High-dose ascorbic acid selectively induces pyroptosis in LKB1-deficient lung cancer and sensitizes immunotherapy

Xiangyu Sun; Xiaoting Cai; Shangbiao Li; Ruozheng Pi; Zeqin Guo; Jiayu Jiang; Pinhao Wang; Jingrong Xiong; Zhuangzhuang Liu; Zixuan Rong; Zihang Yu; Xiaonan Zhang; Jiaqi Chen; Duanduan Han; Yanpei Zhang; Jiale Tan; Yan Lin; Zhuocheng Zou; Haochen Ai; Fangfang Kang; Xuejun Guo; Zhongyi Dong; Dehua Wu; Xue Bai

doi:10.1016/j.xcrm.2025.102291

. 2025 Aug 15;6(9):102291. doi: 10.1016/j.xcrm.2025.102291

High-dose ascorbic acid selectively induces pyroptosis in LKB1-deficient lung cancer and sensitizes immunotherapy

Xiangyu Sun ^1,³, Xiaoting Cai ^1,³, Shangbiao Li ^1,^2,³, Ruozheng Pi ^1,³, Zeqin Guo ^1,³, Jiayu Jiang ¹, Pinhao Wang ¹, Jingrong Xiong ¹, Zhuangzhuang Liu ¹, Zixuan Rong ¹, Zihang Yu ¹, Xiaonan Zhang ¹, Jiaqi Chen ¹, Duanduan Han ¹, Yanpei Zhang ¹, Jiale Tan ¹, Yan Lin ¹, Zhuocheng Zou ¹, Haochen Ai ¹, Fangfang Kang ¹, Xuejun Guo ^1,^∗, Zhongyi Dong ^1,^∗∗, Dehua Wu ^1,^∗∗∗, Xue Bai ^1,^4,^∗∗∗∗

PMCID: PMC12490232 PMID: 40818456

Summary

Liver kinase B1 (LKB1)-deficient non-small cell lung cancers (NSCLCs) exhibit primary resistance to immune checkpoint inhibitors (ICIs). The redox imbalance inherent in these tumors may represent a potential therapeutic vulnerability. High-dose ascorbic acid (AA) could induce cell redox imbalance. Here, we uncover that LKB1 deficiency upregulates the transporter GLUT1, which enables the accumulation of AA, thereby exacerbating redox imbalance in NSCLC cells. This triggers pyroptosis in LKB1-deficient NSCLC cells via the H₂O₂/reactive oxygen species (ROS)-caspase-3-gasdermin-E (GSDME) axis. In pre-clinical models, high-dose AA reverses ICI resistance and remodels the immune microenvironment, characterized by T cell factor 1 (TCF1)⁺CD8⁺ T cell (progenitor-exhausted CD8⁺ T cell [Tpex]) infiltration. Pyroptosis-driven immunogenic cell death (ICD) promotes cross-presenting dendritic cell (DC) maturation, which drives Tpex proliferation. Crucially, in Batf3^−/− mice lacking functional CD103⁺ DC populations, both Tpex expansion and therapeutic benefits are abrogated, confirming DC dependence. In addition, GSDME is validated as a gatekeeper of pyroptosis-driven antitumor immunity. This work provides a rationale for clinical trials combining ICI with high-dose AA.

Keywords: LKB1-deficient lung cancer, immunotherapy resistance, pyroptosis, reactive oxygen species, caspase-3, GSDME, high-dose ascorbic acid, dendritic cells, Tpex

Graphical abstract

Highlights

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GLUT1-mediated AA loading enhances redox stress in LKB1-deficient NSCLC
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High-dose AA induces pyroptosis in LKB1-deficient NSCLC via the ROS-caspase-3-GSDME pathway
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High-dose AA-triggered pyroptosis activates DCs to drive Tpex expansion
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High-dose AA enhances the response of LKB1-deficient NSCLC to anti-PD-1 therapy

Sun et al. report that high-dose ascorbic acid selectively induces pyroptosis in lung cancer with LKB1 deficiency via the ROS-caspase-3-GSDME axis. This promotes dendritic cell activation and progenitor-exhausted T cell expansion, ultimately sensitizing immunotherapy in LKB1-deficient lung cancer.

Introduction

While immune checkpoint inhibitors (ICIs) have transformed the standard of care for patients with non-small cell lung cancer (NSCLC), liver kinase B1 (LKB1) mutations lead to primary resistance to ICI and are inextricably associated with a more aggressive phenotype.¹^,² Notably, deficiency of LKB1 defines a cold tumor subtype of NSCLC, characterized by a paucity of progenitor-exhausted CD8⁺ T cell (Tpex) infiltration.³ Tpex cells stand out as the primary subset responding to programmed cell death protein 1/ligand 1 (PD-1/PD-L1) blockade immunotherapy with high expression of the transcription factor T cell factor 1 (TCF1, encoded by TCF7).⁴^,⁵^,⁶^,⁷ Nevertheless, an effective strategy for Tpex expansion is still lacking. Given that, facilitating Tpex expansion may be crucial in reversing immunotherapy resistance in LKB1-deficient NSCLC.

As a master regulator of AMP-activated protein kinase, LKB1 safeguards metabolic homeostasis under stress.⁸^,⁹ The deficiency of LKB1 in tumor cells cripples mitochondrial redox buffering, triggering reactive oxygen species (ROS) hyperaccumulation and rendering tumors exquisitely sensitive to oxidative perturbations.¹⁰^,¹¹^,¹² Therefore, we postulated that the redox imbalance may be the Achilles’ heel of LKB1-deficient cells and sought strategies to counteract this vulnerability.

Studies have reported that intravenous pharmacological ascorbic acid (AA) primarily inhibits tumors via exacerbating redox imbalance and epigenetic regulators.¹³^,¹⁴^,¹⁵ Noteworthily, high-dose AA could selectively kill cancer cells with specific driver gene mutations, such as KRAS and BRAF.¹⁶ Moreover, high-dose AA activates tumor immune microenvironment (TIME) and synergizes with ICIs in mouse lymphoma models as well as preclinical models of melanoma, breast, pancreatic, colorectal, and renal cancers.¹⁷^,¹⁸^,¹⁹^,²⁰ However, despite the oxidative stress imbalance observed in LKB1-deficient tumor cells, it remains unclear whether AA could selectively kill LKB1-deficient lung cancer cells and whether high-dose AA could synergize with ICIs in this type of immune-desert NSCLC.

Here, we investigated the biological consequences of high-dose AA in LKB1-deficient NSCLC. Our results indicate that LKB1-deficient NSCLC cells predominantly take up AA and show vulnerability to high-dose AA-induced pyroptosis via H₂O₂/ROS-caspase-3-gasdermin-E (GSDME) pathway. Pyroptosis-mediated immunogenic cell death (ICD) could enhance the antigen presentation of dendritic cells (DCs), therefore promoting the expansion of Tpex. Consequently, high-dose AA could reverse immunotherapy resistance in LKB1-deficient NSCLC. Collectively, our data suggested that high-dose AA is a promising therapeutic strategy to overcome the inert TIME observed in LKB1-deficient tumors.

Results

LKB1 deficiency in NSCLC elevates GLUT1-mediated AA uptake and exacerbates redox imbalance

Previous studies have reported increased ROS levels in LKB1-deficient cells.¹¹^,¹²^,²¹ To comprehensively investigate this phenomenon in lung cancer, we extracted primary lung cancer cells from genetically engineered Kras-driven Trp53 mutation (Kras^G12D/+ Trp53^R172H/+) mice, with CRISPR-Cas9-mediated LKB1 knockout (KP-sgLkb1) or not (KP-sgCtrl) (Figures S1A and S1B). We also generated human-derived A549 and H1944 lung cancer cell lines transfected with lentivirus expressing the indicated genes (LKB1) or not (Ctrl) (A549 and H1944 cell lines display a complete loss of LKB1), and murine Lewis lung carcinoma (LLC1) cell line knocking down Lkb1 (LLC1-shLkb1) or not (LLC1-shCtrl) (Figure S1B).

The cellular ROS levels were measured using the DCFH-DA assay. As expected, LKB1-deficient cells showed significantly higher ROS levels than LKB1-proficient cells (Figures 1A and 1B). Considering that high-dose AA can exacerbate redox imbalance,¹⁶ we further treated the cells with 10 mM AA, a high concentration reported to kill cancer cells.²² The addition of AA resulted in a more pronounced increase in ROS levels in LKB1-deficient cells compared to LKB1-proficient cells (Figures 1A, 1B, and S1C).

LKB1 deficiency in NSCLC elevates GLUT1-mediated AA uptake and exacerbates redox imbalance

(A and B) Intracellular reactive oxygen species (ROS) levels in A549 (A) and KP (B) cell lines with LKB1 deficiency or proficiency after AA treatment or not. Left: optical density (OD) quantification by microplate reader (A549: n = 6 per group; KP: n = 5 per group). Right: fluorescence microscope images. Scale bars: 50 μm.

(C) Quantitative reverse-transcription PCR (RT-qPCR) of relative GLUT1 expression in A549, H1944, LLC1, and KP cells with LKB1 deficiency or proficiency.

(D) GLUT1 expressions in A549, LLC1, and KP cells with LKB1 deficiency or proficiency were analyzed by immunoblot.

(E) Heatmap illustrating the GLUT1 expression in murine lung cancer cell lines with or without LKB1 deficiency, based on scaled Gene Expression Omnibus (GEO) RNA sequencing data.

(F) IHC staining of lung adenocarcinoma tissue microarray sections with anti-LKB1 and anti-GLUT1 antibodies. Representative images were displayed (left). The IHC-score of GLUT1 in LKB1-high and LKB1-low group was compared (right). Scale bars, 1 mm, 500 μm. Mann-Whitney test was used for statistical analysis.

(G) The effect of AA on intracellular ROS levels in A549 cells with GLUT1 knocked down.

(H) LC-MS analysis of the intracellular levels of AA in A549 cells with or without LKB1 deficiency. Cells were treated with 10 mM AA.

(I) Intracellular levels of AA in A549 and H1944 cells with or without LKB1 was detected by phosphomolybdic acid colorimetry.

