Thesis Caroline Hochheuser

The bone marrow (BM) plays a vital role in both blood cell production and cancer metastasis, making it a challenging site to treat. In neuroblastoma (NB) – the most common extracranial solid tumor in children – disseminated tumor cells (DTCs) persisting in the BM drive relapse and contribute to the poor 50% survival rate seen in high-risk patients. In some patients, current treatments, including anti-GD2 immunotherapy, fail to fully eliminate these cells. This thesis aimed to improve targeting of BM metastatic disease by investigating the DTCs themselves, their supportive mesenchymal stromal cell (MSC) environment, and the broader impact of metastases on the hematopoietic compartment. A single-cell RNA sequencing based comparison of treatment-naïve matched tumor and BM samples in Chapter 2 uncovered differences in the copy number variation profile between PT cells and BM-DTCs, indicating genetic diversification between primary and metastatic cells, with potential implications for staging and risk assessment. We characterized DTCs predicted to be particularly challenging to target with chemo and immunotherapy, based on cell cycle status and surface expression of immunotherapy target GD2. These findings offer new avenues for developing therapeutic strategies to target all subpopulations within the highly complex metastatic site. Chapters 3-5 examine the role of MSCs in NB metastasis. In metastatic BM, the MSC compartment was found to be altered in both composition and function, with increased cell numbers, enhanced osteoblastic differentiation potential, and the emergence of a distinct CD146posCD271neg subtype. In vitro co-cultures of MSCs with adrenergic (ADRN) or mesenchymal (MES) NB tumoroids demonstrated increased proliferation of ADRN tumoroids via direct contact, and enhanced survival of both ADRN and MES tumoroids following chemotherapy. CRISPR-Cas9-mediated gene knockdown revealed that CD146 was not implicated in these pro-tumorigenic effects, while imaging of BM trephine biopsies (n=6) demonstrated a localization pattern suggestive of alternative tumor-supportive roles. The precise mechanisms and effects will be explored in a follow-up study. Chapter 6 examines hematopoietic stem and progenitor cell (HSPC) mobilization in pediatric autologous stem cell transplantation, finding that ~14% of patients had insufficient HSPC collection. BM metastasis at diagnosis and at mobilization start were key predictors, and patients with NB were 2.5 times more likely to be poor mobilizers than patients with other pediatric malignancies. Understanding the mechanisms by which NB DTCs disrupt HSPC mobilization could improve transplantation success and prevent treatment delays. Taken together, the work in this thesis advances our understanding of BM metastases in NB by highlighting heterogeneity within the DTC population, the pro-tumorigenic role of MSCs, and the impact of metastases on normal BM function such as HSPC mobilization. Developing novel treatment strategies that complement current treatments will require addressing several key knowledge gaps, including the molecular mechanisms of communication between DTCs and niche cells, the role of immunosuppression in the BM, the localization of DTCs within sub-niches, and the resulting availability of cellular interaction partners. A multi-layered approach targeting all DTC subpopulations and their BM interactions early could be most promising for preventing relapse and driving survival rates toward 100%.