The influence of Lactobacillus johnsonii on tumor growth and lymph node metastasis in papillary thyroid carcinoma

doi:10.21203/rs.3.rs-4663618/v1

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The influence of Lactobacillus johnsonii on tumor growth and lymph node metastasis in papillary thyroid carcinoma

https://doi.org/10.21203/rs.3.rs-4663618/v1

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The presence of lymph node metastasis (LNM) is one of the most crucial factors in the prognosis of papillary thyroid carcinoma (PTC). The aim of this study was to investigate the influence of intratumoral bacteria on the LNM in PTC. The intrathyroidal microbiome was analyzed in 55 PTC patients by 16S rRNA gene sequencing. The CCK8 and Transwell assays were conducted to determine the impact of bacteria on the proliferation and migration abilities of PTC cells. Xenograft tumor and bacterial colonization experiments were carried out using nude mice. We showed that Lactobacillus was significantly decreased in PTC lesions from patients with LNM. Lactobacillus johnsonii (L. johnsonii) could suppress the proliferation and migration capability of PTC cells in vitro and in vivo. Bacterial gut colonization of L. johnsonii increased its abundance in tumors and inhibited PTC growth and LNM. These findings suggested that L. johnsonii could be harnessed for the development of innovative therapeutic strategies for PTC.

Thyroid cancer is the most common endocrine malignancy worldwide, of which papillary thyroid cancer (PTC) is the most common subtype.¹ The increasing incidence of PTC is mainly due to the more frequent use of neck ultrasonography and fine-needle aspiration of thyroid nodules.² In spite of the favorable prognosis of PTC, it is reported that over half of patients develop cervical lymph nodes metastasis (LNM) which is a major risk factor for disease recurrence.^3,4 Thus, controlling the LNM of PTC may be an effective measure to further improve the prognosis of patients.

Recent researches have indicated that microbiota, particularly the gut microbiota, is involved in the initiation, progression and dissemination of cancer.⁵ Gut microbiota potentially influence individual's susceptibility to cancer, and certain bacteria have been reported to accelerate tumor growth and metastatic progression.^6,7 Besides, microbiota has ability to modify the microenvironment within tumor and could thereby enhance tumor response to therapeutic drugs. Previous research demonstrated that fecal microbiota transplantation from responders significantly improved tumor-suppressing efficacy of PD-1 blockade, suggesting the potential contribution of the microbiota in tumor treatment.⁸ Specifically, alterations of intestinal microbial community have also been found to correlate with pathogenesis and therapy response of thyroid carcinoma.^9,10 Yet, considering the thyroid's anatomical separation from intestine, the precise association and underlying mechanism between thyroid cancer and bacteria remains uncertain.

The relationship between tumor cell with intratumoral bacteria and the initiation, growth, metastasis, and resistance to chemotherapy of tumors is widely acknowledged.¹¹ A study conducted on PTC patients revealed notable variations in the intratumoral microbiome characteristics based on patients' age and tumor stage.¹² Another study has identified significant differences in microbial diversity and composition between the peritumor and tumor microhabitats of patients with thyroid cancer, with a potential correlation between a higher abundance of Sphingomonas and LNM.¹³ However, there is a scarcity of research examining the involvement of intratumoral bacteria in thyroid cancer. The aforementioned studies solely relied on the analysis of 16S rRNA gene sequencing data and did not conduct experimental investigations to validate their hypotheses.

In this study, we successfully isolated and cultured intrathyroidal bacteria under both anaerobic and aerobic conditions. Additionally, we established a nude mice LNM model of PTC by subcutaneously injecting a fluorescently labeled PTC cell line. Our findings reveal that intratumoral Lactobacillus johnsonii (L. johnsonii) is more prevalent in non-metastatic PTC, and it exerts inhibitory effects on LNM by suppressing the epithelial-mesenchymal transition (EMT) process. Significantly, we demonstrated that the oral administration of L. johnsonii can enhance its colonization in the gut and intratumoral regions, suggesting its potential as a novel therapeutic approach to inhibit LNM in PTC.

Intratumoral Lactobacillus was enriched in Non-metastatic PTC.

To investigate the impact of intrathyroidal microbiome on LNM of PTC, we collected tumor tissues and adjacent non-tumor (normal) tissues from 55 PTC patients and performed 16S rRNA gene sequencing. The demographic and clinical characteristics of the patients with PTC were shown in Table 1. There was no significant difference in community composition at phylum level between tumor and normal tissue, of which the three predominant bacteria were Proteobacteria, Actinobacteriota, and Firmicutes. (Figs. 1A and 1B). Subsequent LEfSe analysis at genus level revealed a significant enrichment of Cutibacterium and Gaiella in the normal tissues, while Herbaspirillum and Coprococcus were enriched in tumor tissues (Figs. 1C and 1D). Moreover, we divided the tumor tissues into two groups by LNM and found that the abundance of Lactobacillus was the most significant difference between the metastasis group (n = 29) and non-metastasis group (n = 26) (Figs. 1E and 1F).