(J) Schematic illustration showing schedules of high-dose AA treatment in the subcutaneous tumor model of LLC-shLkb1 (left). Levels of AA in subcutaneous tumors were detected by phosphomolybdic acid colorimetry (right).

Data depict one representative experiment of three independent experiments. Unpaired t test (C, F, H, I, J), two-way ANOVA (A, B), and one-way ANOVA (G) were used for statistical analysis. Data are shown as mean ± SD. ns: p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

In the cellular growth milieu, extracellular AA is rapidly oxidized to dehydroascorbic acid (DHA) with an approximate half-life of 70 min.¹⁶^,²³ We hypothesized that enhanced AA/DHA transport might underlie this vulnerability. Previous study identified GLUT1, GLUT3, and GLUT4 as DHA transporters,²⁴ and KRAS/BRAF-mutant colorectal cancer cells reportedly upregulate GLUT1 to increase AA uptake.¹⁶ We hypothesized that the exaggerated ROS increase is due to enhanced AA uptake mediated by GLUT1. We found that both mRNA and protein levels of GLUT1 were significantly upregulated in LKB1-deficient cells versus LKB1 wild-type cells (Figures 1C and 1D). Consistently, gene profiles of NSCLC mouse tissue also displayed higher levels of GLUT1 in the LKB1-deficient group (Figure 1E). GLUT3 and GLUT4 expression levels did not show an increase in LKB1-deficient cells (Figure S2A). Patient tissue immunohistochemistry (IHC) confirmed an inverse GLUT1-LKB1 relationship (Figures 1F and S2B). Importantly, knocking down GLUT1, but not GLUT3 or GLUT4, restored the increased levels of ROS (Figure S2D) (the effect of knocking down was detected by qPCR in Figure S2C). Intriguingly, when overexpressing GLUT1 in LKB1-intact lung cancer cells, AA also resulted in increased ROS levels but to a smaller degree compared with cells lacking LKB1 (Figures S2E and S2F). Normally, cells with proficient LKB1 that have low GLUT1 levels take up insufficient AA to induce a lethal ROS buildup, as their antioxidant systems maintain homeostasis. However, when GLUT1 is forcibly overexpressed in these cells, AA uptake surges, and ROS generation may outpace the capacity of the antioxidant defenses, leading to “system overload” and cell death.

To further validate the cells’ capacity for AA uptake, we measured intracellular AA levels in LKB1-deficient and LKB1-intact cell lines treated with 10 mM AA, a high dosage previously reported for cancer therapy. Liquid chromatography-mass spectrometry (LC-MS) assessment indicated that the absence of LKB1 enhanced the cell’s ability to uptake AA (Figures 1H and S2G). Phosphomolybdic acid colorimetry methods yielded similar results (Figure 1I). For in vivo experiments, we administered AA at 4 g/kg via intraperitoneal injection, a high dose chosen for its proven safety and efficacy in preclinical models.¹⁹^,²² Notably, AA accumulation was significantly higher in LKB1-deficient tumor tissue than in those with intact LKB1 (Figure 1J). Collectively, these findings indicate that LKB1 deficiency leads to upregulation of GLUT1, which in turn facilitates increased AA (via DHA) uptake and promotes a further elevation in ROS levels. GLUT3 and GLUT4 play minimal roles in this process.

High-dose AA selectively induces pyroptosis in LKB1-deficient NSCLC cells via activating the cleavage of GSDME

Since we found that LKB1 absence enhanced cells’ ability to absorb AA, we delved deeper into the effects of high-dose AA on LKB1-deficient lung cancer. Previous studies demonstrated that high-dose AA could promote cancer cell death.¹⁸^,¹⁹ Our flow cytometry analysis and propidium iodide (PI) staining also showed a higher level of cell death in LKB1-deficient cells (Figures S3A–S3C), but the specific modality of cell death remained unclear.²⁵ To investigate this issue, we administered high doses of AA to NSCLC cell lines derived from both human and murine sources, with either deficient or intact LKB1. Notably, LKB1-deficient but not LKB1-intact cells in high-dose AA group showed significant morphological alterations, with swelling of the cells and bubble-like protrusions appearing on the surface of the cellular membrane (Figures 2A and S3D). These changes were consistent with the morphology of pyroptosis as described in previous studies.²⁶ Furthermore, transmission electron microscopy revealed that high-dose AA-treated cells exhibited morphological features consistent with pyroptosis, such as membrane rupture, cell swelling, nuclear shrinkage, intact nuclear membrane, and lysis (Figure 2B). It is noteworthy that high-dose AA treatment did not induce significant pyroptosis in LKB1 wild-type lung cancer cells (Figures 2A and 2B).

To further elucidate the mechanism by which high-dose AA induces cell death in LKB1-deficient lung cancer cells, we performed RNA sequencing on cells with deficient or intact LKB1 pretreated with high-dose AA or PBS and analyzed the gene expression profiles associated with different forms of programmed cell death (Table S1). We found that pyroptosis signaling was significantly raised in LKB1-deficient cells after AA treatment (Figure 2C). To support our findings, A549 cells were incubated with ferroptosis inhibitor (Fer-1) or necroptosis inhibitor (GSK-872). The results showed that high-dose AA promoted cell death regardless of inhibitor treatment (Figure 2D), thereby excluding the involvement of ferroptosis and necroptosis.

Pyroptotic cells could release damage-associated molecular patterns, such as ATP, interleukin (IL)-1β, and IL-18.²⁷ Next, we measured tumor cell pyroptosis by assessing the release of these molecules and found them significantly increased in cell supernatant after AA treatment (Figures 2E and 2F). Similarly, high-dose AA remarkably promoted the mRNA expression of IL-1β and IL-18 in LKB1-deficient but not LKB1 wild-type lung cancer cells (Figure 2G). Pyroptosis has the potential to elicit ICD to initiate the inflammatory state of TIME.²⁸ When tumor cells undergo ICD, they tend to secrete high-mobility group box 1 (HMGB1) to extracellular.²⁹ So we detected the level of HMGB1 in cell lines and found it significantly increased after high-dose AA treatment (Figure 2H). Specifically, HMGB1 decreased in the cytoplasm but increased in the cell supernatant in the high-dose AA group, which suggested that HMGB1 was released from the cytoplasm into the extracellular medium (Figure 2I). These results collectively indicate that high-dose AA selectively elicits pyroptosis in LKB1 lung cancer cells but not ferroptosis and necroptosis.

Mediators of pyroptosis mainly involve the gasdermin family, and current studies predominantly focus on GSDMD and GSDME.³⁰^,³¹ We treated A549-LKB1-deficient cells with a range of AA concentrations. Notably, dose- and time-dependent GSDME cleavage was observed, reaching maximal intensity following 4 h of treatment with 10 mM AA (Figure 2J), a dose consistent with reported anticancer efficacy. So this concentration and time point were chosen for our experiments. While we observed obvious cleavage of GSDME (Figure 2K), the cleavage of GSDMD was not observed under these conditions (Figure S3F). Moreover, knockdown of GSDME resulted in a reduced proportion of pyroptosis in the high-dose AA group (Figures S3G and S3H). These results demonstrated that GSDME is the principal mediator of AA-induced pyroptosis. In vivo, intraperitoneal administration of AA at 4 g/kg in murine models similarly induced GSDME cleavage in tumor tissues (Figure 2L). We observed substantial elevations in both serum and intratumoral AA concentrations, confirming successful drug delivery and biological activity (Figure S3I). Collectively, these findings indicate that high-dose AA triggers pyroptosis in LKB1-deficient lung cancer cells via the activation and cleavage of GSDME.

Pyroptosis induced by high-dose AA depends on the H₂O₂/ROS-caspase-3-GSDME pathway

Figure 1 demonstrates that high doses of AA can elevate ROS levels in LKB1-deficient cells, and previous studies indicate that the increase in ROS/H₂O₂ promotes GSDME cleavage.³² We hypothesize that a similar mechanism may be at work in our study. To test this, we pretreated LKB1-deficient cell lines with the H₂O₂ inhibitor (catalase, CAT). Remarkably, the ability of high-dose AA to induce pyroptosis was restored, which confirmed that AA induced pyroptosis via generating H₂O₂ (Figures 3A and 3B). Furthermore, intracellular ROS levels were significantly elevated in the high-dose AA group, which could be reversed by CAT and N-acetylcysteine (NAC), an ROS inhibitor (Figures 3C, 3D, and S4A). The release of ATP, a damage-associated molecule caused by pyroptosis, could also be reversed by NAC (Figure 3E). Moreover, CAT treatment similarly inhibited AA-induced GSDME cleavage (Figure 3G). These results confirm the crucial role of ROS/H₂O₂ in mediating the cytotoxic effects of high-dose AA on LKB1-deficient tumor cells.

It was reported that ROS induces cell pyroptosis through activating caspase-3 to cleave GSDME.³¹ To determine whether high-dose AA degraded GSDME by activating caspase-3, we treated LKB1-deficient cells with the caspase-3 inhibitor (Z-DEVD-FMK) and the pan-caspase inhibitor (Z-VAD-FMK). No obvious pyroptosis phenomenon was observed after AA treatment (Figures 3F, S4B, and S4C). Next, we evaluated the levels of GSDME-F, GSDME-N, cleaved caspase-3, and caspase-3 in LKB1-deficient cells by immunoblotting. The findings implied that caspase-3 cleavage induced by H₂O₂ was responsible for GSDME activation and pyroptosis (Figure 3G). Both the caspase-3 inhibitor (Z-DEVD-FMK) and the pan-caspase inhibitor (Z-VAD-FMK) were able to suppress the cleavage of caspase-3 and the slice of GSDME (Figure 3H). Furthermore, we performed immunohistochemical staining for cleaved caspase-3 in LKB1-deficient tumor tissues and found its expression significantly higher in the AA-treated group compared to the vehicle group (Figure 3I). In summary, our in vivo and in vitro experiments demonstrate that AA activates caspase-3, leading to its cleavage and subsequent activation of GSDME.

