GSDMD is upregulated in intestinal cancer

To investigate GSDMD expression in intestinal cancer, a human tissue microarray consisting of 80 colorectal cancer samples and their corresponding adjacent non-tumor specimens was analyzed for GSDMD protein levels using immunohistochemistry (IHC). Compared to adjacent non-tumor tissues, GSDMD expression was significantly elevated in intestinal cancer (Fig. 1A-E). Although GSDMD was predominantly localized in the cytoplasm (Fig. 1C), nuclear expression was detected in 57.5% (46/80) of tumor samples, whereas only 6.25% (5/80) of adjacent tissues exhibited GSDMD expression in the nucleus (Fig. 1D). Additionally, GSDMD expression was higher in the cytoplasm of tumor cells than in the nucleus (Fig. 1C and D). These findings suggest that GSDMD may translocate from the cytoplasm to the nucleus during the development of intestinal cancer. Fisher’s exact test revealed a significant positive correlation between GSDMD expression and both tumor grade and tumor size, while no significant associations were found with patient sex, age, or tumor stage (Table 1).

Table 1 Correlation between GSDMD expression and clinicopathological characteristics

In addition to human intestinal cancer, GSDMD expression was also increased in small intestinal tumor of Apcmin/+ mice, a model of spontaneous intestinal carcinogenesis (Fig. 1F). To determine whether GSDMD is activated in intestinal cancer, normal small intestine tissue and intestinal tumor tissue from Apcmin/+ mice were analyzed by immunoblot analysis, and we found that GSDMD was significantly activated in tumor tissue but not in normal tissue (Fig. 1F), suggesting that GSDMD is activated in intestinal cancer. Thus, GSDMD is upregulated in both human and mouse intestinal cancer tissues.

Fig. 1

GSDMD is upregulated in intestinal cancer. (A) IHC analysis of GSDMD protein level in a human tissue microarray, including 80 colorectal cancer and matched adjacent tumor specimens. (B) IHC analysis of GSDMD protein level in four colorectal cancer tissue and matched adjacent cancer tissue from (A) (200× magnification). (C-E) GSDMD expression score in cytoplasm (C), nuclear (D), and cytoplasm + nuclear (E) as calculated by multiplying the intensity and positive percentage scores according to (A). (F) Western blot analysis of GSDMD in in small intestine tumor tissue from Apcmin/+ mice (n = 5) and normal tissue from WT mice (n = 3). Data are representative of at least three independent experiments (mean ± SEM). ***p < 0.001 by Student’s t test. FL means full length

GSDMD promotes spontaneous intestinal cancer progression

To explore the functional role of GSDMD in spontaneous intestinal cancer, Gsdmd-deficient mice were crossed with Apcmin/+ mice to generate Apcmin/+Gsdmd−/− mice. Deficiency of Gsdmd dramatically inhibited the formation of small intestinal tumors (Fig. 2A). Histological analysis revealed a marked reduction in tumor size in Apcmin/+Gsdmd−/− mice (Fig. 2B). Both tumor number (Fig. 2C and E) and tumor load (Fig. 2D and F) were substantially reduced in the small intestines and colons of Apcmin/+Gsdmd−/− mice. Tumor size distribution analysis further demonstrated that Apcmin/+Gsdmd−/− mice had a higher proportion of smaller tumors (Fig. 2G). Consistent with severely decreased intestinal tumor development, compared to Apcmin/+ mice, the anemia and thymus atrophy were notably improved in Apcmin/+Gsdmd−/− mice (Fig. 2H and I). Additionally, Apcmin/+ mice had much larger spleen than Apcmin/+Gsdmd−/− mice (Fig. 2J). These data suggest that GSDMD is critical for the development of spontaneous intestinal cancer.