Table 1

The demographic and clinical characteristics of the PTC patients
Patient ID	Sex	Age (year)	TNM stage	Clinical stage	Extrathyroid extension	Vascular invasion	LNM	Concomitant HT
1	Female	29	T1aN1aM0	1	No	No	Yes	No
2	Female	35	T1aN0M0	1	No	No	No	No
3	Female	49	T1aN1aM0	1	Yes	No	Yes	No
4	Female	41	T1aN0M0	1	Yes	No	No	No
5	Female	41	T4aN1bM0	1	Yes	No	Yes	Yes
6	Female	66	T1bN0M0	1	No	No	No	No
7	Male	35	T1bN1aM0	1	No	No	Yes	No
8	Female	34	T1aN0M0	1	No	No	No	No
9	Female	56	T1bN0M0	1	No	No	No	No
10	Female	41	T1aN0M0	1	No	No	No	Yes
11	Female	65	T1aN0M0	1	No	No	No	No
12	Male	38	T1aN1bM0	1	No	No	Yes	No
13	Female	34	T1bN0M0	1	Yes	No	No	Yes
14	Female	38	T1aN0M0	1	No	No	No	No
15	Female	37	T1bN0M0	1	No	No	No	No
16	Female	55	T1aN1aM0	2	No	No	Yes	No
17	Female	34	T1bN1bM0	1	No	No	Yes	Yes
18	Female	33	T1bN1bM0	1	No	No	Yes	No
19	Female	67	T4aN1bM0	3	Yes	Yes	Yes	Yes
20	Female	17	T2N1bM0	1	Yes	Yes	Yes	No
21	Male	38	T2N1bM0	1	Yes	Yes	Yes	No
22	Male	52	T4aN0M0	1	Yes	Yes	No	No
23	Female	53	T1bN0M0	1	No	No	No	No
24	Female	49	T4aN1bM0	1	Yes	No	Yes	Yes
25	Male	59	T4aN1bM0	3	Yes	No	Yes	Yes
26	Male	49	T1bN1aM0	1	No	No	Yes	No
27	Female	56	T1bN1aM0	2	No	No	Yes	Yes
28	Female	56	T3bN0M0	1	Yes	No	No	No
29	Female	49	T1aN0M0	1	No	No	No	No
30	Female	47	T2N1aM0	1	No	No	Yes	Yes
31	Female	21	T2N1bM0	1	Yes	Yes	Yes	Yes
32	Female	70	T1aN1aM0	2	No	No	Yes	Yes
33	Female	52	T1aN1aM0	1	No	No	Yes	No
34	Female	40	T1aN0M0	1	No	No	No	No
35	Female	60	T3bN0M0	1	Yes	No	No	Yes
36	Female	42	T1aN1aM0	1	Yes	Yes	Yes	Yes
37	Female	25	T2N1bM0	1	No	No	Yes	No
38	Female	34	T1bN0M0	1	No	No	No	No
39	Female	41	T1bN0M0	1	No	No	No	No
40	Female	54	T1aN0M0	1	No	No	No	Yes
41	Female	55	T4aN1bM0	3	Yes	Yes	Yes	No
42	Male	66	T4aN1aM0	3	Yes	No	Yes	Yes
43	Male	29	T1bN0M0	1	No	No	No	Yes
44	Male	60	T1bN1aM0	2	No	No	Yes	No
45	Female	31	T2N1bM0	1	No	No	Yes	Yes
46	Female	52	T1aN0M0	1	No	No	No	No
47	Female	33	T1aN0M0	1	No	No	No	No
48	Female	52	T1aN0M0	1	No	No	No	No
49	Female	45	T1bN0M0	1	No	No	No	No
50	Male	49	T4aN1aM0	1	Yes	Yes	Yes	No
51	Male	48	T1bN0M0	1	No	No	No	No
52	Male	33	T1aN0M0	1	No	No	No	No
53	Female	43	T1bN1aM0	1	No	No	Yes	Yes
54	Female	39	T1bN1aM0	1	No	No	Yes	No
55	Female	56	T2N1aM0	2	Yes	Yes	Yes	Yes

Lactobacillus johnsonii was presented in PTC tissues.

