ASH 2017 | Pre-clinical studies to examine immune suppression, surveillance and homing mechanisms in MM

On Saturday 9 December 2017, an oral abstract session was held entitled: Session 651. Myeloma: Biology and Pathophysiology, excluding Therapy: New Insights into the Role of the Immune Microenvironment of Multiple Myeloma. The session was moderated by Qing Yi, from the Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, US and Karin Vanderkerken from the Free University of Brussels, Belgium, and the last three talks from this session are summarized in this article. This is based on data presented at the live session and therefore may supersede information in the pre-published ASH Abstracts. 

Abstract 124: Mesenchymal Stromal Cell Sialylation Enhances Immune Suppression in Multiple Myeloma

Kevin Lynch, from the Regenerative Medicine Institute, National University of Ireland, Galway, Ireland presented the fourth talk in this session with data from the study looking at how sialylation of bone marrow mesenchymal stromal cells (BM-MSC) can influence effector T cell function. The composition of the BM microenvironment is largely comprised of BM-MSCs, endothelial cells, immune cells, and extracellular matrix (ECM). Signals within the tumor microenvironment (TME) influence the phenotype of cells, which can result in drug resistance. Aberrant sialylation has been linked with poor prognosis but the immunological consequences of this are unknown. This study aimed to investigate whether the regulation of BM-MSC sialylation alters their ability to inhibit effector T-cell function in an inflammatory microenvironment.

 Key findings:

•  BM-MSC were isolated from BALB/c mice and extensively characterized in vitro
• iMSC displayed significant phenotypical changes, following treatment with TNF-α/IL-1β and co-culture in mixed lymphocyte reactions (MLRs) for 96 hours:
• Significant increase in CD73 and CD44 levels
• Significant reduction in SCA-1 and CD105 levels
• No changes in cell viability or proliferation were noted after pre-treatment
• TNFα treatment led to a decrease in proliferation in T cells and lymphocytes
• Increased levels of both α2-3 and α2-6 linked sialic acid in iMSC, when compared to BM-MSC, when pre-treated with a sialyltransferase inhibitor (3Fax-Neu5Ac) for 72 hours prior to TNF-α and IL-1β stimulation
• iMSC had an enhanced ability to inhibit lymphocyte proliferation, compared to BM-MSC alone
• iMSC significantly inhibited the proliferation of hyper-stimulated CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes
• Following sialyltransferase inhibition, BM-MSC and iMSC displayed no significant changes in phenotype, viability, proliferation and cell size, although iMSC lost the ability to suppress both CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes, which led to a significant restoration of lymphocyte proliferation
• The absence of CD11c+ and CD11b+ innate immune cells resulted in an enhancement of the restoration of lymphocyte proliferation
• Cell surface sialic acid expression correlated with both CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocyte suppression
• No changes in soluble mediators of BM-MSC immunomodulation (e.g. NO, IL-10 and PGE-2) were observed

In conclusion, it was stated that inflammation in the MM TME is responsible for BM-MSC sialylation and that this modification enhances their ability to suppress activated adaptive and innate immune effectors. This, therefore, identifies a possible means of modifying the TME in MM. The functional importance of the BM stroma and its interaction with MM and immune cells is likely to lead to the identification of sialylation as a new molecular target that warrants further study.

Abstract 125: Apoptotic Osteocytes Promote Myeloma Dissemination to New Bone Sites Via Suppression of Immune Surveillance in Bone Marrow

Xiaoxiao Hao from the Department of Pathology, University of Alabama, Birmingham, AL, US presented data describing mechanisms of suppression of immune surveillance in the bone marrow. In this study, the potential involvement of osteocyte apoptosis at distant bone sites to MM metastasis was investigated. Xiaoxiao Hao and her research group developed a unique syngenic animal model of murine MM in which osteocyte apoptosis was induced through targeted expression of human diphtheria toxin receptor (DTR) in osteocytes and subsequent injection of diphtheria toxin (DT). Initial experiments showed that 5TGM1 murine MM cells home to bone significantly faster and grow larger tumors in the bone of osteocyte-ablated (DTR+DT) mice compared to control (DTR+PBS) mice, suggesting the contribution of osteocyte apoptosis in new bone sites for MM spread and progression in those areas.