Fig. 2

Deficiency of GSDMD suppresses spontaneous intestinal cancer development. (A) Macroscopic view of representative small intestine from 20-week old Apcmin/+ and Apcmin/+Gsdmd−/− mice. (B) Hematoxylin and eosin (H&E) staining of the representative intestinal tumor from the Apcmin/+ and Apcmin/+Gsdmd−/− mice as in (A) (100× magnification). (C-F) Tumor number (C and E) and tumor load (D and F) from the small intestines or colons of 20-week-old Apcmin/+ (n = 7) and Apcmin/+Gsdmd−/− (n = 7) mice. (G) Histogram showing the size distribution of tumors from the small intestines of 20-week-old Apcmin/+ (n = 7) and Apcmin/+Gsdmd−/− (n = 7) mice. (H-J) Hematocrit (H), thymus weight (I) and spleen weight (J) of 20-week-old Apcmin/+ (n = 6–7) and Apcmin/+Gsdmd−/− (n = 6–7) mice. Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test

GSDMD promotes intestinal tumorigenesis via IL-1β release

IL-1β, a cytokine known to promote intestinal cancer, is released upon inflammasome activation, a process regulated by GSDMD [16]. To determine whether GSDMD influences IL-1β release during spontaneous intestinal cancer development, we compared Apcmin/+ and Apcmin/+Gsdmd−/− mice. While the mRNA levels of IL-1β were similar in tumors from both genotypes (Fig. 3A), the protein levels of IL-1β were significantly higher in tumors from Apcmin/+ mice (Fig. 3B). These findings suggest that GSDMD facilitates IL-1β release from small intestinal tumors. Some studies reported that IL-1β could promote intestinal cancer progression by promoting colon cancer growth and regulating tumor microenvironment [9,10,11,12]. Further analysis revealed that chemokine KC, HIF-1α, and pro-proliferative factors (IL-6 and CCND1) were markedly increased in Apcmin/+ tumors compared to Apcmin/+Gsdmd−/− tumors (Fig. 3C-F). The number of Ki67-positive cells, indicative of proliferating cells, was also significantly higher in Apcmin/+ tumors (Fig. 3G), suggesting an increased proliferation in the small intestinal tumor from Apcmin/+ mice. These data demonstrate that GSDMD promotes IL-1β release from intestinal tumor, and suggest that IL-1β may promote tumor progression by increasing KC, HIF-1α expression and epithelial cells proliferation.

Fig. 3

Deficiency of GSDMD suppresses IL-1β release in Apcmin/+ mice. (A) Quantitative mRNA expression of GSDMD from small intestines of WT (n = 4), Gsdmd−/− (n = 4), Apcmin/+ (n = 7) and Apcmin/+Gsdmd−/− (n = 6) mice. (B) ELISA analysis of GSDMD protein level from small intestines of WT (n = 4), Gsdmd−/− (n = 4), Apcmin/+ (n = 7) and Apcmin/+Gsdmd−/− (n = 7) mice. (C-F) Quantitative mRNA expression of KC (C), IL-6 (D), HIF-1α (E), and CCND1 (F) from small intestines of WT (n = 4), Gsdmd−/− (n = 4), Apcmin/+ (n = 7) and Apcmin/+Gsdmd−/− (n = 6) mice. (G) Ki67 staining of small intestine from above mice as in (A) (400× magnification). Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test

Exogenous IL-1β partially rescues intestinal tumor formation in Apc min/+Gsdmd −/− mice

To determine whether GSDMD promotes intestinal tumorigenesis through IL-1β, we administrated recombinant mouse IL-1β (0.5 µg) intraperitoneally twice weekly to 8-week old Apcmin/+Gsdmd−/− mice. We found that exogenous IL-1β significantly increased small intestinal tumor size in Apcmin/+Gsdmd−/− mice (Fig. 4A). The IL-1β-treated Apcmin/+Gsdmd−/− mice exhibited increased tumor number and tumor load of small intestine (Fig. 4B and C). However, both the tumor number and tumor load were lower than those in Apcmin/+ mice. Notably, exogenous IL-1β did not increase the number or load of colon tumors (Fig. 4D and E), but it did increase spleen weight (Fig. 4F) and exacerbate anemia (Fig. 4G). Tumor size distribution analysis showed that exogenous IL-1β reduced the number of small tumors in Apcmin/+Gsdmd−/− mice (Fig. 4H). The data suggest that exogenous IL-1β partially restores intestinal tumor formation in Apcmin/+Gsdmd−/− mice. Furthermore, exogenous IL-1β reinstated KC, HIF-1α, IL-6, and CCND1 expression in these mice (Figure S1A-S1D), and the number of Ki67-positive cells was also increased (Figure S1E). Together, these findings suggest that GSDMD promotes intestinal cancer progression in part by inducing IL-1β release.

