Abstract

   Dedifferentiation of osteosarcoma cells leads to poor prognosis. We
   plan to identify the key molecules that are involved in cell
   dedifferentiation and explore how they promote the pulmonary metastasis
   of osteosarcoma cells. We performed a sphere formation assay and
   confirmed that the spheroid cells could be redifferentiated into
   osteoblasts, adipocytes, and chondrocytes in specific medium, and the
   stem cell-like markers Stro-1 and CD117 were detected on the cell
   surface, which indicated that the spheroid cells were dedifferentiated
   cells. Thrombospondin 1 (THBS1) and ITGAs were identified as the key
   molecules in dedifferentiation through mRNA-seq and analysis, and
   osteosarcoma patients with higher THBS1 expression had a worse
   prognosis than those with lower THBS1 expression. THBS1 promotes the
   accumulation of ITGA1 and ITGA6 on the cell membrane in the early phase
   of dedifferentiation, thereby increasing the phosphorylation of FAK,
   RasGRF1, and MLC2 in the cytoplasm and promoting cytoskeleton
   remodeling. Our results suggest that THBS1 promotes cell
   dedifferentiation and pulmonary metastasis by promoting cytoskeletal
   remodeling and that ITGA1 and ITGA6 play important roles in mediating
   extracellular to intracellular signals; this mediating effect takes
   place mainly in the early phase of dedifferentiation.

   Keywords: osteosarcoma, dedifferentiation, metastasis, cytoskeleton
   remodeling, cell adhesion, THBS1

Introduction

   Osteosarcoma is most commonly diagnosed in children and young adults,
   and the long-term survival rates remain low due to pulmonary
   metastasis[39]^1. The vast majority of patients already show
   micrometastasis at the time of diagnosis[40]^2. The overall survival
   rate for osteosarcoma is approximately 60-70% over a five-year
   period[41]^3. However, metastatic osteosarcoma presents significant
   treatment challenges, leading to overall survival rates of less than
   20%[42]^4^, [43]^5. Numerous studies have demonstrated that proteins
   and signaling pathways—such as ezrin, TGF-β, MMPs, RUNX2, Notch,
   Wnt/β-catenin, and PI3K/AKT—are critical factors in the metastasis of
   osteosarcoma[44]^6. Some of these metastasis related molecules are also
   associated with the stemness of osteosarcoma cells. TGF-β appears to
   enhance stemness. Ma et al. demonstrated that knocking down TGF-β
   reduced the expression of stem cell markers in osteosarcoma
   cells[45]^7. Additionally, there appears to be a connection between
   Notch signaling and stem cells[46]^8^, [47]^9. Many miRNAs associated
   with osteosarcoma metastasis can also target stem cell markers, thereby
   influencing stem cell activity either positively or negatively. One
   notable example is miR-487b-3p[48]^6.

   Recent studies in several types of human cancers showed that tumor
   cells are heterogeneous, and dedifferentiated cells are a subpopulation
   of cancer cells with stem cell-like self-renewal ability and
   multipotency[49]^10. The process by which mature, differentiated cells
   revert to a less differentiated, stem cell-like state is referred to as
   dedifferentiation. Dedifferentiated cells are
   radioresistant/chemoresistant and are associated with distant
   metastasis and poor prognosis[50]^11^-[51]^16. Dedifferentiation occurs
   in osteosarcoma cells and is associated with tumor malignancy,
   including tumor metastasis. Zhang et al. demonstrated that stem cells
   and mature differentiated cells exist in a dynamic equilibrium within
   the osteosarcoma cell population and that cancer stem cells may develop
   de novo from differentiated cancer cells[52]^17. Stemness factors such
   as Sox2, Oct4, and Nanog are found in pathological specimens of
   osteosarcoma patients[53]^18. The dedifferentiation of osteosarcoma was
   proven to account for tumor growth and metastasis[54]^19^, [55]^20.
   CD117 and Stro-1, as markers for MSCs, are also double-positively
   expressed in some human and mouse osteosarcoma cells, which exhibit
   stronger multilineage differentiation potential, invasion, and
   metastasis ability[56]^19. CD133, a marker of dedifferentiation, is
   closely related to pulmonary metastasis and poor prognosis in
   osteosarcoma patients[57]^21. Lamhamedi-Cherradi et al. identified a
   subset of osteosarcoma cells termed derived circulating tumor cells
   (dCTCs) that showed less differentiation and highly expressed
   epithelial-to-mesenchymal transition-associated transcription factors
   (EMT-TFs)[58]^22.

   Therefore, exploring how osteosarcoma cells dedifferentiate and finding
   solutions to inhibit dedifferentiation is necessary to help clinicians
   inhibit tumor pulmonary metastasis in patients with osteosarcoma. Zhang
   et al. found that blocking of TGF-β1 resulted in decrease of the
   dedifferentiation of osteosarcoma cells[59]^17. Additionally, several
   researchers have observed that the upregulation of miR-34a expression
   plays a crucial role in the dedifferentiation of osteosarcoma[60]^23.
   This finding suggests that miR-34a may represent a promising
   therapeutic target for preventing the dedifferentiation of osteosarcoma
   cells.

   However, there are currently no studies that clearly illustrate how
   osteosarcoma cells dedifferentiate and detail which molecules could
   serve as targets to block the dedifferentiation of osteosarcoma cells.
   In this study, we aim to isolate dedifferentiated osteosarcoma cells
   and conduct mRNA sequencing analysis to identify key molecules involved
   in their dedifferentiation. We will explore how these molecules promote
   both dedifferentiation and lung metastasis of osteosarcoma cells. This
   research will serve as a foundation for the future development of drugs
   aimed at inhibiting dedifferentiation and lung metastasis in
   osteosarcoma.

