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. 2026 Jan 23;57:100742. doi: 10.1016/j.jbo.2026.100742

Differential expression of CD74 and C1QB in jaw versus long bone osteosarcoma: Insights from animal models, public datasets and clinical cohorts

Lalan Zhang a, Jiaoyan Liu b, Shengjie Shao c, Xiang Liu c, Liqin Zhang d, Weihong Wang a,⁎
PMCID: PMC12907127  PMID: 41705027

Highlights

  • •

    Novel jaw and long bone osteosarcoma rat models were established.

  • •

    CD74 and C1QB were identified as top hub genes from 414 DEGs.

  • •

    Jaw osteosarcoma exhibits higher CD74/C1QB than long bone osteosarcoma.

  • •

    These markers are potential candidates for functional and mechanistic research.

Keywords: Jaw osteosarcoma, Long bone osteosarcoma, Macrophages, CD74, C1QB

Abstract

Objective

This study aimed to investigate the differential expression of macrophage-related factors between jaw and long bone osteosarcoma, thereby providing potential candidates for future functional and mechanistic research.

Methods

Twenty-four Sprague-Dawley (SD) rats were randomly divided into control, jaw osteosarcoma, and long bone osteosarcoma groups. UMR106 cells were implanted subcutaneously in nude mice, then tumors were transplanted into rat jaw and tibial bone marrow to establish osteosarcoma models. Bone changes were observed using Micro-CT, while histological changes were examined with H&E and Masson’s trichrome staining. Transcriptome sequencing identified differentially expressed genes (DEGs), with the top three genes selected using Cytohubba software. Survival analysis was performed using the GEPIA database. A retrospective analysis was conducted on jaw osteosarcoma patients and long bone osteosarcoma patients from November 2014 to June 2023. Finally, immunohistochemical staining validated the expression levels of relevant proteins in both animal and clinical tissue samples.

Results

Micro-CT revealed significant bone destruction in both osteosarcoma models. Histological analysis revealed high pleomorphism in osteosarcoma cells, predominantly spindle and polygonal shapes, with evident pathological mitosis. Transcriptome sequencing identified 414 DEGs, including the top three: RT1-Da, CD74, and C1QB. Survival analysis showed high expression of CD74 and C1QB correlated positively with overall survival in patients with osteosarcoma. Immunohistochemistry demonstrated higher CD74 and C1QB expression in jaw osteosarcoma compared to long bone osteosarcoma in both rats and humans (P < 0.05).

Conclusion

The expression levels of CD74 and C1QB were significantly higher in jaw osteosarcoma than in long bone osteosarcoma, suggesting that this differential expression may have clinical relevance for patient survival and warrants further investigation.

1. Introduction

Osteosarcoma (OS), originating from primitive osteogenic mesenchymal cells, is the most common primary malignant bone tumor in adolescents [1]. Over the past 30 years, the prognosis for patients with advanced and recurrent osteosarcoma has not improved significantly [2], [3]. Jaw osteosarcoma (JOS) primarily occurs in the mandible and has a lower rate of lung metastasis, while long bone osteosarcoma (LBOS) commonly affects the femur, tibia, and humerus, and is prone to bone or lung metastasis, leading to a poorer prognosis [4], [5]. Studies have shown that the clinical and biological differences between JOS and LBOS are related to variations in their immune microenvironments [5], [6]. The OS immune microenvironment consists of various cells and extracellular components that interact to form an inhibitory immune network [7], [8], [9]. This network is closely associated with tumor immune evasion, abnormal proliferation, and metastatic invasion. Tumor-associated macrophages are the most abundant immune cells in the osteosarcoma microenvironment, promoting osteosarcoma cell proliferation, migration, invasion, angiogenesis, and apoptosis inhibition [10]. However, the regulatory differences in macrophage-associated factors between JOS and LBOS remain unclear. Therefore, this study aims to investigate the differences in the expression of macrophage-associated factors between JOS and LBOS using SD rat models, providing potential candidates for future functional and mechanistic research.

2. Materials and methods

2.1. Experimental animals and cell lines

The study utilized 4-week-old SPF-grade male nude mice (n = 5), each weighing approximately 15 ± 5 g, and 3-week-old SPF-grade male SD rats (n = 24), each weighing approximately 50 ± 5 g, both provided by the Laboratory Animal Center of Kunming Medical University. The SD rats were randomly divided into three groups: control group (n = 4), jaw osteosarcoma group (n = 10), and long bone osteosarcoma group (n = 10). The UMR106 cell line is derived from the SD rat osteogenic osteosarcoma cell line. It is an osteosarcoma cell strain induced by radioactive phosphorus (32P) in SD rats and was purchased from Shanghai Jingxiang Qidian Technology Co., Ltd.

