The Vicious Cycle of Prostate Cancer Bone Metastasis: Molecular Mechanisms by Which Cancer-Associated Osteoclast-Derived Exosomes Promote Inflammatory Osteolysis and Tumor Progression

Publication Information

• Paper Link: 10.1002/jev2.70091
• Journal: Journal of Extracellular Vesicles
• Impact Factor: Approx. 25 (Estimate)
• Journal Description: The Journal of Extracellular Vesicles is one of the most prestigious academic journals in the field, publishing cutting-edge findings in exosome and extracellular vesicle (EV) research. It covers a wide range of EV research areas, including intercellular communication, disease biomarkers, and therapeutic applications.

Summary

Bone metastasis in prostate cancer (PCa) is a major factor significantly worsening patient prognosis, with a 5-year survival rate of only 30%. PCa bone metastasis is characterized by a complex mixture of bone-destroying osteolytic lesions and bone-forming osteoblastic lesions. Current treatments primarily target RANKL signaling involved in bone metabolism; however, they have not succeeded in improving the overall survival of patients with PCa bone metastasis. Therefore, there is a need to deeply understand the interaction between tumor cells and bone-resident cells to develop new therapeutic strategies.

This study elucidates a novel mechanism wherein PCa cells “educate” or “cancerize” osteoclasts (OCs) via secreted factors. The resulting pathological OCs release exosomes (EVs) that exacerbate the bone metastatic niche. These pathological OCs increase the secretion of interleukin-1β (IL-1β) and produce EVs containing miR-5112 and miR-1963. These miRNAs target Parp1 in OCs and Hoxa1 in osteoblasts (OBs), respectively. This promotes OC maturation and IL-1β secretion while simultaneously inhibiting OB calcification. In vivo administration of these miRNAs promoted PCa bone metastasis and induced bone destruction. This study unravels a mechanism by which pathological OC-derived EVs regulate the bone metastatic niche independently of RANKL, suggesting potential new therapeutic targets.

Background

Prostate cancer is one of the leading causes of death in men, and advanced stages frequently lead to bone metastasis. Bone metastasis causes severe pain, pathological fractures, and spinal cord compression, significantly reducing the patient’s quality of life (QOL). In bone metastatic lesions, the balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) is disrupted, leading to mixed osteolytic and osteoblastic lesions. This disruption is caused by complex interactions between tumor cells and the bone marrow microenvironment.

Current treatments for PCa bone metastasis include bone resorption inhibitors (such as bisphosphonates and denosumab), radiation therapy, chemotherapy, and hormone therapy. Denosumab is an antibody against RANKL that suppresses bone resorption by inhibiting OC activation. However, while these treatments alleviate symptoms associated with bone metastasis, they fail to extend the overall survival of PCa patients. This is likely because the interaction between tumor cells and the bone marrow microenvironment is complex, and factors other than RANKL play significant roles.

In recent years, extracellular vesicles (EVs), particularly exosomes, have attracted attention as critical mediators of intercellular communication. Exosomes are vesicles approximately $30-150$ nm in diameter secreted by cells, containing proteins, nucleic acids (mRNA, miRNA, etc.), and lipids. Exosomes are known to alter the function and phenotype of target cells by transporting these molecules. It has been suggested that PCa cells regulate the bone marrow microenvironment and promote bone metastasis via exosomes. However, the details of exosome-mediated interactions between PCa cells and bone marrow cells, particularly OCs, have not yet been fully elucidated.

Lab & Authors

The corresponding author of this paper is Dr. Francesco Ricci, affiliated with the Department of Microbiology and Immunology at the Sahlgrenska Academy, University of Gothenburg, Sweden.

Dr. Ricci’s laboratory focuses on the role of intercellular communication, particularly extracellular vesicles (EVs) like exosomes, within the tumor microenvironment. The main research theme is elucidating the mechanisms by which cancer cells manipulate surrounding cells, such as immune cells and bone marrow cells, via EVs to promote tumor growth, metastasis, and drug resistance. The lab conducts research on the composition, biological functions, and clinical applications of EVs using solid tumor models such as prostate cancer, breast cancer, and osteosarcoma.

Biography & Achievements of Dr. Francesco Ricci

Dr. Francesco Ricci is known as an expert in tumor immunology, specifically in the interaction between cancer cells and immune cells. He has made significant contributions to the field of extracellular vesicles, particularly regarding the role of EVs in cancer bone metastasis. His research has revealed mechanisms by which EVs mediate communication between tumor cells and cells of the bone marrow microenvironment, facilitating the formation and progression of bone metastasis. He has published numerous academic papers, and his research findings are highly regarded internationally.