Materials and Methods

Cell culture

   DMEM (HyClone, Utah, USA) medium contained 10% fetal bovine serum (FBS,
   Gibco, NY, USA), penicillin (100 U/ L, Invitrogen, NY, USA), and
   streptomycin (100 mg/L, Invitrogen, NY, USA). McCoy's 5a (Invitrogen,
   NY, USA) medium contained 15% FBS, penicillin (100 U/L), and
   streptomycin (100 mg/L). DMEM/ F12 (HyClone, Utah, USA) medium
   contained 10% FBS, penicillin (100 U/L), and streptomycin (100 mg/L).
   U-2 OS cells were cultured in McCoy's 5a medium, MG-63 and 143B cells
   were cultured in DMEM, primary osteosarcoma cells were cultured in
   DMEM/F12, and all cells were placed in a humidified atmosphere with 5%
   CO[2] at 37°C.

In vitro dedifferentiation and redifferentiation

   We employed a sphere formation assay to obtain dedifferentiated cells.
   The abovementioned osteosarcoma cells were cultured in 100 mm dishes
   until the cell confluence is approximately 80%-90%. The cells were
   washed with warm PBS, 1 mL of 0.05% trypsin-EDTA was added, and the
   cells were incubated at 37°C for 3 minutes. The mixture was then
   transferred to a 15 mL centrifuge tube, the cells were centrifuged at
   200 × g for 5 minutes, and the cells were resuspended in 1 mL of
   serum-free medium. Matrigel (BD Biosciences, CA, USA) and the cell
   suspension were mixed at a ratio of 1:1, and the mixture was spread
   into the center of a 24-well plate culture dish at 5,000 cells per
   well. The plate was incubated at 37°C for 60 minutes until the Matrigel
   was cured. After solidification, 500 μL of warm serum-free medium
   containing ITS-X (BasalMedia Technologies, Shanghai, China) was added
   to each culture well, and the warm medium was replenished every 3 days.

   We also performed redifferentiation experiments of spheroid cells
   according to the manufacturer's instructions for osteogenic
   differentiation medium, chondrogenic differentiation medium, and
   adipogenic differentiation medium (Cyagen Biosciences, Jiangsu, China).

mRNA-seq and the identification of key molecules

   High-throughput mRNA sequencing was conducted by OE Biotech (Shanghai,
   China). In brief, total RNA of the sarcospheres and residual cells was
   extracted, and magnetic beads with oligo (dT) were used to enrich
   eukaryotic mRNA. Then, cDNA was synthesized, and PCR amplification was
   performed. After quality inspection with an Agilent 2100 Bioanalyzer,
   sequencing was performed using an Illumina HiSeq 4000. Differentially
   expressed genes (DEGs) were identified using the DESeq package, and
   then the DEGs were used for Gene Ontology (GO) and Kyoto Encyclopedia
   of Genes and Genomes (KEGG) enrichment analysis. For protein-protein
   interaction (PPI) analysis, the proteins corresponding to all
   identified DEGs were submitted to the STRING database for to evaluate
   protein interactions. Then, the cytoNCA and MCODE plugins of Cytoscape
   software were used to analyze and cluster networks. We ranked all
   proteins based on their betweenness, and the size of each node in the
   networks represented the betweenness value of the corresponding
   protein. The settings for the MCODE plugin to explore key molecules
   were as follows: degree cutoff = 10, node score cutoff = 0.2, k-core =
   2, and max.depth = 100[61]^24^, [62]^25.

Knocking down or overexpression

   Recombinant lentiviruses designed for the knockdown or overexpression
   of THBS1, as well as the knockdown of ITGA1 or ITGA6, along with a
   control blank lentivirus, were procured from Shanghai GeneChem Co.,Ltd.
   (Shanghai, China). These lentiviruses were subsequently used to
   transfect osteosarcoma cells. Initial preliminary experiments were
   conducted to determine the optimal multiplicity of infection (MOI) for
   viral infections, which guided our formal experimental protocols. To
   increase the proportion of infected cells, appropriate concentrations
   of puromycin or neomycin were incorporated into the culture medium,
   resulting in the selective enrichment of infected cells after a 3-day
   infection period. mRNA and protein extraction were performed once the
   cells reached 70-80% confluence. The effectiveness of the knockdown or
   overexpression was validated through RT-qPCR and Western blotting
   analyses.

Immunocytochemistry (ICC)

   The formed spheres or cells were fixed with 4% paraformaldehyde for 30
   minutes, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, and
   blocked with blocking buffer (0.1% bovine serum albumin (BSA), 0.2%
   Triton X-100, and 10% normal goat serum (NGS) in PBS) for 2 h.
   Spheres/cells were incubated overnight with primary antibody at 4 ℃.
   The primary antibodies used were as follows: anti-Stro-1 (1:100,
   ab214086, Abcam), anti-CD117 (1:100, ab283653, Abcam), anti-THBS1
   (1:100, ab267388, Abcam), anti-ITGA1 (1:50, sc-271034, Santa Cruz),
   anti-ITGA6 (1:50, sc-374057, Santa Cruz), and anti-ITGA10 (1:100,
   PA5-100840, ThermoFisher). The primary antibody was removed after
   incubation, the spheres/cells were gently washed with PBS, and then the
   spheres/cells were incubated with the secondary antibody (all secondary
   antibodies from Beyotime Biotechnology, Shanghai, China). For F-actin
   staining, cells were incubated with Alexa Fluor 647 phalloidin (1:20,
   CST) for 15 minutes at room temperature. Afterward, spheres/cells were
   mounted with DAPI (Sigma Aldrich) after being washed with PBS.
   Spheres/cells were observed and photographed under a confocal
   microscope (LSM900, Carl Zeiss, Germany).

Immunohistochemistry (IHC)

   With institutional review board approval from Shanghai Tenth People's
   Hospital, human osteosarcoma specimens were acquired from patients who
   underwent surgery. This study was performed in accordance with the
   Declaration of Helsinki. Osteosarcoma specimens and mouse lung tissues
   were fixed in 4% paraformaldehyde, embedded in paraffin, and
   subsequently prepared for hematoxylin and eosin (H&E) staining as well
   as IHC staining. We performed IHC staining using an IHC kit according
   to the manufacturer's instructions (Yeasen Biotechnology, Shanghai,
   China). The primary antibodies were utilized as follows: anti-THBS1
   (1:5000, ab267388, Abcam), anti-Stro-1 (1:500, 14-6688-82,
   ThermoFisher), anti-CD117 (1:100, ab283653, Abcam), anti-phospho-MLC2
   (1:50, AF3829, Affinity Biosciences).