To further confirm the role of caspase-3, we used LKB1-deficient A549 cells with knockout caspase-3 by CRISPR-Cas9 (Figure S4D). We observed that high-dose AA induced the release of ATP, lactate dehydrogenase (LDH), and HMGB1 in cells with wild-type caspase-3, which was absent in the caspase-3 knockout group (Figure 3J). Moreover, cells lacking caspase-3 did not exhibit morphological features consistent with pyroptosis (Figure S4E).

Pyroptosis is typically initiated by inflammasomes that activate caspase-1 or caspase-11, leading to GSDMD cleavage.³³ We examined the protein levels of caspase-1 and caspase-11 after AA treatment, and immunoblotting revealed no significant changes, indicating that these caspases are not key drivers of AA-induced pyroptosis (Figure S4F). Besides, as apoptosis signaling pathways can lead to the activation of caspase-3, which then proteolytically activates gasdermins,³¹ we examined the protein levels of BCL2-associated X (BAX) and BCL2 antagonist/killer (BAK) (essential for apoptosis)³⁴ or mixed lineage kinase domain-like protein (MLKL, essential for necroptosis),³⁵ which remained unchanged (Figure S4G). We also used CRISPR-Cas9-mediated BAX/BAK double-knockout and MLKL-knockout A549 cells (which are LKB1 deficient) to directly investigate to assess their involvement in AA-induced cell death (Figure S4H). Notably, sgBAX/BAK, sgMLKL, and sgCtrl cells all displayed typical pyroptotic morphology after high-dose AA treatment (Figure S4I), with PI staining confirming similar levels of cell death across groups (Figures S4J and S4K). Moreover, AA treatment markedly increased ATP and LDH release in sgCtrl, sgBAX/BAK, and sgMLKL cells (Figure S4L). This indicates that apoptosis and necroptosis contribute minimally to AA-induced cell death observed in our study. These findings reinforce the notion that AA-induced pyroptosis is primarily mediated by ROS-induced caspase-3 activation and subsequent GSDME cleavage.

High-dose AA restores PD-1 blockade resistance of LKB1 deficiency in NSCLC in vivo

To test the therapeutic effects of AA, we applied treatment protocols for established aggressive lung cancer mice models. Firstly, Lewis lung cancer (LLC1) and TC1 cell lines with stable knockdown of LKB1 or Ctrl were constructed (Figures S1B and S5A) and then subcutaneously implanted. As expected, LKB1-deficient lung cancer exhibited an immune-desert TIME (Figures S5B and S5C). We treated the mice with 4 g/kg AA five times a week and 200 μg αPD-1 Ab (antibody) three times a week for 2 weeks when tumors became palpable (Figure S5D). Immunoglobulin G was used as vehicle. Mice were sacrificed when any tumor exceeded the allowed size. In the LKB1-shCtrl group, αPD-1 Ab therapy could restrict the tumor growth in a certain level, although it did not improve survival (Figures 4A and 4B). Since AA mainly targets LKB1-deficient tumor cells, its efficacy in this setting was limited. In the LKB1-deficient group, while αPD-1 Ab alone cannot significantly suppress tumors, the combination of AA and αPD-1 Ab exhibited a strong ability to restrict tumor growth and prolong survival (Figures 4C–4F). Moreover, we stably transduced the LLC1-shLkb1 and LLC1-shCtrl cell lines with a luciferase reporter plasmid and implanted them orthotopically into the lung to establish an in situ model. We found that high-dose AA overcame αPD-1 Ab resistance in the LKB1-deficient group, whereas it had no effect in the LKB1-proficient group (Figures 4G–4L). These observations align with the notion that high-dose AA restores PD-1 blockade resistance of LKB1 deficiency in NSCLC in vivo.

The dose used, 4 g/kg, was chosen based on preclinical studies, and we found that it does not cause overt toxicity, as evidenced by normal blood counts, even though these cells express GLUT1 (Figure S6A).³⁶ Moreover, the 4 g/kg AA treatment does not affect the body weight of the mice (Figure S6B) nor compromise the normal organs such as lungs, liver, spleen, and kidneys (Figure S6C).

In conclusion, we have demonstrated that high-dose AA enhances the efficacy of ICI in LKB1-deficient lung tumors while exhibiting minimal toxicity and side effects. Notably, effective tumor control is achieved only when AA is combined with PD-1 inhibition; AA monotherapy demonstrates limited efficacy. We speculate that AA may act as an immune reserve, bolstering the overall immune response.

High-dose AA remodels the TIME characterized by the infiltration of Tpex cells

To evaluate the effect of high-dose AA on the immune landscape of NSCLC, we performed single-cell RNA sequencing on tumors treated with αPD-1 Ab alone or αPD-1 Ab + high-dose AA. After rigorous quality control and cellular filtration, we cataloged 44,745 cells into 10 distinct cellular lineages, annotated with canonical cell markers (Figures 5A and S7A). We found that combination therapy decreased tumor cells and macrophages while increasing T cells relative to αPD-1 Ab monotherapy (Figure 5A).

High-dose AA remodeled the tumor immune microenvironment characterized by the infiltration of TCF1⁺CD8⁺ T lymphocytes

(A) Left: t-distributed stochastic neighbor embedding (t-SNE) plot of 44,745 cells from lung tumor samples treated with αPD-1 Ab (n = 2) and high-dose AA + αPD-1 Ab (n = 2), colored by their 10 major cell types. Right: cell type composition of each cluster.

(B) Cluster composition of T cells.

(C) Volcano plot displaying the differentially expressed genes between cells within cluster 2 and cells outside cluster 2 (red: upregulated in cluster 2). The x axis represents log-fold changes, and the y axis represents log10 adjusted p values. A two-sided Wilcoxon rank-sum test was used.

(D) Expression levels of *Tcf7* in T cells treated with αPD-1 Ab or high-dose AA + αPD-1 Ab. ∗∗∗∗p < 0.0001.

(E) Enrichment (log2 p values) of progenitor-like gene signature in each cell illustrated in t-SNE plots.

(F) Trajectory manifold of T cells annotated by cell type using the Monocle 2 algorithm.

(G) RNA velocity analysis of gene expression in T cells from αPD-1 Ab group and αPD-1 Ab+AA group, predicted by Velocyto/ScVelo.

(H) Visualization and localization images (left) and quantification (right) of TCF1 (red)-expressing CD8⁺ (green) T cells through immunofluorescence (IF) from lung tumor samples of LLC1-shCtrl and shLkb1 mice with indicated treatments (for control immunoglobulin G [vehicle], anti-PD-1 Ab [αPD-1 Ab], high-dose AA [AA], or co-treatment with anti-PD-1 Ab and AA [αPD-1 Ab + AA], n = 5 for per group). Groups compared using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ns: p > 0.05.

Given that T cells have been regarded as the vanguard of antitumor immunity,³⁷ we extracted T cell subpopulations for further analysis. Six distinct clusters were partitioned based on their transcriptomic characteristics (Figures S7B and 5B). We observed that combined treatment primarily augmented the number of T cells within cluster 2 (Figure 5B). Notably, the gene most significantly upregulated in cluster 2 was Tcf7 (Figures 5C and S7C), renowned as a hallmark of progenitor-like CD8⁺ T cells. The combination treatment significantly increased the abundance of Tcf7-expressing T cells compared to αPD-1 Ab treatment alone (Figure 5D). To determine how cells in cluster 2 overlap with progenitor-like cells, we performed a single-cell gene enrichment analysis using 207 progenitor-like signature genes previously identified (Table S2). Cluster 2 showed significant enrichment of progenitor-like signature genes, whereas such characteristics were sparsely observed in other cellular clusters (Figure 5E). Based on its transcriptional signature, we defined cluster 2 as the progenitor-like CD8⁺ (Tpex) population. Meanwhile, we found that the high-dose AA + αPD-1 Ab treatment group owned a higher progenitor-like score than the αPD-1 Ab alone group (Figure S7D).

Trajectory analysis revealed that Tpex cells resided at the root of differentiation, which transited into exhausted effector T cells capable of performing direct tumor cell killing (Figure 5F). RNA velocity analysis revealed that the combination treatment enhanced the exhausted progenitor-like characteristics of T cells compared to αPD-1 Ab monotherapy (Figure 5G), which has the stemness potential of self-renewing, proliferation, and differentiation. These results indicated that high-dose AA treatment could enhance the infiltration of Tpex cells.

We further evaluated myeloid cells in TIME. Tumor-associated macrophages (TAMs) were aggregated and clustered into 7 clusters (Figure S8A), then they were annotated with SPP1⁺ macrophages and CXCL9⁺ macrophages (Figures S8B and S8C). SPP1 defined the polarity of TAMs, which was associated with a poorer prognosis.³⁸ Notably, the combination treatment significantly reduced the abundance of SPP1-expressing macrophages (Figures S8D and S8E).

Next, we conducted immunofluorescence (IF) to further confirm the altered immune microenvironment. In LKB1-deficient lung orthotopic tumors, Tpex cells markedly expanded after AA or AA + αPD-1 Ab treatment, whereas αPD-1 Ab monotherapy had no effect (Figure 5H). In the LKB1-proficient group, where a certain degree of immunogenicity was present, αPD-1 Ab treatment alone could induce Tpex expansion at a certain level, whereas AA exhibited no effect (Figure 5H). This further underscores the selective cytotoxicity of high-dose AA against LKB1-deficient lung tumors. Notably, our findings also revealed that the combination of high-dose AA and αPD-1 Ab effectively reduced the abundance of SPP1⁺ macrophages (Figures S8F and S8G). This effect was observed only in the combination treatment group (Figures S8F and S8G). Taken together, high-dose AA dynamically remodeled the TIME, fostering Tpex cell expansion and reinforcing the immune response.