To confirm the presence of bacteria in the thyroid, we utilized high-resolution electron microscopy (EM) analysis, which revealed the presence of bacteria in both tumor and adjacent normal tissues (Fig. 2A). Subsequently, we collected tumor and adjacent normal tissues from 8 additional PTC patients, and the intrathyroidal bacteria isolated from these tissues were cultured under anaerobic and aerobic conditions. As shown in Fig. 2B, colonized bacteria were found in thyroid tissues of 3 patients both under anaerobic and aerobic conditions. Specifically, in Patient No.60 and No.62, bacteria could be found both in tumor and normal tissues, while in Patient No.63, bacteria could be found only in tumor tissue. To identify the specific bacteria strain in these 3 patients, we picked the single colony and ran colony PCR to get the bacteria sequencing. By aligning to the 16S rRNA sequences database in the NCBI BLAST site, we found out that the main bacteria strain was Escherichia coli (E. coli) in Patient No.60 and No.63 both under anaerobic and aerobic conditions. Comparatively, Lactobacillus johnsonii (L. johnsonii) was mostly presented in the tumor tissue of Patient No.62 under anaerobic condition (Fig. 2B). We further confirmed the presence of L. johnsonii in both tumor and normal tissues through Fluorescence in situ hybridization (FISH) (Fig. 2C).

Lactobacillus johnsonii suppressed proliferation, invasion and migration of human PTC cells via inhibiting Wnt/β-catenin pathway.

To investigate the roles of Lactobacillus in PTC, we examined the correlation between tumor volume and the abundance of Lactobacillus in 55 PTC patients. The 55 PTC patients were divided into two groups according to the median abundance value of Lactobacillus. As shown in Fig. 3A, group with low abundance of Lactobacillus exhibited larger tumor sizes than the other group with high abundance of Lactobacillus. The correlation analysis showed that there was a significant negative correlation between the abundance of Lactobacillus and tumor volume. The representative sequence of each ASV belonging to Lactobacillus was further aligned to the NCBI 16S BLAST database to identify the ASV of L. johnsonii. As shown in Fig. 3B, the abundance of L. johnsonii was significantly lower in tumor tissues compared to normal tissues. Consequently, the 55 PTC patients was divided into another two groups according to the median abundance value of L. johnsonii. Similarly, group with low abundance of L. johnsonii also exhibited larger tumor sizes than that with high abundance of L. johnsonii. (Fig. 3C), indicating a significant negative correlation between the abundance of L. johnsonii and tumor volume (Fig. 3D). We evaluated cell proliferation of IHH4 and TPC1 cells treated with different MOI of L. johnsonii. As shown in Fig. 3E and 3F, the higher MOI of L. johnsonii initially introduced, the more slowly the IHH4 and TPC1 cells proliferated.

According to the findings depicted in Fig. 3G and 3H, there was a significant reduction in the relative abundances of Lactobacillus and L. johnsonii in tumor tissues with LNM compared to those without LNM. To investigate the potential impact of L. johnsonii on the invasion and migration of PTC cells, a Transwell invasion and migration assay was conducted using IHH4 and TPC1 cells co-cultured with L. johnsonii. As shown in Figs. 3I and 3J, L. johnsonii significantly inhibited the invasion and migration ability of IHH4 and TPC1 cells compared with cells co-cultured with E. coli (isolated from PTC) or 1×PBS as negative controls. Previous study suggested that L. johnsonii could regulate the β-catenin pathway to inhibit the metastasis of colorectal cancer. We wondered whether β-catenin signaling pathway was involved in L. johnsonii suppressing PTC growth and metastasis, and detected the related proteins. The results showed that β-catenin, c-MYC, N-cadherin and Vimentin was down-regulated, accompanied with markedly up-regulated E-cadherin in IHH4 and TPC1 cells after co-cultured with L. johnsonii (Fig. 3K).

L. johnsonii suppressed tumor growth and LNM of PTC in vivo.

A xenograft tumor experiment in nude mice was conducted to verify the potential effect of L. johnsonii on PTC. As shown in Fig. 4A, the IHH4-Luciferase cell line labeled with nano carbon tracer was used to observe the tumor formation and metastasis. As described in the “METHODS” section, the mouse LNM model of PTC was established by L. johnsonii, E. coli or PBS pretreated IHH4-Luciferase cells subcutaneously injection and the subcutaneous tumors were excised on day 28 (Fig. 4B). In the L. johnsonii treated group, the size and weight of xenograft tumor were significantly lower compared with the E. coli and PBS treated groups (Figs. 4C and 4D). Furthermore, after removal of subcutaneous tumor, the L. johnsonii treated group showed a significant lower LNM rate compared with the E. coli and PBS treated groups (Figs. 4E and 4F). These results indicated that the L. johnsonii was capable of suppressing tumor growth and metastasis of PTC in vivo.

