Journal: Frontiers in Immunology

Article Title: Non-invasive radiogenomic mapping of the SMARCAL1-driven ferroptotic niche is associated with longitudinal MRD-negative surveillance in early-stage NSCLC

doi: 10.3389/fimmu.2026.1835413

Figure Lengend Snippet: CRISPR-Cas9 loss-of-function validation of the SMARCAL1–ferroptosis–chemokine axis and integrated mechanistic model. (a) SMARCAL1 knockout validation in A549 and H1299 NSCLC cell lines: anti-SMARCAL1 immunoblot (115 kDa; GAPDH loading control 37 kDa) demonstrating absence of SMARCAL1 protein in sgSMARCAL1-1 and sgSMARCAL1-2 versus sgScramble; Sanger sequencing chromatograms of indel-introduced sgSMARCAL1 target locus within SMARCAL1 exon 3 region (~150 bp) versus wild-type reference; TIDE indel size distribution (sgSMARCAL1-1 38% efficiency, sgSMARCAL1-2 22% efficiency at predominant indel sizes −5 to −1, +1, +5). (b) Ferroptosis phenotyping after erastin (5 µM, 24 h) in A549 (left) and H1299 (right), expressed as relative value (% sgScramble): cell viability, BODIPY-C11 oxidized/reduced ratio, intracellular Fe 2+ (FerroOrange MFI), malondialdehyde (MDA), reduced glutathione (GSH), and TEM ferroptotic mitochondrial percent. SMARCAL1-KO cells (sgSMARCAL1-1, -2) show resistance to erastin-induced ferroptosis; ***P < 0.001 versus sgScramble. Lower rescue panel (A549): wild-type SMARCAL1 reconstitution fully rescues ferroptosis susceptibility (viability 31% sgScramble baseline); helicase-dead R764Q mutant fails to rescue (viability 74%, no rescue). (c) Chemokine secretion assays. ELISA of CXCL9 and CXCL10 (pg/mL) and qRT-PCR mRNA fold-change versus baseline in sgScramble (sgScr) versus sgSMARCAL1 (sgSMAR) ± wild-type or R764Q rescue: SMARCAL1 knockout reduces CXCL9 by 62% (1240 → 471 pg/mL) and CXCL10 by 57% (842 → 362 pg/mL) under IFN-γ stimulation; full rescue by wild-type, partial rescue (45% efficiency) by R764Q. ***P < 0.001, **P < 0.01, ns = not significant. (d) Western blot of cGAS-STING-IRF3 pathway activation: phospho-STING Ser366, phospho-TBK1 Ser172, phospho-IRF3 Ser396, with total STING/TBK1/IRF3 and GAPDH loading controls, in sgScr versus sgSMAR ± IFN-γ; quantified bar plots (arbitrary units): p-STING 4.7 → 1.5, p-TBK1 3.9 → 1.4, p-IRF3 5.2 → 1.5, all ***P < 0.001. diABZI rescue panel (10 µM, 6 h): STING agonist diABZI partially restores CXCL9 (64% recovery, 471 → 792 pg/mL) and CXCL10 (58% recovery, 362 → 591 pg/mL) secretion in SMARCAL1-KO cells. (e) Primary human CD8 + T cell transwell chemotaxis (transmigrated CXCR3 + CD8 + cells × 10 3 , mean ± SD across three donors): condition 1 unstimulated (2.1); condition 2 IFN-γ-stimulated sgScramble supernatant (9.8); condition 3 IFN-γ-stimulated sgSMARCAL1 supernatant (4.1, 58% reduction, ***P < 0.001); condition 4 sgScramble supernatant + AMG 487 (1 µM CXCR3 antagonist, 0.9, > 90% blockade); condition 5 isotype control (2); condition 6 H1299 IFN-γ sgScramble (8.2); condition 7 H1299 IFN-γ sgSMARCAL1 (3.9, 52% reduction, ***P < 0.001). ***P < 0.001, ns = not significant. (f) Integrated mechanistic model schematic: replication stress in APOBEC-enriched (high Rad-Score) tumors → SMARCAL1 upregulation (helicase activity-dependent) → ACSL4-dependent phospholipid remodeling and ferroptotic susceptibility → cGAS-STING-IRF3 axis activation amplifying IFN-γ-induced CXCL9/CXCL10 transcription → chemokine gradient recruiting CXCR3 + CD8 + T cells to peritumoral invasive margin → systemic immune priming and sustained post-operative MRD-negative surveillance. Evidence sources annotated per arrow: bulk RNA-seq (transcriptomic), Visium spatial, mIF/IHC (protein), CRISPR (causal), chemotaxis (functional).

Article Snippet: Knockout was validated by: (i) Sanger sequencing of the target locus (PCR-amplified genomic DNA); (ii) Western blot demonstrating absence of SMARCAL1 protein; (iii) TIDE analysis (Brinkman et al., NAR 2014) of indel profiles.

Techniques: CRISPR, Biomarker Discovery, Knock-Out, Western Blot, Control, Sequencing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activation Assay, Chemotaxis Assay, Activity Assay, RNA Sequencing, Functional Assay