The expression and clinical significance of tumor stem cell markers CD34 and CD40 in osteosarcoma tissue

doi:10.21203/rs.3.rs-4884389/v1

Objective: To investigate the expression and clinical significance of tumor stem cell markers CD34 and CD40 in osteosarcoma tissue.

Methods: A total of 106 osteosarcoma patients who underwent surgical treatment were selected as the study subjects. The expression of CD34 and CD40 in tumor and adjacent tissues was detected by immunohistochemistry. Clinical and pathological data were collected, and the impact of CD34 and CD40 expression on clinical and pathological parameters and prognosis of osteosarcoma patients was statistically analyzed.

Results: (1) RT-qPCR and Western blot assay showed that the mRNA and protein expression levels of CD34 and CD40 in osteosarcoma tissues were significantly higher than those in adjacent tissues. (2) CD34 and CD40 were mainly located on the cell membrane of tumor cells. The positive rates of CD34 in adjacent and tumor tissues were 17.9% (19/106) and 60.4% (64/106), respectively, and the positive rates of CD40 in adjacent and tumor tissues were 23.6% (25/106) and 64.1% (68/106), respectively. The positive rates of CD34 (χ²=40.095, P＜0.001) and CD40 (χ²=35.420, P＜0.001) in osteosarcoma tissues were significantly higher than those in adjacent tissues. (3) CD34 and CD40 positive expression was closely related to Histological Grading, Tumor metastasis, and TNM staging. The Kaplan-Meier survival analysis showed that the survival time of patients in CD34-positive group (χ²=29.33, P＜0.01) and CD40-positive group (χ²=8.690, P=0.003) was significantly shorter than that in corresponding negative control group.

Conclusion: The tumor stem cell markers CD34 and CD40 are highly expressed in osteosarcoma tissues and have a significant negative impact on clinical and pathological parameters and prognosis of osteosarcoma patients.

Osteosarcoma

Tumor stem cells

CD34

CD40

Osteosarcoma is one of the most common primary bone tumors in children and adolescents, accounting for 20% of primary malignant bone tumors, with an annual incidence rate of 2 persons/1 million[1, 2]. The site of occurrence is mostly at the metaphysis of long bones, rarely in the spine or pelvis, and often presents as a solitary lesion. Patients usually present with local pain and swelling, and rarely pathological fractures, making it easy to be confused with other diseases[3]. Osteosarcoma has a high degree of malignancy and is prone to lung metastasis, with a poor prognosis once lung metastasis occurs. With the popularization of the new adjuvant chemotherapy regimen proposed by Rosen et al. in 1976, the 5-year survival rate of patients has been increased from 20% with simple surgical treatment to 60%, greatly improving the quality of life and survival time of patients[4]. However, in recent decades, the survival rate of osteosarcoma patients has remained at 60–70%. One of the main reasons for treatment failure today is that patients often develop resistance to anticancer drugs. So far, doxorubicin, cisplatin, and methotrexate are commonly used chemotherapy drugs for osteosarcoma. Multidrug resistance is the main obstacle to chemotherapy for osteosarcoma and is also an important challenge for successfully treating cancer[5, 6]. To improve the treatment effect, the analysis of the mechanism of chemotherapy resistance in osteosarcoma cells is crucial for exploring new treatment strategies for this disease[7]. Tumor stem cells are a special type of cell that could self-renew and differentiate into various tumor cells and is the major driving force for tumor growth and metastasis. CD34 and CD40 are two molecular markers associated with tumor stem cells and have important significance for the study of malignant tumors[8]. However, there have been no reports on the expression of CD34 and CD40 in osteosarcoma tissue, so this study aims to use immunohistochemistry to investigate the expression of CD34 and CD40 in osteosarcoma and corresponding adjacent tissues and analyze the correlation between their expression and clinical pathological parameters and prognosis of patients.