Pyroptosis induces ICD and enhances cross-presenting DC maturation, thereby driving Tpex expansion

Next, we investigate why high-dose AA leads to the expansion of Tpex cells. We have confirmed that AA induces pyroptosis in vitro, prompting us to validate these findings in vivo. The pyroptosis score based on 57 genes associated with pyroptosis (Table S3) was greatly higher in the high-dose AA + αPD-1 Ab group versus the αPD-1 Ab group (Figure S9A). Additionally, single-cell analysis revealed a marked increase in Hmgb1 expression in the combination group (Figure S9B). To confirm that high-dose AA could induce ICD in vivo, we subcutaneously injected pyroptotic high-dose AA-treated LKB1-deficient LLC1 cells into immunocompetent mice as the tumor vaccine and then challenged them 8 days later with untreated LKB1-deficient LLC1 cells into the opposite flank of mice (Figure 6A). Immunization with AA-treated LKB1-deficient LLC1 markedly protected mice from tumor challenge. Three of 7 mice vaccinated with AA-treated cells remained tumor-free for the duration of the study, and the remaining mice had tiny tumors, while only one of seven mice remained tumor-free in the vehicle group (Figure 6A). Moreover, tumors that developed in mice immunized with AA-treated cells exhibited a significantly slower growth rate compared to the control group (Figure 6B).

Pyroptosis induces immunogenic cell death and enhances cross-presenting DC maturation, thereby driving Tpex expansion

(A) Left: schematic of the vaccination protocol. Right: tumor incidence in vaccinated versus control mice (n = 7 per group).

(B) Tumor growth in vaccine (n = 4) vs. vehicle (n = 6) groups.

(C and D) The percentages of mature DCs (CD80⁺CD86⁺), CD103⁺ DCs among total CD45⁺CD11c⁺ cells (C), and TCF1⁺ TIM3⁻ T cells among total CD45⁺CD3⁺CD8⁺PD1⁺ cells (D) in tumor-draining lymph nodes isolated from TC1-shLkb1 mice models with indicated treatments (n = 5 per group).

(E) C57BL/6J background *Batf3*^−/− mice (n = 4 per group) and *Batf3*^+/+ (wild-type [WT]) mice (n = 5 per group) were injected with TC1-shLkb1 tumor cells. Tumor size was monitored. Two-way ANOVA was performed. Data are shown as mean ± SE.

(F) The percentages of TCF1⁺ TIM3⁻ T cells among total CD45⁺CD3⁺CD8⁺PD1⁺ cells in tumor DLNs.

(G) Visualization and localization images (left) and quantification (right) of TCF1⁺ (red) CD8⁺ (green) T cells through immunofluorescence (IF) in tumors from *Batf3*^−/− mice (n = 4 per group) and WT mice (n = 5 per group).

One-way ANOVA (C, D, F, G) and two-way ANOVA (B, E) were performed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns: p > 0.05.

To further investigate the pyroptosis-mediated ICD induced by high-dose AA treatment, we performed flow cytometry analyses on tumor-draining lymph nodes (dLNs) to assess the changes in immune cell populations. It is known that DCs are essential for antigen uptake and the subsequent regulation of immune responses in vivo. We found that both the AA group and the combination group exhibited significant increases in mature DCs (CD80⁺CD86⁺ DCs) (Figure 6C). Moreover, a significantly increased percentage of CD103⁺ DCs in dLNs was observed (Figure 6C), which is critical for enabling the presentation of tumor antigen to CD8 T cells. These findings corroborate our hypothesis that ICD promotes DC maturation and antigen presentation. Meanwhile, we observed a marked increase in Tpex cells (TCF1⁺ PD-1⁺ TIM3⁻) in the AA group and the combination group (Figure 6D). This expansion was absent in Batf3^−/− mice, which lack functional CD103⁺ DCs that are essential for cross-priming,³⁹ demonstrating the necessity of antigen presentation for Tpex cell generation (Figure 6F). Notably, in Batf3^−/− mice, the combination of high-dose AA with αPD-1 Ab therapy failed to control tumor growth (Figure 6E). Immunofluorescence of tumor tissues revealed that the combination treatment markedly increased Tpex cell infiltration. This increase was not observed in Batf3^−/− mice (Figure 6G). These findings collectively demonstrate that ICD induced by high-dose AA facilitates DC maturation and antigen presentation in dLNs, which drives Tpex cell proliferation, ultimately responding to immune checkpoint blockade.

Inner blockage of GSDME reversed the synergistic effect of high-dose AA and αPD-1 Ab therapy

In light of our in vitro findings, we subsequently investigated whether high-dose AA-induced LKB1-deficient pyroptosis depended on GSDME in vivo. Firstly, we knocked out GSDME in LLC1-shLkb1 cells using CRISPR (Figure S10A). Then we transplanted LLC1-shLkb1-sgGsdme and LLC1-shLkb1-sgCtrl cells subcutaneously and orthotopically in a murine model, followed by treatment with vehicle or high-dose AA combined with αPD-1 Ab treatment. Notably, Gsdme deletion significantly exacerbated the tumor burden in the combination treatment group (Figures 7A–7C). To further elucidate the impact of blocking GSDME on TIME subjected to combination treatment, we profiled the cell atlas of orthotopic tumors by single-cell RNA sequencing. A total of 14,260 cells were selected after quality control, and they were cataloged into 8 distinct cell lineages annotated with canonical cell markers (Figures 7D and S10B). Cell composition analysis showed reduced T cells and increased cancer cells in the sgGsdme group compared with the sgctrl group (Figure 7D).

Inner blockage of GSDME reversed the synergistic effect of high-dose AA and anti-PD-1 therapy

(A) LLC1-shLkb1-sgctrl or LLC1-shLkb1-sgGsdme cells were injected subcutaneously, followed by indicated treatments (vehicle: control immunoglobulin G, Comb: αPD-1 Ab + AA). Tumor growth curves and tumor appearance were shown (n = 5 per group). Two-way ANOVA was performed to analyze the tumor growth curves, ∗p < 0.05, ∗∗∗p < 0.001, ns: p > 0.05.

(B) LLC1-shLkb1-sgctrl or LLC1-shLkb1-sgGsdme cells were injected into the left chest of mice followed by co-treatment with αPD-1 Ab and AA. Tumor formation was detected using micro-CT scan.

(C) Waterfall plot showing tumor volume response to the treatment (related to B). Each column represents one mouse. One-way ANOVA was performed. ∗p < 0.05.

(D) Left: Uniform manifold approximation and projection plot of 16, 217 cells from control (n = 2) and sgGsdme (n = 2) tumors treated with the combination of high-dose AA and αPD-1 Ab. Eight cell types were identified. Right: percentages of cells from each cluster in each sample.

(E) Violin plot of pyroptosis score in tumor cells from the control group and the sgGsdme group. ∗∗∗∗p < 0.0001.

(F) The expression level of *Hmgb1* in tumor cells from the control group and the sgGsdme group detected from single-cell RNA sequencing and visualized through a feature plot.

(G) Expression level of *Tcf7* in T cells from lung tumor samples of the control group and the sgGsdme group after αPD-1 Ab + AA treatment. One-way ANOVA was performed. ∗∗∗∗p < 0.0001.

(H) mIHC assay of LLC1-shLkb1-sgctrl (left) or LLC1-shLkb1-sgGsdme (right) orthotopic mouse models with αPD-1 Ab and AA treatment. The representative image with CD8 (green), TCF1 (purple), F4/80 (yellow), SPP1 (red), and DAPI (blue). n = 5 per group. Scale bars, 40 μm.

(I) Quantitative analysis of the proportion of TCF1⁺ CD8⁺ T among total CD8⁺ T and SPP1⁺ macrophages in TAMs (n = 5 per group). Groups compared using one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001.

We proceeded to investigate the effect of GSDME inhibition on the functionality of these cells. Notably, tumor cells in the sgGsdme group exhibited a lower pyroptosis score (Figure 7E). Pyroptosis-related cytokine HMGB1 was also significantly reduced in tumor cells from the sgGsdme group (Figure 7F). Analysis of the enriched pathways among tumor cells revealed that the pyroptosis and immune effector processes were markedly upregulated in the control group compared to the sgGsdme group (Figure S10C). This meant that tumor cell pyroptosis induced by high-dose AA was restored.

Besides, we found that the sgGsdme group showed a decreased abundance of Tcf7-expressing T cells and an increased abundance of Spp1-expressing macrophages (Figures 7G and S10D). In line with our findings from transcriptomic data, quantitative multiplex IHC (mIHC) assay of orthotopic murine tumors revealed a reduced number of TCF1⁺ CD8⁺ T cells and an increased number of SPP1⁺ macrophages in the sgGsdme group after combination therapy (Figures 7H, S10E, and S10F). These results demonstrate that inner blockage of GSDME reversed the synergistic effect of high-dose AA and αPD-1 Ab therapy.

Discussion

This study proposes a strategy to reverse resistance to immune checkpoint inhibitors therapy by leveraging the unique features of LKB1-deficient NSCLC. By exploiting the inherent redox imbalance in LKB1-deficient NSCLC cells, we discovered that high doses of AA can further intensify this imbalance, selectively eliminating these tumor cells. Mechanistically, we found that LKB1-deficient NSCLC cells express high levels of GLUT1, which leads to increased uptake of AA. High-dose AA induces cell pyroptosis via the H₂O₂/ROS-caspase-3-GSDME pathway. Pyroptosis elicited ICD and activates DCs for effective antigen presentation, thereby promoting the expansion of progenitor-exhausted T cells (Tpex) and conferring sensitivity to αPD-1 Ab therapy.

Our study demonstrates that high-dose AA exhibits selective lethality in LKB1-deficient NSCLC for two critical reasons. Factor 1 (transport): high GLUT1 expression enables AA uptake and accumulation (via its oxidized form, DHA) in LKB1-deficient cells. Factor 2 (redox vulnerability): LKB1 deficiency creates a pre-existing redox imbalance, which synergizes with DHA’s pro-oxidant effects to overwhelm antioxidant defenses. In normal cells that express GLUT1 (e.g., activated T cells),⁴⁰ intact LKB1 signaling maintains redox homeostasis, and it possesses the capacity to metabolize ROS, thereby ensuring their survival. In LKB1-deficient NSCLC cells, high GLUT1 expression combined with an inherent redox imbalance renders them more susceptible to AA. Consequently, AA drives ROS levels beyond the cytotoxic threshold, eliciting cell pyroptosis.