Western blotting and Co-Immunoprecipitation (Co-IP)

   Cells from each group were collected, and membrane/membrane-associated
   proteins, along with cytosolic proteins, were isolated using a Mem-PER
   Plus Membrane Protein Extraction kit (Pierce, IL, USA). The protein
   concentration was determined by a BCA Protein Assay kit (Pierce) and
   equal amounts of cell extracts were run on SDS PAGE gels. Separated
   protein bands were transferred onto polyvinylidene fluoride (PVDF,
   Millipore, MA, USA) membranes and blocked in 5% BSA (Sigma-Aldrich, St.
   Louis, MO, USA). Membranes were incubated overnight at 4°C with the
   appropriate primary antibodies. The following antibodies were utilized:
   anti-THBS1 (1:1000, ab267388, Abcam), anti-ITGA1 (1:100, sc-271034,
   Santa Cruz), anti-ITGA6 (1:100, sc-374057, Santa Cruz), anti-ITGA10
   (1:500, PA5-100840, ThermoFisher), anti-Na^+/K^+-ATPase (1:500,
   ab58475, Abcam), anti-FAK (1:500, A11131, ABclonal), anti-phospho-FAK
   (1:100, AP0302, ABclonal), anti-RasGRF1 (1:1000, DF8026, Affinity
   Biosciences), anti-phospho-RasGRF1 (1:1000, 82309-1-RR, Proteintech),
   anti-MLC2 (1:1000, 8505, CST), anti-phospho-MLC2 (1:1000, 3671, CST),
   anti-GAPDH (1:1000, AC001, ABclonal). We chose a loading control
   according to Abcam's recommendation. The relative protein level in
   different groups was normalized to Na^+/K^+-ATPase or GAPDH
   concentration. Membranes were then incubated with secondary antibodies
   (ABclonal) for 1 h at room temperature. The immunoreactive bands were
   visualized using BeyoECL Plus (Beyotime Biotechnology, Shanghai,
   China).

   RhoA-GTPase activity was evaluated according to the manufacturer's
   instructions using the RhoA Pull-Down Activation Assay Biochem Kit
   (Cytoskeleton, USA). Subsequently, the protein samples were analyzed
   through Western blotting, as previously described.

   Co-IP was applied using the Immunoprecipitation Kit with Protein A+G
   Agarose Gel according to the manufacturer's instructions (Beyotime
   Biotechnology, Shanghai, China). Immunoprecipitates were subjected to
   Western blotting analysis as described above.

Wound-healing assay

   Osteosarcoma cells from each group were cultured in 6-well culture
   plates and grown to 90% confluence. Wounds were created using a 1000 μL
   micropipette tip. The cells were then maintained in a serum-free
   medium. The migration of cells toward the wound was monitored and the
   wounds were photographed. The calculated cell migration rate = (0 h
   width - 12 h or 24 h width)/0 h width × 100%.

Transwell migration and invasion assays

   The migration and invasion abilities of cells were determined in a 6.5
   mm transwell (Corning, NY, USA). For the invasion assay, the filter of
   the top chamber was coated with 50 μL diluted Matrigel. Then, 5×10^4
   osteosarcoma cells were suspended in serum-free medium and plated on
   the top chambers, and the lower chambers were filled with 500 μL medium
   containing FBS. The cells were cultured for 24 h, and the invading
   cells were fixed with 4% paraformaldehyde for 30 minutes and then
   stained with a crystal violet staining solution (Sigma Aldrich). Then,
   the non-invading cells were removed, and the number of invading cells
   was counted and analyzed by Image J software (NIH Image, MD, USA).

Animal model and treatment

   All experimental animal procedures were performed according to the
   protocols approved by the Animal Research Ethics Committee of the
   Shanghai Tenth People's Hospital. For the tail vein pulmonary
   metastasis model, 1×10^6 143B cells of each group were injected through
   the tail vein into 6-week-old female nude mice. For the orthotopic lung
   metastasis model, 2×10^6 143B cells from each group were injected into
   the proximal tibia of 6-week-old female nude mice. At 6 weeks after
   injection of cells, the mice were then sacrificed, and the harvested
   lungs were fixed in formaldehyde for histopathology analysis.

Statistical analysis

   The results were expressed as the mean ± standard deviation (SD).
   Differences between means were analyzed using a two-tailed t-test for
   comparisons between two groups and ANOVA for comparisons involving more
   than two groups. SPSS 20.0 (IBM, USA), and GraphPad Prism 9 (San Diego,
   USA) were used for all statistical analyses. Kaplan‒Meier survival
   curves were used to assess overall survival in osteosarcoma patients,
   and the results were then evaluated using the log-rank test.
   Statistically significant results are labeled as follows in all
   figures: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Results