1.1 Research subjects

The research subjects were 106 osteosarcoma patients admitted to Xuzhou Medical University Affiliated Hospital from January 2015 to January 2022. Inclusion Criteria: ① All patients received surgical resection. ② Postoperative pathological diagnosis was confirmed as osteosarcoma. ③ Cancer tissue and para-cancer tissue were kept in the laboratory of our hospital. ④ All osteosarcoma patients were first diagnosed and had not undergone any antitumor treatment, including radiotherapy, chemotherapy, traditional Chinese medicine treatment, targeted therapy, and immunotherapy before surgery. ⑤ Patient clinical data were complete. Exclusion criteria: ① Coexisting with other malignant tumors. ② Presence of coagulation dysfunction. ③ In-hospital death cases. Among all patients, there were 58 males and 48 females, with an age range of 23–49 years and an average age of (28.6 ± 3.72).

All methods in this study were carried out in accordance with relevant guidelines and regulations. The experimental protocols were approved by the Ethics Committee of Xuzhou Medical University Affiliated Hospital (Approval Number: XYFY2023-KL251-01). Informed consent was obtained from all subjects and/or their legal guardian(s) prior to their participation in the study.

1.2 RT-qPCR assay

Total RNA was extracted from frozen tissue samples using the Qiagen RNeasy Mini Kit along with the QIA shredder and RNase-Free DNase Set, according to the manufacturer's instructions. SuperScript® VILO™ MasterMix (Invitrogen) was used to generate cDNA. Real-time RT-qPCR reactions were carried out using SYBR® Select Master Mix (Life Technologies) and the BioRad CXF96 Real-Time PCR Detection System. The reaction conditions were 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing/extension at 60°C for 1 minute. Gene expression fold change was determined by the 2^−ΔΔCT, using GAPDH as the housekeeping gene.

1.3 Western blot

Tumor tissue and adjacent tissues were homogenized in lysis buffer containing 1% protease inhibitors. The resulting cell lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentrations were measured using the Bradford kit (Thermo Fisher Scientific, Waltham, USA). Proteins with varying molecular weights were separated using 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes that were pre-treated with methanol. The PVDF membranes were then blocked for 2 hours with 5% nonfat dry milk in TBS-T before being probed with the anti-UBE2S primary antibody overnight at 4°C. After washing the PVDF membrane with PBS, it was incubated with horseradish peroxidase-labeled rabbit secondary antibody for 1 hour at 26°C. Following another round of washing with PBS, the western blot on the PVDF membrane was developed using ECL developer, and imaging was carried out with a LAS4000 charge-coupled camera (Fujifilm, Tokyo, Japan).

1.4 Immunohistochemical staining

The tissue slices were baked in a 60℃ oven for 2 hours, and then dewaxed and hydrated. Endogenous enzymes were immobilized with 3% hydrogen peroxide, followed by a rinse with flowing water and heat antigen retrieval. PBS was used for washing 1–2 times. The slices were then incubated with a 5% BSA blocking solution for 20 minutes. After removing the blocking solution, CD34 and CD40 antibodies were added to the slices, which were then placed at 4℃ overnight. The next day, the slices were rinsed three times with PBS, and the secondary antibody was added and incubated for 30 minutes at room temperature. PBS was used to wash the slides three times, followed by DAB staining. Observation under a microscope and photography were then performed.

1.5 Statistical Analysis

All data were analyzed using SPSS 22.0 statistical software. The comparison of count data was performed using the chi-square test or Fisher's exact probability method. Correlation analysis was conducted using Spearman's correlation analysis. The survival analysis was conducted using the Kaplan-Meier. P < 0.05 was considered statistically significant.