2.2. Cell culture

UMR106 rat osteosarcoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. For routine culture, cells were seeded in T25 cm2 flasks and passaged at approximately 80–90% confluence using trypsin-EDTA dissociation at a split ratio of 1:3.

2.3. Osteosarcoma model

The tissue block transplantation method was employed to establish the animal models. Specifically, the JOS model was based on our prior establishment, the data are reused or partially overlapping with the cohort reported in [11], whereas the LBOS model was newly generated for this comparative study. (Fig. 1A).

Fig. 1.

Fig. 1

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Establishment of the JOS and LBOS models in SD rats. (A) Flowchart of establishing osteosarcoma animal models using tissue block transplantation. (B) Tumor tissue samples for JOS and LBOS models. (C) Lung of JOS model. (D) Lung of LBOS model.

2.4. Micro-CT scanning

High-resolution Micro-CT scans of SD rat jaw and tibia specimens were performed using a large-volume scanning chamber (CT model: NMC-100) to observe the changes in bone density.

2.5. Transcriptome sequencing

Fresh tumor tissue samples from SD rat JOS and LBOS models were quickly frozen in liquid nitrogen and stored at −80°C. Transcriptome sequencing was outsourced to the Novogene Bioinformatics Technology. DEGs were identified using the criteria of Padj < 0.05 and |log2Fold Change|≥1, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The top three genes were selected using the Cytohubba software, and survival analysis of DEGs was conducted using the GEPIA database.

2.6. Clinical JOS and LBOS samples

A retrospective analysis was conducted on paraffin-embedded JOS tissues from the Affiliated Stomatological Hospital of Kunming Medical University and paraffin-embedded LBOS tissue samples from the First People's Hospital of Yunnan Province, collected between November 2014 and June 2023. Paraffin-embedded samples were provided by the pathology departments.

2.7. Histological analysis

Tumor specimens from nude mice and SD rats were fixed, decalcified, dehydrated, paraffin-embedded, sectioned, and deparaffinized. They were subjected to H&E staining, Masson’s trichrome staining, and immunohistochemical staining to observe histopathological changes and expression of CD74 and C1QB proteins.

H&E staining: Paraffin-embedded mandibular sections (4 µm) were deparaffinized, rehydrated, stained with hematoxylin and eosin, dehydrated, cleared, and mounted.

Masson’s trichrome staining: Sections were stained sequentially with Weigert’s hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic acid, and aniline blue to visualize collagen, followed by dehydration, clearing, and mounting.

Immunohistochemistry and quantification: Following antigen retrieval and blocking, sections were incubated with anti-CD74/C1QB (1:300, 4°C overnight) and HRP‑conjugated secondary antibody (1:300), developed with DAB, counterstained, and mounted. Protein expression was quantified using Image‑Pro Plus 6.0. For each case, five representative tumor ROIs were delineated by a blinded pathologist. The mean optical density of tumor cells per ROI was automatically measured, and the sample‑level expression was calculated as the mean optical density across the five ROIs.

2.8. Statistical analysis

Statistical analyses were performed using IBM SPSS 22.0. The primary methods included independent-sample t-tests and one-way ANOVA. Results are presented as the mean ± standard deviation. Differences were considered statistically significant at P < 0.05.

3. Results

3.1. Tumor growth and pulmonary status

Using the tissue block transplantation method, we successfully established JOS and LBOS SD rat models (Fig. 1B). As reported in reference [11], the JOS group demonstrated significant right-sided facial swelling and substantial weight loss at the 2 week postoperative interval, achieving a tumor formation rate of 80% (8/10). No significant abnormalities were observed in the dissected lung tissues (Fig. 1C). In the LBOS group of SD rats, a noticeable swelling at the right tibial implantation site appeared 1 week post-surgery, accompanied by lameness. The tumor formation rate was 90% (9/10) and the lung metastasis rate was 50% (5/10), with the lung tissue surface covered in millet-sized nodules (Fig. 1D).

3.2. Micro-CT examination

In the control group, the cortical bones of the mandible and tibia appeared intact and smooth. In the JOS group, the tumor images showed severe bone destruction with unclear boundaries and occasional Godman triangle images. The LBOS group also exhibited tumor images with significant bone destruction, unclear boundaries, and pathological fractures (Fig. 2).

Fig. 2.

