Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression and chemoresistance by enhancing the secretion of chemotherapy-induced senescence-associated secretory phenotype via activation of DNA damage response pathway

Figures & data

Figure 1. CDDP induced senescence and a robust SASP in p53 wild-type ESCC cells and in human ESCC cancerous tissues.

(a) ESCC cells treated with or without CDDP at the indicated concentrations for 96 h were examined for SA-β-Gal activity. Original magnification, 200 ×. Scale bar = 50 μm. (b) Quantification of SA-β-Gal-positive cells. (c) The same as (a), but cells were assayed for the ability of cell proliferation using EdU incorporation assay. Blue fluorescence indicated nuclear staining with Hoechst 33342, and green fluorescence reflected EdU staining. Original magnification, 200 ×. Scale bar = 50 μm. (d) Quantification of EdU-positive cells. (e) The same as (a), but cells were measured for γH2AX formation using immunofluorescent staining. Blue fluorescence indicated nuclear staining with DAPI, and red fluorescence reflected γH2AX immunostaining. Original magnification, 400 ×. Scale bar = 20 μm. (f) Quantification of γH2AX-positive cells. (g) Western blot analysis of γH2AX and a loading control GAPDH in ESCC cells under the same conditions as (a). (h) RT-qPCR analysis of the expression of p53 and CDKN1A genes in ESCC cells under the same conditions as (a). (i) Western blot analysis of the protein expression of p53, p21 (encoded by the CDKN1A gene), and GAPDH in ESCC cells under the same conditions as (a). (j) RT-qPCR analysis of SASP gene expression in KYSE-150 cells under the same conditions as (a). (k) The levels of SASP-related factors in the CM from CDDP-treated and untreated KYSE-150 cells were measured using ELISA. (l and m) The sections of formalin-fixed and paraffin-embedded cancerous tissues of ESCC patients who had received CDDP-based neoadjuvant chemotherapy before surgical resection and that of patients who had not received chemotherapy prior to surgical resection were examined for the expression of p21 and p16 using immunohistochemistry staining. Original magnification, 200 ×. Scale bar = 50 μm. Original magnification, 400 ×. Scale bar = 20 μm.The results are presented as mean ± standard deviation. ***p < 0.001.

Table 1. The expression of p21 and p16 in cancerous tissues of ESCC patients treated with or without CDDP-based neoadjuvant chemotherapy.

Factors	CDDP-based neoadjuvant chemotherapy		χ^2	p value
	Treated (n = 25)	Untreated (n = 20)
p21 positive	15 (60%)	3 (15%)	9.375	0.002**
p21 negative	10 (40%)	17 (85%)
p16 positive	11 (44%)	2 (10%)	6.252	0.012*
p16 negative	14 (56%)	18 (90%)

Data were analyzed by Pearson χ² test. *p < 0.05 and **p < 0.01.

Figure 2. CDDP-induced SASP promoted tumorigenesis in the xenograft model and ESCC cell growth, migration, invasion, and chemoresistance.

(a) CCK-8 assay was used to assay the proliferation of ESCC cells in vitro. ESCC cells were treated with Sen CM or n-Sen CM. Note that Sen CM and n-Sen CM were additionally supplemented with 5% FBS. For the negative control group, cells were incubated in RPMI-1640 medium containing 5% FBS. (b) KYSE-150 cells were subcutaneously inoculated into male BALB/c nude mice. The mice were intraperitoneally injected every other day with 5-fold concentrated Sen CM (n = 5) or n-Sen CM (n = 5), four times in total. When the tumors reached a volume of 50 mm³, the mice were intratumorally injected every other day with Sen CM or n-Sen CM, four times in total. Left panel: the macroscopic features of nude mice tumors formed by different treatments. Right panel: the tumor volume was measured every 4 days, and the tumor growth curve was drawn. (c) Left panel: the excised xenograft tumors. Right panel: quantification of tumor weight. (d) Left panel: representative H&E staining and Ki-67 immunostaining of xenograft tumor tissues. Original magnification, 200 ×. Scale bar = 50 μm. Right panel: quantification of Ki-67-positive cells. (e) The transwell assay was used to analyze the migration and invasion ability of ESCC cells. ESCC cells were treated with Sen CM or n-Sen CM for 36 h. Note that Sen CM and n-Sen CM were additionally supplemented with 1% FBS. For the negative control group and the positive control group, cells were incubated in RPMI-1640 medium containing 1% FBS and medium containing 10% FBS, respectively. Original magnification, 200 ×. Scale bar = 50 μm. (f) Quantification of migratory and invasive cells after different treatments. (g) ESCC cells cultured with Sen CM or n-Sen CM for 48 h were measured for the protein expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug, and a loading control GAPDH by western blot. (h) ESCC cells incubated with Sen CM or n-Sen CM were treated with or without 5 μg/ml CDDP for 24 h and then examined for cell apoptosis by flow cytometry. For the negative control group, cells incubated in serum-free RPMI-1640 medium were treated with 5 μg/ml CDDP for 24 h. The results are presented as mean ± standard deviation. ns, no significance. **p < 0.01 and ***p < 0.001.

