Characterizing the Multiple Myeloma Bone Marrow Microenvironment at the Single-Cell Level in a Preclinical Model

Introduction: Multiple myeloma (MM) is a hematological malignancy characterized by the proliferation of plasma cells within the bone marrow (BM), facilitated by a supportive microenvironment that promotes tumor growth and survival. The immune dysregulation spans both myeloid and lymphoid compartments, further complicating treatment. Immunotherapy, which aims to stimulate the immune system to attack tumor cells, has shown promising results but MM remains incurable due to persistent disease relapse. A deeper understanding of the immune cells' heterogeneity at the cellular level and their dynamic interactions in the microenvironment is urgently needed. Here, aim to provide a comprehensive characterization of the BM niche, focusing on macrophages, CD4+ T cells, and CD8+ T cells, by performing scRNA-sequencing and potentially identifying novel targets for therapeutic intervention.

Methods: We used immunocompetent BALB/cByJ mice inoculated with the murine MM cell line MOPC315.BM, which faithfully replicates the major characteristics for human disease. BM cells were obtained from flush of femurs upon the appearance of disease symptoms given by hind limb paralysis. We performed a plate-based scRNA-seq (SORT-Seq) on immune populations from the BM of control and MOPC315.BM-bearing mice to investigate molecular determinants of MM development within the BM niche. We analyzed sorted macrophages (n=1853 cells, 3 control plates and 3 MOPC315.BM-bearing mice plates), CD8+ T cells (n=1399 cells, 3 control plates and 3 MOPC315.BM-bearing mice plates), and CD4+ T cells (n=1432 cells, 3 control plates and 3 MOPC315.BM-bearing mice plates), identifying significant differences in subpopulations numbers and gene expression. To functionally validate potential new targets in vivo, we generated nanoparticles containing siRNA molecules against our gene of interest and evaluated the microenvironment modulation by flow cytometry.

Results: We identified eight distinct macrophage subpopulations, including two novel pro-tumoral subpopulations, Vcan+ myeloma-associated macrophages (MAMs) and Spon1+ osteomacs, specific to MOPC315.BM-bearing mice. Notably, macrophages from MOPC315.BM-bearing mice exhibited increased expression of Vcan, an extracellular matrix proteoglycan, suggesting a pro-tumoral role. Immunofluorescence confirmed higher Vcan expression at the protein level in MOPC315.BM-bearing mice compared with controls and showed high levels of Vcan in newly diagnosed MM patients. CD8+ T cells were categorized into six subpopulations. Significant differences were found in cell numbers in one cluster, particularly in immunosuppressive Pdcd1+ cells, which were present in higher number in MOPC315.BM-bearing mice compared to controls, suggesting their role in the immunosuppressive environment of MM. CD4+ T cells were divided into five distinct clusters. Differences in cell numbers were observed in clusters of migrating cells, central memory/naive cells, and cytotoxic cells. However, gene expression profiles revealed minimal differences between MOPC315.BM-bearing mice and controls.

Lastly, we performed in vivo experiments using anti-Vcan molecules, which induced changes in immune cells including macrophages and the T-cell compartment, confirming Vcan importance in the MM microenvironment and its validation as a potential therapeutic target. Interestingly, modulation of the levels of Vcan led to a decrease on the expression of the immune checkpoint molecules PD-L1 in macrophages and PD-1 in both CD8+ and CD4+ T cells.

Conclusions: Vcan emerges as a promising target for MM therapy due to its differential expression patterns and in vivo modulated impact indirectly on both PD-L1-expressing macrophages and PD-1-expressing T cells. Overall, these findings highlight the complex interplay between MM cells and their microenvironment, underscoring the potential of targeting specific immune cell populations to improve therapeutic outcomes and pave the way for more effective, innovative and personalized treatments.

Queirós:Janssen: Research Funding. Florindo:CURAPATH: Consultancy. João:Amgen: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Lilly: Other: Lecture fees; GSK: Other: Lecture fees; Amgen: Other: Lecture fees; Pfizer: Other: Lecture fees; BMS: Other: Lecture fees.