The KISS1 Receptor as an In Vivo Microenvironment Imaging Biomarker of Multiple Myeloma Bone Disease

Multiple myeloma is one of the most common hematological diseases and is characterized by an aberrant proliferation of plasma cells within the bone marrow. As a result of crosstalk between cancer cells and the bone microenvironment, bone homeostasis is disrupted leading to osteolytic lesions and poor prognosis. Current diagnostic strategies for myeloma typically rely on detection of excess monoclonal immunoglobulins or light chains in the urine or serum. However, these strategies fail to localize the sites of malignancies. In this study we sought to identify novel biomarkers of myeloma bone disease which could target the malignant cells and/or the surrounding cells of the tumor microenvironment. From these studies, the KISS1 receptor (KISS1R), a G-protein-coupled receptor known to play a role in the regulation of endocrine functions, was identified as a target gene that was upregulated on mesenchymal stem cells (MSCs) and osteoprogenitor cells (OPCs) when co-cultured with myeloma cells. To determine the potential of this receptor as a biomarker, in vitro and in vivo studies were performed with the KISS1R ligand, kisspeptin, conjugated with a fluorescent dye. In vitro microscopy showed binding of fluorescently-labeled kisspeptin to both myeloma cells as well as MSCs under direct co-culture conditions. Next, conjugated kisspeptin was injected into immune-competent mice containing myeloma bone lesions. Tumor-burdened limbs showed increased peak fluorescence compared to contralateral controls. These data suggest the utility of the KISS1R as a novel biomarker for multiple myeloma, capable of targeting both tumor cells and host cells of the tumor microenvironment.


Introduction
The aim of this study was to test whether KISS1R and kisspeptin are expressed in MM cells and cells of the tumor microenvironment, whether interactions between MM cells and skeletal precursors resulted in up-regulation of the KISS1R-kisspeptin system, and whether these changes in gene expression signature could be used as a tool to develop a novel biomarker for the MM microenvironment in myeloma bone disease suitable for diagnostics and therapy.

Primary cells and cell lines
Primary human MSCs were obtained from the cancellous bone from the acetabulum of patients that received a total hip arthroplasty. Cancellous bone was used in accordance with the local Ethics Committee (Medical Faculty of the University of Würzburg), which approved this study, and after receiving the written consent from the patients of the Orthopedic clinic. The isolation of MSCs was performed as previously described [21] and according to the following procedure: The cancellous bone was washed once with DMEM/Ham´s F-12 medium, 1:1 mixture (Life Technologies GmbH, Darmstadt, Germany). After centrifugation at 270 g for 5 min, the supernatant was removed and the cancellous bone was washed several times with DMEM/Ham´s F-12 medium, 1:1 mixture. The supernatant of each washing step was collected and the cells were pelleted by centrifugation (270 g, 5 min). The cell pellet was reconstituted in propagation medium, consisting of DMEM/Ham´s F-12 medium, 1:1 mixture, supplemented with 10% (v/v) heat inactivated FCS (Biochrom, Berlin, Germany), 50 μg/ml L-Ascorbic acid 2-phosphate (Sigma-Aldrich Chemie GmbH, Schnelldof, Germany), 100 U/ml penicillin (Life Technologies GmbH), and 100 μg/ml streptomycin (Life Technologies GmbH). Cells were plated at a density of 4x10 7 vital cells/ml in 175 cm 2 cell culture flasks (total volume: 25 ml) and were incubated for 48 h to 72 h at 37°C, 5% CO 2 , followed by washing with PBS. Adherent cells were expanded in propagation medium for 10 to 14 days in total to confluency. At confluency, MSCs were detached using 0.05% trypsin-EDTA (Life Technologies GmbH) and reseeded as required.

Staining of INA-6 cells with CellTracker™ Green
Myeloma cells were stained with CellTracker™ Green 5-chloromethylfluorescein diacetate (CMFDA) (Lonza Group AG, Basel, Switzerland) according to manufacturer´s instructions. Briefly, 4.5x10 7 cells were resuspended in 12.8 ml serum-free propagation medium containing 5 μmol/l CMFDA. After 15 min of incubation, cells were pelleted (270 g, 5 min) and resuspended in 16 ml RPMI complete medium. After another 30 min incubation, cells were washed once with PBS, resuspended at a cell density of 3x10 5 cells per ml RPMI 1640 complete medium and incubated overnight. The next day, cells were prepared for co-culturing with MSCs and OPCs by washing them once with PBS and resuspending them in mixed media 1:1 (v/v), consisting of 1 part of MSC or OPC propagation medium and 1 part of RPMI complete medium.