THBS1 and ITGAs are key molecules in the dedifferentiation of osteosarcoma
cells

   Previous studies confirmed the presence of osteosarcoma stem cells by
   the sphere formation assay or cell sorting according to cell surface
   markers (CD117^+/Stro-1^+)[63]^19^, [64]^26. Osteosarcoma sarcospheres
   were formed in Matrigel-constructed 3D medium in 24-well plates (Figure
   [65]1A). After repeated cultivation, we observed that osteosarcoma
   cells formed spheres in Matrigel (Figure [66]1B-C). The spheroid cells
   were enriched in the expression of CD117 and Stro-1 when compared to
   the residual cells (Figure [67]1D). These spheroid cells could also
   redifferentiate into osteoblasts, chondrocytes, and adipocytes (Figure
   [68]1E-G). The results suggest that osteosarcoma cells underwent
   dedifferentiation during the process of sphere formation. Later,
   mRNA-seq was employed to explore the DEGs between residual cells and
   sarcospheres, and identify the key molecules in the dedifferentiation
   of osteosarcoma cells. In total, 2794 genes had significant differences
   in expression in sarcospheres compared to residual cells (p < 0.05 and
   |logFC| > 1); 1395 genes had markedly upregulated expression, and 1399
   genes had downregulated expression. Hierarchical clustering clearly
   showed the landscape of genomic differences between sarcospheres and
   residual cells (Figure [69]1H). Functional annotations of GO enrichment
   indicated that these genes were significantly associated with cell
   adhesion, extracellular matrix, extracellular space, and extracellular
   matrix structural constituent (Figure [70]1I, red arrows). KEGG
   enrichment analysis results demonstrated that these genes were also
   related to ECM-receptor interaction (Figure [71]1J, red arrow). Then,
   we constructed a PPI network with the proteins corresponding to the
   DEGs and ranked all proteins based on their betweenness. The proteins
   arranged in the orange inner ring were relatively closely related to
   dedifferentiation, among which THBS1 had the strongest correlation
   (Figure [72]1K, red arrow). We determined a subnetwork module with
   significant prognostic values; of the molecules in the subnetwork,
   THBS1, ITGA1, ITGA6, ITGA10, COL4A4, TLN2, and LAMA2 were selected as
   key molecules based on their high degree of connectivity (Figure
   [73]1L). Among them, direct interactions had been shown between THBS1
   and ITGA1, and between THBS1 and ITGA6 (Figure [74]1L). Furthermore,
   THBS1 is the most differentially expressed DEGs of these key molecules
   in dedifferentiation (Figure [75]1M, black arrow).

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Identification of the key molecules involved in the dedifferentiation
   of osteosarcoma cells. (A) Scheme of the experimental procedure of
   sphere formation assay. (B) Sarcospheres of U-2 OS and MG-63 cells are
   shown. (C) The three-dimensional shape of a sphere is shown. (D)
   Immunocytochemistry was performed to determine Stro-1 (green) and CD117
   (green) expression in residual cells and sarcospheres. Residual cells
   and sarcospheres were counterstained with DAPI (blue). (E-G)
   Osteogenic, chondrogenic and adipogenic experiments were performed. (H)
   Hierarchical clustering of the differentially expressed genes between
   formed spheres and residual cells was performed. Red indicates the
   upregulated genes and blue indicates downregulated genes. Each column
   represents a sample, and each row represents a differentially expressed
   gene. (I) GO functional enrichment analysis for differentially
   expressed genes was performed. (J) KEGG pathway enrichment analysis for
   differentially expressed genes was performed. (K) A PPI network among
   differentially expressed genes was constructed. The red arrow indicates
   THBS1. (L) The relationship among the seven key molecules at the
   protein level is shown. (M) A volcano plot was constructed with the
   cutoff criteria q < 0.05 and |logFC > 1. Red indicates upregulated
   genes, and blue indicates downregulated genes. Each circle represents a
   gene, and the identified key molecules are marked with different
   colors. The black arrow indicates THBS1.

THBS1 leads to worse prognosis and promotes dedifferentiation via ITGA1 and
ITGA6

   The above results indicated that THBS1 and three ITGAs were key
   molecules in the dedifferentiation of osteosarcoma cells. We further
   employed Western blotting to confirm the above results in protein
   level, and found that in U-2 OS, 143B, MG-63, Saos-2 and MNNG/HOS
   cells, THBS1 was more highly expressed in sarcospheres relative to that
   in residual cells (Figure [78]2A). Then, we performed IHC on 79 human
   osteosarcoma specimens and observed that patients with higher
   expression of THBS1 had a worse prognosis (Figure [79]2B, log-rank P =
   0.0018). Lentiviral transfection was employed to knockdown or
   overexpress THBS1 in the 143B and MG-63 osteosarcoma cell lines. The
   Western blotting results indicated that the expression of ITGA1 and
   ITGA6 changed after knocking down or overexpressing of THBS1, but no
   similar significant changes occurred in terms of ITGA10 expression
   (Figure [80]2C). We also attempted to confirm whether knockdown or
   overexpression of THBS1 would affect osteosarcoma cell
   dedifferentiation, and a sphere formation assay showed that knocking
   down THBS1 significantly reduced the number and diameter of
   sarcospheres, while overexpression of THBS1 increased the diameter of
   sarcospheres, indicating that THBS1 affects the dedifferentiation of
   osteosarcoma cells (Figure [81]2D).

Figure 2.

   [82]Figure 2
   [83]Open in a new tab

   THBS1 leads to worse prognosis and promotes dedifferentiation via ITGA1
   and ITGA6. (A) The expression of THBS1 between formed spheres and
   residual cells in different osteosarcoma cell lines was determined. (B)
   Representative IHC staining images of THBS1 in osteosarcoma specimens
   are shown and the Kaplan-Meier plot of overall survival based on THBS1
   expression in osteosarcoma patients is shown. (C) The expression of
   THBS1, ITGA1, ITGA6 and ITGA10 in THBS1 knockdown and overexpression
   cells is shown. (D) The sphere formation ability of THBS1 knockdown and
   THBS1 overexpression cells was evaluated. ***P < 0.001, **P < 0.01, *P
   < 0.05.

