Induced Tumor-Suppressing (iTS) Cell-Based Approach for Protecting the Bone from Advanced Prostate Cancer

TL;DR

This paper introduces a new cell-based approach to protect bones from advanced prostate cancer by using tumor-suppressing cells and proteins.

Contribution

The study introduces the iTS cell-based approach and identifies Moesin (MSN) as a novel extracellular tumor-suppressing protein.

Findings

Lrp5 and β-catenin overexpression can convert various cells into iTS cells.

Moesin (MSN) acts as an extracellular tumor-suppressing protein by interacting with CD44.

Extracellular MSN reduces Src tyrosine kinase activity and β-catenin nuclear localization.

Abstract

Advanced prostate cancer frequently metastasizes to bone, but no effective therapy exists. To seek a novel treatment option and identify a new drug target, we took an induced tumor-suppressing (iTS) cell-based approach and produced tumor-suppressing proteins and conditioned medium (CM). Notably, the overexpression of Lrp5 and β-catenin, as well as the pharmacological Wnt activator, converted osteocytes, Murine mesenchymal stem cells, mononuclear cells, and monocytes into iTS cells. While Lrp5 conditional knockout mice presented severe bone loss, Lrp5-overexpressing osteocyte-derived CM rescued tumor-induced bone damage. Whole-genome proteomics analysis revealed that Moesin (MSN), which acted as an oncogene in tumor cells, was enriched in CM as an extracellular tumor-suppressing protein. Its anti-tumor action was mediated primarily by the interaction with CD44. Consistently, FRET live-cell imaging demonstrated that extracellular MSN reduced Src tyrosine kinase activity and nuclear localization of β-catenin. Collectively, we demonstrated herein the iTS cell-based approach to protect bone from prostate cancer and showed MSN as a potent extracellular tumor-suppressing protein.

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Full text

1. Introduction

Prostate cancer is the most common cancer in men, particularly those over the age of 50. The increased risk in this age group is associated with age-related hormonal changes, cumulative genetic mutations, and reduced immune surveillance [1,2]. Despite increased screening and a steady decline in mortality, it remains the second leading cause of cancer deaths among men in the US, with heart disease being the leading cause [3]. Bone provides a hospitable microenvironment, and advanced stages of the disease nearly always involve bone dissemination. Metastasized prostate cancer induces pain, pathological bone fractures, and spinal cord compression, significantly decreasing survival rates [4]. Treatment options include bisphosphonates to inhibit bone-resorbing osteoclasts, radiation therapy to alleviate pain and reduce tumor progression, and palliative surgery to stabilize collapsed bones [5]. Notably, metastasis-directed stereotactic body radiotherapy has shown promise in oligometastatic disease, offering a targeted approach to reduce tumor burden [6]. While these treatments can help reduce medical complications, an effective therapy that improves survival rates without inducing life-threatening side effects is still an unmet need.

Prostate and breast cancers preferentially metastasize to bone, but it is not well understood whether the same mechanism governs their seed-soil communications. Over 90% of prostate and breast cancers are adenocarcinomas, establishing a vicious cycle that exacerbates tumor-induced bone degradation [7]. A key distinction lies in the nature of bone lesions: most breast cancers induce osteolytic lesions, whereas prostate cancer is associated with osteoblastic lesions and reduced bone density [8,9]. Osteocytes orchestrate bone homeostasis, in which Wnt signaling is activated via Lrp5, a Wnt co-receptor [10], with β-catenin acting as a signal transducer. Wnt signaling is tightly associated with the progression of many types of cancer [11,12,13], but the role of Lrp5-mediated Wnt signaling in prostate cancer-associated bone destruction has not been fully elucidated.

In our previous cancer studies, we demonstrated that the overexpression of pro-tumorigenic genes can convert many types of tumor and non-tumor cells into induced tumor-suppressing (iTS) cells [14]. For example, the overexpression of Akt in PI3K signaling, Snail in the induction of EMT, and Lrp5 and β-catenin in Wnt signaling converted osteocytes and Murine mesenchymal stem cells (MSCs) [15], as well as breast cancer cells, pancreatic cancer cells [16], and osteosarcoma cells [17], into iTS cells [15,18]. The whole-genome proteomics analysis and the subsequent cell culture assays identified the enrichment of unique tumor-suppressing proteins such as Hsp90ab1, Calreticulin, Enolase 1, Aldolase A, Histone H4, and polyubiquitin C [14,19,20]. The question herein was whether these iTS cell-derived tumor-suppressing proteins can effectively inhibit the progression of prostate cancer and its associated bone destruction.

Many lines of evidence indicate that iTS cells are competitive cells with a high level of survival fitness [21]. In Drosophila wing discs, iTS-like cells with a high rate of protein synthesis are reported to eliminate neighboring cells with a low rate of protein synthesis. Likewise, cells with higher Myc levels in mouse embryos eliminate those with lower Myc levels [22]. We have previously shown that the overexpression of c-Myc transformed MSCs into iTS cells [23]. These examples suggest the possibility of eliminating cancer cells with metabolically active iTS cells.

We examined the effects of iTS cell-derived CM on three processes: (a) bone degradation by prostate cancer cells, (b) bone resorption by osteoclasts, and (c) bone formation by osteoblasts. The goal herein was to inhibit tumor progression without blocking bone homeostasis by osteocytes, osteoclasts, and osteoblasts [24]. To cope with the potential differences in prostate and breast cancers, we employed both male and female mice as well as osteocyte-specific Lrp5 conditional knockout mice.