Fig. 2

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Micro‑CT analysis of JOS and LBOS model. (A-B) Normal jaw bone exhibiting a smooth and intact cortical surface. JOS model at the tumor site showing a bulging lesion (C) and pronounced osteolytic destruction (D) at the tumor site. (E-F) Normal tibia, displaying a smooth and even cortical surface. (G-H) LBOS model, demonstrating extensive osteolytic damage and disruption of the normal bone architecture. White arrows indicate the primary tumor regions.

3.3. H&E and Masson's trichrome staining

In the subcutaneous osteosarcoma of nude mice and in the JOS and LBOS groups of SD rats, the tumor cells exhibited diffuse growth with varying cell sizes. The cells were spindle-shaped or polygonal, with single or multiple nuclei, enlarged nucleoli, reduced intercellular stroma, and a high nucleus-to-cytoplasm ratio. Pale pink osteoid-like tissue, granular calcification, and necrotic tumor cells were observed, along with occasional giant tumor cells (Fig. 3A-C). The lung tissues of the control and JOS groups displayed a uniform reticular structure with clear edges, thin and evenly distributed alveolar walls, and no signs of thickening, congestion, or edema in the alveoli. In contrast, the lung tissues of the LBOS group showed disappearance of lung septa, multiple metastatic tumors, spindle or polygonal infiltration, partial calcification, and necrosis of tumor tissues (Fig. 3D-F).

Fig. 3.

Fig. 3

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Histopathological analysis by H&E and Masson staining. H&E staining: (A) Subcutaneous osteosarcoma model in nude mice; (B) JOS model in SD rats; (C) LBOS model in SD rats; (D) Normal lung; (E) Lung of JOS model; (F) Lung of LBOS model. Masson staining: (A) Subcutaneous osteosarcoma model in nude mice; (B) JOS model in SD rats; (C) LBOS model in SD rats; (D) Normal lung; (E) Lung of JOS model; (F) Lung of LBOS model (Magnification:×40, ×200).

3.4. Transcriptome sequencing

Differentially expressed gene (DEG) analysis between JOS and LBOS identified a total of 414 DEGs, with 315 upregulated and 99 downregulated genes (Fig. 4A). GO enrichment analysis showed that the upregulated DEGs were closely related to adaptive immune response, complement activation, T cell activation, positive regulation of leukocyte activation, and immune response activation (Fig. 4B). KEGG pathway enrichment analysis indicated that the upregulated DEGs were positively associated with antigen processing and presentation, complement and coagulation cascades, Th1 and Th2 cell differentiation, and Th17 cell differentiation (Fig. 4C). DEGs were imported into the STRING database, and with a default combined score threshold of ≥0.4, a protein–protein interaction (PPI) network was generated (Fig. 4D). Further analysis using the cytoHubba plugin in the Cytoscape software identified the top three key genes as RT1-Da, C1QB, and CD74 (Fig. 4E). Kaplan-Meier survival analysis using the GEPIA database revealed that high expression of CD74 and C1QB correlated with longer overall survival and disease-free survival in patients with osteosarcoma, indicating better prognosis (Fig. 4F).

Fig. 4.

Fig. 4

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Transcriptome sequencing results of JOS and LBOS in SD rats. (A) Volcanic map of DEGs. (B) Bubble chart for GO enrichment analysis of up-DEGs between the JOS group and the LBOS group. (C) Bubble chart for KEGG enrichment analysis of up-DEGs between the JOS group and the LBOS group. (D) Protein-protein interaction networks of DEGs in JOS and LBOS group. (E) Cytohubba analysis of the core gene nodes in PPI network. (F) Kaplan-Meier analysis of the hub genes.

3.5. Immunohistochemical staining

Immunohistochemical staining revealed CD74 and C1QB protein expression in both JOS and LBOS samples (Fig. 5). The expression levels of CD74 in JOS and LBOS from SD rats were 27.77 ± 7.03 and 4.92 ± 1.10, respectively, and the expression levels of C1QB were 69.27 ± 18.96 and 16.13 ± 5.49, respectively. The expression of both CD74 and C1QB was significantly higher in JOS compared to LBOS, with statistically significant differences (P < 0.01). In the clinical study, we analyzed 19 osteosarcoma samples, comprising 10 JOS and 9 LBOS cases. JOS patients were older (mean age, 37.2 ± 18.2 years) than LBOS patients (21.8 ± 9.4  years). The JOS group comprised 2 males and 8 females, while the LBOS group had 5 males and 4 females. During follow-up, no distant metastases were documented in the JOS group; however, 4 cases experienced local recurrence, and 2 patients died. In contrast, the LBOS group presented with 1 case of metastasis, 1 recurrence, and 1 death (Table S1). No lung metastasis was observed in JOS patients, while 1 case of lung metastasis was observed in LBOS patients. The expression levels of CD74 in JOS and LBOS patients were 21.19 ± 11.24 and 9.55 ± 3.95, respectively, and the expression levels of C1QB were 77.48 ± 22.90 and 27.53 ± 18.13, respectively. The expression of both CD74 and C1QB was significantly higher in human JOS compared to human LBOS, with statistically significant differences (P < 0.05).

