Design on What Was Actually Done

 Quick Analysis Plan Copied

 Long Analysis Plan

Experimental Design: FTIN–AHR–TERT Axis in SNU-C4 Xenografts

The experiment is a 2×2 factorial design: bacterial strain (WT Fn vs ΔtnaA) × treatment (PAS-B inhibitor vs vehicle), yielding 4 groups per bacterial condition (WT+vehicle, WT+inhibitor, ΔtnaA+vehicle, ΔtnaA+inhibitor) plus a no-bacteria vehicle baseline (n=5 groups total). SNU-C4 is selected because it is AHR-responsive to FTIN (unlike HCT116 or HT-29) and shows >3× TERT mRNA upregulation with FTIN treatment FTIN-AHR-TERT axis paper.

Sample size: n=8 mice per group (40 total) provides ~80% power at α=0.05 to detect a 1.5-fold change in tumor volume given the reported n=5 per group in the original Fn xenograft experiment Xenograft n=5 per group. Increasing to n=8 accounts for xenograft dropout and enables mixed-model repeated-measures analysis of tumor growth curves.

Bioinformatics Analysis Pipeline

RNA-seq processing: FastQC → Trimmomatic v0.36 → STAR v2.5.2b → featureCounts → DESeq2 for differential expression (as used in the source study RNA-seq pipeline tools). Primary endpoint: TERT log2 fold-change and AHR pathway gene set enrichment (CYP1A1, CYP1B1, NQO1, AHRR) using fgsea/GSEA against Hallmark_Aryl_Hydrocarbon_Receptor_Signaling.

Key contrast pairs: (1) WT+vehicle vs ΔtnaA+vehicle → isolate FTIN-dependent transcription; (2) WT+inhibitor vs WT+vehicle → PAS-B inhibitor effect on AHR/TERT signature; (3) Interaction term (strain × treatment) in a DESeq2 LRT model tests whether the inhibitor specifically blocks FTIN-driven transcription.

TRAP assay analysis: Telomerase activity normalized to protein input; comparison across groups by one-way ANOVA with Tukey post-hoc; primary prediction: WT+vehicle > WT+inhibitor ≈ ΔtnaA groups.

LC-MS FTIN quantification: Targeted MRM on triple-quadrupole; internal standard isotope-labeled FTIN; report nmol/g tissue; LOD established from blank tumor homogenates.

Falsification Criteria

• ΔtnaA null: If ΔtnaA tumors show equivalent FTIN levels, TERT expression, telomerase activity, and tumor growth to WT+vehicle, the FTIN-specific mechanism is falsified (tnaA pleiotropy confound must be excluded by complemented strain rescue ΔtnaA null phenotype).
• Inhibitor occupancy-without-transcription: If PAS-B inhibitor achieves AHR binding occupancy (demonstrated by PAL competition assay) yet fails to suppress TERT/AHR-responsive gene expression in RNA-seq, the transcriptional mechanism is falsified—suggesting either insufficient intratumoral drug concentration or non-PAS-B AHR activation pathways.

Critical Limitations and Blindspots

The tnaA deletion has pleiotropic effects on L-tryptophan metabolism beyond FTIN, potentially confounding phenotypic differences tnaA pleiotropy limitation. A complemented strain (ΔtnaA/ptnaA) is essential as a third bacterial condition to attribute effects specifically to FTIN rather than other tryptophan metabolites. Additionally, FTIN is produced by 9 bacteria across 7 species in 5 genera (notably Porphyromonas spp.), so other intratumoral microbes may contribute FTIN independently of Fn FTIN produced by other bacteria. 16S rRNA sequencing of excised tumors (DADA2 pipeline) is recommended to control for endogenous microbiome composition.