To determine the regulatory mechanism underlying the iTS cell-based approach, we conducted two sets of mass spectrometry-based proteomics analyses. We first identified a group of proteins that were enriched in Wnt-activated osteocyte-derived CM. Moesin (MSN) was identified as one of such tumor-suppressing protein candidates. MSN can regulate a variety of inflammation-related events to enhance immune lymphocyte infiltration [25]. Immuno-precipitation followed by in vitro assays, together with FRET (fluorescent resonance energy transfer)-based live-cell imaging, revealed that MSN interacted with CD44, a cell-adhesion molecule and cancer stem-cell marker. Although MSN is reported to be pro-tumorigenic in tumor cells [26], our findings demonstrate that it acted as an extracellular tumor-suppressing protein. A dichotomous role of MSN has already been reported [27], as other proteins such as high mobility group protein B1 are also reported to have both oncogenic and tumor-suppressing roles [28].

The generation of iTS cells can be achieved through the activation of proliferative signaling pathways such as PI3K and PKA [14], as well as biophysical stimulations like low-intensity vibration [29]. In this study, we highlight the dual role of Wnt signaling in both promoting the progression of prostate cancer and building the tumor-suppressive bone microenvironment via osteocytes. Additionally, we explore the potential of identifying druggable protein targets, such as CD44, which interacts with iTS cell-derived tumor-suppressing proteins.

2. Materials and Methods

2.1. Cell Culture

TRAMP-C2ras prostate tumor cells (ATCC, CRL-2731) were cultured in DMEM/F-12 medium as described [30]. EO771 mouse mammary tumor cells (CH3 BioSystems, Amherst, NY, USA), 4T1.2 mouse mammary tumor cells (obtained from Dr. R. Anderson at Peter MacCallum Cancer Institute, Melbourne, Australia), MDA-MB-231 breast cancer cells (ATCC, Manassas, VA, USA), and J774A.1 mouse monocytes were cultured in DMEM. MC3T3 osteoblast-like cells (Sigma-Aldrich, St. Louis, MO, USA), MLO-A5 osteocytes (obtained from Dr. L. Bonewald at Indiana University, Indianapolis, IN, USA), and RAW264.7 pre-osteoclast cells were grown in αMEM. PC-3 human prostate cancer cells (ATCC, CRL-1435), Jurkat T lymphocytes, and human monocytes were cultured in RPMI-1640 (Gibco, Carlsbad, CA, USA). Murine mesenchymal stem cells (MSCs), isolated from the bone marrow of C57BL/6 mice (Envigo RMS, Inc., Indianapolis, IN, USA), were cultured in MesenCult medium (Stem Cell Technology, Cambridge, MA, USA). All culture media were supplemented with 10% fetal bovine serum (FBS) and antibiotics (50 units/mL penicillin and 50 µg/mL streptomycin), and cells were maintained at 37 °C in a 5% CO_2_ incubator. Recombinant TRAIL proteins (752904, BioLegend, San Diego, CA, USA) were administered to tumor cells.

2.2. Preparation of Conditioned Medium (CM)

CM for in vitro assays was prepared by centrifuging culture media at 2000 rpm for 10 min, and the cell-free supernate was further centrifuged at 4000 rpm for 10 min, followed by filtration with a 0.22 µm polyethersulfone membrane (Sigma). CM for in vivo experiments was prepared in serum-free media. The centrifuged CM was cleaned by a filter with a cutoff molecular weight of 3 kDa and condensed in PBS 10-fold for intravenous injection from the tail vein.

2.3. Generating the CM from Human Peripheral Blood

The study to use human peripheral blood samples was approved by the ethics committee of Osaka University (protocol #21344, approval date 26 August 2022) and conducted according to the guidelines of the Declaration of Helsinki. All samples were collected with prospective informed consent from donors, and all participants were informed of the study purpose, procedures, potential risks, and their right to withdraw at any time without penalty. From 5 healthy volunteers, 8 mL of peripheral blood was collected and diluted with an equal volume of 0.9% NaCl solution. Mononuclear cells were isolated using a lymphoprep kit (# 1114544, Abbott Diagnostics Technologies AS, Oslo, Norway). After centrifugation at 800× g for 30 min at room temperature, the mononuclear cell fractions were collected with a Pasteur pipette. The AlyS705 medium (Cell Science & Technology Institute, Inc., Tendō, Japan) was used to culture mononuclear cells in the presence of 0.2 µM BML284 for 1 day. The culture medium was then changed to a fresh medium, and cells were grown for 1 day. The supernatant was collected and subjected to sequential centrifugation steps (1000× g for 10 min, 14,000× g for 2 min, and 14,000× g for 60 min) to isolate CM. Using a centrifugal evaporator, CM was concentrated approximately 10-fold.

2.4. MTT Assay, EdU Assay, Transwell Invasion Assay, and Scratch Assay

Cell viability and proliferation were examined using an MTT assay and fluorescence-based EdU assay (Thermo-Fisher, Waltham, MA, USA). In the MTT assay, optical density for assessing cell metabolic activities was measured at 570 nm using a multi-well spectrophotometer. In the fluorescence-based EdU cell proliferation kit, we counted the number of fluorescently labeled cells and determined the ratio to the total number of cells [31]. A transwell invasion assay was conducted to detect cell motility, and a wound-healing scratch assay was utilized to evaluate 2-dimensional cell motility [32].

