<i>LACTOCOCCUS LACTIS </i>SUPPRESSES COLORECTAL TUMORIGENESIS BY RESTORING GUT MICROBIOME COMPOSITION AND GENERATING BENEFICIAL ALPHA- MANNOSIDASE

Background: Through shotgun metagenomic sequencing, we identified the depletion of probiotic species Lactobacillus acidophilus in stools of mice with non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC). Here, we aimed to investigate the prophylactic potential and protective mechanism of L. acidophilus against NAFLD-HCC.

Methods: NAFLD-HCC tumorigenesis models were established in mice with a single injection of diethylnitrosamine (DEN) at day 14 and feeding of high-fat high-cholesterol (HFHC) diet for 8 months, or choline-deficient high-fat diet (CD-HFD) for 6 months. Orthotopic NAFLD-HCC xenografts were established in mice with intrahepatic injection of mouse HCC cell line Hepa 1-6 and feeding of HFHC diet for 4 months. Metabolomic profiling of stool, portal vein serum and liver tissue of mice, and culture medium of L. acidophilus (LA-CM) were evaluated by non-targeted and targeted liquid chromatography-mass spectrometry. Human NAFLD-HCC cell lines (HKCI-2, HKCI-10) and mouse NAFLD-HCC organoids were used for bio-functional assays of L. acidophilus and its metabolite. RNA-sequencing was performed on NAFLD-HCC cells treated with candidate metabolite.

Results: Oral gavage of L. acidophilus suppressed NAFLD-HCC tumorigenesis in mice induced by DEN and HFHC diet, and also in orthotopic mouse xenografts. L. acidophilus was undetectable in liver tissues but was detected in stools of L. acidophilus-treated mice, implicating that the antitumor function of L. acidophilus is attributed to its products. Co-culture with LA-CM significantly reduced viability (P<0.0001) and proliferation (P<0.01) of human NAFLD-HCC cells, and suppressed mouse NAFLD-HCC organoid growth (P<0.0001). The tumor-suppressing effect of L. acidophilus was attributed to its non-protein small molecules with a molecular weight of <3kDa. By metabolomic profiling, valeric acid which is a short-chain fatty acid, was identified as the top differential metabolite in LA-CM, and its upregulation was confirmed in stool and portal vein serum of L. acidophilus-treated mice. Oral gavage of valeric acid significantly suppressed tumor number (P<0.01) and tumor load (P<0.05) in DEN-treated mice fed with HFHC diet, accompanied with improved gut barrier integrity. This was confirmed in DEN-treated mice fed with CD-HFD diet. Valeric acid also reduced viability (P<0.01) and proliferation (P<0.001) of human NAFLD-HCC cells, and inhibited mouse NAFLD-HCC organoid growth (P<0.0001). Mechanistically, valeric acid bound to G protein-coupled receptor (GPR)41 and GPR43 of hepatocytes to induce their expression, which in turn inhibiting the oncogenic Rho-GTPase pathway to ablate NAFLD-HCC development.

Conclusion: L. acidophilus protects against hepatocarcinogenesis by secreting antitumor metabolite valeric acid. Probiotic supplementation is therefore a potential prophylactic of NAFLD-HCC.

Background
Malnutrition amongst under-fives remains common in resource-poor countries and is resistant to current interventions. New opportunities have emerged to target environmental enteric dysfunction, which refers to the abnormal gut structure and function that compromises nutrition and growth in early life. Dietary supplements of pro- or synbiotics may build the resilience of the gut microbiome against environmental factors and boost colonization resistance against ingested pathogens.
Methods
We conducted an open label, randomized, controlled trial to assess whether administering pro/synbiotics to infants in western Kenya improved gut health and reduced systemic inflammation. Newborns less than four days old were enrolled at the Homa Bay County Teaching and Referral Hospital, western Kenya. They were randomly allocated, stratified by HIV exposure, in a 1:1:1:1 ratio to one of 4 study arms to receive either
Labinic synbiotic^[1]; Biofloratech Ltd, Walton-on-Thames, UK
Lab4b synbiotic^[2]; Cultech Ltd., Port Talbot, UK
Lab4b probiotic (as above)
No supplement
Interventions were given daily for ten days and then weekly until six months of age. Biomarkers of systemic inflammation (plasma alpha 1-acid glycoprotein (AGP) concentration, a biomarker of longer-term systemic inflammation; primary outcome), gut health and growth (secondary outcomes) were measured in blood and stool samples collected at 6 weeks, 3, and 6 months.
Results
Between 28 October, 2020 and 13 January, 2022, 600 newborns were enrolled. Urban vs. rural residence, place and mode of delivery, sex, gestational age, birthweight, HIV exposure, antibiotic exposure and socio-economic and hygiene/sanitation variables at recruitment were similar in the four study arms.
Median plasma AGP concentration, increased progressively at 3 and 6 months in the controls. This increase was almost completely abrogated in all the intervention arms (Figure). At 6 months, plasma AGP was raised (>1g/L) in 56/134 (41.8%) infants in the control arm but in <1.5% infants in each of the intervention arms (P<0.001). Gut health biomarkers were significantly higher at 3 and/or 6 months in the control arm than in any intervention arm. Growth hormones were slightly, but significantly, lower at 6 months in the control arm than in any intervention arm (Table).
Conclusion
Pro/synbiotics offer a novel approach to improving gut health and reducing systemic inflammation in young infants in a low-resource setting.


