response, leading to regression of untreated, multifocal intrahepatic tumors and reversal of immunotherapy resistance	Lucas et al[92], 2024
Phase II study (preliminary results) NCT04724226	Advanced HCC with PVTT; first-line therapy	Cryoablation (to inactivate lesions as much as possible) –within 48 hours, start combination therapy with camrelizumab (anti-PD-1) + apatinib	ORR (mRECIST): 71.4%; ORR (RECIST v1.1): 14.3%; mPFS (mRECIST): 4.63 months; mOS: 19.0 months	Tumor burden: Limited (“up-to-7” criteria); PVTT: Yes (100% of patients, 57.1% with Vp3/4); liver function: Child-Pugh A (all patients, scores of 5 or 6)	Increase in CD8+ T cells; decrease in regulatory T cells and myeloid-derived suppressor cells	Gao et al[82], 2025
CASTLE-01 single-arm, phase II NCT05010668	Locally advanced or metastatic ICC; post first-line gemcitabine + cisplatin (second-line)	Partial cryoablation of one intrahepatic lesion, followed by combination therapy with sintilimab (anti-PD-1) + lenvatinib	ORR: 75.0% (21/28, including 2 CR); disease control rate: 100%; mPFS: 16.8 months; mOS: 25.4 months (RECIST v1.1)	Tumor burden: High (79% TNM stage IV, advanced/metastatic); PVTT: Not specifically reported; liver function: Predominantly well-preserved (all enrolled had Child-Pugh class A; 11% had cirrhosis)	Cryoablation triggered recruitment and clonal expansion of CD8+ PD-1hi effector T cells into the TME and enhanced tumor immunogenicity (increased antigen presentation and IFN signaling). Lenvatinib promoted tumor vasculature normalization (increased postcapillary venule ECs, decreased angiogenic tip/stalk ECs), facilitating the influx of novel T cell clones. Combination therapy reshaped a “cold” TME into an inflamed “hot” one, increased CD4+ CXCL13+ T follicular helper cells, and was associated with tertiary lymphoid structures formation	Gu et al[83], 2026
Single-arm study NCT03183219	Advanced HCC/ICC	Locoregional therapy (incl. cryoablation) + allogeneic γδ T-cell adoptive transfer	HCC: The mPFS 8.0 months vs 4.0 months (combo vs mono), mOS 13.0 months vs 8.0 months; ICC: MPFS 8.0 months vs 4.0 months	Mixed population (HCC and ICC); detailed burden/function: NR	Expansion/persistence of donor γδ T cells; elevated serum IFN-γ, tumor necrosis factor-alpha	Zhang et al[93], 2022
Retrospective study	Metastatic HCC; advanced patients unsuitable for or refusing surgery/chemotherapy (not first-line, mostly after prior treatments)	Four groups compared: (1) Comprehensive cryoablation + immunotherapy (cryo-immunotherapy): Complete cryoablation of intrahepatic primary and accessible extrahepatic metastatic lesions, followed by adoptive dendritic cell and cytokine-induced killer cell immunotherapy cell immunotherapy (4 infusions); (2) Cryotherapy only group; (3) Immunotherapy only group; and (4) Untreated/supportive care group	The mOS: Cryo-immunotherapy group: 32.0 months; cryotherapy only group: 17.5 months; immunotherapy only group: 4.0 months; untreated group: 3.0 months; statistical significance: Overall survival in the cryo-immunotherapy group was significantly longer than in the cryotherapy only group (P = 0.024) and the untreated group (P < 0.01; assessment criteria: Revised RECIST 1.1)	Tumor burden: High. All patients had metastatic disease (bone, lung, or multiple organs). High intrahepatic primary tumor burden (24 cases with single lesion, avg. diameter 6.5 cm; 21 cases with multiple lesions, total 71 lesions). PVTT: Not explicitly mentioned. Liver function: All patients were Child-Pugh class A (25 cases) or B (18 cases); 43/45 (95.6%) had cirrhosis	In the combination therapy group (cryo-immunotherapy), patients showed an increased proportion of CD3+ CD4+ T cells in peripheral blood and elevated serum levels of interleukin-2 and IFN-γ	Niu et al[94], 2013

Efficacy was assessed according to modified Response Evaluation Criteria in Solid Tumors for hepatocellular carcinoma unless stated otherwise. Sample size (n) indicates the number of patients or evaluable lesions. Survival outcomes (median progression-free survival, median overall survival) are expressed in months. CR: Complete response; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; ICC: Intrahepatic cholangiocarcinoma; IFN: Interferon; mOS: Median overall survival; mPFS: Median progression-free survival; mRECIST: Modified Response Evaluation Criteria in Solid Tumors; NR: Not reported; ORR: Objective response rate; PD-1: Programmed death-1; PVTT: Portal vein tumor thrombus; RECIST: Response Evaluation Criteria in Solid Tumors; TME: Tumor microenvironment.