Modulating gut microbiota to enhance anti-PD-1/PD-L1 immunotherapy in hepatocellular carcinoma: the role of Lactobacillus kefiranofaciens and SOCS3 regulation.

Lactobacillus kefiranofaciens (LK) was successfully isolated from kefir grains and identified through genomic sequencing.

• LK was isolated specifically from kefir grains as the source material.
• Identification was confirmed via genomic sequencing methodology.
• The organism was then used in subsequent in vitro and in vivo experimental models.

LK significantly inhibited HCC cell growth, migration, and invasion while promoting apoptosis in vitro.

• Experiments were conducted using Hepa1-6 hepatocellular carcinoma cells.
• Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay.
• Proliferation was measured using 5-Ethynyl-2'-deoxyuridine (EdU) staining and colony formation assays.
• Migration and invasion were evaluated using Transwell assays.
• Apoptosis was measured using apoptosis detection assays.

LK treatment reduced tumor size and improved immune cell infiltration in vivo in HCC mouse models.

• Both subcutaneous and orthotopic HCC mouse models were used.
• LK treatment led to measurable reductions in tumor size.
• Improved infiltration was observed particularly for T cells and NK cells.
• These findings were observed across two distinct in vivo model systems.

Transcriptomic analysis revealed that LK upregulates SOCS3 expression in HCC.

• Both single-cell RNA sequencing (scRNA-seq) and Bulk RNA-seq analyses were performed.
• SOCS3 (Suppressor of cytokine signaling 3) was identified as a key transcriptional target upregulated by LK.
• SOCS3 expression was further validated by manipulation via lentiviral transfection.
• Lentiviral transfection experiments confirmed SOCS3's role in immunotherapy enhancement.

LK suppressed the JAK-STAT signaling pathway in HCC cells.

• Transcriptomic analysis identified JAK-STAT signaling pathway suppression as a key mechanism of LK action.
• Suppression of JAK-STAT signaling was associated with upregulation of SOCS3.
• JAK-STAT pathway suppression was identified through scRNA-seq and Bulk RNA-seq analyses.
• This pathway modulation was identified as a key signaling event linking LK treatment to immune changes.

LK treatment reduced PD-L1 expression, enhancing T cell-mediated immune responses.

• PD-L1 (programmed cell death-ligand 1) expression was reduced following LK treatment.
• Reduction in PD-L1 expression was associated with enhanced T cell-mediated immune responses.
• This mechanism is consistent with improved responsiveness to anti-PD-1/PD-L1 immunotherapy.
• PD-L1 downregulation was linked mechanistically to SOCS3 upregulation and JAK-STAT pathway suppression.

SOCS3 manipulation via lentiviral transfection validated its role as a critical mediator of LK-enhanced immunotherapy response.

• Lentiviral transfection was used to experimentally manipulate SOCS3 expression levels.
• This approach was used to validate the functional role of SOCS3 in the observed immunotherapy enhancement.
• Results confirmed SOCS3 as a key therapeutic target in the LK-mediated mechanism.
• SOCS3 modulation was shown to affect both JAK-STAT signaling and PD-L1 expression.

Gut microbiota modulation through LK represents a novel approach to enhancing anti-PD-1/PD-L1 immunotherapy efficacy in HCC.

• The study positions LK-based gut microbiota modulation as a strategy to overcome limited immunotherapy efficacy in HCC.
• The molecular mechanism involves the SOCS3-JAK-STAT-PD-L1 axis.
• Authors describe these findings as providing 'a new therapeutic approach for improving immunotherapy outcomes in HCC.'
• The study frames gut probiotics as modulators of the immune microenvironment to enhance anti-PD-1/PD-L1 responses in liver cancer.