Gut colonization of L. johnsonii inhibited PTC metastatic progression.

Considering that the main colonization site of L. johnsonii was gastrointestinal tract and the oral intake was the main way that human get L. johnsonii, we collected fecal tissues from those 55 PTC patients and performed qPCR to detect the abundance of L. johnsonii. The qPCR results indicated that the abundance of L. johnsonii in the feces of metastatic group was significantly lower than that of non-metastatic group (Fig. 5A). The correlation analysis revealed a significant positive correlation between the abundance of L. johnsonii in patients' tumors and their feces (Fig. 5B). Then we performed gut colonization mice experiment to investigate the impact of L. johnsonii on LNM of PTC. Mice implanted with subcutaneous IHH4 cell lines were gavaged with MRS, E. coli, or L. johnsonii after 5-day treatment of antibiotic cocktails (Fig. 5C). Indeed, the xenograft tumor volume and weight (Fig. 5D) were significantly lower in L. johnsonii gavaged group compared with the E. coli and MRS gavaged groups, supporting that L. johnsonii significantly inhibited tumor growth of PTC in vivo.

To certify that the tumor suppression was modulated by gut colonization of L. johnsonii, mice fecal and xenograft tumors were collected after 28 days of colonization gavage. Compared with the E. coli and MRS gavaged groups, the abundance of L. johnsonii in mice fecal was significantly increased in L. johnsonii gavaged group (Fig. 5E), and similar results were observed in xenograft tumors (Fig. 5F). Moreover, the LNM of PTC was evaluated by in vivo bioluminescence imaging system, and the fluorescence was significantly decreased in L. johnsonii gavaged group compared with the E. coli and MRS gavaged groups (Figs. 5G). The above results suggested that L. johnsonii was able to colonize in the gastrointestinal tract and translocate to PTC tissue to further suppress the proliferation and metastasis of PTC cell. To disclose the mechanism underlying this phenomenon, expressions of β-catenin pathway-related proteins in xenograft tumors were examined by Western blot. Consistently, β-catenin, c-MYC, N-cadherin and Vimentin was downregulated accompanied with markedly upregulation of E-cadherin in xenograft tumors of L. johnsonii gavaged group compared with the MRS gavaged group (Fig. 5H).

Several studies have shown the correlation between gut microbiota and thyroid disease. One study demonstrated that untreated primary hypothyroidism patients can be differentiated from healthy individuals based on the presence of four specific intestinal bacteria (Veillonella, Paraprevotella, Neisseria, and Rheinheimera).¹⁴ Another study revealed that individuals with hyperthyroidism exhibit reduced levels of Bifidobacteria and Lactobacillaceae.¹⁵ A separate study found a notable increase in Bacteroides and a significant decrease in Bifidobacterium, while patients not receiving thyroid hormone replacement therapy displayed higher levels of Lactobacillus species compared to those taking oral levothyroxine.¹⁶ In recent years, there has been an expansion of research on the human microbiome, extending beyond the gut, which is known for its abundance of microorganisms, to previously presumed sterile organs such as the bladder and lungs.^17,18 In our study, we utilized 16S rRNA gene sequencing and provided preliminary evidence for the presence of bacteria in the thyroid and PTC. To further validate this discovery, we cultured intrathyroidal bacteria isolated from tumor and normal tissues of an additional 8 PTC patients under both anaerobic and aerobic conditions. Recent evidence suggested that intratumoral bacteria were primarily derived from the digestive tract.¹⁹ Our result showed that a comparable abundance of L. johnsonii was observed in the subcutaneous PTC tumor gavage with L. johnsonii, suggesting that the intestinal microorganism might translocate to extraintestinal tumor tissue. Bender et al. also reported that probiotic Lactobacillus reuteri translocated to, colonized, and persisted within melanoma.²⁰ However, the possibility of direct bacterial infection at the tumor site via blood vessels could not be completely dismissed.