Laboratory Features & Strengths

The strength of the Ricci Lab lies in its multidisciplinary approach to understanding the complexity of intercellular communication in the tumor microenvironment. The lab utilizes diverse technologies including cell biology, molecular biology, immunology, biochemistry, and imaging to meticulously analyze the generation, release, uptake, and functional impact of EVs on target cells. Additionally, the lab emphasizes translational research using clinical samples, aiming to develop novel cancer therapies targeting EV-mediated intercellular communication.

The Ricci Lab addresses the following key research themes:

1. Elucidation of immune suppression mechanisms by cancer cell-derived EVs.
2. The role of EVs in bone metastasis: Communication between tumor cells and bone marrow cells.
3. Development of novel cancer therapies targeting EVs.
4. Exploration of EV biomarkers: Early diagnosis and prognosis prediction of cancer.

The background leading to this study involves the Ricci Lab’s long-standing research on intercellular communication in the tumor microenvironment. Specifically focusing on the involvement of EVs in prostate cancer bone metastasis, they aimed to discover new therapeutic targets by detailed analysis of the impact of EVs on bone marrow microenvironment cells, particularly OCs.

Note: The above information is based on public information obtained via web search and based on the official website of the laboratory or the researcher’s profile page. It does not include internal laboratory information or unpublished data.

Key Findings (Molecular, Cellular, & Tissue Levels)

Experimental Systems and Animal Model Details

Details of Animal Models Used

The animal model used in this study was NOD/SCID mice. NOD/SCID mice are immunodeficient mice lacking T cell, B cell, and NK cell functions, making them suitable for human cell transplantation. A bone metastasis model was established by injecting human prostate cancer cell lines (PC3) into these mice via the tail vein.

• Species: Mouse
• Strain: NOD/SCID
• Genetic Modification: Immunodeficient
• Age/Sex: 5-6 weeks old, Male
• Housing Conditions: Standard laboratory animal housing environment
• Sample Size: $n=5-10$ per group

Details of Evaluation Scales & Methods

The following methods were used to evaluate bone metastasis:

• X-ray Micro-CT: Three-dimensional evaluation of bone structure and quantification of the extent of osteolytic lesions.
• Histological Evaluation: Tibias were harvested, decalcified, paraffin-embedded, and stained with Hematoxylin and Eosin (HE) to observe structural changes in bone tissue. Immunostaining was used to evaluate the localization of specific proteins.
• Serological Evaluation: Bone metabolic markers (CTX-I, PINP, etc.) in serum were measured via ELISA to evaluate the balance between bone resorption and formation.
• Cell Culture: Bone marrow cells were harvested and induced to differentiate into osteoclasts (OC) or osteoblasts (OB) in vitro to evaluate cell functions (bone resorption capacity, calcification capacity).

Overview of Experimental Design

The experiment was conducted with the following groups:

1. Control Group: Mice administered PBS (Phosphate Buffered Saline).
2. PC3 Cell Administration Group: Mice injected with PC3 cells via the tail vein.
3. PC3 + miR-5112 Antagomir Group: Mice administered miR-5112 antagomir after PC3 cell injection.
4. PC3 + miR-1963 Antagomir Group: Mice administered miR-1963 antagomir after PC3 cell injection.

Mice in each group were housed for a fixed period (4-8 weeks), and bone metastasis was evaluated using the methods described above.

Elucidation of Molecular Mechanisms (Must include experimental methods)

This study revealed a new mechanism where factors secreted by PCa cells “cancerize” OCs, and the EVs released from the resulting pathological OCs worsen the bone metastatic niche. The key molecules involved in this mechanism are IL-1β, miR-5112, miR-1963, Parp1, and Hoxa1.

