Cell culture

The B16F10 mouse melanoma cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin under standard conditions of 37 °C and 5% CO2. The MLO-Y4 murine osteocytes were cultured on plates pre-coated with rat tail collagen type I (Corning, Cat. #354236, 0.15 mg/mL), generously provided by Erwin F. Wagner from the Department of Dermatology and Department of Laboratory Medicine, MedUni Wien, Vienna. The cells were cultured in α-MEM supplemented with 2.5% FBS, 2.5% bovine calf serum (BCS, VWR, Cat. #HYCLSH30072.3), and penicillin-streptomycin at 37 °C and 5% CO2, following established protocols.59

Murine osteoclast differentiation

Bone marrow cells from wildtype mice aged six weeks were isolated by flushing the femur and tibia. The cells were plated overnight at 37 °C with 5.5% CO2 in a 100 × 20 mm dish in osteoclast medium. This consisted of αMEM and GlutaMAX (Gibco), 10% fetal calf serum (FCS), and 1% penicillin/streptomycin (Gibco), supplemented with 5 ng/mL macrophage colony-stimulating factor (M-CSF) (PeproTech). On the subsequent day, the nonadherent bone marrow macrophages (BMMs) were collected, washed, and subsequently cultured in osteoclast medium with 20 ng/mL M-CSF and 10 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) (PeproTech) in 96-well plates (200 μL/well for TRAP staining) or 48-well plates (500 μL/well for RNA analysis) at a concentration of 1 × 106 cells/mL at 37 °C with 5.5% CO2. The medium was replaced every two days. In the experimental design, bone marrow cells treated with M-CSF but without RANKL served as the undifferentiated control group; cells treated with M-CSF and RANKL served as the differentiated experimental group; and cells treated with M-CSF, RANKL, and B16F10 conditioned medium (CM) served as the B16F10-treated experimental group. The fully differentiated osteoclasts (on days 5) were washed with PBS, the cells in 96-well plates were fixed with fixation buffer (MilliporeSigma), and they were stained with TRAP solution. The cells in 48-well plates were prepared for further RNA isolation.

Mice

Male C57BL/6 N mice, aged six weeks, were obtained from Charles River Laboratories (Sulzfeld, Germany) and housed in the animal facility of the Friedrich-Alexander-Universität Erlangen-Nürnberg Faculty of Medicine. The mice were kept in a controlled environment with a 12-h light and dark cycle at 25 °C.

The injection of B16F10 melanoma cells was performed intracardially by administering 1×105 cells in a 100 μL volume after inducing anesthesia with isoflurane (Abbott; IsoFlo®, Cat. #05260-05), following established protocols. Mice without tumors received PBS injections as controls and were continuously monitored for 24 h.

The treatment group received B16F10 melanoma cells injected as previously described. Subsequently, daily intraperitoneal injections of Ferrostatin-1 (Fer-1, 1 mg/kg) (MedChemExpress, Cat. # HY-100579), Zinc Protoporphyrin (Znpp, 10 mg/kg) (MedChemExpress, Cat. # HY-101193), and Roxadustat (10 mg/kg) (Invivochem, Cat.# V0293-500mg) were administered for 13 consecutive days. The control group received an equal volume of DMSO in PBS.

All mice were euthanized 14 days after B16F10 cell inoculation. The animal experiments followed the approved protocols set by the government of Franconia in Germany (license numbers: mouse models 54-2532.1).

Bulk RNA sequence

The bulk RNA sequence analysis was conducted by acquiring bone tissues and MLO-Y4 cells as specified in the Figure legends. To ensure enrichment of osteocytes and minimize contamination from other cell types, the bone tissue samples underwent the following treatments before RNA-seq analysis: First, bone tissues were placed in centrifuge tubes containing cold PBS and centrifuged (3 000 g for 5 minutes) to remove bone marrow. After bone marrow removal, bone tissues were further processed to eliminate cells attached to the bone surface. The tissues were washed with PBS, gently shaken in PBS containing 1% Triton X-100 for 10 minutes, and then washed several times with PBS to remove surface cells. Finally, the epiphyses of the bones were removed during the process, retaining only the diaphysis for subsequent RNA extraction. This step helps reduce interference from joint cartilage and other non-osteocyte cells.