Multiple studies have provided evidence indicating that intratumoral bacteria possess diagnostic significance for various forms of cancer and show intricate correlation with patient prognosis.^21,22 Extensive researches have elucidated the influence of intratumoral microbiome composition on patient outcomes,²³ responses to cancer therapies,²⁴ and the promotion of cancer metastasis.²⁵ Within the realm of genitourinary cancers, investigators have reported a noteworthy decrease in the abundance of Lactobacillus within prostate cancer tissues compared to normal prostate tissues.²⁶ Furthermore, previous research has demonstrated a correlation between the scarcity of Lactobacillus in the lower female reproductive tract and the development of cervical cancer.²⁷ These findings suggested that Lactobacillus might function as a bacterium that suppressed tumor progression. However, Fu et al. reported an enrichment of Lactobacillus in breast cancer cells, which were found to inhibit the RhoA-ROCK signaling pathway and induce cytoskeletal remodeling.²⁵ This mechanism enables tumor cells to withstand mechanical stress within blood vessels, thereby facilitating tumor metastasis. Moreover, through experimentation with germ-free and immunodeficient mice, it has been demonstrated that intracellular bacteria within tumors can promote metastasis autonomously, independent of the intestinal flora and immune system.²⁵ In our study, analysis of the 16S rRNA gene sequencing data revealed no notable disparities in community composition at the phylum level between tumor and normal tissue of PTC patients. However, a more detailed examination revealed a significant reduction in the abundance of Lactobacillus in tumors with LNM compared to tumors without LNM. These results suggested that Lactobacillus may play a role in inhibiting the LNM of PTC, thereby acted as a tumor suppressor in this context.

Until now, the effect of intratumoral bacteria on tumor metastasis has been widely discussed but the specific mechanism has not been well-established. L. johnsonii has been identified as a potential beneficial bacterium that may improve memory impairment through the brain-gut axis²⁸ and regulate metabolic-related diseases.²⁹ Wu et al. have reported that L. johnsonii could alleviate colitis by inhibiting M1 macrophage polarization through modulation of the MAPK pathway.³⁰ Furthermore, oral administration of L. johnsonii has been found to enhance levels of tight junction proteins ZO-1 and occludin in colitis.³³ It has been observed that L. johnsonii acts as a repair agent for gut mucosal immunity and barrier in the gastrointestinal tract. In our study, we established an in vitro coculture system with L. johnsonii stimulation on IHH4 cells to investigate its effects on human PTC cells. Our findings indicated that L. johnsonii can suppress the proliferation and migration of PTC cells. Additionally, we conducted a xenograft tumor experiment in nude mice to confirm that L. johnsonii can inhibit the LNM of PTC in vivo. Activation of the Wnt/β-catenin pathway plays a crucial role in tumor growth and metastasis. Research indicates that L. johnsonii inhibits the progression of colorectal cancer by suppressing the β-catenin/c-MYC pathway³². Wnt/β-catenin pathway can regulate the transcription of c-MYC to promote tumor growth and facilitate tumor metastasis by regulating EMT-related genes. EMT was known to enhance invasiveness and metastatic potential in solid tumors.³³ We observed a significant inhibition of the EMT in PTC cells when co-cultured with L. johnsonii. Furthermore, our results showed that after co-cultured with L. johnsonii, there was a decrease in the expression of β-catenin, c-MYC, N-cadherin, and Vimentin, concomitant with a notable increase in E-cadherin levels in IHH4 and TPC1 cells. These findings mark the initial steps in understanding how L. johnsonii influences the LNM in PTC, providing a foundation for further investigation into its mechanisms.

The presence of bacterial communities in the tumor environment, which were also commonly found in the gut microbiome, indicated the potential occurrence of bacterial translocation from the gut to tumors in other locations.³⁴ To explore the potential clinical application of treating PTC, we conducted a gut colonization experiment to examine the effect of L. johnsonii on LNM in PTC. However, the absence of mouse PTC cell lines prevented the establishment of an allograft tumor model, thus presenting a methodological constraint in this study. Interestingly, our experiment involved xenograft tumors in nude mice coincidentally revealed that administering L. johnsonii through gavage significantly increased the presence of intratumoral L. johnsonii and inhibited the LNM of PTC. This finding was significant as previous studies have assumed that Lactobacillus colonization in the intestine exerts anti-tumor effects by promoting T-cell activation.^20,35 However, our results demonstrated that intestinal colonization of L. johnsonii could directly impact tumor cells and inhibit LNM of PTC even in the absence of T-cell involvement. Therefore, the oral consumption of L. johnsonii may hold potential as a treatment modality for PTC.

In conclusion, this study employed 16S rRNA gene sequencing alongside in vitro and in vivo experiments to elucidate the prevalence of L. johnsonii and substantiate its involvement and mechanisms in the LNM of PTC. Our results provided evidence that intratumoral L. johnsonii possesses the ability to impede tumor growth and metastasis in PTC, suggesting that oral administration of L. johnsonii could be a promising therapeutic approach for PTC.