Fig. 4

Exogenous IL-1β promotes intestinal tumor development in Apcmin/+Gsdmd−/− mice. (A) 8-week-old Apcmin/+Gsdmd−/− mice simultaneously received injection of IL-1β or PBS twice a week, while age and sex-matched Apcmin/+ mice were injected with PBS (n = 5–6/group). H&E staining of the representative intestinal tumor from the 20-week old above mice (100× magnification). Tumors are circled with dashed lines. (B-E) Tumor number (B and D) and tumor load (C and E) from the small intestines or colons of 20-week-old above mice as in (A). (F-G) Spleen weight (F), and hematocrit (G) of 20-week-old above mice as in (A). (H) Histogram showing the size distribution of tumors from the small intestines of 20-week-old above mice as in (A). Data are representative of at least three independent experiments (mean ± SEM). **p < 0.01 by Student’s t test. N.S. means no significance

Apc min/+Gsdmd −/− mice exhibit increased Lactobacillus abundance

Previous studies have shown that gene modifications can alter the composition of the gut microbiota in mice, and some of these alterations are associated with intestinal cancer development [5]. To investigate the differences in the gut microbiota between Apcmin/+ mice and Apcmin/+Gsdmd−/− mice, we performed 16S ribosomal RNA gene sequencing on fecal samples. A Venn diagram indicated that Apcmin/+ mice had 2,058 specific operational taxonomic units (OTUs), while Apcmin/+Gsdmd−/− mice had 1,230 specific OTUs, with 1,125 OTUs shared between both groups (Fig. 5A). The Simpson index revealed altered alpha diversity in Apcmin/+Gsdmd−/− mice compared to Apcmin/+ mice, although the Chao 1 index was similar between the two groups (Fig. 5B). The community structure of the fecal microbiota also differed markedly between Apcmin/+Gsdmd−/− mice and Apcmin/+ mice (Fig. 5C). A heatmap illustrated differences in the gut microbiota composition between the two groups (Figure S2), which were further analyzed at the phylum, class, order, family, and genus levels (Fig. 5D-I). At the phylum level, Firmicutes were significantly increased, while Bacteroidetes were decreased in Apcmin/+Gsdmd−/− mice compared to Apcmin/+ mice (Fig. 5D). Consistent with these changes, Bacilli (class level), Lactobacillales (order level), Lactobacillaceae (family level), and Lactobacillus (genus level) were significantly enriched in Apcmin/+Gsdmd−/− mice, while Bacteroidia (class level), Bacteroidiales (order level), and Lachnospiraceae (family level) were reduced (Fig. 5E- G). At the genus level, Apcmin/+Gsdmd−/− mice exhibited significantly higher levels of Lactobacillus compared to Apcmin/+ mice (Fig. 5H and I). Linear discriminant analysis effect size (LEfSe) further confirmed the enrichment of Lactobacillales, Lactobacillaceae, and Lactobacillus in Apcmin/+Gsdmd−/− mice (Fig. 5J). These findings suggest that GSDMD deficiency alters the gut microbiota composition, as Apcmin/+Gsdmd−/− mice display an increased abundance of Lactobacillus.

Fig. 5

Deficiency of GSDMD alters gut microbiota composition of Apcmin/+ mice. (A) Gut microbiota composition of fecal samples from 10-week-old Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice were assessed with 16S rRNA sequencing. Venn diagram of 16S rRNA sequencing data. WT means Apcmin/+ mice; KO means Apcmin/+Gsdmd−/− mice. (B) Chao 1 and Simpson index of 16S rRNA sequencing data as in (A). (C) PCoA analysis of 16S rRNA sequencing data as in (A). (D-H) Relative abundance of top 20 phylum (D), class (E), order (F), family (G), and genus (H) from the gut microbiota of Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. (I) Relative abundance of unidentified_S24_7 and Lactobacillus from the gut microbiota of Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. (J) LEfSe analysis of the relative abundance from the gut microbiota of Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01 by Student’s t test

