LIM and SH3 protein 1 deficiency confers resistance to cytotoxic lymphocyte-mediated lysis and immunotherapy in gastric cancer by disrupting cytoskeleton dynamics

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  Figure 1. LASP1 has been identified as a cytoskeleton-related protein associated with tumor immunotherapy. (A) Different cell types from four different kinds of tumor were projected onto the UMAP plot. (B) Epithelials from responder and non-responder to immunotherapy were projected onto the UMAP plot. (C) Differently expressed gene in epithelials from responder and non-responder were analyzed. (D) The common differentially expressed genes were intersected with cytoskeleton-related genes. (E) Different expression level of LASP1 in epithelials from responder and non-responder were analyzed. (F) The expression level of LASP1 in different organs was analyzed. (G) Different expression level of LASP1 in different kinds of tumor tissues and paired normal tissues. All values are presented as the mean±SEM. ***p<0.001. COAD, colon adenocarcinoma; CRG, cytoskeleton-related gene; ESCC, esophageal squamous cell carcinoma; FC, fold change; LIHC, liver hepatocellular carcinoma; NK, natural killer; STAD, stomach adenocarcinoma; TPM, Transcripts Per Million; UMAP, uniform manifold approximation and projection.

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  Figure 2. The deficiency of LASP1 prevents patients from benefiting from cytotoxic lymphocyte infiltration and affects the efficacy of anti-PD-1 immunotherapy. (A–C) Kaplan-Meier analysis of overall survival in patients was performed based on the LASP1 expression level using own cohort (A), TCGA cohort (B) and GSE84437 cohort (C). (D,E) Univariate Cox analysis of overall survival in patients based on the stratified LASP1 expression level or CL infiltration using own cohort (D) and GSE84437 cohort (E). (F,G) Different expression levels of LASP1 in gastric cancer tissue were detected by IHC and analyzed according to immunotherapy response. (H) The progression-free survival of gastric cancer patients with different LASP1 expression levels was analyzed. (I) Subcutaneous and orthotopic tumors in the indicated groups were photographed. (J) Growth curves of subcutaneous tumors in indicated groups of mice. (K,L) Subcutaneous and orthotopic tumors were weighed and their weights were calculated. All values are presented as the mean±SEM. *p<0.05; **p<0.01; ***p<0.001. CL, cytotoxic lymphocyte; CR, complete response; IHC, Immunohistochemistry; NK, natural killer; PD, progressive disease; PD-1,…

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  Figure 3. The deficiency of LASP1 reprograms the immune environment. (A) Immune cells from subcutaneously transplanted tumors in the mock and LASP1-KO groups were analyzed using CyTOF (n=3). Cell populations were clustered, defined manually, projected onto the t-SNE space, and assigned specific colors. (B) Heatmap showing differential marker expression in CD45+ TIL clusters identified by analysis of CyTOF data. (C) The frequency of T cells, B cells, NK cells, macrophages, DCs, neutrophils, eosinophils, and other cells in total cells were analyzed. (D) The proportion of different lymphocyte subsets in total cells was analyzed based on differential marker expression. (E–G) Subtypes of CD4+T cells (E), CD8+ T cells (F), and NK cells (G) were projected onto the UMAP plot. (H) Patients were ranked according to the different expression levels of LASP1 in epithelial cells. (I) Portions of different subtypes of CD4+T cells, CD8+ T cells, and NK cells were analyzed according to LASP1 levels. (J–L) The intensity and proportion of exhausted CD8+ T cells in gastric cancer tissue were detected and analyzed. All values are presented as the mean±SEM. *p<0.05; **p<0.01. CyTOF, cytometry by time-of-flight;…

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  Figure 4. The deficiency of LASP1 in tumor cells enables the tumor cells to resist cytotoxic lymphocyte-mediated lysis while effectively activating immune cells in vitro. (A) LASP1 knockout HGC-27 and SGC-7901 cell lines were constructed using the Crispr-Cas9. LASP1 was overexpressed or complemented in wild-type or LASP1-KO HGC-27 and SGC-7901 cell lines. (B–D) After co-culture with PBMC for 16 hours, the apoptosis of cancer cells in the mock group and the LASP1-KO group was analyzed by flow cytometry (n=3). (E,F) After co-culture with PBMC for 16 hours, the apoptosis of wild-type (E) or LASP1-KO (F) cancer cells with overexpressed or compensated LASP1 was analyzed by flow cytometry (n=3). (G–I) The accumulation of GZMB in mock or LASP1-KO cancer cells co-cultured with PBMC for 6 hours was analyzed by flow cytometry (n=3). (J–L) The degranulation levels of CD8+ T cells and NK cells after 6-hour co-culture with mock or LASP1-KO cancer cells were analyzed by flow cytometry (n=3). (M–O) The activation levels of CD8+ T cells after 24-hour co-culture with mock or LASP1-KO cancer cells were analyzed by flow cytometry (n=3). (P–R) The exhaustion levels of CD8+ T cells after 72-hour co-culture…

