The emergence of drug resistance remains one of the most formidable challenges in the effective treatment of solid tumors.(p1) Despite significant advances in targeted therapies, immunotherapies, and combination therapy, many cancers develop mechanisms of therapeutic evasion, ultimately leading to treatment failure and disease progression.(p1) A growing body of evidence implicates exosomes, a subtype of small extracellular vesicles (EVs) (30–150 nm), as key mediators of this resistance.(p2) These vesicles, secreted by tumor cells and cells of the surrounding microenvironment, shuttle a diverse cargo of proteins, nucleic acids, lipids, and metabolites, facilitating intercellular communication and promoting tumor survival, immune evasion, metabolic adaptation, and therapy resistance.

Traditionally, exosomal studies have relied on bulk analyses that pool vesicles from heterogeneous sources, masking the contribution of region-specific or cell-type-specific vesicle populations.(p3) This overlooks a crucial aspect of tumor biology: spatial heterogeneity. Solid tumors are composed of functionally distinct zones, such as hypoxic cores, invasive edges, nutrient-rich perivascular niches, and fibrotic stromal interfaces.(p4),(p5) Each of these tumor zones presents a unique microenvironment shaped by gradients in oxygen, pH, nutrient availability, immune cell infiltration, and extracellular matrix (ECM) composition.(p6) Recent developments in single-cell sequencing and multi-omics analysis have led to understanding of the spatial distribution of this microenvironment.(p7) These environmental cues not only influence tumor cell behavior but also govern the biogenesis, cargo selection, and functional specialization of the exosomes they release.(p8) Over the past decade, research has increasingly focused on exosomes derived from distinct tumor states, which carry state-specific cargo and possess the ability to reprogram recipient cells by transferring molecular signatures from their cells of origin.(p9) Further, exosomes derived from different spatial regions of a tumor might carry distinct molecular cargo reflective of their zone of origin, thereby exhibiting compositional heterogeneity that mirrors the spatial zonation within the TME. Crucially, this functional heterogeneity of exosomes is not just a passive consequence of spatial diversity; it is also a driver of it. Exosomes act as communication vectors between spatial zones, reinforcing niche-specific identities and enabling coordination across tumor compartments. For instance, exosomes from hypoxic regions might transmit hypoxia-adapted signals to normal tumor cells, prompting them to adopt more resistant phenotypes under stress; those from invasive fronts might precondition stromal or immune cells in adjacent zones to support invasion and immune escape. In this way, exosome-mediated signaling might serve as both a product and a propagator of spatial heterogeneity, helping to maintain the adaptive architecture of the tumor.(p10) This bidirectional relationship, where spatial zonation governs exosome function and exosomes reinforce zonal identity and resistance phenotypes, underscores a new paradigm in tumor biology. It suggests that overcoming drug resistance will require not just an understanding of genetic and cellular heterogeneity, but also an appreciation of exosomal geography, namely, the spatial logic behind vesicle-mediated communication within the tumor landscape. Recognizing and decoding this spatial exosomal communication is critical for the advancement of precision medicine in oncology.(p11) However, most current exosome-based diagnostics and therapeutic strategies overlook the intratumoral spatial variation in vesicle content and function. Integrating the understanding of zonal exosome profiling into clinical workflows has the potential to revolutionize this field, offering more accurate biomarkers for spatially informed liquid biopsies and opening avenues for zone-specific therapeutic interventions, such as inhibitors targeting exosome biogenesis or uptake in resistant niches.

In this review, we present a comprehensive framework for understanding exosomal heterogeneity through the lens of tumor spatial architecture. We explore how the TME shapes exosome function and aids in drug resistance, introduce emerging technologies for spatial EV and exosome profiling, and highlight the clinical significance of mapping functional exosome zonation for overcoming drug resistance. By integrating spatial biology with exosome profiling, this review aims to shift the current paradigm and push the boundaries of precision oncology toward a future where therapies are not only tumor-specific, but also zone-specific, reflecting the dynamic, compartmentalized, and adaptive nature of cancer itself.