Key Methods:

• Male and female 8-week-old syngenic DTR mice were separated into two groups (n = 12 per group):
  • intraperitoneal injection with DT (12.5 µg/kg) resulting in osteocyte-ablated (DTR+DT) mice
  • intraperitoneal injection with PBS for control mice
• Injection of 2×10⁶ luciferase-expressing 5TGM1 MM cells 7 days later
• Harvest of MM, Bone marrow (BM), blood and spleen cells (n =6 from each group) and bones (tibia and femurs from the other 6 mice from each group) 4 days later for staining and analysis by flow cytometry

 Key Findings:

• Apoptotic osteocytes create an immunosuppressive microenvironment in BM
  • Significant increase in MDSC stimulating cytokines (IL-6, VEGF, GM-CSF and IL-10) in BM of DTR+DT mice
  • Significant increase in immunosuppressive cells in BM of DTR+DT mice (BM of DTR+DT mice vs BM of DTR+PBS mice)
    • MDSCs (38.8% vs1%, P <0.05)
    • Tregs (13.7% vs3%, P <0.05)
    • Bregs (47.0% vs0%, P <0.05)
  • Apoptotic osteocytes reduce the numbers of NK cells and pDC in BM compared to controls
    • Significant reduction in NK cells in BM of DTR+DT mice (0.93% vs53%, P <0.05)
    • Significant reduction in pDC cells in BM of DTR+DT mice (0.66% vs46%, P <0.05)
  • Apoptotic osteocytes inhibit the tumor killing function of T cells and prevent MM cells from apoptosis as evidenced by changes in MM cells of DTR+DT mice in comparison to control mice:
    • Significant reduction in granzyme B, IFNγ, and perforin
    • Significant increase in BCL2 and Ki67, indicative of higher rate in cell proliferation in DTR+DT mice
    • Significant reduction in the % of cleaved caspase 3
  • Tumor infiltration of total T cells and cytotoxic T cells is significantly decreased in osteocyte apoptotic mice

Xiaoxiao Hao concluded that apoptotic osteocytes in distant bone sites create an immunosuppressive bone microenvironment, compromising immune reactivity and response to MM metastasis, which promotes MM dissemination and progression. These findings provide novel insights into the mechanisms of MM progression, and could potentially inform prognosis in patients with MM.

Abstract 126: Runx2 Deficiency in Committed Osteoblasts Promotes Myeloma Cell Homing to and Progression in New Bone Sites

 The final talk in this series was given by Xiaoxuan Xu, from the Department of Hematology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China and was entitled ‘Runx2 Deficiency in Committed Osteoblasts Promotes Myeloma Cell Homing to and Progression in New Bone Sites’. It aimed to elucidate the effect of inhibition of the critical Runt-related transcription factor 2 (Runx2) in osteoblast (OB) progenitors and committed OBs at distant bone sites on MM dissemination and progression to these sites.

Key findings:

• Generation of a syngeneic mouse model of MM in which Runx2 was specifically deleted in the committed OBs of C57BL6/KaLwRij mice (OB^-Runx2^-/^- mice)
• Bone-homing and growth of luciferase-expressing 5TGM1 (5TGM1-Luc) MM cells was tested by tail vein injection in 5-week-old:
  • OB-Runx2+/+ mice (controls, n=7)
  • OB-Runx2-/- mice (n=7)
• Significant enhanced bone homing of MM cells and increased tumor growth in bone in OB-Runx2-/- mice, confirmed by weekly luciferin imaging and ELISA for serum level of IgG2bк
• Increased levels of pro-metastatic cytokines in the BM supernatants of OB-Runx2-/- mice including: EGF, GM-CSF, M-CSF, ICAM-1, IGF-1, MMP-3, OPN, SDF-1, TGF- β, RANKL, VCAM-1, and IL-10
• Significant increase in immune inhibitory cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells, in the BM of OB-Runx2^-/^- mice
• Significant increase in proliferating and non-apoptotic MM cells in the BM of OB-Runx2^-/^- mice
  • Increase in Ki-67, a marker of cell proliferation
  • Increase in Bcl-2, an anti-apoptotic protein
• Runx2 deficiency in committed OBs resulted in a decreased immune response to MM cells
  • Significant decrease in CD8⁺ T cells in OB-Runx2^-/^- mice vs control mice (21.4% vs 32.6%, n =3, P <0.05)
  • Significant decrease in T cell activation in OB-Runx2^-/^- mice vs control mice
  • Higher expression of CD8⁺ T cell inhibitory molecules (Arginase 1, iNOS, IL-10) in MDSCs in OB-Runx2^-/^- mice
  • Decreased production of tumor-killing factors (Perforin, Granzyme B, and IFN-γ) in T cells in OB-Runx2^-/^- mice

It was concluded that Runx2 deficiency in committed OBs induces an inflammatory and immunosuppressive pre-condition in the BM microenvironment, which attracts MM cells and allows their invasion into the bone tissue. This study provides insight into the mechanism of MM dissemination and progression, and could help determine the prognosis of patients with MM.