Oncopediatric Revolution: Advancing Therapeutic Insights with Fusion-Negative Rhabdomyosarcoma 3D Organoids

Beyond Traditional Models: The Challenge of Fusion-Negative Rhabdomyosarcoma

Pediatric soft-tissue sarcomas, specifically rhabdomyosarcomas (RMS), have long resisted therapeutic advances. Among these, fusion-negative RMS (FNRMS) presents the most formidable challenge due to its biological complexity and heterogeneity. This subgroup, defined by the absence of PAX3/7-FOXO1 translocations, lacks the defining genetic signature of fusion-positive RMS (FPRMS). Consequently, the classification is based on exclusion, leading to significant variability in treatment outcomes.

Although initial localized cases of FNRMS show promise with survival rates reaching 60–80%, these numbers plummet dramatically for relapsed or metastatic cases, hovering around a grim 20%. This stark disparity underscores an unmet need for innovative therapeutic strategies. The crux of the issue lies in the lack of reliable preclinical models. While protocols for tumor-derived organoids, or tumoroids, have gained traction, FNRMS organoids have historically failed to capture the genetic, epigenetic, and molecular intricacies of the original tumors.

A recent study has broken this impasse by introducing next-generation FNRMS-derived organoids. These models, cultivated in an optimized three-dimensional (3D) environment, promise to replicate the tumor’s microenvironment with unparalleled precision. Their ability to persist and expand over months positions them as invaluable tools for preclinical research.

Constructing Tumoroids: Bridging Complexity and Precision

The development of the next-generation FNRMS organoids began with a rigorous exploration of the signaling cascades that sustain tumor cell growth. Researchers formulated a novel M3 culture medium tailored to support the establishment of 3D organoids directly from tumor biopsies. These organoids, derived from both adult and pediatric samples, boast a 100% derivation efficiency, a stark contrast to the sub-20% success rate of previous methods.

What distinguishes these organoids is their fidelity in preserving the histological and molecular features of the original tumors. Unlike their two-dimensional (2D) counterparts, which exhibit artificial enrichment in cell adhesion markers, the organoids retain a functional hierarchy of tumor cell states. Transcriptomic analysis confirms that these organoids closely cluster with their parental tumors, preserving not only genetic profiles but also the nuanced epigenetic and methylation landscapes critical for understanding tumor behavior.

This breakthrough is more than a technical achievement; it is a paradigm shift. The ability to sustain long-term 3D cultures opens avenues for iterative drug testing and the exploration of tumor evolution under therapeutic pressure. This capability is particularly relevant for FNRMS, where intra- and inter-tumor heterogeneity plays a critical role in treatment resistance.

Unlocking Intra-Tumor Heterogeneity: A Deep Dive

Treatment resistance in FNRMS is often fueled by its complex intra-tumoral hierarchy. Using single-cell RNA sequencing, researchers identified six distinct cellular clusters within the organoids, reflecting the tumor’s hierarchical organization. These clusters trace a developmental trajectory from quiescent satellite-like cells to proliferative myoblast-like cells, a sequence reminiscent of normal myogenesis.

Key markers such as PAX7, MYOD1, and CRYAB reveal a spectrum of differentiation states, while others, like MEOX2, underscore the mesodermal origins of FNRMS. Importantly, these findings validate the organoids as faithful representations of their tumors of origin, maintaining the dynamic interplay of quiescent and proliferative states.

The implications are profound. By accurately modeling this hierarchy, researchers can investigate how different cell populations respond to treatment. For instance, the study demonstrated that organoids reproduce patient-specific resistance patterns to vincristine, a frontline chemotherapeutic agent. Moreover, the 3D cultures enable researchers to mimic post-treatment relapse scenarios, providing a powerful tool to anticipate and counteract resistance mechanisms.

Targeting Apoptosis: A New Hope in Drug Discovery

FNRMS tumors are notorious for their evasion of apoptosis, the programmed cell death essential for eliminating cancerous cells. Transcriptomic analysis of the organoids pinpointed Survivin, encoded by the BIRC5 gene, as a central player in this resistance. Survivin acts as a downstream inhibitor of apoptosis, preventing the activation of cell death cascades.

To exploit this vulnerability, researchers tested YM-155, a small-molecule inhibitor of Survivin expression. The results were compelling: YM-155 induced significant cell death in FNRMS organoids at nanomolar concentrations. However, a subset of cells remained viable, underscoring the need for combination therapies to address the tumor’s heterogeneous cell states.

Building on this insight, the team identified VDAC2, a gene expressed in the quiescent satellite-like population, as an additional target. Combining YM-155 with Erastin, a VDAC2 inhibitor, produced a synergistic effect, eradicating both proliferative and quiescent cell populations. This dual-target strategy prevented regrowth in long-term cultures, demonstrating the potential of organoids to refine combination therapies.

Drug Screening at Scale: The Organoid Advantage

The robustness and scalability of these organoids position them as ideal candidates for high-throughput drug screening. Using image-based high-content assays, researchers screened a library of apoptosis-inducing compounds alongside conventional chemotherapeutics. The organoids not only confirmed known vulnerabilities, such as sensitivity to YM-155, but also revealed novel resistance mechanisms.

Unlike traditional 2D models, which often overestimate drug efficacy, the organoids provide a more accurate reflection of clinical outcomes. For instance, the weaker response to YM-155 observed in 3D cultures aligns with clinical data, highlighting the organoids’ translational relevance. Moreover, their stability and expandability enable longitudinal studies, making them indispensable for modeling chronic drug exposure and resistance development.

Toward Personalized Medicine: A Transformative Tool

The creation of FNRMS-derived organoids marks a turning point in pediatric oncology. By faithfully replicating the genetic, epigenetic, and molecular landscapes of their tumors of origin, these models offer a new lens to study this elusive disease. Their ability to sustain long-term cultures and preserve tumor heterogeneity makes them uniquely suited for personalized medicine.

However, challenges remain. Expanding the biobank of FNRMS organoids to capture the full spectrum of tumor heterogeneity is a priority. Integrating stromal and immune components to recreate the tumor microenvironment will further enhance their predictive power. Despite these hurdles, the potential of these organoids to accelerate drug discovery and optimize therapeutic strategies is undeniable.

As the field advances, FNRMS-derived organoids may bridge the gap between bench and bedside, offering renewed hope for patients with this devastating disease. Their development represents not just a technical achievement but a beacon of possibility in the fight against pediatric cancers.

Study DOI: https://doi.org/10.1016/j.xcrm.2023.101339

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES