E De Bruyne

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells

Neighboring adipocytes participate in the bone marrow microenvironment of multiple myeloma cells

Multiple myeloma induces the immunosuppressive capacity of distinct myeloid-derived suppressor cell subpopulations in the bone marrow.

Multiple myeloma induces the immunosuppressive capacity of distinct myeloid-derived suppressor cell subpopulations in the bone marrow

Tumor-initiating capacity of CD138- and CD138+ tumor cells in the 5T33 multiple myeloma model.

Tumor-initiating capacity of CD138− and CD138+ tumor cells in the 5T33 multiple myeloma model

Dll1/Notch activation accelerates multiple myeloma disease development by promoting CD138+ MM-cell proliferation.

Dll1/Notch activation accelerates multiple myeloma disease development by promoting CD138+ MM-cell proliferation

Endothelial cell-driven regulation of CD9 or motility-related protein-1 expression in multiple myeloma cells within the murine 5T33MM model and myeloma patients.

The cell surface expression of CD9, a glycoprotein of the tetraspanin family influencing several processes including cell motility and metastasis, inversely correlates with progression in several solid tumors. In the present work, we studied the expression and role of CD9 in multiple myeloma (MM) biology using the 5T33MM mouse model. The 5T33MMvitro cells were found to be CD9 negative. Injection of these cells in mice caused upregulation of CD9 expression, while reculturing them resulted in downregulation of CD9. Coculturing of CD9-negative 5T33MMvitro cells with BM endothelial cells (BMECs) resulted in a partial retrieval of CD9. Laser microdissection followed by real-time polymerase chain reaction and immunohistochemistry performed on bone sections of 5T33MMvivo diseased mice demonstrated strong local expression of CD9 on MM cells in contact with BMEC compared to MM cells further away. These findings were also confirmed by immunohistochemistry in MM patients. Neutralizing anti-CD9 antibodies inhibited transendothelial invasion of CD9-expressing human MM5.1 and murine 5T33MMvivo cells. In conclusion, we provide evidence that CD9 expression by the MM cells is upregulated in vivo by close interaction of the cells with BMEC and that CD9 is involved in transendothelial invasion, thus possibly mediating homing and/or spreading of the MM cells.

Imaging and radioimmunotherapy of multiple myeloma with anti-idiotypic Nanobodies