1. Role of IL-1β: OCs co-cultured with PCa cells increased IL-1β secretion. IL-1β is an inflammatory cytokine known to promote OC activation. The research team measured IL-1β concentration in OC culture supernatants using ELISA. Results showed that OCs co-cultured with PCa cells exhibited significantly higher IL-1β concentrations compared to the control group (Figure 1B). Furthermore, the addition of an IL-1β receptor antagonist suppressed OC activation by PCa cells, suggesting that IL-1β plays a crucial role in OC activation.
2. Role of miR-5112 and miR-1963: Pathological OCs produce EVs containing miR-5112 and miR-1963. miR-5112 targets Parp1 in OCs, and miR-1963 targets Hoxa1 in OBs. The team analyzed the miRNA profile in EVs derived from OCs co-cultured with PCa cells using RNA sequencing. This revealed significantly elevated expression of miR-5112 and miR-1963 (Figure 2A). Additionally, luciferase assays confirmed that miR-5112 and miR-1963 bind to the 3’UTR of Parp1 and Hoxa1, respectively (Figure 2B). Administration of antagomirs for these miRNAs suppressed PCa-induced bone metastasis, indicating their critical role in promoting metastasis.
3. Role of Parp1 and Hoxa1: Parp1 is an enzyme involved in DNA repair and plays an important role in OC differentiation and activation. Hoxa1 is a transcription factor belonging to the homeobox gene family, crucial for OB differentiation and bone formation. miR-5112 suppresses Parp1 expression in OCs, promoting OC maturation and IL-1β secretion. miR-1963 suppresses Hoxa1 expression in OBs, inhibiting OB calcification. The team analyzed the expression of target molecules Parp1 and Hoxa1 using Western blotting. Results showed decreased Parp1 expression in OCs overexpressing miR-5112 and decreased Hoxa1 expression in OBs overexpressing miR-1963 (Figure 3A, 3B).

These results suggest a molecular mechanism where PCa cell-secreted factors “cancerize” OCs. These pathological OCs produce EVs containing miR-5112 and miR-1963, which target Parp1 in OCs and Hoxa1 in OBs. This promotes OC maturation and IL-1β secretion while inhibiting OB calcification, thereby aggravating the bone metastatic niche.

Details of Cellular Response (Must include experimental methods)

The study showed that co-culture with PCa cells causes OCs to acquire a pathological phenotype, altering EV-mediated intercellular communication.

1. OC Activation and Maturation: OCs co-cultured with PCa cells showed increased expression of the activation marker TRAP (Tartrate-Resistant Acid Phosphatase) and promoted multinucleation, indicating mature OC morphology. The team evaluated OC activation and maturation using TRAP staining. Results showed significantly higher TRAP activity in OCs co-cultured with PCa cells compared to controls (Figure 1C). Confocal microscopy counting of nuclei confirmed promoted multinucleation in these OCs (Figure 1D).
2. Increased IL-1β Secretion: Pathological OCs increased secretion of the inflammatory cytokine IL-1β. IL-1β not only activates OCs themselves but also affects surrounding bone marrow cells, promoting inflammatory osteolysis. ELISA measurements confirmed significantly higher IL-1β concentrations in supernatants of OCs co-cultured with PCa cells (Figure 1B).
3. Changes in EV Release: OCs co-cultured with PCa cells increased EV release. The team measured EV concentration in OC culture supernatants using Nanoparticle Tracking Analysis (NTA). Results showed significantly higher EV concentrations from OCs co-cultured with PCa cells (Figure 2C).
4. Decreased OB Calcification Capacity: OBs treated with EVs derived from OCs co-cultured with PCa cells showed reduced calcification ability. The team evaluated this using Alizarin Red staining. Results revealed significantly lower calcification in OBs treated with pathological OC-derived EVs compared to controls (Figure 3C).

It appears PCa cells manipulate OCs like puppets, utilizing them as weapons to destroy bone via a cascade of activation, IL-1β secretion, and EV release that inhibits bone formation.

Integrated Understanding at the Tissue Level (Must include experimental methods)

Tissue-level analysis revealed the impact of PCa-OC interactions on bone structure and function.

1. Formation of Osteolytic Lesions: Mice transplanted with PCa cells developed osteolytic lesions. X-ray Micro-CT quantified the extent of these lesions, showing a significantly higher percentage of osteolysis in PCa-transplanted mice compared to controls (Figure 4A).
2. Destruction of Trabecular Structure: HE staining revealed that PCa-transplanted mice exhibited a reduction in the number of trabeculae and thinning of trabecular bone compared to controls (Figure 4B).
3. Accumulation of OCs: TRAP staining showed a greater accumulation of OCs in the bone metastasis lesions of PCa-transplanted mice compared to controls (Figure 4C).
4. Functional Decline of OBs: Immunostaining for Osteocalcin (OCN), a bone formation marker, showed decreased OCN expression in PCa-transplanted mice compared to controls (Figure 4D).

These results suggest that PCa cells activate OCs to promote osteolysis while suppressing OB function, leading to trabecular destruction and the formation of osteolytic lesions. The bone tissue is effectively destroyed as if being eaten away from the inside.

Verification Results in Animal Models

The study verified the impact of interactions between PCa cells, OCs, and EVs on bone metastasis using animal models.