Next, total RNA was extracted using the QIAGEN RNeasy kit, followed by RNA-seq analysis performed by Novogene in London, UK. The ‘DESeq2′ R package was used for differential gene expression analysis, which enabled the identification of Differentially Expressed Genes (DEGs) between distinct experimental groups.60

To annotate biological functions and elucidate enriched pathways associated with these DEGs, we conducted Gene Ontology (GO) functional analysis and KEGG pathway enrichment analysis using the ‘clusterProfiler’ R package.61 Furthermore, Gene Set Enrichment Analysis (GSEA) yielded additional information on pertinent biological pathways.62 Volcano diagrams were used to generate visual representations of differential gene expression patterns with the ‘ggplot2′ R package.63 Additionally, a heatmap was created using the ‘pheatmap’ R package to display the expression profiles across samples.64

The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus65 and are accessible through GEO Series accession number GSE276370 (Bone samples) and GSE276373 (MLO-Y4 samples).

RNA extraction and real-time quantitative PCR

The cells and bone tissues, treated as described in the Figure legend, were lysed in RNA-Solv (VWR, Cat. #R6830-02) according to the manufacturer’s protocol for RNA extraction. The extracted RNA was then converted into complementary DNA (cDNA) through reverse transcription using the Life Technologies kit (Life Tech, Cat. #4368813) following the provided guidelines.

Quantitative PCR analysis was performed using the Select Master Mix (Applied Biosystems, Life Tech, 447290). The internal control β-Actin was used to normalize the expression levels of target genes (Table 1), ensuring the accuracy and reliability of the analysis.

Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling Staining (TUNEL staining)

TUNEL staining was performed on MLO-Y4 cells and bone tissues following specific guidelines outlined in the figure legend. To quantify osteocyte death percentages, both in vivo and in vitro, the One-step TUNEL In Situ Apoptosis Kit (biomol, Cat# E-CK-A320) protocol was followed as per the manufacturer’s instructions.66

PI/Annexin V staining

MLO-Y4 cells exposed to B16F10-derived conditioned medium, with or without specific inhibitors as described in the figure legends, were stained using Annexin V in Annexin V binding buffer (BioLegend, Cat# 422201) at a 1:200 dilution.67 This staining process was performed in the dark at room temperature for 20 minutes. The cells were then washed twice with PBS and stained with a 1:1 000 dilution of propidium iodide (PI) (Thermo Fisher Scientific, Cat# 00-6990-50) for 5 minutes. Flow cytometry was used to analyse the stained cells, and the data obtained were processed and analysed using FlowJo software, version 10.4.

Ferroptosis probes

MLO-Y4 cells exposed to B16F10-derived conditioned medium, with or without specific inhibitors as described in the figure legends, were assessed for ferroptosis using C11-BODIPY 581/591 fluorescent sensors68 (Invitrogen, Cat# D3861) and H2DCFDA69 (MedChemExpress, Cat# HY-D0940). After treatment, cells were washed twice with PBS and incubated with 2 μmol/L C11-BODIPY 581/591 and H2DCFDA for 30 minutes in the dark. The cells were then washed with PBS and the stained cells were analysed by flow cytometry. Data were analysed using FlowJo software (version 10.4).

Cell Transfection and Lentiviral Infection

Specific shRNAs against HMOX1 (shHMOX1) and negative control (shNC) were obtained from VectorBuilder (Cat# No. Ecoli(VB900046-5163hfx) for shRNA1; Ecoli(VB900046-5169gxz) for shRNA2; Ecoli(VB900046-5173muu) for shRNA3; Ecoli(VB010000-0009mxc)-P for negative control. HIF1α overexpression plasmid (pcDNA3 mHIF-1α, addgene, Cat# 44028), pcDNA3 was performed as an empty vector (addgene, Cat# 13032). Specific shRNAs against HIF1α (shHIF1α) and negative control (shNC) were obtained from VectorBuilder (pLV[shRNA]-mCherry>mHif1a[shRNA#1]; Cat# VB900122-2420djn) for shRNA1; pLV[shRNA]-mCherry>mHif1a[shRNA#2] (Cat# VB900122-2421ngn) for shRNA2; mCherry lentiviral control vector pLV-mCherry (Cat# VB010000-9390nka-P) for negative control. Lentiviral transfer plasmids were co-transfected into HEK293T cells using a Lipofectamine 3000 transfection reagent. Cell culture media containing lentiviral particles were collected 48 h after transfection, filtered through a 0.45 μm syringe filter and concentrated by ultracentrifugation at 12 000 r/min for 1 h. The viral pellet was resuspended in phosphate-buffered saline (PBS) and stored at -80 °C until further use.

MLO-Y4 cells were cultured in appropriate media. For lentiviral infection, cells were plated at the desired density and exposed to lentiviral vectors. Polybrene (Sigma, Cat# TR-1003-G) (5 μg/mL) was added to increase transduction efficiency. Cells were incubated with lentivirus for 48 h and then maintained in fresh culture media. Puromycin (Invivogen, Cat# ant-pr-1) (5 μg/mL) was used to sort the infected cells. Western blotting was performed to further determine the knockdown efficiency.

Chromatin immunoprecipitation

ChIP experiments were performed using ChIP-IT Express Kit (Active motif, Cat# 53040). Cells were sonicated at 40% power for 15 cycles of 4 pulses (20 sec with 30 sec breaks on ice between each pulse). The following reaction components were used: Protein G Magnetic Beads 25 μL, ChIP buffer 110 μL, sheared chromatin 60 μL, PIC 3 μL, anti-HIF1α (Novus, Cat# NB100-105) 2 μL. On the second day, magnetic beads were washed and chromatin was eluted for qPCR (Table 2). Sheared chromatin was used as input to normalize DNA loading.

Dual-Luciferase AssayPlasmid construction

The wild-type (WT) and mutant (MUT) Hmox1 promoter regions were cloned into the pGL4.10 vector using BglII and HindIII restriction sites. The following plasmids were constructed: pGL4.10 (Empty Vector); pGL4.10-Hmox1 promoter (WT); pGL4.10-Hmox1 promoter (MUT).

Cell culture and transfection

HEK 293 T cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO2 incubator. For transfection, cells were seeded into 96-well plates and incubated overnight. The cells were divided into six groups for transfection: 1. pGL4.10 + pcDNA3.1( + )-MSC-6xHis + pRL-CMV (Empty Vector Control); 2. pGL4.10 + pcDNA3.1(+)-Hif1α-6xHis + pRL-CMV (HIF1α Overexpression Control); 3. pGL4.10-Hmox1 promoter (WT) + pcDNA3.1( + )-MSC-6xHis + pRL-CMV (WT Promoter + Empty Vector); 4. pGL4.10-Hmox1 promoter (WT) + pcDNA3.1(+)-Hif1α-6xHis + pRL-CMV (WT Promoter + HIF1α Overexpression); 5. pGL4.10-Hmox1 promoter (MUT) + pcDNA3.1( + )-MSC-6xHis + pRL-CMV (MUT Promoter + Empty Vector); 6. pGL4.10-Hmox1 promoter (MUT) + pcDNA3.1(+)-Hif1α-6xHis + pRL-CMV (MUT Promoter + HIF1α Overexpression). Each well was transfected with a total of 1 μg DNA using Lipo8000™ Transfection Reagent. The DNA mixture was gently mixed with the transfection reagent according to the manufacturer’s protocol and added directly to the cells. The cells were incubated for 30 h post-transfection.

Dual-Luciferase Assay

After 30 h of transfection, the dual-luciferase reporter assay was performed using the Dual Luciferase Reporter Gene Assay Kit (Beyotime RG027). Firefly and Renilla luciferase activities were measured sequentially using a luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency.

Immunofluorescence

Immunofluorescence staining of bone tissue involves incubation of sections or cell slides with specific primary antibodies, including anti-HIF1α (Cayman Chemicals, Cat# 10006421-1), anti-HIF2α (Novus, Cat#NB100-122), anti-HMOX1 (Proteintech, Cat# 10701-1-AP) and anti-GPX4 (Proteintech, Cat# 67763-1-Ig), all the first antibodies were diluted at a concentration of 1:300 in 2% BSA in PBS. This was followed by incubation with DyLight 594-conjugated (1:200, Vector, Cat. #Dl-1094) or Alexa Fluor 488-conjugated secondary antibodies (1:200, Vector, Cat. #Dl-2488-1.5). Sections or cell slides were mounted with fresh DAPI (Biozol, Cat# H-1200) and imaged using a Keyence fluorescence microscope. Image quantification was performed using ImageJ.