Inevitably, this study has a few limitations. While our sample size provided valuable insights into the involvement of intratumoral bacteria in PTC, the results must be validated by future study with larger sample size. Furthermore, the absence of a control group consisting of individuals with benign nodules may hinder the interpretation of the results. Comparing the microbial disparities in thyroid tissues between patients with benign nodules and those with PTC would further bolster the observations made in this study.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-E-Cadherin	CST	Cat#3195T
anti-Vimentin	CST	Cat#GAPDH
GAPDH	CST	Cat#2118T
anti-N-Cadherin	CST	Cat#13116T
anti-β-catenin	CST	Cat#8480T
anti-c-MYC	CST	Cat#5605T
Bacterial strains
L. johnsonii	This paper	N/A
Biological samples
Human PTC tumor tissues	the First Affiliated Hospital of Nanchang University	N/A
Human fecal samples	the First Affiliated Hospital of Nanchang University	N/A
Chemicals, peptides, and recombinant proteins
Trypsin-EDTA	Thermo	Cat#25200072
DMEM	Gibco	Cat#C11995500BT
FBS	HyClone	Cat#SV30160
penicillin–streptomycin	Gibco	Cat#15140122
MRS(Man Rogosa Sharpe)	Oxoid	Cat#CM1175B
BHI (brain heart infusion)	BD	Cat#237500
luciferin	Meilunbio	Cat#MB1834-2
vancomycin	Sangon Biotech	Cat#A600983-0001
ampicillin	Sangon Biotech	Cat#A100339-0025
neomycin	Sangon Biotech	Cat#A610366-0025
metronidazole	Sigma	Cat#M3761-100G
RIPA buffer	Beyotime	Cat#P0013B
Matrige	Corning	Cat#356234
CCK8	Meilunbio	Cat#CCK8
brucella broth agar	BD	Cat#211088
Critical commercial assays
DNeasy Blood & Tissue Kit	QIAGEN	Cat##69504
HiPure Stool DNA Kit	Magen	Cat#D3141-02
TB Green® Premix Ex Taq™	TAKARA	Cat#RR420A
steriled enzyme-free water	Solarbio	Cat#R1600-500ml
Transwell chambers	Corning	Cat#3422
Cy3-conjugated probe	servicebio	Cat#G3045
BCA Protein Assay Kit	Thermofisher	Cat#
Taq plus MasterMix II	Vazyme	P213-03
Deposited data
16S amplicon sequencing	BioProject: PRJCA022672	https://ngdc.cncb.ac.cn/gsa
Experimental models: Organisms/strains
BABL/C nude	GemPharmatech	Cat#D000521/Nude/Nu
Software and algorithms
GraphPad Prism, version 8	SCR_002798	https://www.graphpad.com/scientific-software/prism/

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Xiang Min ([email protected]).

Materials availability

All requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact ([email protected]).

All reagents including antibodies, bacteria and plasmid may be available on request after completion of a Materials Transfer Agreement.

Data and code availability

16S amplicon sequencing has been deposited at SRA and are publicly available as of the date of publication. Project number and accession links are listed in the key resources table.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice experiments

Mice experiments were carried out in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Nanchang University (CDYFY-IACUC-202308QR020). Female BABL/C nude mice were purchased from Guangzhou GemPharmatech Co.,Ltd (Guangzhou, China).Animals were housed in a specific pathogen-free conditions and fed standard mouse chow.

IHH4-Luc cells were plated for 12 hours followed by medium replacement with penicillin–streptomycin (P/S) free DMEM (10% FBS) and then co-cultured with bacteria (L. johnsonii or E. coli) at a multiplicity of infection (MOI) of 10:1 for another 12 hours under anaerobic conditions at 37°C. Cells were washed with sterile 1×PBS three times and cultured in DMEM (1% P/S, 10% FBS) medium for 8 hours to kill any remaining bacteria. Finally, the cells were collected and injected subcutaneously into BABL/C nude mice (2×10⁶ cells/mice). On day 28, the subcutaneous tumor was surgically resected and the wound was sutured. The metastasis of inguinal lymph nodes was evaluated on day 42 by measuring luciferase fluorescence via In Vivo Imaging System (AniView). Each mouse received an intraperitoneal (i.p.) injection of 200 µL luciferin (15 mg/mL; Meilunbio, #MB1834-2) and the luciferase signal was captured after 10 minutes. Mice were then sacrificed.

For bacterial colonization in gut, mice were given an antibiotic cocktail containing vancomycin (100 mg/L), metronidazole (200 mg/L), ampicillin (200 mg/L) and neomycin (200 mg/L) in the drinking water for 5 days. After 48 hours washout period, mice were gavaged with bacteria (1×10⁹ CFU/mouse) or 1×PBS every other day until the end of the experiment. IHH4-Luc cells were injected subcutaneously into BABL/C nude mice on day 0, and the subcutaneous tumor was surgically resected and the wound was sutured on day 28. On day 42, the progression of the metastatic lymph nodes was evaluated as described above.