2.5. Western Blot Analysis

Cultured cells were lysed in a radio-immunoprecipitation assay buffer with protease inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phosphatase inhibitors (Calbiochem, Billerica, MA, USA). The protein concentration was determined using a BCA Protein Assay kit (Thermo-Fisher). Proteins were fractionated by 10–15% SDS gels and electro-transferred to polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA). After blocking for 1 h with a blocking buffer (Bio-Rad, Hercules, CA, USA), the membrane was incubated overnight with primary antibodies and then with secondary antibodies conjugated with horseradish peroxidase for 45 min (Cell Signaling, Danvers, MA, USA). The level of proteins was determined using a SuperSignal west femto maximum sensitivity substrate (Thermo Scientific), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities. We used antibodies against CD44 (37259T), cleaved caspase 3 (9661S), Lrp5 (5731S), MSN (3726T), Snail (3879S), TGFβ (3711S), Vimentin (5741T) (Cell Signaling), Fibronectin 1 (sc-271098), cathepsin K (sc-48353), MMP9 (sc-393859), NFATc1 (sc-7294) (Santa Cruz Biotechnology), LIMA1 (Nbp1-87947), TRAIL (NB500-220) (Novus, Centennial, CO, USA), ANXA6 (ab199422) (Abcam, Cambridge, UK), p53 (MA5-12557) (Invitrogen, Carlsbad, CA, USA), and β-actin (Sigma).

2.6. Plasmid Transfection, RNA Interference, and Cytokine Analysis

Using 2 × 10^6^ cells, Lrp5, β-catenin, p53, and MSN plasmids (40 ng/µL) were transfected. Conditioned medium (CM, 9 mL) was prepared with antibiotics and a fraction of FBS consisting of factors with 3 kDa or smaller. After one day of incubation, the medium was ultra-centrifuged to remove exosomes and condensed 10-fold by filtering (Amicon, Sigma, Saint Louis, MO, USA) to collect proteins larger than 3 kDa. For overexpressing plasmids, Lrp5 (#115907, Addgene, Watertown, MA, USA), β-catenin (#31785, Addgene), p53 (#69003, Addgene), and MSN (#11637, Addgene) were transfected into A5 osteocytes and RAW264.7 pre-osteoclasts separately, while a blank plasmid vector (FLAG-HA-pcDNA3.1; Addgene) was used as a control. Osteocytes were treated with shRNA specific to Lrp5 (sc-149050-V, Santa Cruz), with control GFP shRNA (sc-108084, Santa Cruz). Cells were transfected with lipofectamine^®^3000 (L300015, Thermo Scientific) [33], and stable transfectants were selected using puromycin (Sigma). Using a Lipofectamine™ RNAiMAX transfection reagent (Thermo, 13778100) and the negative control siRNA (Thermo, 4390843), osteocytes were transfected with TRAIL siRNA (S75440, Thermo Scientific) and MSN siRNA (S70072, Thermo Scientific), RAW264.7 cells with TRAIL siRNA (S75440, Thermo Scientific), and TRAMP cells with CD44 siRNA (S63659, Thermo Scientific), and Vimentin siRNA (S75963, Thermo Fisher).

2.7. 3D Spheroid Assay and Ex Vivo Tissue Assay

Cells were cultured in ultra-low attachment 96-well plates (S-BIO, Hudson, NH, USA) at 1 × 10^4^ cells/well for TRAMP cells. Spheroid images were captured every 24 h, and areas were quantified using Image J software (v1.52p). For the ex vivo tissue assay, the usage of human prostate cancer tissues was approved by the Indiana University Institutional Review Board. A sample (1 g), received from Simon Cancer Center Tissue Procurement Core, was manually fragmented with a scalpel into small pieces (0.50.8 mm in length). These pieces were grown in DMEM with 10% fetal bovine serum and antibiotics for a day. CM^Oy-Lrp5^ was then added for two additional days, and changes in the fragment size were determined.

2.8. Live Cell Imaging

To evaluate tension force at a focal adhesion and migratory capacity of tumor cells in response to CM^Oy-Lrp5^, a plasmid expressing a vinculin tension sensor (VinTS, #26019, Addgene) was transfected. The fluorescence lifetime images were acquired by a custom-made microscope built on a laser scanning confocal microscope (FluoView 1000, Olympus; Center Valley, PA, USA), using the procedures previously described [34]. Of note, a decrease in the tension force of the vinculin sensor implies an increase in fluorescence efficiency. To visualize Src activity, a Src biosensor (#78302, Addgene) based on fluorescence resonance energy transfer (FRET) was used. FRET images were acquired by a Nikon Ti-E microscope equipped with a charge-coupled device camera (Evolve 312, Photometrics, Huntington Beach, CA, USA) using the procedures previously described [34]. An enhanced GFP-β-catenin (#16071, Addgene) was used to monitor the translocation of β-catenin to the nucleus. The GFP images were quantified by the procedures previously described [35].

2.9. Animal Models

The experimental procedures using animals were approved by the Indiana University Animal Care and Use Committee and complied with the Guiding Principles in the Care and Use of Animals endorsed by the American Physiological Society. C57BL/6 mice lacking Lrp5 in osteocytes (Dmp1-Cre; Lrp5f/f; conditional Lrp5 knockout mice) were created by breeding Dmp1-Cre transgenic mice with Lrp5 floxed mice, both of which have been described earlier [36]. Mice were housed in five per cage and provided with mouse chow and water ad libitum.