Apc min/+Gsdmd −/− mice exhibit reduced kynurenine levels

To determine whether changes in the microbiota composition contribute to intestinal tumor development of Apcmin/+Gsdmd−/− mice through microbiota-associated metabolites, we further investigated the metabolites in the feces of Apcmin/+Gsdmd−/− mice and Apcmin/+ mice using mass spectrometry. Partial least squares discriminant analysis (PLS-DA) revealed distinct metabolomic profiles between Apcmin/+Gsdmd−/− mice and Apcmin/+ mice (Fig. 6A). A total of 781 metabolites were identified in the feces, with 25 downregulated metabolites and 61 upregulated metabolites in Apcmin/+ mice compared to Apcmin/+Gsdmd−/− mice (Fig. 6B). The upregulated metabolites (Fig. 6C) and downregulated metaboiltes (Figure S3) were showed using heatmaps. The correlation of different metaboiltes was showed with correlated heatmap (Figure S4). Z-score analysis highlighted significant differences in metabolite abundance, with L-Kynurenine (Kyn) being particularly elevated (Fig. 6D). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified tryptophan (Trp) metabolism as the pathway most affected by GSDMD deficiency (Fig. 6E). A network diagram revealed that Kyn, indolepyruvate, quinolinic acid, and 3-methoxyanthranilate were elevated, while 3-hydroxyanthranilate and 2-aminobenzoic acid were reduced in Apcmin/+ mice compared to Apcmin/+Gsdmd−/− mice (Fig. 6F), which was confirmed by intensity analysis (Fig. 6G). Kyn has been implicated in intestinal cancer development [21, 22], and its reduction in Apcmin/+Gsdmd−/− mice may contribute to the reduced tumor formation observed in these animals.

Fig. 6

Deficiency of GSDMD alters gut metabolites of Apcmin/+ mice. (A) PLS-DA scores for fecal metabolites of Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. (B) Statistics of differentially expressed fecal metabolites in Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. (C) Heatmap analysis of upregulated metabolites in Apcmin/+ (n = 10) mice compared to Apcmin/+Gsdmd−/− (n = 16) mice. WT means Apcmin/+ mice; KO means Apcmin/+Gsdmd−/− mice. (D) Top 15 metabolites of Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice based on Z-score. (E) Top 20 enriched KEGG pathways of differentially expressed fecal metabolites in Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. (F) Network of KEGG enriched pathway. Blue dot: pathway, red dot: upregulated metabolite, purple dot: downregulated metabolite. Metabolites from Apcmin/+ mice vs. metabolites from Apcmin/+Gsdmd−/− mice. (G) Log2(intensity) of Trp pathway metabolites in Apcmin/+ (n = 10) and Apcmin/+Gsdmd−/− (n = 16) mice. Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01 by Student’s t test. N.S. means no significance

Kynurenine levels negatively correlate with Lactobacillus abundance

To determine the correlation of microbiota and metabolites, correlated heatmap was carried out with Pearson correlation analysis. We found that Kyn was positively correlated with Bacteroidia (class level) and negatively correlated with Bacilli (class level) (Figure S5A). Consistent with the class level, Kyn was positively correlated with Bacteroidiales (order level), Lachnospiraceae (family level), and unidentified_S24-7, but negatively correlated with Lactobacillales (order level), Lactobacillaceae (family level), and Lactobacillus (genus level) (Figure S5B-5D). Correlation scatter plots showed that fecal microbiota from Apcmin/+Gsdmd−/− mice had more Bacilli (class level) and fewer Bacteroidia (class level), which were negatively and positively correlated with Kyn respectively (Figure S5E). These trends were consistent across the order, family, and genus levels (Figure S5F-5H). The data suggest that increased Lactobacillus may promote intestinal tumor formation through suppressing Kyn production in Apcmin/+Gsdmd−/− mice.