Preparation of RNA samples
A total of 5x10 4 MSCs were seeded per cm 2 in 6 well plates or 175 cm 2 cell culture flasks and allowed to attach for one day. For experiments with OPCs, differentiation of MSCs followed as described above. One day before co-culturing, medium was replaced with mixed media 1:1 (v/v). Afterwards, 2x10 6 (6 well plate) or 3.5x10 7 (175 cm 2 flask) CMFDA + myeloma cells were added to MSCs or OPCs in a final density of 4x10 5 cells/ml. As a control, respective MSCs or OPCs were incubated in the same volume of mixed media 1:1 (v/v). MSCs/OPCs were purified after 24 h of co-culture by fluorescence activated cell sorting (FACS) using a BD FACS Aria TM III cell sorter (Becton Dickinson GmbH, Heidelberg, Germany) or by magnetic activated cell sorting (MACS) using CD45 MicroBeads (for INA-6 cells, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or CD38 and CD138 MicroBeads (for all remaining myeloma cell lines). Purity of FACS-or MACS-sorted fractions was controlled by re-analysis of sorted fractions using the BD FACS Aria TM III cell sorter or Axio Vert.A1 fluorescence microscope (GFP filter) (Carl Zeiss AG, Jena, Germany), respectively.
Indirect co-culture was performed for 24 h using a trans-well culture system (6 well format, 0.4 μm pore) (Corning, Inc., purchased from VWR International GmbH, Darmstadt, Germany) and equal cell densities as described above. As a control, MSCs or OPCs were incubated in mixed media, 1:1 (v/v).
Alexa 633-kisspeptin probe conjugation, purification, and quality control The 54 amino acid kisspeptin, modified to include an N-terminal cysteine residue for site-specific fluorescent labeling, was synthesized by Genecust (Dudelange, Luxembourg). 1 mg peptide was dissolved in 1.6 ml PBS (final concentration 100 μM) and treated with 10-fold molar excess of tris(2-carboxyethyl)phosphine (Life Technologies GmbH) for 3 min at room temperature. Reduced peptide was then incubated with 10-fold molar excess (1 mg dye in 830 μl PBS to generate a 1 mM working stock) of Alexa Fluor 633 C5 Maleimide (Life Technologies GmbH). Incubation was carried out at room temperature in the dark for 2 h. Conjugated peptide was purified twice using a Sephadex PD-10 column (GE Healthcare Europe GmbH). Assessment of conjugation efficacy was performed by reverse phase high performance liquid chromatography (HPLC) using a MultoHigh-Bio200-5uC18 column (CS Chromatographie Services GmbH, Langerwehe, Germany) in a Shimadzu prominence HPLC machine (Duisburg, Germany). Samples were diluted in water containing 0.1% trifluoroacetic acid (Carl Roth, Karlsruhe, Germany). Samples were measured at 640 ms intervals for 50 min.

Animal experiments
In vivo binding of Alexa 633-kisspeptin was tested in immune competent BALB/c and CD-1 nude mice, inoculated with MOPC cells and MCF-7 cells, respectively. All experiments were performed according to the German regulations for animal experimentation. The study was approved by the Regierung von Unterfranken as the responsible authority (Permit Number 55.2-2531.01-76/10 and -103/11) and by the institutional authorities of the Ethics Committee for Animal Experiments at the Christian Albrechts University of Kiel (V242-7224.121-17). 1x10 6 MCF-7 cells transfected with the near-infrared fluorescent protein [31] were suspended in 5 μl PBS and injected directly into the right tibia, with equal amounts of PBS injected into contralateral control limbs, of 3 female CD-1 nude mice (Charles River). Tumor progression was monitored using the NightOwl planar imaging system (Berthold Technologies, Bad Wildbad, Germany). 1x10 5 MOPC cells, suspended in 100 μl PBS, were injected i.v. via the lateral tail vein into immune-competent female BALB/c mice (Charles River). 19 days after the injection of the MM cells, mice were imaged by in vivo bioluminescence (BLI) to identify 6 mice which contain lesions in only one of the hind legs. All animals were kept in temperature and humidity-controlled, pathogen-free environment, with a 12 h light/dark cycle, and access to food and water ad libitum. All experiments were conducted before disease burdened resulted in reduced appetite, decreased body weight or increased sensitivity to touch.
In vivo imaging BLI was performed with an IVIS Spectrum (Caliper-Xenogen, Alameda, CA, USA) as previously described [32]. Briefly, mice were anesthetized i.p. with a mixture of ketamine (100 mg/ kg) and xylazine (10 mg/kg) in PBS. Luciferin (150 mg/kg) was co-injected and BLI measurements were started 10 min later. For MCF-7 mice, animals were imaged every minute following injection of 100 μl of Alexa 633-kisspeptin (40 μM) using the NightOwl. For MM-containing mice, a fluorescence pre-scan (excitation filter: 605 nm, emission filter: 660 nm; illumination time: 1 s) was performed using the IVIS Spectrum. Next, mice were injected with 100 μl of Alexa 633-kisspeptin (40 μM) in the lateral tail vein and immediately imaged every two minutes for up to 20 min using the fluorescence settings as described above. In addition, fluorescent images were acquired after 30, 45, and 60 min. At the end of the experiment mice were then euthanized, organs were prepared, and fluorescence ex vivo imaging of the organs was performed. Imaging data was analyzed with Living Image 4.0 (Caliper-Xenogen, Alameda, CA, USA) and Prism 5 software (GraphPad, La Jolla, CA, USA).