For cell immunofluorescence staining, cells were seeded on 12 mm circular coverslips and treated with anti-HMOX1, anti-FTL1 (Proteintech, Cat# 10727-1-AP) and anti-LC3 (Proteintech, Cat# 14600-1-AP) antibodies, all the first antibodies were diluted at a concentration of 1:300 in 2% BSA in PBS. This was followed by incubation with DyLight 594-conjugated (1:200, Vector, Cat. #Dl-1094) or Alexa Fluor 488-conjugated secondary antibodies (1:200, Vector, Cat. #Dl-2488-1.5). Cells were then mounted with fresh DAPI and imaged using a Keyence fluorescence microscope and a confocal laser scanning fluorescence microscope (LSM700; Zeiss). Quantitative analysis of the images obtained was performed using ImageJ.

Western blot

For Western blot analysis, cells subjected to the treatments indicated in the figure legend were lysed on ice using RIPA buffer containing protease and phosphatase inhibitors for 30 minutes. Lysates were centrifuged at 12 000 g for 10 minutes at 4 °C. BCA normalization was performed to ensure equal loading of the cell lysates. Equal amounts of protein were then loaded and separated on SDS-PAGE gels, followed by transfer to 0.2 μm PVDF membranes (Thermo Scientific, Cat. No. 88520). The membranes were incubated with primary antibodies including anti-HIF1α, anti-HIF2α, anti-HMOX1, anti-GPX4, anti-FTL1 and anti-LC3, all the antibodies were diluted at a concentration of 1:1 000 in TBS-T buffer (Tris-buffered saline with 0.1% Tween20). Quantification of protein bands was performed using ImageJ.

μCT analysis

Long bones were fixed in 4% PFA overnight prior to assessment. All proximal tibial metaphyses were scanned using the SCANCO Medical μCT 40 cone beam desktop micro computer tomograph. Settings were optimized to visualize calcified tissue at 55 kVp with a current of 145 μA and 200 ms integration time for 500 projections/180°.

An isotropic voxel size of 8.4 μm was used for 3D volume segmentation. Customized greyscale thresholds (threshold in the white region >100) were applied using the Open VMS operating system (SCANCO Medical). Bone structure analysis focused on the proximal tibial metaphysis, starting 0.43–0.42 mm from a defined landmark in the growth plate and extending 1.68 mm distally (200 tomograms).

To assess bone structure within fractured femoral calluses, the callus center was identified and bone volume (BV) was measured over a 2.10 mm span (250 tomograms).

Transmission electron microscopy

For transmission electron microscopy (TEM), MLO-Y4 cells were prepared as described in the figure legends. The cells were harvested and initially fixed with 2.5% glutaraldehyde in cacodylate buffer (pH 7.4, containing sucrose). After washing, the cells were post-fixed with 2% osmium tetroxide in cacodylate buffer and subsequently embedded in 2% agarose. During ethanol dehydration, the cells were treated with 1.0% uranyl acetate in 70% ethanol. The samples were then embedded in Araldite and sectioned using an ultramicrotome (Reichert Ultracut, Vienna, Austria). Image acquisition was performed with a Zeiss EM10 electron microscope (Carl Zeiss AG) equipped with a Gatan SC1000 Orius™ CCD camera, utilizing Digital Micrograph™ software (Gatan GmbH, Munich, Germany).

Histological analysis

Histological analysis was performed on serial paraffin sections. An H&E staining kit (Carl Roth) was used to assess general morphology. For lung tissue, PAS staining was employed to further clarify tumor progression. Histomorphometric evaluation was performed using a Zeiss Axioskop 2 microscope (Carl Zeiss) equipped with the OsteoMeasure image analysis system (Osteometrics). Hematoxylin and eosin-stained sections were used specifically to assess the degree of tumor invasion and the rate of osteocyte death. The Leukocyte Acid Phosphatase Kit (Sigma) was used to detect osteoclasts. All histological analyses were performed using a Zeiss Axioskop 2 microscope (Carl Zeiss), equipped with a digital camera and the OsteoMeasure image analysis system (Osteometrics).

Statistical analysis

Statistical analyses were performed with GraphPad Prism v9. Two-tailed Student’s t-tests compared two groups, while one-way or two-way ANOVA evaluated multiple groups. Pearson’s correlation test was used for correlation analysis, which was assessed by linear regression F-test. A significance threshold of P < 0.05 was used to indicate statistical significance.