Multiple myeloma (MM) is characterized by the monoclonal expansion of malignant plasma cells in the bone marrow (BM) and the production of monoclonal protein (M-protein). With the implementation of autologous stem cell transplantation and highdose chemotherapy using dexamethasone, bortezomib, thalidomide and lenalidomide, the survival rate has improved but MM patients still relapse, even if they achieve complete remission (CR). Therefore, new therapeutic strategies are needed to target residual malignant cells and eliminate minimal residual disease (MRD) in order to improve patient outcome. Nanobodies are the smallest (15 kDa) antigen-binding fragments derived from camelid heavy-chain-only antibodies. Because of their biochemical characteristics including high stability, solubility and target affinity, they are ideal therapeutic and diagnostic tools. Nanobodies can also recognize epitopes that remain undetected by conventional antibodies. Previous work has already demonstrated that Nanobody conjugates are able to reach, bind and kill cancer cells. Moreover, Nanobodies conjugated with radionuclides have been successfully used with the single-positron emission tomography (SPECT) technology combined with micro-computed tomography (micro-CT) for imaging purposes. Despite the extensive Nanobody-based research, there is so far only little evidence about their potential for diagnostic and/or therapeutic applications in MM. Here, we take advantage of the M-protein present in the murine 5T2MM model to prove the potential use of Nanobodies in MM. The 5TMM models are syngeneic, immunocompetent models that resemble human MM clinically and biologically. The best characterized are the 5T33MM and the 5T2MM models. The former represents an aggressive tumor, which develops in a short period of time (4 weeks), whereas the latter represents a more moderate tumor that develops over a period of 3 months. Both express different idiotypes (5T33MMid and 5T2MMid, respectively) on the cell membrane surface. By immunization of a dromedary with purified 5T2MM M-protein and a simple selection method (see Supplementary Materials and Methods), we were able to select, produce and purify a panel of very specific anti-5T2MM-idiotype Nanobodies (a5T2MMid-Nbs) that recognize nearby epitopes on the idiotype (Supplementary Figure 1). After in vitro characterization of these Nanobodies, R3B23 came up as the better binder (see Supplementary Figures 2, 3 and 4) and was therefore selected for in vivo testing. R3B23 was labeled with radionuclides Technetium (Tc) and Lutetium (Lu) using previous established protocols. Tc (half-life: 6 h) is used in SPECT for nuclear medicine imaging techniques, whereas Lu (half-life: 6.7 days) is mainly used for therapeutic applications due to the emission of low-energy b-minus particles. Several radiotherapeutic monoclonal antibodies (MoAbs) are currently under (pre)clinical evaluation in cancers, including B-cell disorders. In MM MRD, which is characterized by a small tumor load in BM, radiotherapeutic MoAbs might not be ideal as radiotoxicity levels to healthy tissues are high, providing a rationale for the use of highly specific and rapidly cleared radiotherapeutic Nanobodies instead. First, we studied the specificity of R3B23 in vivo. At 1 h post injection (p.i.), anesthetized mice were imaged using pinhole SPECT and micro-CT, as described previously. At 30 min after imaging, the mice were killed, different organs were removed, weighed and the radioactivity was measured. Fused SPECT/microCT images obtained from naive mice with nontargeting control Nanobody Tc-cAbBCII10 showed tracer uptake only in the bladder and kidneys (Figure 1a). Biodistribution experiments (Figure 1d) confirmed a high tracer uptake in both kidneys (4200%IA/g) and only marginal levels of uptake in other organs (ranging from 0.20±0.04%IA/g in muscle tissue to 1.02±0.26%IA/g in lungs) as expected for unbound tracers that are eliminated from the body through renal filtration. Importantly, similar results were observed in naive mice injected with Tc-R3B23 (Figures 1e and h) indicating that R3B23 does not bind to circulating immunoglobulins or other in vivo targets. SPECT/micro-CT scan images and biodistribution studies of terminally diseased 5T33MM mice injected with either Tc-cAbBCII10 (Figures 1b and d) or Tc-R3B23 (Figures 1f and h) and 5T2MM mice injected with Tc-cAbBcII10 (Figures 1c and d) showed analogous patterns with a high tracer uptake in kidneys and bladder and a low uptake in all other organs, demonstrating that MM disease does not influence Nanobody uptake. SPECT/micro-CT scan images of 5T2MM mice injected with Tc-R3B23 (Figure 1g) revealed a systemic tracer uptake, which was confirmed by biodistribution studies (Figure 1h). The up to 100-fold-elevated tracer levels in blood (44.56±2.54%IA/g) can be attributed to binding of the antiidiotypic Nanobody to the high levels of circulating M-protein in this late-stage disease model. The elevated tracer blood-pool activity accounts for the decreased uptake observed in the kidneys (circa 8%IA/g) and is responsible for the elevated uptake in other organs (ranging from 0.91±0.04%IA/g in the muscle to 11.63±2.35%IA/g in the lungs). It is noteworthy that the in vivo increase in circulating M-protein can be monitored over time using SPECT/micro-CT scans with Tc-R3B23 (Supplementary Figure 5). In conclusion, the biodistribution studies demonstrate that R3B23 does not bind to any target in healthy mice or to M-protein with a different idiotype, and it is therefore truly antiidiotypic. Finally, we evaluated the effect of Nanobody R3B23 conjugated with Lutetium (Lu-R3B23) on tumor growth. One week after inoculation of 5T2MM cells into naive mice, we started weekly treatments with intravenously administrated Lu-R3B23 or negative control Lu-cAbBCII10. After 5 weeks of treatment, animals were imaged using SPECT/micro-CT scans with TcR3B23 (Figure 2). On the basis of micro-CT images, an ellipsoid region of interest was drawn around the heart. Tracer uptake in heart, as a measurement of blood-pool activity, is expressed as the counts in the tissue divided by the injected activity/cubic centimeter (%IA/cm). The %IA/cm detected in the heart of mice treated with Lu-R3B23 (5.55±1.42) was significantly lower than the values measured in untreated mice (10.03±0.27; Po0.005) and mice treated with control Nanobody Lu-cAbBcII10 (9.19±0.84; Po0.05). The lower blood value of Tc-R3B23 uptake in mice treated with Lu-R3B23 is not due to in vivo competition with the therapeutic Lu-labeled Nanobody, as the latter was already systemically cleared. Indeed, the 10 mg Tc-R3B23 injection and subsequent SPECT/micro-CT scanning were performed 5 days after injection with 10 mg Lu-Nanobody.

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response

Chromosomal region 1p22 is deleted in ⩾20% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients.

Leptin receptor antagonism of iNKT cell function : a novel strategy to combat multiple myeloma

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Here, we report the role of the adipokine leptin as master regulator of anti-myeloma tumor immunity by modulating the invariant natural killer T (iNKT) cell function. A marked increase in serum leptin levels and leptin receptor (LR) expression on iNKT cells in MM patients and the 5T33 murine MM model was observed. MM cells and leptin synergistically counteracted anti-tumor functionality of both murine and human iNKT cells. In vivo blockade of LR signaling combined with iNKT stimulation resulted in superior anti-tumor protection. This was linked to persistent IFN-γ secretion upon repeated iNKT cell stimulation and a restoration of the dynamic antigen-induced motility arrest as observed by intravital microscopy, thereby showing alleviation of iNKT cell anergy. Overall our data reveal the LR axis as novel therapeutic target for checkpoint inhibition to treat MM.