2.1 CD34 and CD40 levels detected by RT-qPCR and Western blot

The results of the RT-qPCR showed that the levels of CD34 mRNA and CD40 mRNA were significantly higher in osteosarcoma tissues compared to adjacent tissues. To further confirm these findings, protein was extracted from the same set of 8 randomly selected patients' adjacent tissues and osteosarcoma tissues and subjected to Western blot assay. The results showed that the protein levels of CD34 and CD40 were generally increased in osteosarcoma tissues compared to adjacent tissues. As shown in Fig. 1, the mRNA and protein levels of CD34 and CD40 were significantly higher in osteosarcoma tissues compared to adjacent tissues.

2.2 The expression of CD34 and CD40 was detected by immunohistochemical staining.

The results showed that CD34 and CD40 were mainly expressed on the cell membrane of tumor cells, with some expression in the cytoplasm. Figure 2 illustrates the immunohistochemical staining of CD34 and CD40, showing that these markers are mainly expressed on the cell membrane of tumor cells. The positive rates of CD34 in adjacent and tumor tissues were 17.9% (19/106) and 60.4% (64/106), respectively, while the positive rates of CD40 were 23.6% (25/106) and 64.1% (68/106), respectively. The positive rates of CD34 (χ²=40.095, P < 0.001) and CD40 (χ²=35.420, P < 0.001) were significantly higher in osteosarcoma tissues than in adjacent tissues.

2.3 Impact of CD34 and CD40 on clinical pathological parameters of patients

The univariate analysis indicated a significant correlation between the positive expression of CD34 and CD40 and histological grading, tumor metastasis, and TNM stage in patients. Specifically, patients with a poor histological grading, presence of distant metastases, and higher TNM staging showed a relatively higher rate of positive expression of CD34 and CD40. These findings suggest that the positive expression of CD34 and CD40 is closely associated with the severity and progression of tumors. Therefore, CD34 and CD40 may serve as potential prognostic markers for the assessment of the prognosis and treatment response in patients with cancer.

2.4 Correlation analysis of CD34 and CD40 expression in osteosarcoma tissue

The Spearman correlation analysis revealed a significant positive correlation between the expression of CD34 and CD40 in osteosarcoma tissues (r = 0.613, P<0.01). This indicates that the expression levels of CD34 and CD40 are positively associated in osteosarcoma, suggesting that these markers may have a similar biological function or signaling pathway in the development and progression of tumors.

2.5 The impact of 2.5 CD34 and CD40 on the prognosis of patients with osteosarcoma

The Kaplan-Meier analysis was used to analyze the survival time of patients, and the results showed that the survival time of patients in the CD34-positive group (χ² = 29.33, P<0.01) and CD40-positive group (χ² = 8.690, P = 0.003) was significantly shorter than that of the corresponding negative control groups. As depicted in Fig. 3, the survival time of patients in the CD34-positive and CD40-positive groups was significantly shorter than that of the corresponding negative control groups. This suggests that the expression of CD34 and CD40 may be associated with the poor prognosis of osteosarcoma patients. Therefore, CD34 and CD40 expression levels may serve as potential prognostic biomarkers for osteosarcoma patients and may be useful in predicting survival outcomes and making treatment decisions.

2.6 Cox analysis of prognostic factors affecting patients with osteosarcoma

The Cox proportional hazards regression analysis showed that Tumor metastasis, TNM staging, CD34(་), and CD40(་) were all independent risk factors that significantly influenced the prognosis of osteosarcoma patients. This suggests that these factors should be considered when developing individualized treatment plans for patients with osteosarcoma to maximize the chances of a favorable outcome.

