Proteomic Characterization of the Multiple Myeloma Bone Marrow Extracellular Matrix

Background

The extracellular matrix (ECM) is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM), interactions between MM cells and the bone marrow (BM) microenvironment, of which the ECM forms a major component, are critical to the pathogenesis of the disease and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Until recently ECM proteins have proven difficult to characterize due to their biochemical properties and large size. Recent advances in proteomics have led to the characterization of the ECM and ECM-associated proteins (“matrisome”) in normal human tissues and tumors using a systematic and comprehensive approach.

Methods

Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x10⁶) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after 2 weeks in order to mimic early tumor development (luminescence = 1x10⁵ p/sec/cm²/sr) or 5 weeks (1x10⁸ p/sec/cm²/sr) to model more advanced MM. Human bone marrow aspirates; Whole bone marrow was obtained from newly diagnosed MM patients (n=9) and healthy human donors (ND) (n=4) following written informed consent. ECM proteins were enriched from bone marrow samples obtained from MM patients, NDs and mice according to previously published methods.Tandem Mass Spectrometry (LC-MS/MS): Peptides were run using reversed-phase microcapillary liquid chromatography – tandem mass spectrometry (LC-MS/MS) on a high resolution hybrid Orbitrap Elite mass spectrometer. MS/MS data were searched against the UniProt Human database using MASCOT to identify proteins. Spectral counts were used as a semi-quantitative measure of abundance. ECM proteins were defined according to the in-silico definition of the matrisome. Validation of expression of ECM mRNA in MM cell lines (MM1s, RPMI-8226 and U266) and in CD138+ cells and bone marrow stromal cells (BMSC’s) from MM patients in comparison to NDs was performed using qRT-PCR.

Results

Primary myeloma sample ECM; Using a spectral count of 2 as a cutoff of peptide abundance we identified a total of 536 unique proteins in ND bone marrow of which 35 are defined as matrisome proteins. 982 unique proteins were enriched from whole bone marrow samples of newly diagnosed MM patients of which 26 are defined as matrisome proteins, 7 unique proteins were identified as ECM or ECM-associated in newly diagnosed patients which were not detected in the ND samples including PRG3, FGG, LEG10, TLN1 and PLEC. Critical ECM components such as laminins, matrix metalloproteinases and collagens were also found to be significantly altered in newly diagnosed MM with evidence of destruction of ECM components in active disease.

Tumor Xenograft ECM; In mice with an earlier phase of human MM1s cell tumor burden we detected a total of 329 unique proteins of which 48 were defined as matrisome proteins, 23 of these proteins were unique to the earlier phase of MM in these mice. Mice with more advanced tumor development had unique ECM proteins which were not detected in the earlier disease stage including collagens, laminins and matrix metalloproteinases, indicating that these ECM components may be critical for re-modelling the ECM in MM. Interestingly, in our xenograft model of MM we were able to detect both human and mouse ECM components indicating that the tumor ECM is secreted from both the murine stroma and the human MM cells and allowing delineation of the source of individual ECM components. This indicates that as MM progresses certain ECM components, including FBN1, which were initially derived from stroma are later derived from MM cells. Differential expression of ECM components, including FBN1 between normal and malignant plasma cells was confirmed using qRT-PCR.

Conclusions

We have performed proteomic profiling of the unique tumor ECM in MM using mass spectrometry with a view to determining the specific components that may be altered with disease progression. Through this approach plasma-cell-derived ECM can be identified with a view to developing therapeutic strategies in this disease.