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  Figure 5. The absence of LASP1 in tumor cells leads to dysfunction of the cytoskeleton during synaptic formation. (A) The differentially expressed genes in mock and LASP1-KO SGC-7901 cells were identified through RNA-seq analysis, and the pathway enrichment analysis of these differentially expressed genes was conducted. (B,C) The conjugate formation experiment was performed to evaluate the adhesion of mock or LASP1-KO cancer cells to effector cells (CD8+ T cells and NK cells) (n=3). (D–G) The synaptic localization of adhesion molecules CD2 (D) and effect molecules perforin-1 (E) of CD8+ T cells (F) or NK cells (G) that form conjugates with mock or LASP1-KO cancer cells was detected and analyzed by immunofluorescence (n=10). (H–K) Lifeact labeling live cell imaging was used to analyze the reorganization of the cytoskeleton at the synaptic junctions of cancer cells after conjugates with CD8+ T cells (J) or NK cells (K) (n=10). All values are presented as the mean±SEM. ***p<0.001. CFSE, 5(6)-Carboxyfluorescein diacetate succinimidyl ester; KO, knock outl; NK, natural killer; ns, no significance; RNA-seq, RNA sequencing.

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  Figure 6. LASP1 acts as a scaffold protein to regulate Arp2/3 complex and remodel cytoskeletal at immunological synapses. (A) siRNA was used to knock down the expression of ARPC2. (B) After co-culture with PBMC for 16 hours, the apoptosis of ARPC2-knockdown cancer cells in the mock group and the LASP1-KO group was analyzed by flow cytometry (n=3). (C) LASP1 mutants with the deletion of different functional domains were constructed. (D) Co-immunoprecipitation experiments were conducted to identify the specific functional domain of LASP1 responsible for its interaction with the ARP2/3-actin complex. (E) Co-localization between LASP1 mutants and ARP2 was detected by immunofluorescence. (F–H) Apoptosis mediated by CLs (F), GZMB accumulation (G), and the induction of CD8+ T-cell exhaustion (H) in cancer cells with different LASP1 mutant expression was analyzed using flow cytometry (n=3). (I,J) Lifeact-labeled live cell imaging was used to analyze the reorganization of the cytoskeleton at the synaptic junctions of cancer cells with different LASP1 mutants expressing after coagulating with CD8+ T cells (I) or NK cells (J) (n=10). (K) Subcutaneous tumor in the indicated groups were photographed.…

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  Figure 7. LASP1-mediated cytoskeletal remodeling at the immunological synapse drives cell membrane lipid alterations. (A–C) Laurdan staining was used to analyze the packing of lipid membrane at the synaptic junctions of cancer cells after conjugates with CD8+ T cells (B) or NK cells (C) (n=10). (D–F) Laurdan staining was used to analyze the packing of lipid membrane at the synaptic junctions of cholesterol or 7-Kc-treated cancer cells after conjugates with CD8+ T cells (E) or NK cells (F) (n=10). (G) The correlation between the remodeling of the cytoskeleton (F-actin intensity ratio) and lipid (ΔGP value) was analyzed in cholesterol or 7-Kc-treated cancer cells. (H) The accumulation of GZMB in cholesterol or 7-Kc-treated cancer cells co-cultured with PBMC for 6 hours was analyzed by flow cytometry (n=3). (I) Subcutaneous tumor with indicated treatment were photographed. (J) Growth curves of subcutaneous tumors with indicated treatment (n=5). (K) Subcutaneous tumor were weighed and their weights were calculated (n=5). All values are presented as the mean±SEM. ***p<0.001. 7-Kc, 7-ketocholesterol; GP, generalized polarization; GZMB, granzyme B; KO, knock out; NK, natural killer; ns, no…