IntroductionLung cancer ranks among the malignancies with the highest global incidence and mortality rates, posing a substantial threat to human health1. As the predominant pathological subtype of lung cancer, adenocarcinoma accounts for over 40% of all cases2. Although radical surgery yields favorable outcomes in early-stage patients, a significant proportion are diagnosed at advanced stages, thereby losing eligibility for curative resection3. While systemic therapies for advanced lung adenocarcinoma, including targeted agents and immunotherapies, have undergone continuous advancements, persistent challenges such as acquired drug resistance and immune evasion continue to limit clinical benefits. Therefore, optimizing the treatment strategy for advanced lung adenocarcinoma has important clinical significance.Cryoablation, as a physically targeted ablation technique, demonstrates multidimensional therapeutic advantages through unique cryobiological mechanisms4. Compared with conventional surgery, cryoablation exhibits superior minimally invasive characteristics with reduced intraoperative bleeding and postoperative complications. In contrast to radiotherapy and chemotherapy, this modality achieves precise tumor ablation via percutaneous or imaging-guided localization5. Rapid freeze-thaw cycles induce ice crystal-induced necrosis and microvascular thrombosis in tumor tissues while maximally preserving surrounding pulmonary function, combining precision with safety. Cryoablation has been widely applied in treating solid tumors, including lung, liver, prostate, and breast cancers6,7,8,9.Our previous studies demonstrated that cryoablation suppresses lung adenocarcinoma invasiveness by inhibiting the MAPK/ERK. Residual tumor cells at the ablation margin exhibited a decreased Bcl-2/Bax ratio, suggesting delayed apoptosis may contribute to long-term therapeutic efficacy. Recent investigations further revealed cryoablation modulates Treg cell differentiation via the TGF-β/Smad axis to remodel the immunosuppressive microenvironment10. However, existing research predominantly focuses on protein-level signaling pathway analysis, while the whole transcriptomic dynamics and regulatory mechanisms induced by cryoablation remain unexplored. Non-coding RNAs (ncRNAs), including miRNAs, lncRNAs, and circRNAs, govern gene regulation through chromatin remodeling, transcriptional interference, and post-transcriptional modifications, critically participating in tumor proliferation, metabolic reprogramming and immune evasion11,12. Notably, physical stressors like hypothermia can alter ncRNA expression profiles, yet their regulatory roles in cryoablation-mediated antitumor effects remain uncharted.This study employed the LLC mice model to validate cryoablation’s antitumor efficacy and performed the first RNA-seq analysis to characterize coding/non-coding RNA profiles in post-ablation tissues. Systematic identification of DEGs revealed key therapeutic targets and core regulatory networks, thereby establishing a theoretical framework for optimizing cryoablation protocols in lung adenocarcinoma.Materials and methodsCell culture and animal modelsMouse lung adenocarcinoma cells (Lewis lung carcinoma, LLC) were obtained from Jiangsu Kaiji Bio-Technology Co. Ltd (Nanjing, China). Cell culture was performed with high-glucose DMEM (HyClone, USA) medium containing 10% FBS (Gibco, USA), and 1% penicillin/streptomycin (Thermo Fisher Scientific, USA). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere.All the animal experiments were complied with the guidelines of the Medical Experimental Animal Care, and animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. (Approval No.YSY-DWLL-2022647), as showed in Fig. S1. 15 male C57BL/6 mice (6–8 weeks old) purchased from SPF (Beijing) Biotechnology Co., Ltd. (Number of Animal License: SCXK 2019–0027). The mice were housed at the animal center, with specific pathogen-free conditions (temperature: 20–26℃, humidity 40% − 70%). A 7-day environmental acclimation protocol was implemented before experimental procedures.Tumor model establishment and experimental designLLC cells suspension (2 × 106 cells/mL) were engrafted via subcutaneous administration in the right lower abdomen of mice, 0.2 mL/mouse. Successful xenograft establishment was validated upon achieving target tumor volume (5 mm diameter, caliper measurements) within 5–7 days post-post-inoculation. All mice were randomly divided into three groups (n = 7/group) by random number table method: (1) Model group: sham operation, the skin of mice was cut and fixed under anesthesia, the tumor was exposed, immediately sutured, with intraperitoneal (i.p.) saline injection (0.1 mL/10 g) on days 1 and 7. (2) Cryoablation group: Tumor cryoablation was performed, and saline was injected at the same time as the model group. (3) DDP group: i.p. injection of cisplatin (3 mg/kg, Biyuntian Biotechnology Co. Ltd, Shanghai, China) at 0.1 mL/10 g on days 1 and 713.Procedure of cryoablation: Preoperative preparation