Tracking human multiple myeloma xenografts in NOD-Rag-1/IL-2 receptor gamma chain-null mice with the novel biomarker AKAP-4

Background Multiple myeloma (MM) is a fatal malignancy ranking second in prevalence among hematological tumors. Continuous efforts are being made to develop innovative and more effective treatments. The preclinical evaluation of new therapies relies on the use of murine models of the disease. Methods Here we describe a new MM animal model in NOD-Rag1null IL2rgnull (NRG) mice that supports the engraftment of cell lines and primary MM cells that can be tracked with the tumor antigen, AKAP-4. Results Human MM cell lines, U266 and H929, and primary MM cells were successfully engrafted in NRG mice after intravenous administration, and were found in the bone marrow, blood and spleen of tumor-challenged animals. The AKAP-4 expression pattern was similar to that of known MM markers, such as paraproteins, CD38 and CD45. Conclusions We developed for the first time a murine model allowing for the growth of both MM cell lines and primary cells in multifocal sites, thus mimicking the disease seen in patients. Additionally, we validated the use of AKAP-4 antigen to track tumor growth in vivo and to specifically identify MM cells in mouse tissues. We expect that our model will significantly improve the pre-clinical evaluation of new anti-myeloma therapies.


Background
According to the American Cancer Society, more than 20,000 patients were diagnosed with multiple myeloma (MM) in the US in 2010. Among hematologic malignancies, MM ranks second in prevalence and has the shortest 5-year survival rate [1]. Multiple myeloma (MM) is an age-related cancer caused by the accumulation of antibody-producing malignant plasma cells and leads to progressive osteolysis, defective hematopoiesis and renal failure [2]. Recent progresses in understanding the molecular bases of MM have lead to the use of innovative drugs, such as bortezomib, thalidomide and lenalidomide [3]. Unfortunately, although these therapies afforded a significant improvement in the disease course, MM remains invariably fatal because of the high rate of multidrug-resistant relapse [4]. On these bases, constant efforts are dedicated to the evaluation of more effective treatment strategies [5][6][7].
Similarly to other malignancies [8], virtually any innovative treatment for MM requires a pre-clinical assessment, which largely relies on the use of animal models to evaluate the anti-tumor potential and possible toxicities [9][10][11][12]. To this goal, sub-lethally irradiated immunodeficient NOD/SCID mice have been extensively used since they allow for human MM cell line xenografting after intravenous injection [13][14][15][16][17][18][19][20][21][22][23]. More recently, it has been shown that NOD/SCID mice carrying nonfunctional IL-2 receptor gamma chain (NOD/SCID/γc null , NOG) are more permissive recipients than NOD/SCID and can be easily xenografted with human MM cell lines to produce a disease similar to that seen in patients, including multiple metastatic sites and bone lesions [24,25]. A further modification of the NOD strain, carrying double genetic disruptions of the Rag1 and the IL-2 receptor gamma chain genes, namely NOD-Rag1 null IL2rg null (NRG), has been reported to tolerate higher levels of radiation compared with NOD/ SCID and NOG strains and to allow for efficient engraftment of human hematopoietic stem cells [26].
The development of successful animal models for MM also relies on the choice of the biomarkers used to track the disease course and to identify tumor cells in mouse tissues [27][28][29][30][31][32]. The A-kinase anchor protein 4 (AKAP-4) [33] is a scaffolding protein that participates in the intracellular signaling of protein kinase-A [34]. AKAP-4 is a cancer/testis antigen (CTA), a class of tumor associated antigens characterized by high expression in germ cells and cancer, strong immunogenicity and very low expression or absence in normal tissues [35,36]. We have previously shown that AKAP-4 is abnormally expressed at the mRNA and protein levels in MM cell lines and patients' MM primary cells, but absent in normal tissues, and therefore it is a potential novel biomarker for MM [37].
In this study, we used for the first time the NRG strain to establish an innovative model of MM, allowing for the growth and the spread of MM cell lines and primary patients' cells as well. Additionally, we provide evidence that the CTA AKAP-4 is a reliable and specific biomarker that can be used to track the growth of MM cell lines and primary cells in vivo.