Figure 3. Gut microbiota composition was altered in ESCC tissues and F. nucleatum was correlated with poor prognosis and chemoresistance in ESCC patients.

(a) The microbial α diversity analysis between the Cancer and Normal groups via Simpson index. (b) The α diversity analysis between the two groups via Shannon index. (c) The comparison of differences in the main bacterial taxa at the phylum level between the two groups. (d) The comparison of differences in the main bacterial taxa at the genus level between the two groups. (e) FISH with a Cy3-conjugated probe was applied to detect F. nucleatum in cancerous and matched adjacent normal tissues of ESCC patients. Blue fluorescence indicated nuclear staining with DAPI, and red fluorescence reflected F. nucleatum immunostaining. Original magnification, 200 ×. Scale bar = 50 μm. (f) Quantification of the abundance of F. nucleatum in cancerous and adjacent normal tissues based on the results of FISH. (g) The relationship between F. nucleatum abundance in cancerous tissues and tumor infiltration depth. (h) The relationship between F. nucleatum abundance in cancerous tissues and lymph node metastasis. (i and j) The relationship between F. nucleatum abundance in cancerous tissues and tumor TNM stage. (k) The overall survival for ESCC patients with F. nucleatum-high or F. nucleatum-low abundance were calculated via Kaplan–Meier analysis, and the survival differences between the two groups were compared using the log-rank test. The results are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.001.

Table 2. Clinicopathological features in F. nucleatum-low vs. F. nucleatum-high ESCC patients.

Factors	F. nucleatum abundance		p value
	Low (n = 75)	High (n = 32)
Mean age ± SD, year	62.01 ± 6.78	61.84 ± 8	0.305
Sex			1
Male	62	27
Female	13	5
Location			0.352
Upper	9	1
Median	36	17
Lower	30	14
Mean tumor size ± SD, cm	3.47 ± 1.83	4.14 ± 1.89	0.597
Differentiation			0.344
Well	23	9
Moderately	40	14
Poor	12	9
T stage			0.193
T1	15	2
T2	18	8
T3–4	42	22
N stage			<0.001***
N0	57	10
N1–3	18	22
UICC pTNM stage
I-II	58	10	<0.001***
III-IV	17	22

UICC, Union for International Cancer Control. ***p < 0.001.

Figure 4. F. nucleatum stimulation aggravated the SASP-mediated malignant phenotypes and chemoresistance of ESCC cells.