In the mammary tumor model, we conducted a proof-of-principle study, defined as an initial exploratory assessment to validate the therapeutic feasibility of osteocytes by injecting osteocyte cells into the mammary fat pads. This model was specifically selected for its utility in early-stage feasibility evaluation, enabling us to preliminarily confirm whether osteocytes can exert anti-tumor effects through their secreted factors. C57BL/6 female mice (~8 weeks, Envigo RMS, Inc., Indianapolis, IN, USA) received subcutaneous injections of TRAMP cells or EO771 cells (3.0 × 10^5^ cells in 50 µL PBS) to the mammary fat pad. Also, Lrp5-overexpressing osteocytes (3.0 × 10^5^ cells) were co-injected at the seventh mammary fat pad, and osteocyte-derived CM was injected into the intraperitoneal cavity from day 2 to day 18. The mice were euthanized on day 18 for tumor weight measurement. For the osteolysis mouse model, ten C57BL/6 male mice per group received intra-tibial injections of TRAMP cells (3.0 × 10^5^ cells in 20 µL PBS). CM^Oy^ with and without Lrp5 overexpression was also injected at the proximal tibia from day 2 to day 18. Roughness was calculated by fitting an ellipse to the spheroid and summing its non-overlapping areas. In the extravasation assay, EO771 cells (1.0 × 10^6^ cells) were labeled with a green fluorescent dye and injected with and without CM^iTSC^ via a lateral tail vein in C57BL/6 female mice (5 mice per group). Fluorescently labeled EO771 cells were prepared by culturing them with a green fluorescent dye (#4705, Sartorius, Gottingen, Germany) for 20 min at 37 °C. Cells were then centrifuged at 1000 rpm for 5 min to harvest the pellet. The pellet was re-suspended in PBS (placebo group) or CM^Oy^. Mice were sacrificed after 48 h for histological identification of extravascular tumor cells in the lung.

2.10. X-Ray, CT Imaging, and Histology

A whole-body X-ray image was taken using the Faxitron radiographic system (Faxitron X-ray Co., Tucson, AZ, USA). The tibia was harvested for µCT imaging and histology. Micro-computed tomography was performed using Skyscan 1172 (Bruker-MicroCT, Kontich, Belgium) with a voxel size of 8.99 µm, and the images were reconstructed and analyzed using CTan v1.13 software. In histology, H&E staining was conducted as described previously.

2.11. Whole-Genome Proteomics

Q-exactive high-field hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher) was employed to identify proteins in CM with the Dionex UltiMate 3000 RSLC system. Using trypsin/LysC. Proteins were separated using a trap and 50-cm analytical columns. Raw data were processed using MaxQuant (v1.63.3) against the Uniprot mouse protein database at a 1% false discovery rate. Protein quantitation was based on MS/MS counts, and we analyzed proteins that were identified with at least 1 unique peptide and 2 MS/MS counts.

To evaluate the tumor-suppressing capability of the predicted candidates, we employed ten recombinant proteins, such as Actg1 (H00000071-P01, Novus), Aldoa, Eno1, Calm1, Eef1a1, Lmna, MSN, Pkm, Eef2, and Ywhaz (MBS8248528, MBS2009113, MBS2018713, MBS2033168, MBS143846, MBS2031729, MBS8249600, MBS1213669, MBS143242, MyBioSource, SanDiego, CA, USA). In the MTT assay, 5 µg/mL of each recombinant protein was added to assess tumor cell viability.

2.12. Immunoprecipitation

Immunoprecipitation was conducted with an immunoprecipitation starter pack kit (Cytiva, Marlborough, MA, USA) following the manufacturer’s protocol. Briefly, 20 µL of protein A sepharose was washed twice with PBS and incubated with 2 µg of rabbit-raised antibodies for MSN. Rabbit IgG was prepared as a negative control. The antibody-cross-linked beads were incubated overnight with 600 µL protein samples on a shaker. The beads were collected by centrifugation, washed three times with PBS, and resuspended for Western blotting. The protein samples before immunoprecipitation were used as positive controls.

2.13. Statistical Analysis

For cell-based experiments, three or four independent experiments were conducted, and data were expressed as mean ± SD. In animal experiments, the sample size in the mouse model was chosen to achieve a power of 80% with p < 0.05. The primary experimental outcome was tumor weight for the mammary fat pad experiment and the bone volume ratio (BV/TV) for the tibia experiment. The secondary experimental outcome was tumor size for the mammary fat pad experiment and the trabecular number (Tb.N) for the tibia experiment. All data were tested for normality (Shapiro–Wilk normality test) before statistical tests. Statistical significance was evaluated using a one-way analysis of variance (ANOVA). Post hoc statistical comparisons with control groups were performed using Bonferroni correction with statistical significance at p < 0.05. A nonparametric Kolmogorov-Smirnov test was applied to evaluate FRET efficiency in live-cell imaging. The single and double asterisks in the figures indicate p < 0.05 and p < 0.01, respectively.