Human specimens collection

The fecal samples of PTC patients and PTC paired tumor and normal specimens were collected from the First Affiliated Hospital of Nanchang University. PTC patients with LNM (Metastasis group) or without LNM (Non-metastasis group) were recruited in our study. The exclusion criteria included the use of antibiotics within one month, oral and neck infectious diseases, and other concurrent severe organic diseases. Finally, 55 patients were recruited in our study (Metastatic group, n=29; Non-metastasis group, n=26). Postoperative pathological results showing positive lymph nodes were defined as the presence of LNM.

The protocol of human sample usage and the informed consent were approved by the Ethical Review Board of the First Affiliated Hospital of Nanchang University [(2023)CDYFYYLK(07-008)].

METHOD DETAILS

Cell lines and bacterial strains culture

IHH4, TPC1 and IHH4-Luciferase (Luc) cell lines were cultured in DMEM medium (Gibco, #DMEM) supplemented with 10% fetal bovine serum (HyClone, #SV30160) and 1% penicillin–streptomycin (Gibco, #15140122). The L. johnsonii strains were cultured on Man Rogosa Sharpe (MRS, Oxoid, #CM1175B) plates under anaerobic conditions at 37°C for 48 hours. E. coli was cultured on Brain Heart Infusion (BHI, BD, #237500) plates under aerobic conditions at 37°C.

qPCR quantification

The colonization of L. johnsonii in feces and its translocation in tumor were assessed by qPCR as previously described.²⁵ L. johnsonii DNA was used to plot a standard curve to calculate L. johnsonii DNA concentration in the sample and the steriled enzyme-free water served as negative control (NTC) for the reactions.^25,36 Tumor and feces were weighted followed by total DNA extraction using the DNeasy Blood & Tissue Kit (for tumor, QIAGEN, #69504) or HiPure Stool DNA Kit (for feces, Magen, #D3141-02).

For qPCR quantification, briefly, 10 uL reaction mix containing 3 uL steriled enzyme-free water (Solarbio, #R1600-500ml), 5 uL TB Green® Premix Ex Taq™ (TAKARA, #RR420A), 0.5 uL forward primer (5ʹ- TCGAGCGAGCTTGCCTAGATGA-3ʹ), 0.5 uL reverse primer (5ʹ- TCCGGACAACGCTTGCCACC-3ʹ), and 1 uL sample DNA, was loaded on the Applied Biosystems 7500 Real-time PCR system. Raw cycle threshold (Ct) values were normalized according to a bacterial standard curve produced with L. johnsonii DNA.

Fluorescence in situ hybridization (FISH)

Paraffin sections were deparaffinized and then incubated in lysozyme solution (10 mg/mL) at 37℃. L. johnsonii probes were Cy3-conjugated specific probe (5'-AGCTTCAATCTTCAGGAT-3') and were manufactured by Wuhan servicebio Technology. Slides were incubated with probe diluted in prewarmed hybridization buffer overnight at 40℃ in a humid chamber. Slides were subsequently washed three times with pre-warmed (37℃) washing buffer and Tris buffer, respectively. Tissues were stained by DAPI, and fluorescent signal capture using a confocal microscope (NIKON ECLIPSE CI, Japan).

CCK8 assay

IHH4 and TPC1 cells were stimulated by bacteria as described above. Then cell growth was assessed using the CCK8 assay. Briefly, IHH4 and TPC1 cells were seeded in 96-well plates (2×10³ cells/well). The culture medium was removed the next day and 100 uL fresh medium containing 10 µL CCK8 solution were added to each well. After 1 hour incubation, the absorbance was measured at 450 nm.

Migration and invasion assays

Tumor cells invasion and migration assays were carried out in Transwell chambers (Corning, #3422) with or without Matrigel® Basement Membrane Matrix (Corning, #356234). 200 µL cells in DMEM (FBS-free, 1% P/S) were seeded in the upper chamber (2×10⁵ cells /well), with the bottom chamber filled with 600 μL DMEM (10% FBS, 1% P/S). After 24 hours, cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.2% crystal violet for 30 minutes. Images of invaded or migrated cells were captured from five random fields per condition with a microscopy. The bound crystal violet was eluted by adding 33% acetic acid into each insert and shaking for 10 minutes. The eluent from the lower chamber was transferred to a 96-well clear microplate, and the absorbance value (optical density, OD) at 570 nm was measured using Thermo Scientific Microplate Reader.