THBS1 promotes migration and invasion of osteosarcoma cells through
cytoskeleton remodeling

   Some research suggests the relationship between cell dedifferentiation
   and tumor metastasis[84]^19^, [85]^21^, [86]^22. In this section, we
   aimed to investigate whether THBS1 promotes osteosarcoma cell migration
   and invasion in ivtro. The wound-healing assay using 143B and MG-63
   cells suggested that THBS1 affects cell migration; knockdown of THBS1
   reduced cell migration, while overexpression of THBS1 increased cell
   migration (Figure [87]3A). Transwell migration and invasion assays also
   showed that knocking down THBS1 reduced cell migration and invasion,
   while overexpressing of THBS1 increased cell migration and invasion
   (Figure [88]3B). We found a significant change in cell morphology after
   THBS1 overexpression, which was demonstrated by F-actin staining
   (Figure [89]3C). These results suggest a cytoskeleton remodeling exist
   in the cells with THBS1 expression. Therefore, combining enrichment
   results from mRNA-seq, we assessed cell adhesion and
   cytoskeleton-related factors and found that the expression of p-FAK,
   p-RasGRF1, p-MLC2 and GTP-RhoA was positively correlated with the
   expression of THBS1 (Figure [90]3D-E).

Figure 3.

   [91]Figure 3
   [92]Open in a new tab

   The role and mechanism of THBS1 in osteosarcoma cell dedifferentiation
   and tumor malignancy. (A) Wound-healing assay results of THBS1
   knockdown and overexpression cells are shown. (B) Transwell migration
   and invasion assay results of THBS1 knockdown and overexpression cells
   are shown. (C) Cell morphological changes after expression of THBS1 in
   different osteosarcoma cell lines are shown. Cells were stained for
   F-actin (magenta), DAPI (blue). (D) The expression of p-FAK, FAK,
   p-RasGRF1, RasGRF1, p-MLC2 and MLC2 in THBS1 knockdown and
   overexpression cells was determined. (E) The expression of GTP-RhoA and
   total RhoA in each group was shown. ***P < 0.001, **P < 0.01, *P <
   0.05.

THBS1 promotes dedifferentiation via ITGA1 and ITGA6 in the early phase

   The results of the immunocytochemistry assay demonstrated that there
   was no obvious colocalization phenomenon between THBS1 and ITGAs in
   residual cells, but colocalization of THBS1 with ITGA1/ITGA6 was
   observed in sarcospheres, suggesting that there may be interactions
   between THBS1 and ITGA1, and between THBS1 and ITGA6 in sarcospheres
   (Figure [93]4A-B). In addition, during the sphere formation of
   osteosarcoma cells, the signal intensity of ITGA1 and ITGA6 on the cell
   membrane surface at the outer edge of the sphere was higher than that
   inside the sphere, and colocalization with THBS1 also occurred in cells
   at the outer edge of the sphere. In contrast, the ITGA10 signal did not
   show similar changes and did not colocalize with THBS1 (Figure [94]4B).
   As expected, the colocalization between THBS1 and ITGA1/ITGA6
   disappeared after knocking down THBS1 (Figure [95]4C). Co-IP
   experiments confirmed the interaction between THBS1 and ITGA1/ITGA6 in
   the dedifferentiation of osteosarcoma cells (Figure [96]4D). We defined
   phase 0 cells as cells that had not yet formed spheres, phase 1 cells
   as cells that had just participated in sphere formation and were at the
   outer edge of the sphere, and phase 2 cells as cells that had already
   participated in sphere formation and were inside the sphere. We
   observed that the colocalization of THBS1 and ITGA1, or THBS1 and ITGA6
   only occurred in phase 1 (Figure [97]4E-G, white arrows indicate the
   typical cells in phase 1). These results suggest that THBS1 promotes
   dedifferentiation via ITGA1 and ITGA6 in the early phase, but this
   promotion may no longer be functional in the mature sarcosphers (Figure
   [98]4H).

Figure 4.

   [99]Figure 4
   [100]Open in a new tab

   The role and timing of THBS1 and ITGAs in osteosarcoma cell
   dedifferentiation. (A-C) Immunocytochemistry was utilized to evaluate
   THBS1 (green), ITGA1 (red), ITGA6 (red) and ITGA10 (red) expression in
   residual cells and sarcospheres. Residual cells and sarcospheres were
   counterstained with DAPI (blue). (D) Co-IP experiments were performed
   to determine the interaction between THBS1 and ITGAs. (E-G) The
   colocalization of THBS1 and ITGAs in different phases of osteosarcoma
   cell dedifferentiation was assessed. (H) Scheme of the colocalization
   of THBS1 and ITGAs in different phases of osteosarcoma cell
   dedifferentiation.

THBS1 and ITGAs do not play a role in promoting dedifferentiation inside
mature sarcospheres

   To determine the specific stage at which THBS1 primarily influences the
   dedifferentiation of osteosarcoma cells, we analyzed the expression
   levels of THBS1 and ITGAs in osteosarcoma cells across various
   dedifferentiation time points. We found that the expression of THBS1,
   ITGA1, and ITGA6 in the cell membrane was relatively high from week 1
   to 2 in the sphere formation process, indicating that these molecules
   were highly expressed in the early phase of dedifferentiation, but we
   also observed lower expression of THBS1, ITGA1 and ITGA6 in later phase
   of dedifferentiation (Figure [101]5A). To explain why the expression of
   THBS1, ITGA1, and ITGA6 in the cell membrane decreased in the late
   phase of sphere formation, we also assessed the expression pattern on
   the surface and inside of mature spheres that were subjected to a
   sphere formation assay for 4 weeks (Figure [102]5B-C) and found that
   THBS1, ITGA1, and ITGA6 had a strong signal only on the surface of
   mature spheres (Figure [103]5D-E).

Figure 5.

   [104]Figure 5
   [105]Open in a new tab

   The expression of THBS1 and ITGAs in mature sarcospheres. (A)
   Expression changes in THBS1 and ITGAs on the cell membrane during
   different phases of osteosarcoma cell dedifferentiation are shown. A
   schematic diagram of obtaining immunofluorescence images of cells on
   the surface (B) or inside the sphere (C). The red arrows indicate the
   position of the sphere where the image is located. (D) The
   colocalization of THBS1 and ITGAs on the surface of mature sarcospheres
   was assessed. (E) The colocalization of THBS1 and ITGAs inside the
   mature spheres was assessed. Sarcospheres were stained for THBS1
   (green), ITGA1 (red), ITGA6 (red), ITGA10 (red) and DAPI (blue).

