Introduction:
Oncofetal gene reactivation is a hallmark of cancer plasticity, but its spatial dynamics and integration with tumor regulatory circuits remain unclear in colorectal cancer (CRC). We investigated how a fetal-like state, defined by an oncofetal signature (OnFS), contributes to intratumoral heterogeneity and how it is shaped by silencing AP-1 transcription factors (FOS and JUND), using single-cell and spatial transcriptomics to map its organization within the tumor microenvironment (TME).

Methods:
Single-cell RNA sequencing and spatial transcriptomics were applied to CRC patient-derived organoids (PDOs) and matched tumor biopsies, including shFOS and shJUND lines. Analyses included UMAP, Louvain clustering, SingleR-based cell annotation, differential expression, pathway enrichment (ssGSEA), and trajectory inference (PAGA and Monocle3). Spatial co-localization assessed OnFS-high enrichment in TME niches (e.g., invasive fronts, hypoxic cores, and immune-excluded regions). OnFS activation was quantified with AddModuleScore and its association with EMT, stemness, and AP-1 disruption evaluated.

Expected Outcomes:
We anticipate a gradient of OnFS activation across PDOs and tumor sections, enriched at invasion zones and stromal interaction sites. OnFS-high cells are expected to act as hubs in phenotypic trajectories and localize near immunosuppressive and hypoxic niches. Co-expression analysis should reveal modules linked to EMT, hypoxia, and therapy resistance, with strong overlap between OnFS-high and AP-1–silenced profiles, suggesting convergent spatial mechanisms.

Discussion:
Fetal-like reprogramming in CRC emerges as a spatially organized and dynamic driver of tumor heterogeneity, functionally tied to AP-1 regulation. Spatial mapping shows OnFS-driven plasticity concentrates in biologically active niches, providing potential biomarkers and therapeutic targets to disrupt tumor adaptation and resistance.