3. Results

3.1. Inhibition of TRAMP-C2ras Prostate Tumor Cells by CMOy-Lrp5

We first examined the response of TRAMP-C2ras (TRAMP) prostate tumor cells to MLO-A5 osteocyte-derived CM (CM^Oy^). In the MTT-based viability, EdU-based proliferation, CM^Oy^ did not significantly alter tumor cell behaviors. When Lrp5 was overexpressed, however, CM^Oy-Lrp5^ reduced the viability and proliferation of TRAMP cells (Figure 1a,b). Fluorescent live-cell imaging showed that CM^Oy-Lrp5^ increased the FRET efficiency of the vinculin biosensor in TRAMP cells (Figure 1c). The observed increase implies a decrease in vinculin-mediated molecular force at focal adhesions, in agreement with the inhibition of migratory behaviors. Consistent with the suppression of cell motility, the scratch-based migration and transwell invasion of prostate cancer cells were also inhibited by CM^Oy-Lrp5^ (Figure 1d,e). CM^Oy^ slightly but significantly inhibited the growth of 3-dimensional tumor spheroids, and CM^Oy-Lrp5^ enhanced the growth inhibition (Figure 1f).

3.2. Inhibition of PC-3 Human Prostate Tumor Cells and Breast Cancer Cells by CMOy-Lrp5

Consistent with the response of TRAMP cells, the inhibitory effects of CM^Oy-Lrp5^ were also detected in PC-3 human prostate tumor cells in the MTT-based viability, EdU-based proliferation (Figure 2a,b), scratch-based migration, transwell invasion, and tumor spheroid assays (Figure 2c–e). Besides the tumor-suppressing capability of CM^Oy-Lrp5^ in prostate cancer cells, we employed freshly isolated human prostate cancer tissues to correlate with clinical significance. The tissue was manually fragmented, and the fragments were cultured in CM^Oy-Lrp5^. The result revealed that compared to the placebo (control CM^Oy^), the CM^Oy-Lrp5^ group significantly reduced the size of cancer fragments (Figure 2f).

To compare the effects of CM^Oy-Lrp5^ between prostate and breast cancer cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells were examined. Consistently, the overexpression of Lrp5 inhibited the proliferation and migration of EO771 and MDA-MB-231 cells (Figure S1a,c). To further evaluate the efficacy of Lrp5-overexpressing osteocytes, TRAMP cells were injected into the mammary fat pad of female C57BL/6 mice. Notably, the weight of tumor tissues was significantly reduced by a local injection of Lrp5-overexpressing osteocytes (Figure S2a).

3.3. Inhibition of the Tumor Progression in the Tibia by CMOy-Lrp5

We next examined the in vivo effect of CM^Oy-Lrp5^ on the extravasation to the lung and osteolysis of the tibia. The intravenous injection of CM^Oy-Lrp5^ with TRAMP cells markedly reduced the number of tumor cells that invaded into the lungs of C57BL/6 male mice (Figure 3a). In the male and female mouse models, tumor cells invaded the tibia by the inoculation of TRAMP and EO771 cells, respectively. In the TRAMP cell-invaded tibia and fibula, the tibia was swollen, and the fibula was enlarged with an increase in surface roughness (Figure 3b,c and Figure S2b). In response to the inoculation of EO771 cells in female mice, however, the tibia was not swollen, and the fibula was not enlarged, although the surface roughness of the fibula was elevated (Figure 3d,e).

Three-dimensional CT reconstruction of the proximal tibia and histological examination supported the tumor-suppressing action of CM^Oy-Lrp5^ in male mice. Compared to the placebo that received the inoculation of TRAMP cells alone, the CM^Oy^ and CM^Oy-Lrp5^ injected groups reduced trabecular bone loss in the proximal tibia with the elevation of bone mineral density, bone volume ratio, and trabecular number, with a decrease in trabecular separation (Figure 3f). H&E-stained histological sections revealed that the tumor-invaded area was markedly reduced by the local injection of CM^Oy^ and CM^Oy-Lrp5^ (Figure 3g).

3.4. Increased Tumor-Driven Bone Loss in Lrp5-Deleted Mice

Consistent with the tumor-suppressing effects of the overexpression of Lrp5, Lrp5-deleted conditional knockout mice showed a more severe bone loss in trabecular bone in the TRAMP cell-inoculated tibia than their wild-type littermates (Figure 4a–c). Furthermore, the injection of CM^Oy-Lrp5^ to the proximal tibia significantly improved the bone microstructure, with an increase in bone mineral density, bone volume ratio, and the trabecular number, together with a decrease in the trabecular separation (Figure 4d). H&E-stained histological sections also exhibited a notable increase in the tumor-invaded area in Lrp5-deleted mice and a pronounced decrease by the administration of CM^Oy-Lrp5^ (Figure 4e).

3.5. Inhibition of Osteoclast Development by CMOy-Lrp5

The results obtained so far support the tumor-suppressing and bone-protective capability of CM^Oy-Lrp5^. Since osteoclasts are bone-resorbing cells, we examined whether Lrp5 overexpression in osteocytes might affect the development of osteoclasts. The result showed that the RANKL-driven development of RAW264.7 pre-osteoclasts was inhibited by CM^Oy-Lrp5^, with the downregulation of NFATc1 and cathepsin K (Figure 5a,b).

