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A.G. Solimando et al.
lower vessel density (calculated as number of vessels/mm2) than the group treated with the isotype control, which had a higher number of vessels with well-lit lumina (9.77±2,63 and 6.48±0,631 in the groups treated with isotype control vs. anti-JAM-A, respectively; P<0.0001).
To assess the activity of the anti-JAM-A blocking anti- body on angiogenesis on a solitary plasmacytoma in vivo and to monitor MM-cell growth at extra-osseous sites non- invasively with a caliper, we employed a subcutaneous MM xenograft model. This approach enables dissection of the endothelial bystander effect on BM-independent extramedullary MM.21 Thus, we employed a second in vivo xenograft model engrafting RPMI-8226 cells subcu- taneously into the flanks of NOD/SCID mice.21 The ani- mals were randomized at day 3 after engraftment and treated with either anti-JAM-A monoclonal antibody or a non-specific isotype control antibody intraperitoneally for 3 days/week for 40 days. Subsequently, we measured the vascular area, tumor volume and hemoglobin content of the MM mass. Blocking JAM-A reduced the vascular area in the soft tissue MM masses compared to that in animals treated with an isotype control (difference between medi- ans -0.015; P<0.0001). No adverse events occurred upon continuous anti-JAM-A treatment.
Notably, after 40 days, the vascular area increased signif- icantly in tumors and MM disease progressed more in con- trols than in the group treated with the monoclonal anti- body against JAM-A (Figure 6A, CD31 staining, and 6B). In isotype-treated control mice, tumors grew exponentially, contrasting with the only limited tumor growth in anti- JAM-A-treated animals (Figure 6B, Online Supplementary Figure S4A). Lower hemoglobin content confirmed poor MM vascularization in the anti-JAM-A-treated mice (8.4±0.04 in the isotype-treated control mice vs. 5.5±0.04 in the anti-JAM-A-treated group; P<0.0001, 95% CI: -3.02 to -2.8 (Figure 6C). Ki-67-staining, vascular area and vessel count confirmed that blocking JAM-A strongly reduced MM vascularity and disease progression. Furthermore, JAM-A blocking significantly reduced pro-angiogenic fac- tors such as FGF-2 and VEGF-A in the peripheral blood of MM-bearing mice (Online Supplementary Figure S4B-D).
Discussion
Angiogenic switching is a key process during transition from premalignant asymptomatic MGUS to full-blown MM. Angiogenic parameters in the BM at the time of diag- nosis were widely considered to be able to predict MM pro- gression.22 In solid tumors, such as breast, lung, head and neck, and brain cancers, JAM-A activation promotes tumor progression, while its inhibition by anti-JAM-A2 agents reduces tumor growth.11 We demonstrated in four inde- pendent experimental settings that JAM-A essentially stim- ulates MM-associated angiogenesis. In the CAM assay, a monoclonal antibody neutralizing JAM-A caused a strong reduction of the number of vessels, implying that JAM-A exerts an essential angiogenic stimulus that could not be replaced by any other compensating factor contained in the MMEC conditioned medium.23 Our new findings pinpoint JAM-A as an attractive target in MM patients.
JAM-A appears pivotal in MM evolution, which can be explained by several angiogenic mechanisms.24,25 First, we demonstrated significantly higher JAM-A levels on MMEC from NDMM patients than on MGEC. Furthermore, we
could link the high JAM-A surface expression on MMEC with a significantly shorter overall survival in both NDMM and RRMM and, at even more advanced disease stages, higher JAM-A expression levels also correlated with reduced progression-free survival. We therefore examined the pathophysiological basis responsible for favoring MM progression. As already described for the HGF/cMET axis,26,27 JAM-A acts within the BM microenvironment, sus- taining the neoplastic clone and promoting MM-related angiogenesis both directly and indirectly by priming MMEC. Thus, JAM-A and its soluble isoform sJAM-A appear to feed into a vicious cycle involving MMEC, gen- erating a malignant environment favorable for MM pro- gression. Although JAM-A is expressed in several solid can- cers,3 to our knowledge this is the first report of the role of endothelial JAM-A expression in the MM tumor microen- vironment. Homophilic interactions between recombinant sJAM-A and membrane JAM-A have been demonstrated biochemically.10 Homophilic JAM-A interactions can be inhibited by an anti-JAM-A monoclonal antibody that binds to an epitope close to the N-terminus of the mature protein10 as well as by a peptide that corresponds to the N- terminal 23 residues of the mature protein.28 This suggests that the homophilic trans-interaction is mediated through the membrane-distal V-type Ig-like JAM-A domain at the N-terminus of the molecule. Targeting this domain of the JAM-A molecule on MMEC in our in vitro co-culture sys- tems suggested that this type of interaction mediates the MM-MMEC crosstalk. In line with previous reports about MMEC sustaining MM growth,29,30 our disease models showed that during the transition from the pre-angiogenic to the angiogenic phase, proliferation of tumor cells and neovascularization intensely involve over-expression of JAM-A on MMEC. MMEC were responsive to the pres- ence of sJAM-A in the surrounding microenvironment, which increased their JAM-A surface protein expression. sJAM-A directly and indirectly upregulated JAM-A on the bystander MM-cells, independently of their basal JAM-A expression status. These observations support the concept that cellular components of MM BM, including MMEC, can release JAM-A to sustain disease progression and pre- pare a tumor-"friendly" niche, exerting significant modula- tion on FGF-2, VEGF-A and PLG/ENO1 downstream effects.
JAM-A has been described to interact with CD9, a well- known driver of MM-related drug resistance31 and clinical prognosis.32 We found significant expression of FGF-2, a potent stabilizer and activator of a ternary complex involv- ing JAM-A, CD9 and avβ3 integrin, a novel potential ther- apeutic target.33 Peddibhotla et al. described that the aggre- gation of this ternary complex can activate downstream pathway cascades to induce proliferation, migration and an angiogenic stimulus to endothelial cells.34 Our in silico vali- dation shed more light on this pathophysiological process. ENO1 encodes a-enolase, which in the cytoplasm works as a plasminogen receptor and has been described to show upregulated membrane expression in several types of can- cer.35,36 Of note, plasminogen upregulation had been corre- lated with tumor invasion and angiogenesis;37 its activation, derived from the interaction with a-enolase, prompted acti- vation of downstream signaling such as the MEK-ERK pathway, which was able to promote cell invasion and angiogenesis further. a-enolase can also modulate antitu- mor immune responses. Cappello et al. described that a- enolasehigh myeloid-derived suppressor cells could not
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haematologica | 2021; 106(7)