TCF1-expressing CD8⁺ T cells are instrumental in mounting protective anticancer immunity and are indispensable for anti-PD-1/PD-L1 efficacy.⁴^,⁷ On this ground, how to expand Tpex and maintain T cell stemness has attracted substantial attention. Previous study indicated that intratumoral stem-like CD8⁺ Tpex mainly accumulated via two ways: tumor in situ differentiation and expansion, and migration from local dLNs, with the latter being the dominant source.⁴¹ ICD could promote DC maturation and migration to dLNs to carry out antigen presentation. This is a critical pathway for Tpex cell expansion in dLNs.⁴² Our results further demonstrate that by eliciting ICD, AA treatment promotes the maturation of conventional type 1 dendritic cells (cDC1s), which are essential for cross-presenting tumor antigens and priming cytotoxic T cell responses.⁴ The dependency of AA’s efficacy on cDC1s, as demonstrated in Batf3^−/− mice, emphasizes the centrality of DC activation in bridging innate and adaptive immunity.

Our study reveals a significant decrease in SPP1⁺ macrophages when receiving high-dose AA in conjunction with αPD-1 Ab immunotherapy via GSDME-dependent pyroptosis (Figures S8, S10D, and S10E). Since SPP1 is instrumental in establishing the polarity of TAMs, the prevalence of SPP1⁺ macrophages is indicative of a pro-tumorigenic pathway, which correlates with a more unfavorable prognosis.³⁸ We observed decreased SPP1⁺ macrophages following combination therapy, and this event appears to occur in parallel with Tpex expansion. The latter is primarily driven by the activation of antigen presentation by cDC1s, whereas the former may be associated with the reprogramming of macrophage phenotypes within the tumor microenvironment, such as an increase in pro-fibrotic activity or a reduction in immunosuppressive subpopulations.⁴³^,⁴⁴ Furthermore, AA monotherapy leads to Tpex expansion without a corresponding reduction in SPP1⁺ macrophages, suggesting that factors other than SPP1⁺ cell elimination are at play. Future research is necessary to delineate the precise mechanisms underlying the depletion of SPP1⁺ macrophages mediated by GSDME-dependent pyroptosis.

Pharmacological AA has emerged as a hopeful anticancer agent due to its low financial cost and low toxicity in both preclinical and clinical trials.¹⁴^,²² Schoenfeld et al. reported no grade 3 or 4 toxicities in 14 patients with advanced-stage NSCLC receiving chemotherapy and pharmacological ascorbate.²⁵ Subsequently, a phase 2 clinical trial in 2022 indicated that adding high-dose ascorbate to platinum-based chemotherapy enhanced tumor response in advanced-stage NSCLC.⁴⁵ Thus far, the first-line standard treatment regimen for patients with NSCLC with LKB1 mutations is still immunotherapy combined with platinum-based double chemotherapy, yet these patients typically derive limited benefit.¹⁴ Our results provide evidence that high-dose AA could potentiate ICI efficacy in LKB1-deficient NSCLC. In light of the safety and efficacy of high-dose AA and our preclinical research evidence, we are applying for a phase 2 clinical study to explore the efficacy and safety of pharmacological ascorbate in combination with immunotherapy and chemotherapy as a first-line treatment in LKB1-mutant advanced-stage NSCLC.

Limitations of the study

First, the in vivo findings are constrained by the limited number of murine lung cancer cell lines to establish isogenic mouse models, and the therapeutic efficacy of AA monotherapy is modest. Besides, the effectiveness of high-dose AA combined with αPD-1 Ab in patients with cancer remains to be confirmed. We plan to pursue a clinical trial to assess its therapeutic potential. In addition, further investigation is required to clarify the detailed mechanisms by which high-dose AA combined with αPD-1 Ab leads to the reduction of SPP1⁺ macrophages.

Resource availability

Lead contact

Requests for further information, resources, and reagents should be directed to and will be fulfilled by the lead contact, Xue Bai (baixue1990@i.smu.edu.cn).

Materials availability

This study did not generate any new reagents.

Data and code availability

•
The RNA-seq data for the LKB1-deficient mouse model are accessible in the GEO database under accession codes GSE175479,⁴⁶ GSE193895,⁴⁷ GSE137396,⁴⁸ GSE137244,⁴⁸ and GSE133895. The bulk RNA-seq data used in Figure 2C have been deposited in the National Center for Biotechnology Information’s GEO under the accession number GSE269742. Single-cell RNA data for LLC1-shLkb1 orthotopic lung tumor tissue from this paper used in Figures 5 and 6 have been deposited in GSA under the accession number CRA026496. Single-cell RNA data for LLC1-sgGsdme orthotopic lung tumor tissue from this paper used in Figure 7 have been deposited in GSA under the accession number CRA026608.
•
All original code has been deposited at Zenodo. DOI is https://doi.org/10.5281/zenodo.15745003.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (grant no. 82272731), the Natural Science Foundation of Guangdong Province (grant no. 2024A1515011372), the Regional Joint Fund Project of Basic and Applied Basic Research Foundation of Guangdong Province (2022B1515120035), and the Science and Technology Projects in Guangzhou (grant no. 2024A04J5204). We are grateful to Professor Liang Chen for technical guidance. We also extend our thanks to Kunheng Du and Wenhao Wu for technical support.

Author contributions

Conceptualization, X.S., X.C., X.B., X.G., J.J., Z.D., and S.L.; data curation, X.S., X.C., J.J., and D.H.; formal analysis, X.S., X.G., X.C., J.X., Z.L., Z.R., and Z.Y.; funding acquisition, D.W., X.G., and X.B.; investigation, X.S., X.C., R.P., Z.R., and J.C.; methodology, X.S., X.G., X.C., R.P., J.J., P.W., Z.L., X.Z., Z.Z., H.A., F.K., and Z.D.; project administration, X.S.,Y.Z., X.G., R.P., Z.D., and S.L.; resources, Z.D., S.L., and X.B.; visualization, X.S., P.W., X.Z., and Z.Z.; writing – original draft, X.S., X.G., and X.C.; writing – review and editing, Z.D., D.W., S.L., and X.B.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

anti-LKB1 (27D10)	CST	Cat#3050, RRID:AB_823559
anti-LKB1 (D60C5F10)	CST	Cat#13031, RRID:AB_2716796
anti-β-actin	Fdbio science	Cat#FD0060-100, RRID:AB_2687938
anti-GLUT1	Proteintech	Cat#66290-1-Ig, RRID:AB_2881673
anti-GAPDH	abmart	Cat#P60037S, RRID:AB_2937024
anti-GSDMD	Abcam	Cat#ab210070, RRID:AB_2893325
anti-DFNA5/GSDME	Abcam	Cat#ab215191, RRID:AB_2737000
anti-DFNA5/GSDME	HUABIO	Cat#HA723251, RRID:AB_3697047
anti-F4/80	Abcam	Cat#ab300421, RRID:AB_2936298
anti-SPP1	Abcam	Cat#ab218237, RRID:AB_2732079
anti-CD8	Abcam	Cat#ab217344, RRID:AB_2890649
anti-TCF7	CST	Cat#2203T, RRID:AB_2199302
anti-Caspase 11	Affinity Biosciences	Cat#AF5130, RRID:AB_2837616
anti-phospho-MLKL(Ser358)	Affinity Biosciences	Cat#AF7420, RRID:AB_2843860
anti-MLKL	Proteintech	Cat#66675-1-I, RRID:AB_2882029
anti-BAK	Proteintech	Cat#29552-1-AP, RRID:AB_2923596
anti-BAX	Proteintech	Cat#60267-1-Ig, RRID:AB_2848213
anti-Caspase 1	Proteintech	Cat#81482-1-RR, RRID:AB_2935555
anti-Caspase-3	Proteintech	Cat#19677-1-AP, RRID:AB_10733244
APC/Cy™7-conjugated CD45 (557659)	BD Biosciences	Cat#557659, RRID:AB_396774
BV510-conjugated CD3 (563024)	BD Biosciences	Cat#563024, RRID:AB_2737959
PE/Cy™5-conjugated CD8a (553034)	BD Biosciences	Cat#553034, RRID:AB_394572
APC/R700-conjugated PD1 (565815)	BD Biosciences	Cat#565815, RRID:AB_2739366
PE/CF594-conjugated TIM-3 (566998)	BD Biosciences	Cat#566998, RRID:AB_2869998
BV421-conjugated TCF-1 (566692)	BD Biosciences	Cat#566692, RRID:AB_2869822
BV605-conjugated CD11c (563057)	BD Biosciences	Cat#563057, RRID:AB_2737978
PE-Cyanine7 conjugated CD80 (104734)	BioLegend	Cat#104734, RRID:AB_2563113
PE-conjugated CD86 (553692)	BD Biosciences	Cat#553692, RRID:AB_394994
APC-conjugated CD103 (566717)	BD Biosciences	Cat#566717, RRID:AB_2869828

Bacterial and virus strains

LV-Stk11-sgRNA(08480-1)	Genechem	Cat#1343B79
LV-Stk11-RNAi(72695-1)	Genechem	Cat#133F0EB
LV-Stk11(65515-2)	Genechem	Cat#13461BE
LV-Gsdme-sgRNA(12997-1)	Genechem	Cat#GCEL0359643
pLKO.1-U6-GSDME(human)-shRNA1-Puro	MiaoLingBio	Cat#P78528