3.6. Generation of iTSCs from MSCs and Peripheral Blood Cells

Besides osteocytes, we examined whether MSCs and peripheral blood cells could be converted into induced tumor-suppressing cells (iTSCs) and generate tumor-suppressive CM by the activation of Wnt signaling. Notably, we observed that Lrp5-overexpressing MSC-derived CM (CM^MSC-Lrp5^) decreased the viability and invasion of TRAMP tumor cells (Figure 5c–e). Furthermore, β-catenin-overexpressing CM (CM^MSC-βcat^) reduced the viability and invasion of TRAMP tumor cells (Figure 5f,g). The inhibition was also observed in BML284-treated MSC-derived CM (CM^MSC-BML^), in which BML284 is a pharmacological activator of Wnt signaling.

Regarding peripheral blood cells, we examined three cell types: mononuclear cells isolated from human peripheral blood samples, two monocyte cell lines (J774A.1 and THP-1), and a Jurkat T lymphocyte cell line. Of note, mononuclear cells refer to blood cells that have a single, round nucleus, such as lymphocytes and monocytes. First, BM284-treated mononuclear cell-derived CMs (CM^mononuclear-BML^), which were generated from 5 independent sources, decreased the viability of PC-3 prostate cancer cells (Figure 5h). Second, CM^J774A.1-BML^ and CM^THP1-BML^ also suppressed the viability of PC-3 and TRAMP tumor cells (Figure 5i and Figure S3a,b). However, CM^Jurkat-BML^ did not present an inhibitory effect on these prostate cancer cells (Figure S3c).

We should note that the activation of Wnt signaling by Lrp5 or β-catenin is a double-edged sword. Their overexpression in TRAMP tumor cells presented two contrasting effects, which highlighted the tumorigenic role of Wnt signaling in tumor cells and the anti-tumorigenic role of Wnt-activated cell-derived CM. In TRAMP cells, the overexpression of Lrp5 elevated EdU-based proliferation, associated with the increase in MMP9, Snail, and TGFβ (Figure S4a). However, β-catenin-overexpressing TRAMP-derived CM (CM^TRAMP-βcat^) decreased MTT-based viability and transwell invasion of initial TRAMP tumor cells (Figure S4b,c).

3.7. Identification of Tumor-Suppressing Proteins

In vitro and in vivo results so far support the tumor-suppressing capability of CM^Oy-Lrp5^. Western blot analysis revealed that the overexpression of Lrp5 in osteocytes elevated the levels of known tumor suppressors in both osteocytes and CM^Oy-Lrp5^, including p53, ANXA6, and LIMA1, as well as TRAIL, an apoptosis-inducing factor (Figure S5a,b). As expected, CM^Oy-Lrp5^ downregulated tumor-promoting proteins, including MMP9, Snail, and TGFβ, in TRAMP tumor cells, while this downregulation was suppressed by Lrp5-silenced CM (Figure S5c). The same trend of gene regulation was observed with breast cancer cell lines (Figure S5d).

We herein focused on 10 potential tumor suppressors based on our previous whole-genome proteomics analysis (Figure 6a) [18], and the viability of TRAMP tumor cells was evaluated in response to their recombinant proteins (Figure 6b). Among 10 candidates, we focused on the role of MSN since its administration induced a significant decrease in MMT-based viability. MSN is a cross-linker between plasma membranes and actin-based cytoskeletons. In response to the application of 5 µg/mL recombinant MSN proteins, MTT-based viability and transwell invasion were reduced, and MMP9, Snail, and TGFβ were downregulated, with the upregulation of cleaved caspase 3 (Figure 6c–e).

3.8. Dichotomous Role of Extracellular and Intracellular MSN

In agreement with the action of recombinant MSN protein, MSN-overexpressing osteocyte-derived CM (CM^Oy-MSN^) showed anti-tumor capability by reducing MTT-based viability, transwell invasion, and reducing pro-tumorigenic proteins such as MMP9, Snail, and TGFβ (Figure 6f–h). Consistently, MSN-silenced CM reversed these responses (Figure 6i and Figure S6a,b). The level of MSN was increased in CM^Oy-Lrp5^ (Figure 6j), and in CM^Oy-MSN^ the levels of p53, ANXA6, LIMA1, and TRAIL were elevated (Figure 6k). By contrast, the overexpression of MSN in TRAMP cells elevated MMP9, Snail, and TGFβ, and reduced cleaved caspase 3 in TRAMP cells (Figure 6l). We also observed the elevation of MTT-based viability and transwell invasion of MSN-overexpressing TRAMP prostate cancer cells (Figure S6c,d). Collectively, these results demonstrated that extracellular and intracellular MSN proteins acted oppositely, as tumor-suppressive and tumor-enhancing proteins, respectively.

3.9. Tumor Selectivity by CMOy-Lrp5 and MSN

We next examined whether CM^Oy-Lrp5^ and MSN may selectively inhibit the progression of tumor cells without damaging non-tumor cells. The tumor selectivity, λ, was defined as the MTT-based ratio of the reduction in the viability of tumor cells to that of non-tumor cells. A value λ larger than one indicates that the inhibition is more selective to tumor cells than non-tumor cells. We observed that the tumor selectivity of CM^Oy^, CM^Oy-Lrp5^, and MSN was above 1 based on two tumor cell lines (TRAMP and EO771) and two non-tumor cell types (MSCs and MC3T3 osteoblasts) (Figure 7a). Although the MTT-based viability depends on the types of tumor and non-tumor cells, the result was consistent with the selective anti-tumor actions of CM^Oy^, CM^Oy-Lrp5^, and MSN.