Knocking down of THBS1 inhibits pulmonary metastasis in vivo

   To further clarify the ability of THBS1 to promote osteosarcoma
   metastasis, we first injected tumor cells through the tail vein to
   establish a nude mouse pulmonary metastasis model (Figure [106]6A).
   And, in order to simulate the clinical reality of tumor development
   more closely, we also established an osteosarcoma pulmonary metastasis
   model by injecting tumor cells into the proximal tibia of nude mice
   (Figure [107]6A). The results of the tail vein injection groups
   indicated that the knockdown of THBS1 reduced pulmonary metastasis in
   osteosarcoma (Figure [108]6B-D). Additionally, it decreased the
   expression levels of p-MLC2, Stro-1, and CD117 in the metastatic
   nodules (Figure [109]6B, 6E). The lung weights in subjects with THBS1
   knockdown were significantly reduced, primarily due to the decrease in
   metastatic nodules (Figure [110]6D). Similar results can be seen in the
   proximal tibia injection groups (Figure [111]6F-K).

Figure 6.

   [112]Figure 6
   [113]Open in a new tab

   Knocking down of THBS1 inhibits pulmonary metastasis in vivo. (A) The
   scheme for establishing pulmonary metastasis model in nude mice by tail
   vein injection or proximal tibia injection are shown. (B) A nude mouse
   pulmonary metastasis model was established by injecting tumor cells
   into the tail vein, showing the general morphology of the lungs, H&E
   staining, p-MLC2 staining, Stro-1 staining and CD117 staining. (C) The
   number of metastatic nodules was evaluated and analyzed in osteosarcoma
   cells transfected with lenti-shTHBS1 and the negative control group.
   (D) The weights of the lungs in shCtrl and shTHBS1-1 groups were
   determined. (E) Analysis of expression changes of p-MLC2, Stro-1 and
   CD117 in metastatic nodules following THBS1 knockdown. (F) A nude mouse
   pulmonary metastasis model was established by injecting tumor cells
   into the proximal tibia, showing the in situ tumors, general morphology
   of the lungs, H&E staining, p-MLC2 staining, Stro-1 staining and CD117
   staining. (G-J) The volume and weight of in situ tumors, as well as the
   number of metastatic nodules and lung weights, were determined in both
   the shCtrl and shTHBS1-1 groups. (K) Analysis of expression changes of
   p-MLC2, Stro-1 and CD117 in metastatic nodules. ***P < 0.001, **P <
   0.01, *P < 0.05.

THBS1 promotes osteosarcoma pulmonary metastasis via ITGA1 and ITGA6 in vivo

   The results of the western blotting indicated that the expression of
   ITGA1 and ITGA6 was knocked down in cells overexpressing THBS1 (Figure
   [114]7A). We established pulmonary metastasis model in nude mice again
   by tail vein injection or proximal tibia injection (Figure [115]7B).
   The results of tail vein injection groups showed that overexpression of
   THBS1 promoted osteosarcoma metastasis and then increased the lung
   wights (Figure [116]7C-E). Additionally, THBS1 enhanced the expression
   of p-MLC2, Stro-1, and CD117 in vivo, indicating its role in
   facilitating cytoskeleton remodeling and the dedifferentiation of
   osteosarcoma cells (Figure [117]7C, 7F). However, the role of THBS1 in
   promoting osteosarcoma pulmonary metastasis could be reversed by ITGA1
   or ITGA6 knockdown (Figure [118]7C-F). Similar outcomes were observed
   in the groups receiving proximal tibia injections (Figure [119]7G-L).

Figure 7.

   [120]Figure 7
   [121]Open in a new tab

   THBS1 promotes pulmonary metastasis via ITGA1 and ITGA6 in vivo. (A)
   Western blotting was employed to show the knockdown of ITGA1 or ITGA6
   in cells that overexpress THBS1. (B) The scheme for establishing
   pulmonary metastasis model in nude mice by tail vein injection or
   proximal tibia injection are shown. (C) The general morphology of the
   lungs, H&E staining, p-MLC2 staining, Stro-1 staining and CD117
   staining of tail vein injection groups are shown. (D-E) The number of
   tumor nodules and the weights of the tail vein injection groups were
   evaluated and analyzed. (F) Analysis of the effects of THBS1
   overexpression and ITGA1/ITGA6 knockdown on the expression of p-MLC2,
   Stro-1 and CD117 in metastatic nodules of tail vein injection groups.
   (G) The in situ tumors, general morphology of the lungs, H&E staining,
   p-MLC2 staining, Stro-1 staining and CD117 staining of proximal tibia
   injection groups are shown. (H-K) The volume and weight of in situ
   tumors, and the number of metastatic nodules and the weights of the
   proximal tibia injection groups were evaluated and analyzed. (L)
   Analysis of the effects of THBS1 overexpression and ITGA1/ITGA6
   knockdown on the expression of p-MLC2, Stro-1 and CD117 in metastatic
   nodules of proximal tibia injection groups. ***P < 0.001, *P < 0.05.

THBS1 promotes dedifferentiation and the cytoskeletal remodeling of primary
osteosarcoma cells

   Next, we investigated the role of THBS1 in the dedifferentiation of
   primary osteosarcoma cell. We isolated primary osteosarcoma cells from
   osteosarcoma surgical samples and cultured them in vitro (Figure
   [122]8A-B). Then, we performed the sphere formation assay to obtain
   dedifferentiated primary osteosarcoma cells (Figure [123]8C), and the
   ability of these cells to redifferentiate into osteoblasts,
   chondrocytes, and adipocytes was determined (Figure [124]8D-F). To
   confirm that the obtained cells were indeed tumor cells, we injected
   those cells into the proximal tibia of nude mice, and the results
   showed that the dedifferentiated primary osteosarcoma cells could form
   tumors in the tibia of nude mice (Figure [125]8G-H, red arrows indicate
   the formed tumors). The spheroid cells also had stem cell-like
   characteristics, showing that the spheroid cells were enriched in the
   expression of CD117 and Stro-1 when compared to the residual cells
   (Figure [126]8I). We also found that in the primary cells derived from
   3 osteosarcoma patients, THBS1 was more highly expressed in
   sarcospheres relative to that in residual cells (Figure [127]8J). In
   addition, similar to osteosarcoma cell lines, which was demonstrated by
   F-actin staining, the overexpression of THBS1 in primary osteosarcoma
   cells promoted cytoskeletal remodeling (Figure [128]8K).