Biological samples

Lung cancer TMA involving 76 samples	Outdo Biotech Co, Ltd	Cat#HLugA120PG01

Chemicals, peptides, and recombinant proteins

DNase I(DeoxyribonueleaseⅠ)	solarbio	Cat#D8071
CollagenaseⅠ	solarbio	Cat#C8140
CollagenaseⅣ	solarbio	Cat#C8160
CollagenaseIII	solarbio	Cat#C8490
Opti-MEM™	Thermo Fisher	Cat#31985062
Fetal Bovine Serum	JYK	Cat#BS1105
0.25%Trypsin-EDTA,1X	NCM Biotech	Cat#C100C1
methylene blue	AmBeed	Cat#A151225
CELLSAVING TM	NCM Biotech	Cat#C40100
Penicillin-streptomycin (100X)	NCM Biotech	Cat#C100C5
ascorbic acid	Sigma-Aldrich	Cat#A4034
D-luciferin	Glpbio	Cat#GC43496
Ferrostatin-1	MedChemExpress	Cat#HY-100579
GSK-872	MedChemExpress	Cat#HY-101872
Propidium iodide	FLUORESCENE	Cat#17515-10mg
Z-DEVD-FMK	RHAWN	Cat#R055677-1mg
Z-VAD-FMK	Beyotime	Cat#C1202-0.1mL
catalase	Solarbio	Cat#C8070
N-acetylcysteine	Solarbio	Cat#IA0050
genOFF h-SLC2A1_siRNA	RiboBio	Cat#SR1005
genOFF h-SLC2A3_siRNA	RiboBio	Cat#SIGS0007879-13
genOFF h-SLC2A4_siRNA	RiboBio	Cat#SIGS0007880-13
hSLC2A1 (NM_006516) in pcDNA3.1-3xflag	HanYi	Cat#HY241390
PolyShooterTM HP Transfection Reagent	LeapWal	Cat#P19110
5x DualColor Protein Loading Buffer	Fudebio	Cat#FD002
Protease inhibitor cocktail 100x	Fudebio	Cat#FD1001
Al1-in-One,100x	Fudebio	Cat#FD1002
RIPA lysis buffer	Fudebio	Cat#FD009

Critical commercial assays

BCA kit	Thermo Fisher Scientific	Cat#23227
Fc receptor blocking agent	Biolegend	Cat#101302
Reactive oxygen species assay kit	Solarbio	Cat#CA1420
Reactive oxygen species assay kit	Beyotime	Cat#S0033S
Pentuple-Fluorescence kit	Immunoway	Cat#RS0038
Human IL-1β ELISA kits	Shanghai mlbio	Cat#ml058059-2
Human HMGB1 ELISA kits	Shanghai mlbio	Cat#ml364107-2
ENLITEN™ ATP assay system kit	Promega	Cat#FF2000
anti-mouse Polymer-HRP kit	AiFang biological	Cat# AFIHC002
HMGB1 ELISA kits	JINGMEI BIOTECHNOLOGY	Cat#JM-05108H2
ATP Content Kit	AIDISHENG	Cat#ADS-W-A007-48
Lactate dehydrogenase (LDH) detection kit	AIDISHENG	Cat#ADS-W-FM003
Sunview® SYBR qPCR SuperMix (Universal)	Sunview	Cat#QE010-02
Mouse Vitamin C ELISA kits	Shanghai youyi Biotechnology	Cat#LDQB-28144
Vitamin C test kit	BestBio	Cat#101302
SevenFast Total RNA Extraction Kit for Cells	SevenFast	Cat#SM130-02
Annexin V-APC/PI Apoptosis kits	LIANKE	Cat#AP107
Sunview® Ⅲ Reverse Transcriptase kit(with dsDNase)	Sunview	Cat#RT008-02
NEBNext Ultra RNA Library Prep Kit	New England Biolabs, Ipswich	Cat#NEB 7530
15mL centrifuge Tubes	Jet	Cat#CFT511150
XKL Prestained Protein Ladder(8-180kDa)	XKL	Cat#xkl0901

Deposited data

Bulk RNA-seq data	This paper	GSE269742
Sing-cell RNA-seq data in Figures 5 and 6	This paper	CRA026496
Sing-cell RNA-seq data in Figure 7	This paper	CRA026608
RNA-seq data for Lkb1-deficiency mouse model	Gao et al.⁴⁶ Best et al.⁴⁷ Deng et al.⁴⁸ Deng et al.⁴⁸ Murray et al., 2019	GSE175479 GSE193895 GSE137396 GSE137244 GSE133895

Experimental models: Cell lines

LLC1	ATCC	Cat#CRL-1642
TC1	ATCC	Cat#CRL-2493
A549	ATCC	Cat#CCL-185
H460	ATCC	Cat#HTB-177
H1944	ATCC	Cat#CRL-5907
KP	This paper	N/A
hCASP3 knockout A549	Suzhou Haixing Biosciences	Cat#CGKO-M2487
hBAX&hBAK1 knockout A549	Suzhou Haixing Biosciences	Cat#CGKO-M3107
hMLKL knockout A549	Chongqing Kunlun biotech limited	Cat#CLK200200

Experimental models: Organisms/strains

Mouse: C57BL/6	the Laboratory Animal Center of Southern Medical University	N/A
Mouse: B6.129S(C)-Batf3tm1Kmm/J (Batf3^−/−)	Shanghai Model Organisms Center	NM-KO-190447

Oligonucleotides

See Table S4 for primers sequences	This paper	N/A

Software and algorithms

R4.1.1	The R Project for Statistical Computing	https://www.r-project.org/
Graphpad Prism 9	GraphPad Software, Inc.	http://www.graphpad.com/
Flowjo	Flowjo, L.L.C.	https://www.flowjo.cn/

Open in a new tab

Experimental model and study participant details

Animals

C57BL/6 mice (5–8 weeks old) were purchased from the Laboratory Animal Center of Southern Medical University (Guangzhou, China); Kras^G12D/+ Trp53^R172H/+ mice (5–8 weeks old) were gifts from Professor Shuan Rao at thoracic surgery department, Nanfang hospital (Guangzhou, China). Batf3^−/− mice (5–8 weeks old) were purchased from Shanghai Model Organisms Center, Inc. Animals were randomly assigned to different experimental groups. All the animals were administered according to the guidelines approved by the Institutional Animal Care and Use Committee of Southern Medical University (#IACUC-LAC-20230802-006, IACUC-LAC-20241107-002). Experiments were conducted using both male and female mice without bias toward either sex.

Cell lines

LLC1 cell line was purchased from Guangzhou Jennio Biotech Co., Ltd, who obtained this cell line from American Type Culture Collection (ATCC). TC1 cell line was purchased from Zhejiang Nobo Biological Products Co., Ltd, who obtained this cell line form Chinese Academy of Sciences. A549, H460, H1944 cells were provided by Guangdong Lung Cancer Institute, who purchased these cells from ATCC. Authentication of Human Cell Lines Reports of these cell lines were provided. KP cells were isolated by from Kras^G12D/+ Trp53^R172H/+ mice. The KP-sgCtrl and KP-sgLkb1 cell lines were established by transfecting with CRISPR/Cas9-expressing lentivirus targeting Stk11 (1343B79, Genechem). The LLC1-shCtrl, LLC1-shLkb1, TC1-shCtrl, TC1-shLkb1, A549-Ctrl, A549-LKB1, H1944-Ctrl, H1944 LKB1 cell lines were developed by transfection with lentivirus (13461BE, 133F0EB, Genechem), and selecting for puromycin resistance. LLC1-shLkb1-sgGsdme, LLC1-shLkb1-sgCtrl cell lines were established by transfecting with CRISPR/Cas9-expressing lentivirus targeting Gsdme (P78528, MiaoLingBio). hCASP3 knockout A549 cells (CGKO-M2487) and hBAX&hBAK1 knockout A549 cells (CGKO-M3107) were kindly provided by Suzhou Haixing Biosciences Co., Ltd. hMLKL knockout A549 cells (CLK200200), A549-sgCtrl cells (mC30001) were kindly provided by Chongqing Kunlun Biotech Limited. All cells were maintained in a humidified incubator at 37°C with 5% CO₂, and grown in 1640 or DMEM supplemented with 10% FBS (BS1105, Inner Mongolia Jinyuankang Biotechnology Co., Ltd) and 100 IU/mL penicillin/streptomycin. All cell lines used were negative for mycoplasma. An updated detection report of mycoplasma was also provided.

Method details

Antibodies and reagents

The antibodies used were as below: anti-LKB1 (27D10) (CST, #3050), anti-DFNA5/GSDME (Abcam, #ab215191), anti-Caspase-3 (Proteintech, #19677-1-AP), anti-GSDMD (Abcam, #ab210070), anti-β-actin (Fdbio science, #FD0060-100), anti-LKB1 (D60C5F10) (CST, #13031), anti-GLUT1 (Proteintech, #66290-1-Ig), anti-GAPDH (abmart, #P60037S), anti-F4/80 (Abcam, #ab300421) anti-SPP1 (Abcam, #ab218237), anti-CD8 (Abcam, #ab217344), anti-TCF7(CST, # 2203T), anti-phospho-MLKL (Affinity, #AF7420), anti-Caspase 11 (Affinity, #AF5130), anti-MLKL (Proteintech, #66675-1-I), anti-BAK (Proteintech, #29552-1-AP), anti-BAX (Proteintech, #60267-1-Ig), anti-Caspase 1 (Proteintech, #81482-1-RR), Flow cytometry antibodies used were as following: APC/Cy7-conjugated CD45 (557659), BV510-conjugated CD3 (563024), PE/Cy5-conjugated CD8a (553034), APC/R700-conjugated PD1 (565815), PE/CF594-conjugated TIM-3 (566998), BV421-conjugated TCF-1 (566692), BV605-conjugated CD11c (563057), PE-Cyanine7 conjugated CD80 (104734), PE-conjugated CD86 (553692), APC-conjugated CD103 (566717).

The inhibitors and the concentration used were as follows: high-dose ascorbic acid (Sigma-Aldrich, #A4034) 10 mM, Caspase-3 specific inhibitor (Z-DEVD-FMK, R055677, RHAWN) 25 μM, pan-Caspase inhibitor (Z-VAD-FMK, C1202–0.1, Beyotime) 25 μM, catalase (CAT, C8070, Solarbio) 100 μM and N-acetylcysteine (NAC, IA0050, Solarbio) 50 μM, iron death inhibitor (Ferrostatin-1, HY-100579, MCE (MedChemExpress) 10 μM, necrotic apoptosis inhibitor (GSK-872, HY-101872, MCE (MedChemExpress) 10 μM. If it is not specified, the incubation time is 4 h.