Detection of tumor growth in vivo by ELISA
Indirect ELISA was used to determine the concentration of human paraproteins (IgE and IgG) and AKAP-4 in the sera of tumor-bearing mice ( Figure 1). Anti-human IgE antibodies were used to monitor the growth of U266 and H929 [38], since they are IgE-producing cell lines. For MM primary cells, IgG was used as a paraprotein marker [39]. Figure 1 shows that paraprotein and AKAP-4 levels became evident starting 21 days after injection, and that a progressive increase was detectable over time. Although AKAP-4 levels were on average 20% lower than IgE and IgG, no significant difference between AKAP-4 and paraprotein mean levels was detected at any time analyzed point (two-way ANOVA and Bonferroni's post-test p > 0.05).

Flow-cytometry identification of MM cells from mouse tissues
Six weeks after initiation of tumor challenging, tumorbearing and healthy mice were euthanized, and tissues were processed as described in the Methods section.  Tables 1, 2). IgE was used as a marker for U266 and H929 [38], while primary MM cells were identified by CD38 and CD54 [39]. AKAP-4 was expressed by MM cell lines ( Figure 2; Tables 1, 2) and primary MM cells ( Figure 3; Table 3); therefore it was tested for the detection of both cell types. Results showed that IgE + U266 and H929 were present in mouse bone marrow, blood and spleen (Figure 2; Tables 1, 2). Similarly, primary CD38 + and CD54 + primary MM cells were detected in bone marrow, blood and spleen ( Figure 3; Table 3). The expression pattern of AKAP-4 was comparable to that of IgE, CD38 and CD54 (Figures 2 and 3; Tables 1, 2, 3). The specificity of the assay was confirmed by the failure to detect positive cells in tumor-free mice ( Figure 4).

Analysis of AKAP-4 expression at the mRNA and protein levels in MM xenografts
RT-PCR was performed to evaluate AKAP-4 mRNA expression in tumor-challenged or tumor-free mice. Results ( Figure 5) show that the AKAP-4 transcript was present in MM cell lines, primary MM cells, bone marrow, peripheral blood and spleens of tumor-bearing mice, but undetectable in tumor-free mice (healthy controls). Specificity of results was also confirmed by PCR reactions carried out without cDNA template or without retrotranscribed RNA.
AKAP-4 protein was detected by Western blot analysis ( Figure 5) in MM cell lines, primary MM cells, bone marrow, peripheral blood and spleens of tumor-bearing mice, but not in tumor-free mice (healthy controls).

Discussion
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][41][42][43][44][45] or measurement of MM-derived paraptrotein in the serum [29][30][31][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][51][52], or immunocompromised mice, namely NOD/SCID [13][14][15][16][17][18][19][20][21][22][23], SCIDhu [53][54][55][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][51][52]58,59]. Therefore, it is evident that preclinical 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][61][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 SCIDhu model was developed, in which MM cells are located in subcutaneously implanted human bone chips [53][54][55][56]. Although SCID-hu mice allow for the growth of MM cells in a bone niche of human origin [53][54][55][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][66][67][68][69][70][71][72][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][75][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.

Conclusions
We presented here the proof-of-principle for the use of NRG mice as a new model supporting the metastatic growth of human MM cell lines and primary cells. Additionally, we propose the use of AKAP4 as a universal biomarker to track tumor cells in vivo. We foresee that our results will significantly contribute to the improvement of the pre-clinical evaluation of new antimyeloma therapies. Because our model sustains the growth of primary MM cells, further investigations are warranted to study the suitability of this system to assess the efficacy of personalized therapies directly on patient's cells.

Animals
Six-week-old female NOD.Cg-Rag1 tm1Mom IL2rg tm1Wjl / SzJ (NRG) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.). All mice were maintained in filtered-air laminar-flow cabinets under specific pathogen-free conditions. Treatment and care of the Mean fluorescent intensities (a) and SEM (b) were calculated by flow cytometry from cells and tissues of 5 U266-challenged mice.  Mean fluorescent intensities (a) and SEM (b) were calculated by flow cytometry from cells and tissues of 5 primary MM-challenged mice.
animals were in accordance with the Institutional Guidelines and the Animal Welfare Assurance Act. The mice were checked daily and euthanized 6 weeks after tumor challenge or if they showed signs of excessive discomfort (hind leg paralysis, inability to move, eat or drink).