Figure 8.

   [129]Figure 8
   [130]Open in a new tab

   The function of THBS1 in the dedifferentiation of primary osteosarcoma
   cells. (A) Osteosarcoma primary cells were isolated from osteosarcoma
   tissues. (B) Osteosarcoma primary cell culture was performed. (C)
   Sphere formation of primary osteosarcoma cells was assessed. (D-F) A
   spheroid cell multidirectional differentiation experiment, including
   osteogenic, chondrogenic and adipogenic experiments, was performed.
   (G-H) A tumor formation experiment was performed in nude mice by
   injecting spheroid cells into the proximal tibia. (I)
   Immunocytochemistry was used to evaluate Stro-1 (green) and CD117
   (green) expression in residual cells and sarcospheres. Residual cells
   and sarcospheres were counterstained with DAPI (blue). (J) The
   expression of THBS1 in formed spheres and residual cells in primary
   osteosarcoma cells derived from different patients was determined. (K)
   Changes in cell morphology after overexpression of THBS1 in primary
   osteosarcoma cells. Cells were stained for F-actin (magenta), DAPI
   (blue). ***P < 0.001, **P < 0.01.

Discussion

   Osteosarcoma is the most common primary bone tumor, and it is highly
   malignant, grows rapidly, and is painful. Targeting the
   dedifferentiation process could yield new approaches to solve certain
   problems, such as pulmonary metastasis and unfavorable prognosis.

   To begin our research, we need to obtain dedifferentiated osteosarcoma
   cells. Mature differentiated cells in tumor tissue can serve as a
   reserve of stem cells during the development of tumors. These cells
   acquire stem cell properties under the accumulation of endogenous
   genetic mutations and the stimulation of exogenous environmental
   signals and play a role in tumor initiation and development[131]^27.
   When dedifferentiated tumor cells are cultured in vitro, they are
   typically characterized by a spherical shape suspended in the medium.
   Gibbs' research showed that osteosarcoma cells have the ability of
   forming sarcospheres, and some osteosarcoma cells express mesenchymal
   stem cell markers, such as Stro-1, CD44, and CD105. Compared with
   adherent cells, osteosarcoma spheres significantly highly express the
   embryonic pluripotent stem cell marker molecules Oct4 and
   Nanog[132]^28. Murase et al. demonstrated that sarcospheres can be
   cultured from osteosarcoma cell lines such as the OS2000, KIKU, NY,
   Huo9, HOS, U-2 OS, and Saos2. The formed sarcospheres have stem cell
   characteristics and have tumor-forming ability in vivo in the NOD/SCID
   mice[133]^29. In addition, after long-term treatment of MG-63 cells
   with 3-aminobenzamide (3-AB), a new cell line, 3AB-OS, with stem cell
   characteristics was obtained. It indicated 3AB-OS underwent
   dedifferentiation and these cells had a stronger self-renewal ability
   and tumorigenic ability and highly expressed the stemness genes CD133,
   Oct3/4, Nanog, and ABCG2[134]^30^, [135]^31. In this study, we
   successfully obtained dedifferentiated osteosarcoma cells through
   sphere formation experiments, which provides a material basis for
   subsequent research.

   Exploring how mature differentiated osteosarcoma cells dedifferentiate
   into progenitor cells with stem cell characteristics and why these
   cells are prone to lung metastasis is important. Our results
   demonstrated that THBS1 and ITGAs are the key molecules in osteosarcoma
   cell dedifferentiation. Previously, THBS1 was reported to be an
   inhibitor of angiogenesis and tumor progression[136]^32^-[137]^34.
   However, recent studies have reached the contradictory conclusion that
   THBS1 promotes tumor migration, invasion, and distant metastasis in
   breast cancer, thyroid cancer, colon cancer, prostate cancer, and
   melanoma[138]^35^-[139]^40. Lawler reported that fewer osteosarcomas
   occurred in p53-deficient mice that lack THBS1 than in those that
   express THBS1[140]^41. In addition, the knockout of THBS1 in an animal
   model of breast cancer led to growth of the primary tumor but a
   decrease in the number of metastases[141]^39.

   Integrins interact with many ligands, including ECM proteins, VCAM-1,
   and ADAM family members. THBS1 is a multifunctional protein that
   interacts with several cell surface receptors, including integrins,
   such as α3β1[142]^42^-[143]^45, α4β1[144]^46^, [145]^47, α6β1[146]^48^,
   [147]^49, α9β1[148]^50, ανβ1[149]^51, ανβ3[150]^52. These integrins
   regulate THBS1 to promote the adhesion and migration of tumor cells,
   vascular cells, and T cells[151]^43^, [152]^44^, [153]^47. The RGD
   sequence in the calcium-binding repeats of THBS1 is recognized by
   ανβ3[154]^53. In brief, THBS1 is a cell adhesion-related factor and a
   multifunctional receptor that interacts with ECM proteins through
   integrins. It aligns with our prior analysis, which employed GO, KEGG,
   and PPI to identify factors associated with the dedifferentiation of
   osteosarcoma cells. Our results also demonstrated that ITGA1 and ITGA6,
   two subunits of integrin, play a role in mediating extracellular
   signals in cells and that THBS1 promotes the dedifferentiation and
   metastasis of osteosarcoma cells.