Vaccination assays

LLC1-shLkb1 cells (2 × 10⁶) in vitro treated with high-dose ascorbic acid (A4034, Sigma-Aldrich, 10mM) for 4h were resuspended with PBS and subcutaneously inoculated into the left flank of mice. 8 days later, viable LLC1-shLkb1 cells were subcutaneously injected into the right flank of mice for tumor challenge. Mice in the vehicle group were pre-injected with normal saline. Tumor incidence and tumor size were assessed in both groups.

Measurement of intracellular ascorbic acid by LC-MS

Cells were inoculated in 6-well plates in 1640 involving 10% FBS for 24 h to 70–80% confluency. Then medium was removed and cells were washed by PBS Washing Buffer twice and incubated in conditional medium containing 2 mM glucose with or without 10 mM sodium ascorbate. After 1 h of incubation, cells were washed with PBS and lysed with ice-cold 1 mL CH₃OH: H₂O (80:20) at −80°C for 20 min. Then cells were scraped and collected into 1.5 mL tubes and stored at −80°C overnight. Subsequently, tubes were restored to 4°C and centrifuged at 14000g for 10 min. The supernatant was normalized to total protein and 2 μL solution was injected for LC/MS. LC/MS analysis was performed on a TSQ Quantiva triple quadrupole mass spectrometer coupled to a Prelude SPLCTM System (Thermo Fisher Scientific). Chromatographic column was Amide (Waters) 2.1 × 100mm, 1.7 μm. Mobile phase A was 5 mM ammonium acetate and 1% methanol. Mobile phase B was acetonitrile. The column temperature was 45°C. The chromatographic gradient was set for mobile phase B as follows: 0–0.5 min: 95% B; 0.5 min-4.5 min: from 95% to 70% B; 4.5–5.5 min: from 70% to 50% B; 5.5–6.5 min: from 50% B; from 6.5–8 min: from 50% to 95% B. Spray voltages of 2.5 kV were applied for negative ion. Data were acquired using selected reaction monitoring (SRM). RF lens was 47 V. The ion pairs were obtained based on the chemical structures of the precursors and fragments, and the ion pair (precursor (m/z): 175.274, product (m/z): 115.071, and collision energy (V): 10) was selected for quantification. An AA calibration curve was established in the 10–1000 ng/mL concentration range.

Signatures of programmed cell death

Signatures indicating different forms of programmed cell death were compared between A549 cells with different LKB1 status and with or without ascorbic acid treatment²¹^,²²^,²³^,²⁴ (Table S1). Signature scores were calculated by the R package “GSVA” (1.46.0) and scaled for visualization as a heatmap using the R package “ComplexHeatmap” (2.15.4).

Western Blot

Cells were lysed using RIPA lysis buffer (FD009, Fudebio, Hangzhou, China) supplemented with protease inhibitors (FD1002, Fudebio) and phosphatase inhibitors (FD1001, Fudebio). Protein concentration was quantified using a BCA kit (23227, Thermo Fisher Scientific). For each sample, 20mg protein lysate was loaded onto 10% SDS-PAGE gel. Proteins were separated and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Then the membranes were incubated with appropriate antibodies. Bands were analyzed by using an ECL system (BLT GelView 6000). XKL Prestained Protein Ladder (8-180kDa) (xkl0901, Guangzhou Xinkailai Biotechnology Co., Ltd) was used.

Real-time/quantitative PCR

mRNA was collected using SevenFast Total RNA Extraction Kit (SM130-02, SevenFast) and was reverse transcribed into complementary DNA using Sunview Ⅲ Reverse Transcriptase kit (with dsDNase) (RT008-02, Shenzhen Sunview Technology Co., Ltd). qRT–PCR was performed using TB Green Premix Ex Taq (TAKARA) in a Roche LightCycler 480 System. We used the average 2-ΔΔCT to analyze the data. The qPCR Probes were synthesized by Beijing Tsingke Biotech Co., Ltd. Sequences list of primers are listed in Table S4.

Preparation of single cell suspension

Tumors were dissected from mice, cut into small pieces and digested in RPMI 1640 (PM150110, Pricella) supplemented with 10% FBS, DNase I (0.1mg/ml) and Collagenase I,III,IV (0.5mg/ml) at 37°C for 30 min with gentle shaking. The tumor-draining lymph nodes were visualized 5 min after injecting Methylene Blue (A151225, AmBeed, USA) into the toe, then isolated and dissociated using RPMI 1640 medium. The mixtures were filtered through 75-μm cell strainers and separated by centrifugation (300 g × 5 min) to harvest the single cells. Then the cells were resuspended in PBS supplemented with 2% FBS in 15mL centrifuge tubes (CFT011150, Guangzhou Jet Bio-Filtration Co., Ltd) and used for subsequent experiments.

Flow cytometry

Cells were trypsinized, resuspended in PBS supplemented with 2% BSA, incubated with Fc receptor blocking agent (Biolegend, 101302), and then stained with APC/Cy7-conjugated CD45, BV510-conjugated CD3, PE/Cy5-conjugated CD8a, APC/R700-conjugated PD1, APC/R700-conjugated PD1, PE/CF594-conjugated TIM-3, BV421-conjugated TCF-1, BV605-conjugated CD11c, PE-Cyanine7 conjugated CD80, PE-conjugated CD86, APC-conjugated CD103, followed by incubating on ice for 30 min. After washing with PBS, cells were resuspended in 0.3mL PBS. The data were then analyzed with FlowJo software (version 10.5; Tree Star).

Annexin V-APC/PI fluorescence staining

We used apoptosis kit (Annexin V-APC/PI double fluorescence, AP105, MULTI SCIENCES (LIANKE) BIOTECH CO., LTD) for staining and flow cytometry to detect the number of double-positive cells to reflect cell death according to the manufacturer’s protocol.

siRNA transfection

siRNA was designed and synthesized by Guangzhou RiboBio Co., Ltd. The transfection procedure involved seeding cells into a 24-well plate containing an appropriate amount of complete medium, ensuring the cell density reaches 30–50% at the time of transfection. Each transfection sample was diluted with the siRNA and gently mixed. Next, we prepared the mixture by adding 3μL of riboFECT CP Reagent, gently pipetted to mix, and incubated at room temperature for 0–15 min to form the transfection complex. Then the transfection complex was added into an appropriate volume of complete medium without antibiotics. Finally, the culture plate was placed in a 37°C CO2 incubator for 48 h. Transfection efficiency was assessed by qPCR. The sequences of siRNAs are listed in Table S4.

Overexpression of GLUT1 through plasmid transfection

Firstly, the transfection reagent-nucleic acid complex was prepared by adding 0.5 μg of plasmid (hSLC2A1 in pcDNA3.1-3xflag, HY241390, HanYi) to a 1.5 mL centrifuge tube, followed by adding 2 μL of transfection reagent (P19110, LeapWal) to mix with the plasmid and incubating for 3 min. Next, we added 100 μL of serum-free basic medium to the mixture, gently mixed to form the transfection reagent-nucleic acid complex. Subsequently, we replaced the cell culture medium with fresh pre-warmed complete medium, then added the 100 μL transfection reagent-nucleic acid complex to each well of the cells and gently swirled the plate to mix. After culturing for 24–48 h, transfection efficiency can be assessed by western blot.

PI fluorescence staining

The process was detected by Propidium iodide (PI) staining. Firstly, we prepared a 25 μM PI solution using PBS, then added 1/10 of the culture medium volume of the PI solution into the cell culture medium, incubating the cells at 37°C for 10–20 min. The cells could be observed using a fluorescence microscope equipped with a filter for excitation at 535 nm and emission at 615 nm.

ROS detection

DCFH-DA fluorescence probe kit (Cat#CA1420, Beijing Solarbio Science & Technology Co., Ltd.) was used to detect the level of intracellular reactive oxygen species (ROS) according to the manufacturer’s protocol. The IX33 upright fluorescence microscope (N25014) and the I3x multifunctional microplate reader (N29345) were used for observation.

Bioluminescence imaging (BLI)

Mice were administered with 150 mg/kg D-luciferin (GC43496, Glpbio, Montclair, CA, USA) through intraperitoneal injection and then anesthetized with isoflurane gas. The bioluminescent signals of luciferase-expressing tumors were captured on the AMI HTX imaging system (Spectral Instruments Imaging). The intensities of bioluminescence at different time were analyzed and quantified by Aura software.

Immunohistochemistry (IHC)

Orthotopic and subcutaneous tumors were collected and fixed in 4% PFA at 4°C overnight and paraffin-embedded. The sections were deparaffinized and repaired followed by blocking. Then the slides were probed with primary antibody, anti-cleaved-caspase 3(proteintech, 68773-1-Ig) at 4°C overnight and then incubated with secondary antibodies at room temperature for 1h. The proportion of positive cells and the intensities of positive cells were evaluated. All of them were judged by experienced pathologists.

Lung cancer tissue microarray (TMA)

Lung cancer TMA involving 76 samples was purchased from Outdo Biotech Co, Ltd. (HLugA120PG01, Shanghai, China). The immunohistochemical stainings of GLUT1 (Proteintech, #66290-1-Ig), and LKB1 (CST, #3050) were performed as IHC. High-resolution panoramic pathology scanner (Hamamatsu, NE0N440) was used to scan the slices. The Kaplan-Meier method was used to evaluate the associations between GLUT1 and LKB1 expression.

Immunofluorescence analysis (IF)

Tumor tissues were collected, fixed with 4% PFA at 4°C and paraffin embedded. These sections were deparaffinized and repaired, and then closed. They were then incubated overnight with primary antibody, anti-CD8 (Abcam, #ab217344), anti-TCF1 (Cell Signaling #2203), anti-F4/80 (Abcam, #ab6640), anti-SPP1(Abcam, #218237). Secondary antibody, anti-mouse Polymer-HRP (AiFang biological, #AFIHC002) was used for detection. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Sections were then blocked using fluorescent blocker (Dako) and analyzed using microscopy. All these were judged by an experienced pathologist.