3.10. Involvement of CD44 and Fibronectin 1 (FN1) in the Anti-Tumor Action of MSN

To assess the potential involvement of CD44 and FN1 in the tumor-suppressive action of MSN, we conducted an immunoprecipitation assay. CD44 is a transmembrane adhesion receptor and a cancer stem cell marker that acts as a regulator of the Wnt receptor complex, while FN1 is an extracellular matrix (ECM) protein. Western blotting revealed that CD44 and FN1 were co-immunoprecipitated with MSN using TRAMP protein extracts and TRAMP ECM protein extracts, respectively (Figure 7b). Notably, the anti-tumor effect of 1 μg/mL of recombinant MSN proteins on MTT-based viability was significantly suppressed when CD44 and FN1 were silenced (Figure 7c,d). Furthermore, the silencing of CD44 and FN1 in TRAMP cells suppressed MSN-mediated downregulation of MMP9, TGFβ, and Snail (Figure 7e). The results supported the notion that CD44 and FN1 were involved in MSN’s tumor-suppressive action.

Lastly, we conducted FRET-based imaging analysis to evaluate the role of MSN in the activity of Src, tyrosine kinase, and nuclear localization of β-catenin. The result showed that the administration of MSN decreased Src activity and β-catenin translocation to the nucleus in TRAMP cells. Consistent with the expected role of CD44, the silencing of CD44 reversed MSN-driven effects on Src and β-catenin (Figure 7f).

The survival analysis results showed that patients were divided into two groups based on the median transcript level of MSN (N = 4750 per group). Patients in the MSN high-transcript group (transcript level above the median) exhibited a significantly favorable overall survival outcome compared to those in the MSN low-transcript group (transcript level below the median) (p < 0.00001, Figure 8a). The MTT-based proliferation of PC3 prostate cancer cells and MDA-MB-231 breast cancer cells in response to 0.7, 1, 2, and 5 µg/mL MSN for 2 days (Figure 8b). We also detected the response of PC3 human prostate cells and MDA-MB-231 human breast cancer cells to MSN-overexpressing Jurkat-derived CM (CM^Jur-MSN^), in the MTT-based viability, CM^Jur-MSN^ significantly altered tumor cell behaviors (Figure 8c). In response to the application of 5 µg/mL recombinant MSN proteins, MTT-based viability, and PDL1, p-Src were downregulated (Figure 8d).

4. Discussion

This study showed that Lrp5-overexpressing osteocytes became tumor-suppressing, bone-protecting cells in the prostate tumor-invaded bone microenvironment. CM^Oy-Lrp5^ inhibited the proliferation, migration, and invasion of prostate cancer cells, as well as the ex vivo growth of prostate cancer tissues. Lrp5 knockout mice exhibited severe tumor-induced bone destruction, while Lrp5-overexpressing osteocytes rescued this phenotype. CM^Oy-Lrp5^ also inhibited osteoclast development by downregulating NFATc1 and cathepsin K. Mechanistically, increased TRAIL stimulated tumor cell apoptosis, and p53 contributed to the anti-tumor action. Mass spectrometry identified extracellular MSN as a tumor suppressor interacting with CD44 and FN1. These results suggest that extracellular MSN inhibited tumor progression. Although its overexpression in tumor cells stimulated oncogenic behaviors has been reported [26,37]. Notably, Lrp5 and β-catenin overexpression in Wnt signaling generated anti-tumor proteomes, converting a bone-destructive microenvironment into a tumor-suppressive, bone-protective one. These secreted proteins act as signaling molecules that bind to receptors on or inside the tumor cell, thereby sending a signal to tumor cells to “stop growth” or “start apoptosis”. This profoundly reveals the complexity of cancer: whether a protein acting as a double-edged sword is “good” or “bad” cannot be judged in isolation, but must be combined with its specific molecular environment and functional state. This concept is emphasized in the revised Discussion. Of note, the study focused on the proteome.

Lrp5 and β-catenin in osteocytes activate canonical Wnt signaling, maintaining bone homeostasis [38]. Osteocytes, as mechanical sensors, stimulate bone formation by downregulating Sclerostin, an inhibitory Wnt ligand [39]. While Wnt signaling is known to promote tumor progression, this study showed that its function depends on the context, and overexpressing Lrp5 and β-catenin generates tumor-suppressive secretomes in osteocytes, MSCs, and monocytes. Unlike Lrp5, overexpressing Lrp6 and Fzd7 did not produce breast cancer-suppressive secretomes in osteocytes [14].

The anti-tumor actions of CM^Oy-Lrp5^ involve TRAIL, p53 [40], ANXA6, and LIMA1 [41,42], known tumor suppressors. Proteomics analysis identified extracellular MSN as a unique tumor-suppressing protein. While intracellular MSN is pro-tumorigenic, high levels of extracellular MSN are linked to low metastasis potential [43]. Recombinant extracellular MSN inhibited prostate tumor cell proliferation and invasion. MSN interacted with CD44 and FN1, reducing tumor cell viability. CD44, a cell-adhesion receptor and cancer stem-cell marker, can both promote and suppress cancer growth [44]. FN1, an ECM glycoprotein, is involved in tumor growth and is upregulated in various cancers [45]. The study also found other tumor-suppressing proteins in the secretome, including Enolase 1 (Eno1) and eukaryotic translation elongation factor 2 (Eef2).