   Some signaling pathways have been reported to be involved in cell
   migration that is regulated by THBS1, including the ERK, P38MAPK, and
   FAK pathways[155]^40^, [156]^54^-[157]^57. Hu et al. reported that
   THBS1 promotes pulmonary metastasis of osteosarcoma through FAK
   dependent pathway[158]^58. Our results revealed that THBS1 promotes
   cytoskeleton remodeling and pulmonary metastasis in osteosarcoma, and
   FAK play a role in this process. During the invasion and metastasis of
   tumors, tumor cells at the primary site invade local capillaries or
   lymphatic vessels through adhesion and invasion, spread to distant
   locations, are absorbed by capillaries or lymphatic vessels at a
   specific site, and then undergo subsequent settling and malignant
   proliferation around the blood vessels in secondary sites, eventually
   leading to tumor metastasis[159]^59^-[160]^61. Tumor cells undergo EMT
   to migrate and invade first before distant
   metastasis[161]^62^-[162]^64. During the migration and invasion of
   tumor cells, F-actin is reorganized at the front of the tumor cell to
   promote the formation of protrusions on the cell membrane[163]^65^,
   [164]^66, and the receptors on the protrusions on the cell membrane
   interact with extracellular matrix substrates to combine and recruit
   relevant molecules to form cell-ECM adhesion[165]^67. After forming
   adhesions, protrusions secrete proteases into the ECM to dissolve the
   ECM[166]^68^-[167]^70. Then, the intracellular actin cytoskeleton
   contracts with the stress provided by the adhesions, thereby pulling
   the cell forward[168]^71. Finally, the adhesions of the cell dissolve
   and move forward with the cell body. In summary, cytoskeletal
   remodeling and cell adhesion play an important role in the EMT.
   However, sarcomas, like osteosarcoma, are not epithelial in origin and
   cannot become more “mesenchymal”[169]^72^-[170]^74, so
   Lamhamedi-Cherradi et al. prefer to describe the EMT-like epigenetic
   effect as dedifferentiation[171]^22, which describes rejuvenated cell
   fate as cells shift up the Waddington epigenetic differentiation
   landscape[172]^75.

   The functions of FAK and RasGRF1 are probably closely related and they
   likely play a role in the process of dedifferentiation. Huang et al.
   reported that decreased expression of adhesion and cytoskeleton
   organization-related molecules, such as FAK, RsaGRF1, RhoA, and ROCK,
   was observed with the inhibition of EMT in 5-8F-DLC-1 cells[173]^76.
   Research in 2020 demonstrated that FAK and RsaGRF1 were enriched in
   focal adhesion and expressed differentially when Fat mass- and
   obesity-associated (FTO) gene was overexpressed[174]^77. Baldelli et
   al. reported increased activation of FAK Y576/577 and higher expression
   of RasGRF1 in KRASm/WT^- cells than in KRASm/WT^+ and WT cells[175]^78.
   In addition, researchers have indicated that RasGRF1, RasGFR2, and
   RasGRP are associated with the activation of FAK[176]^79. RasGRF1 is a
   Ras-selective guanyl exchange factor (GEF)[177]^80. GEFs activate
   GTPase by stimulating the release of guanosine diphosphate (GDP) to
   allow the binding of guanosine triphosphate (GTP)[178]^81^, [179]^82.
   Rho GTPase plays a key regulatory role in cell morphology, motility,
   proliferation, and apoptosis through cytoskeleton remodeling and
   dynamics[180]^83. ROCKs are activated when GTP-bound RhoA/C binds to
   the Rho-binding domain (RBD), which is located in the coiled-coil
   region of ROCKs[181]^84^-[182]^86. Subsequently, ROCKs mediate the
   phosphorylation of downstream target proteins, such as MLC, LIMK1/2,
   MYPT1, and adducin, to promote actin contraction[183]^87^-[184]^93. Our
   results showed cytoskeleton remodeling after overexpression of THBS1,
   and the expression of p-FAK, p-RasGRF1, and p-MLC2 was positively
   correlated with the expression of THBS1.

   Osteosarcoma is a malignant bone tumor commonly found in young
   individuals. The survival prognosis for patients is poor due to their
   susceptibility to early pulmonary metastasis. Current treatment
   strategies can effectively eliminate most tumor cells; however, there
   remains a risk of recurrence and lung metastasis. Our research focuses
   on a specific subset of cells exhibiting high metastatic potential,
   particularly those with stem cell-like characteristics that are
   resistant to conventional drugs. We aim to promote the
   redifferentiation of these cells from a poorly differentiated state
   into highly differentiated tumor cells that are more susceptible to
   chemotherapy, while also preventing other tumor cells from undergoing
   dedifferentiation. Our research has identified the key molecule THBS1,
   along with its downstream targets ITGA1 and ITGA6, which play a role in
   the dedifferentiation of osteosarcoma cells. In the future, we aim to
   further explore the binding sites of THBS1 with ITGA1 and ITGA6. We
   also plan to develop competitive binding peptides to disrupt the
   signaling pathway of THBS1, which promotes the dedifferentiation of
   osteosarcoma. This research may contribute to the development of drugs
   that inhibit the dedifferentiation of osteosarcoma cells.

   However, this study is still limited because we did not fully determine
   how THBS1 interacts with ITGA1/ITGA6 and the domains in which they
   interact. Once we identify the structural region where the interaction
   occurs, short peptide drugs that competitively bind to this domain may
   block the function of THBS1 in promoting the dedifferentiation of
   osteosarcoma cells.

   In summary, our results indicated that THBS1 promotes cell
   dedifferentiation and metastasis by promoting cytoskeletal remodeling,
   and that ITGA1 and ITGA6 play an important role in mediating
   extracellular to intracellular signals (Figure [185]9); this mediating
   effect mainly occurred in the early phase of cell dedifferentiation.
   Our finding identified key molecules that promote the dedifferentiation
   of osteosarcoma cells, providing valuable insights for developing drugs
   to inhibit this process and reduce the risk of pulmonary metastasis in
   osteosarcoma.

Figure 9.

   [186]Figure 9
   [187]Open in a new tab

   Graphical abstract.

Acknowledgments