[1] BENEO Orafti Synergy1; 50 oligofructose:50 FOS; 200mg + Lactobacillus acidophilus NCFM, Bifidobacterium infantis Bi-26 and Bifidobacterium bifidum Bb-06; 5 x 10⁹ organisms/day

[2] long-chain fructo-oligosaccharide 150mg + Lactobacillus salivarius CUL61, Lactobacillus paracasei CUL08, Bifidobacterium animalis subspecies lactis CUL34 and Bifidobacterium bifidum CUL20; 10¹⁰ organisms/day

Background & Aims: Lactococcus lactis (L. lactis) was identified to be depleted in the stool of colorectal cancer (CRC) patients in contrast to healthy subjects by fecal shotgun metagenomic sequencing, suggesting its potential protective role in the pathogenesis of CRC and microbiota interaction. Here, we aim to clarify the antitumorigenic role of L. lactis in colorectal tumorigenesis.

Methods: We isolated L. lactis bacterium from stool samples of healthy subject and its complete genome sequence was obtained by PacBio 3^rd generation sequencing. The anti-tumor effects of L. lactis was assessed in patient-derived organoids, and in two murine models of colorectal tumorigenesis (transgenic Apc^min/+ mouse model and AOM-DSS carcinogen-induced CRC mouse model). The effect of L. lactis on the gut microbiome alteration was assessed by shotgun metagenome sequencing. The bio-function of L. lactis conditioned medium (LL-CM) was assessed in CRC cell lines (HCT116 and HT29) and normal colonic epithelial cell (NCM460). Fraction separation of LL-CM with mini-pore filters defines the approximate protein size and the functional component in LL-CM was identified by liquid chromatography-mass spectrometry (LC-MS/MS) analysis.

Results: Oral administration of L. lactis significantly reduced tumor number (P<0.05) and size (P<0.05), along with reduced Ki-67 cells and increased TUNEL-positive cells, compared to E. coli MG1655 and plain culture broth control in AOM/DSS-induced CRC mice. This was confirmed in Apc^min/+ CRC mice. A significant shift of fecal microbial profiling revealed the enrichment of known probiotics, Lactobacillus johnsonii and Limosilactobacillus reuteri, and depletion of pathogenic bacteria, Olsenella uli and Bacteroides eggerthii, in L. lactis treated mice. Co-culturing CRC cells (HCT116 and HT29) with LL-CM decreased cell viability and lowered colony formation, but this was not observed in NCM460 cells. Besides, coincubation of the LL-CM drastically retarded the growth of cultured human-derived CRC organoids (P<0.05). Digesting LL-CM with protease K or heating LL-CM to inactivate protein could abolish the tumor-suppressive effect of LL-CM, indicating the functional fraction in LL-CM should be protein. With further investigation into the fractions of LL-CM, we found that the fraction size >100kDa was identified to exert an anti-CRC effect. Alpha-mannosidase was identified as the critical protein produced by L. lactis by mass spectrometry screening. The tumor suppressive effects of alpha-mannosidase was confirmed in CRC cell lines.

Conclusion: L. lactis protects against colorectal tumorigenesis in mice through restoring the healthy microbial composition. The anti-tumor effect of L. lactis is facilitated by its generated beneficial protein alpha-mannosidase. L. lactis might serve as a novel prophylactic for CRC prevention.