Statistical analyses
All statistical analyses were conducted using Prism 5 software. Comparison between groups was made using either a paired or unpaired two-sample t-tests. Comparison of organ fluorescence was conducted using a 1-way ANOVA and Turkey's multiple comparison test. Alexa 633-kisspeptin binding kinetics were quantified using association then dissociation curves with time 0 = 25 min and NS = 1. P values of <0.05 were considered to be statistically significant.

The KISS1R is upregulated in MSCs and OPCs after direct co-culturing with MM cells
Protein levels of the KISS1R and the kisspeptin precursor KISS1 were analyzed by western blot in primary MSCs and MSC-derived OPCs from healthy donors (Fig 1A and 1B). MSCs and OPCs show low level, heterogeneous expression of both receptor (KISS1R) and ligand (KISS1). KISS1R expressed in skeletal precursor cells was also found to be of a greater molecular weight than that expressed in the MM cell line INA-6, as well as in the breast cancer cell line MCF-7 ( Fig 1C) previously characterized to express high levels of the KISS1R [33]. Both are specific immunoreactive bands, since addition of blocking peptide attenuated antibody detection ( Fig  1C). Therefore, this result may indicate presence of alternative splicing isoforms as has been previously described [34].
To determine if changes in KISS1R expression can be expected following MM cell contact, KISS1R mRNA expression was assessed in MSCs and OPCs after direct or indirect culturing with INA-6 cells. Both MSCs and OPCs showed significantly higher mRNA expression after direct co-culturing with INA-6 cells (Fig 2A and 2B). This upregulation of KISS1R was found to be mediated only by direct INA-6 cell contact, since indirect co-culturing using a trans-well setup failed to show a significant increase in KISS1R mRNA of MSCs relative to controls (p = 0.1764) (Fig 2C). In addition, physical interaction with myeloma cell lines AMO1 and U266 clearly enhanced expression of KISS1R mRNA in bone-forming cells (S1 Fig).

Fluorescently-labeled kisspeptin binds to MSCs in vitro after direct culturing with MM cells
In order to detect upregulation of the KISS1R on MSCs after direct contact with MM cells, kisspeptin was synthesized with an additional N-terminal cysteine residue to permit labeling with the fluorescent dye Alexa Fluor 633 via a maleimide conjugation. Successful conjugation was confirmed by HPLC, which showed shifted peaks at both 633 nm and 280 nm for the conjugated peptide, relative to the unconjugated kisspeptin (Fig 3A and 3B). Shifted peaks were found to overlap, supporting a successful conjugation strategy (Fig 3C). The utility of Alexa 633-kisspeptin was tested first in vitro using cultured MCF-7 cells, and in vivo, Primary MSCs were incubated with either free dye or the conjugated Alexa 633-kisspeptin and imaged by fluorescence microscopy (Fig 4). While addition of free dye revealed no notable fluorescence, addition of Alexa 633-kisspeptin showed low level, heterogeneous binding to MSCs (Fig 4A). Longer exposure images revealed that Alexa 633-kisspeptin does bind to cultured MSCs at basal levels. To determine if co-culturing leads to an up-regulation of KISS1R protein expression, MSCs were cultured either in conditioned media or directly with human (INA-6) or mouse-derived (MOPC) MM cells, incubated with Alexa 633-kisspeptin, and imaged by fluorescence microscopy (Fig 4B). While MSCs cultured in conditioned media showed low level probe binding, similar to levels observed under normal culture conditions, both MSCs and MM cells bound the fluorescent kisspeptin-probe at high levels after direct coculture conditions, supporting mRNA expression data. Alexa 633-kisspeptin shows increased binding in limbs containing MM bone lesions To determine the biodistribution of the Alexa 633-kisspeptin and, importantly, to assess its feasibility as an in vivo biomarker for multiple myeloma, the kisspeptin probe was injected into naïve, immune-competent mice and organ uptake was quantified (Fig 5A and 5B). Along with the liver and kidneys, which showed significantly high levels of fluorescence (S1 Table), a notable amount of probe was also detected in the ovaries, an organ rarely associated with non-specific probe uptake and known to express high levels of KISS1R [35,36]. To assess probe binding in the context of myeloma bone disease, we injected mice intravenously with the syngeneic firefly luciferase-expressing MM cell line MOPC and monitored sites of tumor formation with BLI ( Fig 5C). Mice which had MM bone lesions in the proximal tibia/distal femur region of one leg only were injected with Alexa 633-kisspeptin i.v. and subsequently imaged for 1 h with time-lapse fluorescence imaging (Fig 5D). Significantly greater peak fluorescence levels were reached in tumor-burdened limbs as compared to contralateral sites (Fig 5E), indicating specific probe uptake at sites of MM manifestation.

