This study was aimed to establish and characterize a new murine model of disseminated MM, allowing for the engraftment of human MM cell lines and primary tumor cells derived from MM patients. To this goal, we used the NOD-Rag1
null IL2rg
null (NRG) murine strain, intravenously injected with MM cell lines or with primary MM cells. The lacking of a functional IL-2 receptor makes IL2rg
null mice better xenograft recipients then NOD/SCID animals, because of the absence of NK cells [
38]. In addition, compared with NOD/SCID or NOD/SCID/γc
null (NOG) strains, NRG mice tolerate significantly higher levels of radiation. Differently from SCID mice, the NRG strain carries a functional Prkdc gene, which is essential for the repair of DNA damage induced by radiation in many tissues [
26].
ELISA for serum MM paraproteins showed that xenografted animals supported the growth of both MM cell lines and primary tumor cells. Importantly, AKAP-4 was detectable in the sera of tumor-challenged mice and its levels increased over time, similarly to those of IgE and IgG. This indicates that AKAP-4 is a suitable biomarker for tracking MM progression in murine xenografts. Different techniques have been described to monitor the MM burden in animal models, such as fluorescent tagging of tumor cells [
40‐
45] or measurement of MM-derived paraptrotein in the serum [
29‐
32]. In the clinic, better methods for staging and monitoring the aggressiveness of MM, especially in assessing relapse, are thought to be critical to improve patients' outcomes and develop personalized therapies [
46]. A number of methods are under investigation, including mass spectrometry for the quantification of serum immunoglobulins [
46], and immunohistochemistry for the expression of FGFR3 and cyclin D1 (reported in 15%, and 50% of patients with MM, respectively) [
46,
47]. In this context, the identification of novel tumor antigens in the sera could be instrumental for a more sensitive detection of disease progression [
36]. Here we showed for the first time the use of AKAP-4 as a novel serum biomarker in MM animal models. Further investigations are warranted to evaluate AKAP-4 serum levels in MM patients and the correlation with treatment outcome.
Flow-cytometry analysis confirmed the presence of MM cell lines and primary patient's cells in the bone marrow, blood and spleen of tumor challenged mice, indicating that intravenously injected tumor cells were able to systemically disseminate
in vivo. The specificity of this finding was confirmed by the failure to detect paraprotein- or AKAP-4- positive cells in tumor-free mice. Additionally, we showed that AKAP-4 was expressed in the same tissues at the transcriptional and protein levels in tumor-bearing animals, but absent in healthy controls. Collectively, these results indicate that our model is suitable for the growth and systemic dissemination of human MM cell lines and primary tumors. Currently available murine models for MM include immunocompetent mice, such as the 5TMM series [
48,
49] and genetic models of MM [
50‐
52], or immunocompromised mice, namely NOD/SCID [
13‐
23], SCID-hu [
53‐
56], and NOG [
24,
25,
38,
57]. The 5TMM and the genetic models of MM have the advantage of affording pre-clinical studies in immunocompetent hosts, where possible effects of the therapy on the interaction between tumor cells and the immune system can be evaluated. However, molecular and biological differences exist between murine and human MM cells [
11]. Additionally, the number of available murine genetic models of MM and of 5TMM cell lines is extremely restricted and do not represent the heterogeneity of the human disease [
50‐
52,
58,
59]. Therefore, it is evident that pre-clinical studies on MM cells of human origin are essential [
9], but they are only feasible by using immunodeficient murine xenografts. Among these, subcutaneous inoculation of human MM cells has been extensively described [
13,
17,
45,
60‐
62]. This model affords the possibility to directly assess changing in tumor growth induced by therapies. Yet, tumor cells growing subcutaneously do not interact with the bone marrow microenvironment, which largely accounts for MM drug resistance [
61,
63]. Because we described the presence of tumor cells in the bone marrow of tumor-challenge mice, we propose that our model is suitable to evaluate the protective role played by the bone niche against anti-tumor therapies. Some concerns have been raised about the possibility that the interactions between MM cells and the bone stroma may be partially species-specific [
11,
64]. To address this potential difficulty, the SCID-hu model was developed, in which MM cells are located in subcutaneously implanted human bone chips [
53‐
56]. Although SCID-hu mice allow for the growth of MM cells in a bone niche of human origin [
53‐
56], they do not reproduce the pattern of dissemination and multifocal spread seen in MM patients. Here we described for the first time a murine model supporting the engraftment human MM cells, and allowing for the development of a disease involving multiple sites, similar to that observed in patients [
65‐
73]. This is an important advantage, because the effect of bone resorption in multiple osteolytic lesions is a critical factor for the survival of MM patients [
6,
7]. Pre-clinical studies basing on immunocompromised xenograft models have previously described metastatic spread involving multiple bones, yet they have been limited to human MM cell lines [
40,
41,
43,
44,
74‐
76]. In this study, we show that our model supports the metastatic growth of primary human MM cells. MM cell lines do not adequately represent the heterogeneity of the human disease because they are established from late stage disease and frequently present mutations not seen in patients [
9]. Therefore, the possibility to study not only cell lines, but also primary MM cells in murine models is relevant.