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bioRxiv · 10.64898/2026.07.09.737611

Spatial mitochondrial lineage tracing uncovers a premetastatic niche and microenvironment programmed fate switching in osteosarcoma

Xue, Y.·Su, Z.·Su, J.·Chu, A. S.·Wan, L.·Chen, M.·Cheung, J. P. Y.·Cheung, K. S. C.·Ho, J. W. K.
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Abstract

Osteosarcoma progression involves complex spatiotemporal dynamics, yet the lineage relationships underlying tumor cell fate decisions remain poorly understood. Here we integrate single cell and spatial transcriptomics and mitochondrial variants-based lineage tracing to map the evolution of a late-stage, non-metastatic osteosarcoma. Single cell pseudotemporal reconstruction of malignant cells reveals a stepwise cascade of transcriptional programs that instructs bifurcation of COL3A1 progenitors into ALPL osteoblastic and THY1 mesenchymal lineages. Critically, we demonstrate that the metastatic THY1 mesenchymal cell fate is not governed by cell-autonomous transcriptional programs alone; this pro-tumorigenic state requires strict spatial licensing via direct co-localization with PLVAP dysfunctional endothelia to form a pro-metastatic niche (THY1_Endo) enriched for PTN/NOTCH signaling. To distinguish clonal ancestry from microenvironmental plasticity, we leveraged spatially resolved somatic mitochondrial variants as endogenous lineage tracers. A spatial autocorrelation filter confirmed significant clonal spatial coherence (Z = 10.07, p {approx} 4x10-{superscript 2}). Phylogenetic reconstruction using a Wright-Fisher drift model coupled with a hidden Markov tree (WF-HMT) revealed a striking directional transition rate from COL3A1 to THY1 tumor cells (724.08) versus the reverse (4.09)--a [~]177-fold asymmetry--whereas the COL3A1 to ALPL transition rate was lower (0.47). Notably, drift-agnostic coarse VAF clustering failed to resolve ALPL versus THY1 bifurcation, underscoring that explicit modeling of stochastic mitochondrial drift is essential for uncovering directional fate locking. We propose a "time-space-lineage" relay model where sequential microenvironmental signals program tumor cell fate and pre-metastatic niche assembly through irreversible clonal commitment. This multi-dimensional framework bridges single-cell states, spatial organization, temporal dynamics, and clonal ancestry --underscoring the power of somatic mitochondrial variants to resolve clonal history and intercept cancer evolution.

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Xue, Y., Su, Z., Su, J., Chu, A. S., Wan, L., Chen, M., Cheung, J. P. Y., Cheung, K. S. C., Ho, J. W. K.. 2026-07-11. Spatial mitochondrial lineage tracing uncovers a premetastatic niche and microenvironment programmed fate switching in osteosarcoma. https://doi.org/10.64898/2026.07.09.737611

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