Osteosarcoma is a malignant bone tumor that originates from the bone growth plate or osteogenic tissue. CD34 is a widely distributed molecular marker in vascular endothelial cells, hematopoietic stem cells, and tumor tissues[9]. Many studies have shown that CD34 plays an important role in the occurrence and development of osteosarcoma. CD34-positive osteosarcoma cells have stronger proliferation ability and apoptosis inhibition ability, which can promote tumor cell proliferation and invasion. At the same time, CD34 also participates in the interaction between osteosarcoma cells and the tumor microenvironment, as well as tumor angiogenesis and other processes[10]. CD40 is a cell membrane protein that is widely expressed in immune cells, including B cells, dendritic cells, macrophages, T cells, and is involved in regulating immune responses. CD34 and CD40 are widely recognized markers of cancer stem cells, which are closely related to the occurrence and development of tumors. Cancer stem cells are a subpopulation of tumor cells with self-renewal and tissue reconstruction abilities, which play important roles in the occurrence, growth, metastasis, and drug resistance of tumors[11]. In osteosarcoma cells, CD40 and CD34 may regulate the proliferation and differentiation of cancer stem cells and participate in the invasion and metastasis of osteosarcoma cells, in addition to regulating immune response. Therefore, future research can develop more effective treatment methods by regulating the expression and function of CD40 and CD34 to inhibit the proliferation and invasion of cancer stem cells.

We found that a correlation between the high expression of CD34 and CD40 in osteosarcoma tissue and poor histological differentiation. This correlation may be attributed to their involvement in various biological behaviors of tumors such as growth, metastasis, and invasion. CD34 is a common endothelial cell marker that is widely expressed on the surface of other tissues and tumor cells. In osteosarcoma, high expression of CD34 is associated with angiogenesis, tumor swelling, infiltration, invasion, and metastasis which are typical manifestations of poor histological differentiation. CD40, on the other hand, is a sensitive co-stimulatory molecule that is broadly expressed on the surface of immune system cells and many tumor cells[12]. In osteosarcoma, high expression of CD40 is associated with immune evasion, infiltration, invasion, and metastasis which are also typical manifestations of poor histological differentiation. Thus, the high expression of CD34 and CD40 in osteosarcoma may be related to the malignancy and increased biological behavior of the tumor, leading to poor histological differentiation[13]. The high expression of CD34 and CD40 in osteosarcoma is associated with tumor invasion and metastasis, which may promote the distant metastasis of osteosarcoma patients. In osteosarcoma, high expression of CD34 is associated with angiogenesis, which may promote the formation of new blood vessels by tumor cells, providing them with nutrition and oxygen, and thus enhancing their growth and metastatic potential, promoting the occurrence of distant metastasis#[14]. The high expression of CD40 is associated with immune evasion, which may accelerate the growth and metastasis of osteosarcoma by suppressing the recognition and attack of the immune system towards osteosarcoma cells, leading to the occurrence of distant metastasis. Simultaneously, the high expression of CD34 and CD40 may enhance the malignancy and metastatic potential of osteosarcoma by promoting angiogenesis and suppressing the attack of the immune system, leading to the occurrence of distant metastasis in osteosarcoma patients.

The present study also found a significant positive correlation between the expression of CD34 and CD40 in osteosarcoma tissue. The specific mechanism of mutual promotion between CD34 and CD40 in osteosarcoma tissue has not been reported, but data shows that they may be involved in the following biological processes. Firstly, high level of CD34 promotes the invasion and infiltration of osteosarcoma cells into blood vessels#[15]. Up-regulation of CD34 can stimulate tumor cells to produce hypoxia-inducible factor 1 (HIF-1) and promote the migration of endothelial cells to the inflammatory site of the tumor, thus forming new blood vessels. Secondly, the expression of CD40 is influenced by various types of cells in the tumor microenvironment that secrete pro-inflammatory cytokines and factors[16]. These factors such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) can stimulate the expression and function of CD40, thereby enhancing the immune and inflammatory responses inside the tumor. Additionally, CD34 and CD40 may also interact with each other by regulating the expression of tissue factors. For example, studies have found that under co-culture conditions of CD40L + T cells and osteosarcoma cells, the expression of stem cell factor (SCF) and basic fibroblast growth factor (bFGF) was significantly up-regulated, and these factors can promote angiogenesis and osteosarcoma cell growth[17]. In summary, CD34 and CD40 may interact with each other through multiple mechanisms in osteosarcoma tissue, promoting tumor growth, invasion, and metastasis.