Human MM cell lines
The human MM cell lines U266 and H929 were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.), and cultured in RPMI-1640 medium, supplemented with 10% V/V fetal bovine serum (FBS) and penicillin/streptomycin mix (10 mg/ mL each) in 95% air and 5% CO 2 at 37°C. Prior to injection, cells were washed once in PBS and then resuspended at 10 8 cells/mL in pre-warmed PBS prior to injection.

Primary MM cells
Human material was obtained under informed consent and with the approval from the local ethics committee. Bone marrow aspirate was obtained from a MM patient at diagnosis (Durie-Salmon stage III) from the hip bone. Light density cells were separated by ficoll hypaque centrifugation (Histopaque; Pharmacia, Uppsala, Sweden) [39], washed twice in PBS, counted and adjusted at the final concentration of 10 8 cells/mL in pre-warmed PBS prior to injection.
Xenografts 20 mice were sub-lethally irradiated with a total dose of 550 cGy at 139 cGy/min rate [26]. After 6 hours, mice were assigned to the following groups (5 mice/group): group 1 received U266 cells, group 2 received H929, group 3 was given primary MM cells, while group 4 was left tumor-free and served as a negative control. Each mouse received 10 7 cells by a single intravenous injection in the lateral tail vein (100 μL/mouse).
ELISA for the measurement of serum paraprotein and AKAP-4 concentration Blood (50 μL) was collected weekly from each mouse. Serum was prepared by centrifugation in the absence of anticoagulants and stored at -20°C until use. An enzyme-linked immunosorbent assay (ELISA) was performed on mouse sera for the determination of human paraprotein (IgE and IgG) [39]  Blood (500 μL) was taken by retro-orbital venipuncture immediately after the euthanasia procedure and placed in a heparin-coated tube. Cells were harvested by centrifugation and washed twice in PBS before analyses.

Flow-cytometry
The expression of human IgE, CD38, CD45 and AKAP-4 was analyzed by flow-cytometry 6 weeks after tumor injection as previously described [77]. Specifically, IgE was used to identify U266 and H929 cells [38], while CD38 and CD45 were used as markers for human MM primary cells [39]. AKAP-4 was analyzed in both cell lines and primary cells [37]

RT-PCR and immunoblot
Total RNA was extracted from bone, spleen, blood, or from MM cell lines and primary MM cells by Trizolreagent (Sigma, St Louis, MO, U.S.A.). Purified total RNA was treated with 5 μg RNase-free DNase I (Promega, Madison, WI, U.S.A.) at 37°C for 2 h. mRNA was then isolated using Oligotex mRNA Mini Kit (QIAGEN, Valencia, CA, U.S.A.). First-strand cDNA synthesis was performed using oligo (dT) 15-mers primers. PCR primers for AKAP-4 were as follows: forward 5'-GCGTACTCTGATACTACAATGATG -3' and reverse 5'-GGG GTTTTGGGTAAAGTCA-3' [78]. PCR was performed by 35 amplification cycles at 59°C annealing temperature. For each sample, RNA integrity was checked by amplification of the β-actin cDNA. Successful removal of genomic DNA contamination was confirmed by amplification of the RNA without prior reverse-transcription reaction. All results were confirmed in four independent RT-PCR tests. Immunoblots for AKAP-4 were performed using standard methods, as previously described [78]. Positive controls for immunoblots were proteins extracted from injected MM cells. For healthy mice, protein extracts from human testis were used as positive controls (Applied Biosystems, Foster City, CA, USA) [37].

Statistical analysis
All data are expressed as mean values ± SEM (Standard Error of the Mean). Results were analyzed using GraphPad Prism software (GraphPad Software, Inc., CA, USA.). Statistical analyses were performed by the twoway ANOVA test. A p value < 0.05 was considered statistically significant.