included body weight measurement, and hair removal at the surgical site. Mice were fixed on the operating table after anesthesia, and the surgical area was disinfected with iodophor. A skin incision was made along the longitudinal axis of the tumor to fully expose the tumor mass. CRYO-SL argon-helium knife cryotherapy system (Tianjin, China) was used for ablation concerning the modified double-cycle cryotherapy protocol10,14. Specific parameters: A 1.2 mm-diameter cryoprobe was precisely inserted into the tumor center. Rapid cooling to −120 °C was achieved using argon gas and maintained for 10 s, followed by helium gas-mediated rewarming to 15 °C. Repeat the freeze-thaw cycle once to ensure that the ice ball formed by ablation completely covers the three-dimensional structure of the tumor. Then debridement and suture the wound. After the operation, the mice were placed in the thermal insulation device, and the vital signs and wound healing were closely monitored.Tumor growth and samplingThe duration of treatment was 14 days. The tumor volume and body weight of mice were measured every three days, and the tumor volume of mice was calculated by formula 1/2×L×W2 (L: longest diameter, W: shortest diameter)15. At the experimental endpoint (24 h last administration), the mice were anaesthetized by Sodium Pentobarbital (50 mg/kg) and euthanized by cervical dislocation. Remove and weigh the tumors, liver, kidney, lung, spleen, thymus. Organ index (the ratio of organ weight to body weight) was calculated to determine the safety of treatment. The blood samples were collected and the biochemistry indicators in blood samples were examined by automated biochemistry analyzer (Mindray, Shenzhen, China). Part of the tumor tissue was frozen in liquid nitrogen followed by −80 °C storage, and the other part was fixed in 4% paraformaldehyde fixing solution (Servicebio, Wuhan, China), all of which were used for follow-up detection.Histopathological and immunohistochemical analysisFollowing fixation, the tumor tissues were dehydrated, paraffin-embedded, and sectioned. Hematoxylin and eosin dye solution (Wuhan Servicebio, China) were successively used for staining, and the pathological morphology of tumor tissues was observed under an optical microscope and images were collected.For immunohistochemistry (IHC), paraffin-embedded sections were dewaxed, rehydrated, and subjected to antigen repair. Slides were incubated sequentially with a Ki-67 primary antibody (1:300, ab15580, Abcam) and a corresponding secondary antibody. Chromogenic development was performed using DAB, followed by dehydration, transparent after sealing. Finally, the results were observed under a microscope (BX51, Olympus, Japan), after obtaining images and processed with Image J software.High-throughput RNA sequencingTranscriptomic sequencing was performed on tumor tissues from the model and cryoablation groups. At first, total RNA was extracted from tumor tissues using Trizol kit (Servicebio), and evaluated on Agilent2100 Bioanalyzer (Agilent Technologies, USA). Strandspecific libraries and small RNA libraries were constructed by removing ribosomal RNA and sequenced using Illumina Novaseq 6000 from GeneDenovo Biotechnology Co., LTD. (Guangzhou, China). Raw sequencing data underwent quality control (FastQC) and normalization. The Fragments Per Kilobase Million (FPKM) were used to identify DGEs and analyzed using DESeq2 and edgeR software16,17. The gene |log2FC|≥1.2 and P 0.5 were selected. Cytoscape software (version 3.9.1) was used to visualize the co-expression networks.Enrichment analysesGene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used for functional analysis, and the main biological functions of DEGs and significantly enriched metabolic pathways or signal transduction pathways were identified18,19.Western blot analysisWestern blot analysis was conducted as mentioned before20. Three tumor tissue samples were randomly selected from each group for protein expression detection. Mice tumor tissue lysates were first prepared. Then, protein samples were quantified using the BCA Protein Assay Kit (Kaiji, Nanjing, China). Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Then, membranes were incubated with specific primary antibodies HIF-1α (1:1000, ab179483, Abcam), VEGF (1:1500, ab36844, Abcam), and GAPDH (1:5000, 10494-1-AP, Proteintech) overnight at 4 °C. Following three TBST washes, the membrane was incubated with an appropriate dilution of HRP-conjugated secondary antibody. Finally, the signals detected by the chemiluminescence (Thermo Fisher Scientific) detection system (ECL, Bio-Rad, USA) were used for quantitative analysis of the relative protein expression levels using Image J software.Statistical analysisStatistical analyses and graphing were performed using GraphPad Prism (version 8.0) software. All experimental data were presented as mean ± standard deviation (SD). Multiple comparisons were analyzed by ANOVA, and comparisons between two groups were conducted using t-test. Differences with P-values