Exogenous kynurenine promotes colon cancer development in Apc min/+Gsdmd −/− mice

We observed reduced Kyn in the feces of Apcmin/+Gsdmd−/− mice. As is known, Kyn is a metabolite derived from Trp metabolism, which includes endogenous (host) Trp metabolism and bacterial Trp metabolism. Kyn is produced by endogenous Trp metabolism, but not by bacterial Trp metabolism [28]. To confirm this in our setting, we measured Kyn and Trp levels in the colon tissues of mice. Consistent with a previous report [22], we found that Apcmin/+Gsdmd−/− mice exhibited lower Kyn but not Trp levels in colon tissue compared to Apcmin/+ mice (Fig. 7A). The ratio of Kyn/Trp was significantly reduced in the colon of Apcmin/+ mice (Fig. 7B). To determine whether reduced tumor formation in Apcmin/+Gsdmd−/− mice was due to lower Kyn levels, we intraperitoneally injected Kyn into 8-week-old Apcmin/+Gsdmd−/− mice and analyzed tumor development after 12 weeks. We found that the administration of exogenous Kyn increased Kyn levels in the serum and feces of Apcmin/+Gsdmd−/− mice (Figure S6). As expected, exogenous Kyn significantly increased tumor size in the small intestines of Apcmin/+Gsdmd−/− mice (Fig. 7C). Additionally, Kyn-treated Apcmin/+Gsdmd−/− mice exhibited an increased tumor number (Fig. 7D and F) and tumor load (Fig. 7E and G) in both the small intestine and colon. Meanwhile, Kyn increased spleen weight (Fig. 7H) and exacerbated anemia (Fig. 7I) in Apcmin/+Gsdmd−/− mice. Tumor size distribution analysis further indicated that exogenous Kyn increased tumor size in Apcmin/+Gsdmd−/− mice (Fig. 7J). These results suggest that exogenous Kyn partially restores intestinal tumor formation in Apcmin/+Gsdmd−/− mice.

Fig. 7

Exogenous Kyn promotes intestinal tumor development in Apcmin/+Gsdmd−/− mice. (A) ELISA analysis of Kyn and Trp from colon of Apcmin/+ (n = 6) and Apcmin/+Gsdmd−/− (n = 6) mice. (B) Kyn/Trp was determined as in (A). (C) 8-week-old Apcmin/+Gsdmd−/− mice simultaneously received injection of Kyn or PBS once a week, while age and sex-matched Apcmin/+ mice were injected with PBS (n = 5/group). H&E staining of the representative intestinal tumor from the 20-week old above mice (100× magnification). (D-G) Tumor number (D and F) and tumor load (E and G) from the small intestines or colons of 20-week-old above mice as in (C). (H-I) Spleen weight (H), and hematocrit (I) of 20-week-old above mice as in (C). (J) Histogram showing the size distribution of tumors from the small intestines of 20-week-old above mice as in (C). Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t test. N.S. means no significance

Kyn is known to promote colon cancer by reducing CD8+ T cell numbers and increasing regulatory T (Treg) cell numbers [22, 24]. In Apcmin/+Gsdmd−/− mice, CD8+ T cell numbers were higher in intestinal tumors, and exogenous Kyn reduced these numbers (Fig. 8A and B). Treg cells, which were reduced in Apcmin/+Gsdmd−/− mice, were increased by Kyn treatment (Fig. 8C and D). These data suggest that Kyn promotes tumor development in Apcmin/+Gsdmd−/− mice, likely by enhancing immunosuppression.

Fig. 8

Exogenous Kyn increases Treg cell number in Apcmin/+Gsdmd−/− mice. (A) 8-week-old Apcmin/+Gsdmd−/− mice simultaneously received injection of Kyn or PBS once a week, while age and sex-matched Apcmin/+ mice were injected with PBS (n = 4/group). Flow cytometry analysis of CD4+ and CD8+ T cell number from intestinal tumor of the 20-week old above mice. (B) Total leukocytes were determined by CD45+ cell. CD4+ T cells % of total leukocyte and CD8+ T cells % of total leukocyte were determined as in (A). (C) Flow cytometry analysis of Treg (CD4+ Foxp3+) cell number from intestinal tumor of the 20-week old mice as in (A). (D) Treg % of CD4+ cells was determined as in (C). Data are representative of at least three independent experiments (mean ± SEM). **p < 0.01, ***p < 0.001 by Student’s t test. N.S. means no significance