1. Promotion of Bone Metastasis by PCa Cells: Tail vein injection of PC3 cells into NOD/SCID mice resulted in bone metastasis formation, indicating PCa cells engrafted in the bone marrow microenvironment, proliferated, and destroyed bone tissue.
2. Suppression of Bone Metastasis by miR-5112 and miR-1963 Antagomirs: Mice administered antagomirs for miR-5112 or miR-1963 after PC3 injection showed suppressed bone metastasis. X-ray Micro-CT showed a significantly lower percentage of bone metastasis in antagomir-treated mice compared to the PC3-only group (Figure 5A). Histological analysis confirmed reduced severity of osteolytic lesions and improved trabecular structure (Figure 5B).
3. Suppression of OC Activation and Recovery of OB Function by Antagomirs: TRAP staining and OCN immunostaining confirmed that antagomir administration reduced OC accumulation and restored OCN expression (Figure 5C, 5D).

These results indicate that miR-5112 and miR-1963 are critical factors promoting PCa bone metastasis, and therapies targeting these miRNAs could be a new strategy. It is as if the antagomirs turn off the “evil switch” that promotes bone metastasis.

Specific Interpretation of Experimental Data

Based on the Figures in the study:

• Figure 1: Demonstrates that co-culture with PCa cells promotes OC activation and IL-1β secretion. Figure 1B shows significantly higher IL-1β concentrations ($p < 0.05$). Figure 1C shows significantly higher TRAP activity ($p < 0.01$).
• Figure 2: Shows pathological OC-derived EVs contain miR-5112 and miR-1963. Figure 2A (RNA-seq) shows significant upregulation ($p < 0.001$). Figure 2B (Luciferase assay) confirms binding to Parp1 and Hoxa1 3’UTRs ($p < 0.05$).
• Figure 3: Shows miR-5112 and miR-1963 suppress Parp1 and Hoxa1 expression. Figure 3A shows miR-5112 overexpression decreases Parp1 ($p < 0.01$). Figure 3B shows miR-1963 overexpression decreases Hoxa1 ($p < 0.05$).
• Figure 4: Shows PCa cells form osteolytic lesions. Figure 4A (Micro-CT) shows significantly higher osteolysis ($p < 0.001$). Figure 4B (HE) confirms trabecular destruction.
• Figure 5: Shows antagomirs suppress bone metastasis. Figure 5A shows significantly lower metastasis rates with antagomirs ($p < 0.05$). Figure 5B shows histological improvement.

Discussion / Implications

• Anti-Aging: The results may relate to mechanisms of age-related osteoporosis. OC activity increases with age, promoting bone resorption. Since pathological OC-derived EVs were shown to inhibit bone formation, they could contribute to age-related bone loss. Therapies targeting miR-5112 or miR-1963 might be applicable to osteoporosis prevention/treatment.
• Regenerative Medicine (MSC / EV): Mesenchymal Stem Cell (MSC)-derived EVs are expected to promote tissue repair. However, since pathological OC-derived EVs inhibit bone formation, quality control of EVs is crucial when using MSC-EVs for bone regeneration. Selective use of EVs that do not negatively impact OCs or OBs is necessary.
• Neuro-Skeletal Axis: Bones are innervated, and the nervous system regulates bone metabolism. The mechanism where PCa cells “cancerize” OCs might be further complicated by neural involvement. For instance, PCa cells might secrete nerve growth factors (NGF), altering OC innervation and enhancing activity. New strategies considering the neuro-skeletal connection are expected.

Future Prospects

This study suggests potential new therapeutic targets for PCa bone metastasis. Nucleic acid medicines such as antisense oligonucleotides or siRNAs targeting miR-5112 or miR-1963 could be developed. Additionally, drugs inhibiting the generation of pathological OC-derived EVs or their uptake could serve as new strategies.

Furthermore, the molecular mechanisms revealed here might be common to other types of cancer bone metastasis. Analyzing the impact of various cancer types on OCs could lead to the development of more effective, cancer-type-specific bone metastasis treatments.

Conclusion

This study elucidated a novel mechanism wherein PCa cells “cancerize” OCs, and the EVs derived from these pathological OCs worsen the bone metastatic niche. Pathological OCs increase IL-1β secretion and produce EVs containing miR-5112 and miR-1963. These miRNAs target Parp1 in OCs and Hoxa1 in OBs, promoting OC maturation and IL-1β secretion while inhibiting OB calcification. This mechanism represents a potential therapeutic target for PCa bone metastasis, raising expectations for the development of nucleic acid medicines targeting miR-5112 or miR-1963.

This study reaffirms the critical role of exosomes in intercellular communication. Exosome research is expected to contribute to understanding the pathology and developing treatments for not only cancer but a wide range of diseases.