Fig. 5.

Fig. 5

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Immunohistochemical validation of CD74 and C1QB expression in animal and clinical tissue samples. (A) JOS; (B) LBOS (Magnification:×100, ×400). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

4. Discussion

Current research on osteosarcoma models predominantly uses immunodeficient animals, with a focus on LBOS. However, JOS remains under-researched [12]. In 2019, Bertin et al. [6] pioneered the development of mouse models for JOS, including orthotopic and heterotopic transplants as well as patient-derived xenografts. Despite this, these models did not compare the immune microenvironments of JOS and LBOS. Comparative studies on the immune microenvironments of JOS and LBOS are scarce [13], [14]. Moreover, the innate and adaptive immune systems of mice differ significantly from those of humans [15], [16]. Although various preclinical mouse models exist, they often do not accurately reflect the heterogeneity of OS, the complexity of its immune microenvironment, or its clinical manifestations such as recurrence, metastasis, and chemoresistance [17]. Yao et al. [18] successfully created an LBOS model in SD rats with a fully functioning immune system. This model effectively simulates the immune microenvironment and replicates the complexity and heterogeneity of the tumor, thus laying a critical foundation for developing new treatments and understanding the immune microenvironment of OS.

Our study adopted the tissue block transplantation method to establish both JOS and LBOS models, thereby circumventing the direct lung tumor seeding associated with cell suspension injections [19], thus yielding models that recapitulate the clinical observation that LBOS exhibits a higher lung metastasis rate than JOS [20]. This orthotopic model retains the natural immune microenvironment, allowing the study of the early stages of tumor formation and metastasis [21]. The tumor formation and lung metastasis rates in osteosarcoma models are influenced by various factors, such as the age and type of rats, the osteosarcoma cell lines used, and the transplantation sites [18], [21], [22]. We used the UMR106 cell line, derived from SD rats through 32P induction, known for its strong tumorigenicity and high lung metastasis rate [23]. It is undeniable that the different microenvironments of the jaw and tibia, where UMR106 cells were transplanted, might affect the tumor formation and lung metastasis rates.

To investigate the differences between the immune microenvironments of JOS and LBOS, we conducted transcriptome sequencing of the animal specimens. These results revealed distinct variations in the immune microenvironment. Tumor-associated macrophages constitute more than 50% of immune cells in OS and play a crucial role in tumor proliferation and metastasis [24], [25]. Macrophage migration inhibitory factors can induce an increase in vascular endothelial growth factor (VEGF), whose expression level is related to microvessel density [26]. The VEGF expression level in JOS was significantly lower than that in LBOS, explaining the lower metastasis rate of JOS and suggesting the differential expression of macrophage-related factors between JOS and LBOS [27]. To further explore the differences in macrophage-related factor expression between JOS and LBOS, we used the Cytoscape software to identify the top three key genes: RT1-Da, CD74, and C1QB. However, we could not find an antibody corresponding to the RT1-Da, and the GEPIA online analysis website did not generate a survival curve for this gene. Immunohistochemistry showed that the expression levels of CD74 and C1QB in SD rat JOS were higher than those in LBOS rats (P < 0.05). Survival analysis indicated that CD74 and C1QB are associated with better OS prognosis, suggesting that they may be potential biomarkers for predicting osteosarcoma prognosis and personalized treatment.

CD74 is expressed in dendritic cells and macrophages and serves as a cell membrane receptor for the macrophage migration inhibitory factor, regulating the transport of MHC class II antigens [28]. CD74 is present in various solid tumors, including glioma [29], melanoma [30], and breast cancer [31], and is associated with patient prognosis. In non-metastatic pediatric osteosarcoma patients, CD74 expression is relatively high [32]. As a marker for tissue-resident macrophages and tumor-associated macrophages, complement C1Q is primarily produced by macrophages. It is a hexamer formed by polypeptide chains encoded by the C1QA, C1QB, and C1QC genes and is a key molecule in the complement system activation pathway, playing a role in promoting or inhibiting cancer progression [33], [34], [35]. Huang et al. [36] indicated that C1Q is related to tumor immune infiltration and serves as a biomarker for predicting metastasis and prognosis in OS patients, consistent with our study's findings.