Besides its tumor-suppressing capability, CM^Oy-Lrp5^ inhibited the differentiation of pre-osteoclasts to multinucleated osteoclasts, with TRAIL playing a role in this inhibition. CM^Oy-Lrp5^ suppressed the expression of NFATc1 and cathepsin K in RANKL-stimulated osteoclasts. While this study focused on prostate cancer, CM^Oy-Lrp5^ also showed tumor-suppressing effects on breast cancer. Notably, CM without Lrp5 overexpression inhibited the proliferation of MDA-MB-231 breast cancer and EO771 mammary tumor cells, but not TRAMP and PC-3 prostate cancer cells. Both cancers reduced bone mineral density in tumor-invaded trabecular bone, but prostate cancer cells induced ectopic bone formation on the cortical surface. The tumor-suppressing action of CM^Oy-Lrp5^ may vary depending on the cancer subtype. Notably, the gender-specific bone responses observed (TRAMP-induced osteoblastic changes in males vs. EO771-induced osteolysis in females) reflect the interplay between tumor-intrinsic metastatic phenotypes and host hormonal/bone structural differences.

While the Lrp5-overexpressing osteocyte-derived proteome was mainly studied here, iTSCs were also generated by the overexpression of β-catenin from MSCs and tumor cells [46]. We previously reported that the overexpression of Akt in PI3K signaling [47] and Snail during the induction of EMT in MSCs can generate tumor-suppressive secretomes [14]. This inhibition was also observed in the administration of BML284, a Wnt activator, which generated tumor-suppressive secretomes from osteoclasts [48]. Regarding MSN’s dichotomous role, its extracellular tumor-suppressive function is driven by secretion from iTS cells and interaction with CD44/FN1, whereas intracellular MSN promotes oncogenic behaviors via cytoskeletal regulation—this switch may also be dependent on subcellular localization and post-translational modifications.

In summary, we demonstrated that Lrp5-overexpressing osteocytes inhibit tumor progression and bone degradation, at least in part, via the MSN-CD44/FN1 regulatory axis. The curative applications and therapeutic properties of CM have been reported in regenerative medicine [49]. Building on the preclinical evidence of prostate tumor selectivity and bone-protective efficacy, the translational potential of iTSC-derived therapies for prostate cancer with bone metastasis could be realized through a phase-adaptive clinical trial. Specifically, standardized CM^Oy-Lrp5^ or recombinant MSN protein could be injected intraosseously into the bone site of tumor invasion to achieve local tumor suppression and osteolysis reversal, while intravenous injection of CM^Oy-Lrp5^ can resolve disseminated micrometastasis. MSN is an ECM-associated protein. One option is to use iTS cell-derived ECM as a therapeutic agent instead of CM, as ECM can be more readily approved by the FDA [50]. The anticancer capabilities of iTS cells have been shown in breast cancer, pancreatic cancer, and osteosarcoma, as well as in bone metastasis and brain metastasis [18]. Furthermore, iTS cells are generated not only from bone cells such as osteocytes, osteoblasts, and osteoclasts but also from MSCs and T lymphocytes [14,51]. Enhancing the anticancer capabilities of T cells is particularly interesting since T-cell-derived iTS cells do not need direct interaction with cancer cells to exert their anticancer actions. Three anticancer peptides, P04, P05, and P18, were also developed from tumor-suppressing proteins, which were enriched in iTS cell-derived CM [52,53,54].

iTSCs therapy is indeed a promising new concept, whose core idea is not to kill cells directly, but to alter the tumor microenvironment by “reprogramming” certain cells to secrete proteins that can inhibit the tumor. iTSCs and their derivatives (conditioned medium or key proteins) offer multiple actionable strategies for clinical use, including engineered cell therapy, exosome-based delivery, or recombinant protein infusion, et al. tumor selectivity is derived from signaling pathway targets and tumor microenvironment. However, other unmodified cells lack the hyperactivated signaling network targeted by its derived factors. Our study has limitations; the exploration of gender mechanisms is limited and needs to be further studied. In addition, the mammary tumor model was not designed to mimic bone metastasis directly, but rather to establish a quick proof-of-concept for the anti-tumor potential of Lrp5-overexpressing osteocytes before transitioning to more complex orthotopic bone models. Further studies are recommended to make iTS cells and their derivatives available as a novel option for cancer treatments.

5. Conclusions

This study demonstrates that Wnt signaling activation via Lrp5 or β-catenin overexpression converts osteocytes, mesenchymal stem cells (MSCs), and monocytes into induced tumor-suppressing (iTS) cells, which secrete a protective conditioned medium (CM) capable of inhibiting prostate cancer progression and preserving bone integrity. The iTS cell-derived CM, particularly from Lrp5-overexpressing osteocytes (CM^Oy-Lrp5^), significantly suppresses tumor cell viability, proliferation, migration, and invasion in vitro, and protects against bone destruction in vivo. Proteomic analysis identifies Moesin (MSN) as a key extracellular tumor-suppressing protein enriched in the CM, which exerts its anti-tumor effects primarily through interaction with CD44, leading to reduced Src kinase activity and β-catenin nuclear translocation. Notably, MSN plays a dichotomous role—acting as an oncogene intracellularly within tumor cells but functioning as a potent tumor suppressor when secreted extracellularly. Importantly, this therapeutic approach shows tumor-selective inhibition without harming non-tumor cells. These findings reveal a novel strategy to transform the bone microenvironment from tumor-permissive to tumor-suppressive, highlighting the potential of iTS cell-derived factors, especially MSN, as promising therapeutic agents for treating advanced prostate cancer and associated skeletal complications.