Western blot

Western blot was performed as previously described.³⁷ Tumor tissues and cell pellets were lysed using RIPA buffer (Beyotime, #P0013B) and protein concentration was quantified using BCA Protein Assay Kit (Thermofisher, #23227). GAPDH was used as an endogenous control. The following antibodies were used: anti-E-Cadherin (CST, #3195T, 1:1000 dilution), anti-Vimentin (CST, #5741T, 1:1000 dilution), GAPDH (CST, #2118T, 1:1000 dilution), anti-N-Cadherin (CST, #13116T, 1:1000 dilution), anti-β-catenin (CST, #8480T, 1:1000 dilution), anti-c-MYC (CST, #5605T, 1:1000 dilution).

Bacteria culture and identification

Isolation of anaerobic or aerobic bacteria was performed as previously described. 100-200 mg tumor tissues were cut into pieces and homogenized in 1 mL ice-cold BHI under sterile conditions. 1×PBS was used as NTC and went through the same workflow to evaluate the environmental contaminants. For aerobic culture, 200 uL sample homogenate was plated on BHI plates at 37℃ aerobically with 5% CO₂.

For anaerobic culture, 200 uL sample homogenate was transferred to 5 mL BHI medium and cultured under anaerobic conditions for 2 days for bacterial enrichment, then 20uL culture medium were plated on brucella broth agar plates (Bruce, BD, #211088) supplemented with 5% sheep citrated blood. The plates were incubated at 37 ℃ for 5 days in anaerobic conditions.

For identification of bacteria strain, colonies were picked and streaked in designated plate and condition for 1-3 days to get single colony. The single colony was picked to grown in plates and run colony PCR subsequently. Briefly, the 20 uL reaction mix contained 1 uL bacteria DNA, 8 uL steriled enzyme-free water (Solarbio, #R1600-500ml), 10 uL 2X Taq plus MasterMix II (Dye plus) (Vazyme, #P213-03) and 0.5 uL forward primer (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’), 0.5 uL reverse primer (1492R: 5’-GGTTACCTTGTTACGACTT-3’). The reaction was programmed according to the reagent instructions. The PCR product was sent out for sequencing and the sequencing results were aligned to the 16S rRNA sequences database in the NCBI BLAST site.

High throughput 16S rRNA amplicon sequencing and analysis

Genomic DNA was extracted using FastDNA Spin Kit for Soil (MP Biomedicals). In brief, barcoded amplicons from the V3-V4 region were generated using PCR. In the first step, 10 ng genomic DNA was used as template for the first PCR with a total volume of 20 μL using the 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') primers appended with Illumina adaptor sequences. Subsequently, PCR products were purified, checked on a Fragment analyzer and quantified, followed by equimolar multiplexing, and sequencing on an Illumina MiSeq PE300 platform (2×300 bp). Quantitative Insights into Microbial Ecology 2 (QIIME2) software was used for microbial analyses. Reads were imported,quality filtered and demultiplexed with the q2-data2 plugin. The sequences were classified using Greengenes (version 13.8) as a reference 16S rRNA gene database. Principal Coordinate Analysis (PCoA), Linear discriminant analysis effect size (LEfSe) and Significant Species were performed using R (v4.1.1). 16S rDNA data are available from the Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa) under accession number PRJCA022672.

Quantification and statistical analysis

Statistical analysis and data visualization carried out in this study was performed by GraphPad Prism 8. Data were presented as means ± SEM. Differences between two groups were compared using unpaired Student’s t-tests. Comparisons among three or more groups were performed by one-way ANOVA test combined with Tukey’s multiple comparison test. Differences in clinical characteristics of patients were determined using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. All p values were two-tailed, and differences with a p value less than 0.05 were considered significant.

ACKNOWLEDGMENTS

This work was supported by the Science and Technology Plan of Jiangxi Provincial Health Commission (202130202) and the Jiangxi Provincial Natural Science Foundation (20224BAB216022, 20232BAB206020).

AUTHOR CONTRIBUTIONS

X.M. and L.F.: Study conception and design. X.M. and M.X.: Study supervision and funding acquisition. M.X., T.Y., Q.L. and Z.N.: Sample processing, data curation, data analysis, data interpretation and manuscript writing. X.M., Q.L. and T.Y.: Patient recruitment and sample collection. M.X. and L.F.: Manuscript editing and review. All authors reviewed the results and approved the final version of the manuscript.

DECLARATION OF INTERESTS

The authors declare no competing interests.

INCLUSION AND DIVERSITY

We support inclusive, diverse, and equitable conduct of research.

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The influence of Lactobacillus johnsonii on tumor growth and lymph node metastasis in papillary thyroid carcinoma

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