According to the clinical data of osteosarcoma patients in this study, the average age of LBOS patients is lower than that of JOS patients (21.8 y vs. 37.2 y). This finding aligns with the studies by Rodriguez et al. [37] and Kontio et al. [38], which reported similar average ages for JOS patients. Among LBOS patients, one case of lung metastasis was observed, whereas no lung metastasis was observed in JOS patients, suggesting a better prognosis for JOS. This observation is consistent with the findings of Brown et al. [4]. Immunohistochemistry showed that CD74 and C1QB expression levels in clinical JOS samples were higher than in LBOS samples (P < 0.05), further validating the association of CD74 and C1QB with better prognosis. These markedly elevated expression levels in JOS offer a plausible biological explanation for the concomitant reduction in metastatic incidence documented in both the experimental model and supporting patient data.

However, this study has several limitations. First, the small sample size (n = 19) of the retrospective cohort constrains statistical power, leaving the study underpowered for robust clinical prognostic modeling. Second, the generalizability of the findings is limited by the homogeneous, single-center patient population (Kunming, Yunnan, China), particularly the significant age disparity between the JOS and LBOS cohorts (37.2 y vs. 21.8 y), which may act as a confounding variable in interpreting the observed immune cell infiltration patterns. Evidence suggests that immune status evolves with age, and that age-related immunosenescence influences immune function, immune cell infiltration, and the tumor microenvironment in osteosarcoma [39], [40], [41]. Besides, Prior cancer therapies (e.g., radiotherapy or chemotherapy) can alter the tumor immune microenvironment, a known constraint when extrapolating results from controlled animal models to heterogeneous patient samples [42], [43]. An additional limitation is the absence of survival analysis in the animal experiments. Consequently, future studies necessitate age-matched cohorts or multivariate analysis, combined with multi-center validation in diverse populations, to dissociate tumor-intrinsic effects from age-related immune changes. Furthermore, incorporating survival endpoints into preclinical models is required to strengthen translational relevance. Finally, establishing the mechanistic roles of CD74 and C1QB will require functional validation through in vitro and in vivo studies.

5. Conclusion

In conclusion, our findings demonstrate that elevated expression of CD74 and C1QB in jaw osteosarcoma may be clinically relevant to survival—a promising yet preliminary association warranting further study. Future functional studies on these markers must control for potential confounders such as age-related immunosenescence and prior treatment exposure.

Availability of data and materials

RNA-seq data that support the findings of this study have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences with accession number CRA019541, which are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Ethical disclosure statement

This study extended our previously established JOS model by introducing a novel LBOS model for systematic comparison (transcriptomic analysis and cross-species validation). While the modeling methodology and baseline characteristics for JOS were referenced from our prior work, all data and images presented for the comparative analyses were newly generated and are not reused from previous publications.

Consent for publication

Not applicable.

Code availability

Not applicable.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors did not use any AI-assisted technology during the preparation of this work.

CRediT authorship contribution statement

Lalan Zhang: Writing – review & editing, Writing – original draft, Methodology, Investigation. Jiaoyan Liu: Writing – review & editing, Methodology, Investigation. Shengjie Shao: Writing – review & editing, Resources, Investigation. Xiang Liu: Writing – review & editing, Resources, Investigation. Liqin Zhang: Writing – review & editing, Methodology. Weihong Wang: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

All animal care and experimental procedures complied with the guidelines for the ethical review of animal welfare and were approved by the Animal Ethics Committee of Kunming Medical University (No. kmmu20231535). This study was approved by the Ethics Committee of the Kunming Medical University (No. KYKQ2024MEC0011), and informed consent was obtained from patients or their relatives.

Funding

This study was financially supported by the Medicine Leading Talent of Yunnan Province Health Care Committee (No.L-2018010), Yunnan Province Xing Dian Talent (No.XDYC-MY-2022-0052) and the Scientific Research Fund Project of Yunnan Provincial Department of Education (No.2024Y241).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Not applicable.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jbo.2026.100742.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Data 1
mmc1.xlsx (11.3KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data 1
mmc1.xlsx (11.3KB, xlsx)

Data Availability Statement

RNA-seq data that support the findings of this study have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences with accession number CRA019541, which are publicly accessible at https://ngdc.cncb.ac.cn/gsa.

Articles from Journal of Bone Oncology are provided here courtesy of Elsevier

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