(a) CCK-8 assay was used to examine the proliferation of ESCC cells in vitro. ESCC cells were treated with Sen CM, Sen+F. nucleatum CM, n-Sen CM, or n-Sen+F. nucleatum CM. Note that these CM were additionally supplemented with 5% FBS. (b) KYSE-150 cells were subcutaneously inoculated into male BALB/c nude mice. The mice were intraperitoneally injected every other day with 5-fold concentrated Sen CM (n = 5), Sen+F. nucleatum CM (n = 5), n-Sen CM (n = 5), or n-Sen+F. nucleatum CM (n = 5), four times in total. When the tumors reached a volume of 50 mm³, the mice were intratumorally injected every other day with Sen CM, Sen+F. nucleatum CM, n-Sen CM, or n-Sen+F. nucleatum CM, four times in total. Left panel: the macroscopic features of nude mice tumors formed by different treatments. Right panel: the tumor volume was measured every 4 days, and the tumor growth curve was drawn. (c) Left panel: the excised xenograft tumors. Right panel: quantification of tumor weight. (d) Left panel: representative Ki-67 immunostaining of xenograft tumor tissues. Original magnification, 200 ×. Scale bar = 50 μm. Right panel: quantification of Ki-67-positive cells. (e) The transwell assay was used to analyze the migration and invasion ability of ESCC cells. ESCC cells were treated with Sen CM, Sen+F. nucleatum CM, n-Sen CM, or n-Sen+F. nucleatum CM for 36 h. Note that these CM were additionally supplemented with 1% FBS. Original magnification, 200 ×. Scale bar = 50 μm. (f) Quantification of migratory and invasive cells after different treatments. (g) ESCC cells treated with Sen CM, Sen+F. nucleatum CM, n-Sen CM, or n-Sen+F. nucleatum CM for 48 h were examined for the protein expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug, and a loading control GAPDH by western blot. (h) ESCC cells incubated with Sen CM, Sen+F. nucleatum CM, n-Sen CM, or n-Sen+F. nucleatum CM were treated with or without 5 μg/ml CDDP for 24 h and then measured for cell apoptosis by flow cytometry. The results are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5. F. nucleatum infection enhanced the secretion of CDDP-induced SASP through the activation of DNA damage response pathway.

(a) KYSE-150 cells treated with or without CDDP for 96 h were washed with PBS and then co-cultured with or without F. nucleatum (MOI = 100) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS for 24 h. Cells were examined for SASP gene expression using RT-qPCR. (b) The levels of SASP-related factors in the Sen CM, Sen+F. nucleatum CM, n-Sen CM, and n-Sen+F. nucleatum CM of KYSE-150 cells were measured using ELISA. (c) The same as (a), but cells were analyzed for the protein expression of SASP-related factors and a loading control GAPDH by western blot. (d) Western blot analysis of ATM, p-ATM, Chk2, p-Chk2, and GAPDH in ESCC cells under the same conditions as (a). (e) ESCC cells treated with or without CDDP for 96 h were washed with PBS and then pre-treated with or without 10 μM KU55933 for 2 h followed by treatment with or without F. nucleatum (MOI = 100) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS for 24 h. Cells were examined for the protein expression of ATM, p-ATM, Chk2, p-Chk2, and GAPDH by immunoblotting. (f) The same as (e), but KYSE-150 cells were assayed for SASP gene expression using RT-qPCR. The results are presented as mean ± standard deviation. ns, no significance. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6. F. nucleatum invaded and survived in ESCC cells and increased DNA damage.

(a) ESCC cells treated with or without CDDP for 96 h were washed with PBS and then co-cultured with or without F. nucleatum (MOI = 100) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS for 24 h. Cells were measured for the formation of γH2AX foci using immunofluorescent staining. Blue fluorescence indicated nuclear staining with DAPI, and red fluorescence reflected γH2AX immunostaining. Original magnification, 400 ×. Scale bar = 20 μm. (b) Quantification of γH2AX-positive cells. (c) The same as (a and c), but cells were harvested for the neutral comet assay. Original magnification, 200 ×. Scale bar = 50 μm. (d) Quantification of the olive tail moment. (e) F. nucleatum invasion into senescent and non-senescent ESCC cells. The growth of intracellular F. nucleatum into colonies on the agar after 72 h under anaerobic conditions. (f) Quantification of the number of colonies formed. The results are presented as mean ± standard deviation. ns, no significance. *p < 0.05, **p < 0.01, and ***p < 0.001.

Data availability statement

All sequencing data associated with this study have been uploaded to the NCBI (Sequence Read Archive) SRA database under the accession number PRJNA866879 (http://www.ncbi.nlm.nih.gov/bioproject/866879).