Multiplex immunohistochemistry (mIHC)

mIHC was performed by Pentuple-Fluorescence kit (RS0038, Immunoway). Firstly, paraffin-embedded tumor tissues from mice were sliced into 5-μm-thick pieces. This process was supported by Hubei BIOSSCI Biotechnology Co., LTD. Following deparaffinization, rehydration, and antigen retrieval with EDTA, the pieces were incubated with 3% hydrogen peroxide and blocked in goat serum for 30 min at room temperature. Next, the tissue slices were incubated with primary antibodies at 4°C overnight, followed by incubation with the secondary antibody (HRP) for 20 minutes at 37°C temperature. Afterward, the slides were incubated with TSA dye at room temperature for 10 min. Between the incubation of different primary antibodies, the slides were immersed in antibody stripping buffer at 95°C for 15 min to denature the primary and secondary antibody complexes. Finally, with all the dying process finished, DAPI was applied and the slides were scanned by high-resolution panoramic pathology scanner (Hamamatsu, NE0N440) after a manual cover-slipping. All the results were judged by an experienced pathologist.

Enzyme-linked immunosorbent assay (ELISA)

The supernatant was collected by centrifugation of cell culture medium at 1500 r/min for 20 min at 4°C. The level of AA was detected with kits (BB47444, BestBio). The levels of IL-1β and HMGB1 were detected with ELISA kits (ml058059–2, ml364107-2, Shanghai mlbio). The level of HMGB1 was also detected with ELISA kits (Jiangsu Jingmei Biological Technology Co.,Ltd.) according to the manufacturer’s instructions. Tumor tissues were isolated through mechanical disruption (mincing and homogenization) of the samples, followed by centrifugation to harvest the supernatant. For mouse peripheral blood serum, whole blood was subjected to centrifugation, after which the serum layer was carefully extracted. All experimental procedures strictly followed the protocols provided by the manufacturers. The levels of serum AA and tumor containing AA were detected with ELISA kits (Shanghai Excellent Doctor Biotechnology (shanghai, China)).

Biochemistry assay

The ATP and LDH levels were detected with microplate method (Jiangsu Jingmei Biological Technology Co.,Ltd.) The ATP level of the supernatant was also detected with ENLITEN ATP assay system kit (Promega, America, FF2000) according to the manufacturer’s instructions.

Micro-CT analysis

Mice were taken micro-CT scan by Skyscan 1176 while breathing freely under anesthesia. The scanner was set at the following parameters for images acquisition: X-ray source 50 kvp, X-ray current 500 μA, filter AI 0.5 mm, pixel size 18 μm, and 40 × 40 mm field of view. Duration of the procedure for each mouse was approximately 7 min. A lung region of interest (ROI) was drawn using threshold segmentation and sweep function while other tissue images such as thorax and mediastinum were removed. The reconstruction of the scans was performed with NRecon software. Tomographic reconstruction of the projections resulted in 1061 slices with an isotropic voxel spacing of 80 μm. Images were converted into bmp format and analyzed with DataViewer to further establish 2D maximum intensity projection slices, and quantify the tumor volume. The reconstructed image stacks were then imported into CTvox software and 3D reconstructions were generated.

Bulk RNA extraction and analysis

Human NSCLC cell lines A549 transduced with wild-type or empty vector with high dose AA or PBS were subjected to RNA-sequencing. Total RNA was extracted using SevenFast Total RNA Extraction Kit for Cells (Cat#SM130-02, SevenFast) according to the manufacturer’s protocol, and then reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). Finally, they were sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).

scRNA-seq and analysis

Sequencing data was filtered, and a gene expression matrix was generated by the DNBelab C Series scRNAanalysis software (https://github.com/MGI-tech-bioinformatics/DNBelab_C_Series_HT_scRNAanalysis-software). In summary, sample de-multiplexing, barcode processing, and single-cell 3′ unique molecular identifier (UMI) counting were conducted on all samples using default settings. The processed reads were then aligned to the GRCh38 genome reference with STAR (v2.5.3). The 'barcodeRanks()' function from the DropletUtils tool was employed to automatically identify valid cells by analyzing the UMI count distribution per cell, thereby eliminating background beads and beads with UMI counts below the threshold. Finally, PISA was utilized to compute the gene expression levels of cells and to construct a gene x-cell matrix for each library.⁴⁹

Subsequently, datasets were subjected to quality control steps using Seurat 4.3.0.For the dataset used in Figures 5 and 6, 44, and 745 cells were obtained for the downstream analysis. Similarly, the dataset presented in Figure 7 yielded 16,217 cells for subsequent analytical procedures. Variable genes were curated based on mean expression and dispersion and these were next used for PCA analysis. Clusters and t-SNE plots, UMAP plots were generated based on selected PCA dimensions. Marker genes were identified by Seurat function FindAllMarkers together with previously reported functional markers. The scaled expression values of the identified marker genes were utilized to construct heatmap visualizations. Normalized data were presented through feature plots or violin plot representations. Differential gene expression analysis between subsetted groups was performed by firstly generating pseudobulk samples, subsequently identified in the DESeq2 (v.1.40.2). Seurat’s FindMarkers was also used. We performed GO enrichment analysis with the clusterProfiler (v4.8.3) package. The enrichment metrics for the chosen GO annotation terms were computed through a hypergeometric distribution statistical evaluation, with the significance cut-off value set at 0.05. Trajectory analysis was performed using Monocle 2.⁵⁰

RNA velocity analysis

To predict the forthcoming dynamics and trajectory of transitioning T cells, RNA velocity analysis was performed. Loom files, encapsulating un-spliced mRNAs, were produced for each sample via Cell Ranger. These files were analyzed using Velocyto (Linux R package) to compute RNA velocity. The resultant loom files were consolidated and aligned with the UMAP coordinates of individual cells.⁵¹

Quantification and statistical analysis

Statistical analyses were performed as described in the figure legend for each experiment. Data are presented as mean ± SEM or mean ± SD as described in legends. Group size was determined based on the results of preliminary experiments, and no statistical method was used to predetermine sample size. The indicated sample size (n) represents biological replicates. Group allocation and outcome assessment were not performed in a blinded manner. All samples that met proper experimental conditions were included in the analysis. Survival was measured using the Kaplan–Meier method. Statistical significance was determined by one- or two-way ANOVA, Student’s t test, log rank test, and Pearson’s correlation using Prism 7 software (GraphPad Software) as indicated. Significance was set at p < 0.05. Bar plots, dot plots, tumor growth curves, and Kaplan-Meier survival curves were made using ggplot2, survival, and survminer packages in R (version 4.1.2).

Published: August 15, 2025

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2025.102291.

Contributor Information

Xuejun Guo, Email: 576718933@qq.com.

Zhongyi Dong, Email: dongzy1317@foxmail.com.

Dehua Wu, Email: 18602062748@163.com.

Xue Bai, Email: baixue1990@i.smu.edu.cn.

Supplemental information

Document S1. Figures S1–S10 and Tables S1–S4

mmc1.pdf^{(3MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(31.6MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S10 and Tables S1–S4

mmc1.pdf^{(3MB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(31.6MB, pdf)}

Data Availability Statement

•
The RNA-seq data for the LKB1-deficient mouse model are accessible in the GEO database under accession codes GSE175479,⁴⁶ GSE193895,⁴⁷ GSE137396,⁴⁸ GSE137244,⁴⁸ and GSE133895. The bulk RNA-seq data used in Figure 2C have been deposited in the National Center for Biotechnology Information’s GEO under the accession number GSE269742. Single-cell RNA data for LLC1-shLkb1 orthotopic lung tumor tissue from this paper used in Figures 5 and 6 have been deposited in GSA under the accession number CRA026496. Single-cell RNA data for LLC1-sgGsdme orthotopic lung tumor tissue from this paper used in Figure 7 have been deposited in GSA under the accession number CRA026608.
•
All original code has been deposited at Zenodo. DOI is https://doi.org/10.5281/zenodo.15745003.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

High-dose ascorbic acid selectively induces pyroptosis in LKB1-deficient lung cancer and sensitizes immunotherapy

Xiangyu Sun

Xiaoting Cai

Shangbiao Li

Ruozheng Pi

Zeqin Guo

Jiayu Jiang

Pinhao Wang

Jingrong Xiong

Zhuangzhuang Liu

Zixuan Rong

Zihang Yu

Xiaonan Zhang

Jiaqi Chen

Duanduan Han

Yanpei Zhang

Jiale Tan

Yan Lin

Zhuocheng Zou

Haochen Ai

Fangfang Kang

Xuejun Guo

Zhongyi Dong

Dehua Wu

Xue Bai

Summary

Graphical abstract

Highlights

Introduction

Results

LKB1 deficiency in NSCLC elevates GLUT1-mediated AA uptake and exacerbates redox imbalance

Figure 1.

High-dose AA selectively induces pyroptosis in LKB1-deficient NSCLC cells via activating the cleavage of GSDME

Figure 2.

Pyroptosis induced by high-dose AA depends on the H2O2/ROS-caspase-3-GSDME pathway

Figure 3.

High-dose AA restores PD-1 blockade resistance of LKB1 deficiency in NSCLC in vivo

Figure 4.

High-dose AA remodels the TIME characterized by the infiltration of Tpex cells

Figure 5.

Pyroptosis induces ICD and enhances cross-presenting DC maturation, thereby driving Tpex expansion

Figure 6.

Inner blockage of GSDME reversed the synergistic effect of high-dose AA and αPD-1 Ab therapy

Figure 7.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental model and study participant details

Animals

Cell lines

Method details

Antibodies and reagents

Vaccination assays

Measurement of intracellular ascorbic acid by LC-MS

Signatures of programmed cell death

Western Blot

Real-time/quantitative PCR

Preparation of single cell suspension

Flow cytometry

Annexin V-APC/PI fluorescence staining

siRNA transfection

Overexpression of GLUT1 through plasmid transfection

PI fluorescence staining

ROS detection

Bioluminescence imaging (BLI)

Immunohistochemistry (IHC)

Lung cancer tissue microarray (TMA)

Immunofluorescence analysis (IF)

Multiplex immunohistochemistry (mIHC)

Enzyme-linked immunosorbent assay (ELISA)

Biochemistry assay

Pyroptosis induced by high-dose AA depends on the H₂O₂/ROS-caspase-3-GSDME pathway