Discussion
Sensitive and early stage imaging of MM bone lesions is an important but challenging aim to improve active disease monitoring and to allow for timely therapeutic interventions in patients at the early onset of progressive disease. While traditional imaging techniques rely on the direct imaging of either the tumor itself or indirectly monitoring the resulting bone loss, an imaging biomarker sensitive to both the tumor and its surrounding microenvironment may provide a highly responsive tool in the diagnosis of MM bone lesions at early stages. In this study, we have identified the KISS1R as a novel biomarker for multiple myeloma bone disease. KISS1R and KISS1 expressions varied in MSC and OPC from different donors, which may be explained by the intrinsic heterogeneity of MSC population. However, levels of KISS1R increased significantly in MSCs and OPCs when cultured with INA-6 cells, with the strongest effects under direct co-culture conditions. We confirmed these in vitro expression analyses with further experiments utilizing a fluorescently-labeled kisspeptin probe. Because of its higher stability [37,38], the 54 amino acid form of kisspeptin was selected, though 10, 13, and 14 amino acid isoforms have also been identified as KISS1R ligands [39,40]. Initial in vitro and in vivo screens using the KISS1R-expressing breast cancer cell line MCF-7 [33] revealed strong binding of the kisspeptin probe, validating its potential as a KISS1R marker. The Alexa 633-kisspeptin probe bound to MSCs only at low levels when cultured alone or with conditioned media derived from MM cells, but specific binding to both MSCs and MM cells increased markedly in a direct co-culture setting. We next sought to take advantage of this increased kisspeptin binding to MM and its microenvironment in vivo. A limited biodistribution analysis revealed uptake by the liver and kidneys, as is commonly observed for intravenously administered probes. Notably, fluorescent signal was also observed in the ovaries, an organ known to express high levels of the KISS1R and not traditionally associated with intravenously-injected probes [35,36]. In the context of progressive multiple myeloma bone disease, probe detection   increased significantly in tumor-burdened limbs when compared to the contralateral MM-free sites. These data provide strong evidence that the KISS1R is highly expressed within tumorburdened regions of the skeleton and that this KISS1R upregulation can be utilized as an efficient biomarker of myeloma bone disease. Because the KISS1R is expressed not only by the MM cells themselves, but also by the surrounding stroma cells upon MM cell contact, this new biomarker has the potential to serve both as an early and sensitive diagnostic tool. Concomitantly, kisspeptin can be developed as a potent therapeutic targeting agent, which could help deliver therapeutic agents not just to the resistant tumor cells, but also to the cells forming the The data presented herein support KISS1R as a novel and promising new biomarker for myeloma bone disease, but still several questions remain. Firstly, while we have described the up-regulation of the KISS1R cells directly interacting with MM cells, little is known so far about the role of kisspeptin and the KISS1R in MM disease progression. It will be of great importance to understand the role of kisspeptin and the KISS1R in both bone marrow cells and MM, and to be aware of what potential side effects, either positive or negative, may be associated with targeting or activation of this system. While KISS1 is noted as a tumor suppressor in melanoma metastasis [14], the role of KISS1R signaling on breast cancer invasion is dependent on the estrogen receptor status of the tumor cells [41]. Secondly, the signal to background ratio obtained in this study could potentially be improved by enhancing the short half-life of kisspeptin. Since kisspeptin is cleaved and inactivated by matrix metalloproteinase 2 [42], an enzyme that plays a role in MM progression [43], imaging may benefit from the use of a noncleavable form of the kisspeptin. Furthermore, rational modifications of kisspeptin have been described which could further increase the stability of the peptide [44][45][46][47].
In conclusion, the KISS1R provides a promising new opportunity for the diagnosis of MM bone disease. As a novel strategy it allows for targeting both the tumor cells and the host response to tumor arrival. This brings about an enhanced potential for monitoring especially early changes in bone microenvironment along disease development and also for treatment response.