The Kaplan-Meier method was used to analyze the survival time of patients, and the results showed that the survival time of patients in the CD34-positive and CD40-positive groups was significantly shorter than that in the corresponding negative control groups. The poor prognosis of osteosarcoma patients with high expression of CD34 and CD40 may be related to the following factors[18, 19]: (1) Tumor invasion and metastasis: CD34 is an endothelial cell marker, and its high expression suggests that osteosarcoma cells have the ability to invade blood vessel endothelial cells. In addition, high expression of CD40 can promote tumor cell metastasis and invasion and reduce patient survival. (2) Changes in tumor microenvironment: The expression of CD40 is influenced by cytokines in the tumor microenvironment, which can change the interaction between tumor cells and the microenvironment, leading to accelerated tumor cell growth, invasion, and metastasis. (3) Suppressed immune response: CD40 plays a role in activating and enhancing immune responses in immune cells. However, in osteosarcoma cells, high expression of CD40 may suppress immune responses, leading to tumor cells being ignored by the immune system and making treatment more difficult. (4) Chemotherapy resistance: High expression of CD34 and CD40 may be related to chemotherapy resistance in osteosarcoma cells. Some studies have shown that the efficacy of chemotherapy is poor in osteosarcoma patients with high expression of CD34 and CD40. High expression of CD34 and CD40 may increase the invasive, metastatic, and chemotherapy-resistant capabilities of osteosarcoma cells, while reducing the immune response of patients, increasing the difficulty of treatment, and therefore may result in a poor prognosis for patients.

In conclusion, this study found that tumor stem cell markers CD34 and CD40 are highly expressed in osteosarcoma tissue and have significant negative impacts on patients' clinical pathological parameters and prognosis.

Data Availability Statement

The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy considerations but are available from the corresponding author on reasonable request (email: [email protected]).

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Table 1 Effect of CD34 and CD40 expression on clinical pathological parameters of patients

Index	CD34		c²	P	CD40		c²	P
Index	Positive	negative	c²	P	Positive	negative	c²	P
Age			0.076	0.783			0.544	0.461
＜25	41	28			46	23
≥25	23	14			22	15
Gender			0.165	0.684			0.007	0.933
Female	34	24			37	21
Male	30	18			31	17
Tumor Size			0.398	0.528			0.254	0.614
＞5 cm	39	23			41	21
≤5 cm	25	19			27	17
Histological Grading			9.217	0.002			9.224	0.002
Highly	39	37			42	34
Middle-low	25	5			26	4
Tumor metastasis			5.962	0.015			7.002	0.008
Yes	32	11			34	9
NO	32	31			34	29
TNM			9.833	0.002			10.612	0.001
Ⅰ	11	19			12	18
Ⅱ～Ⅲ	53	23			56	20

Table 2 Correlation analysis of CD34 and CD40 expression in osteosarcoma tissue

CD34	CD40		r	P
	Negative	Positive
Negative	15	27	0.613	＜0.01
Positive	23	41

Table 3 Cox analysis of prognostic factors affecting patients with osteosarcoma

Index	β	SE	Wald	P	OR	95%CI
Age	0.865	0.362	0.550	0.442	1.035	0.965～1.632
Gender	0.715	0.415	0.339	0.215	0.963	0.632～1.258
Tumor Size	0.635	0.215	0.239	0.119	1.563	1.236～1.778
Histological Grading	0.715	0.195	3.652	0.009	1.636	1.118～2.069
Tumor metastasis	0.412	0.352	4.852	＜0.001	2.035	1.362～2.710
TNM	0.623	0.847	3.965	＜0.001	1.584	1.005～1.894
CD34	0.558	0.532	2.512	0.043	1.462	0.965～1.741
CD40	0.469	0.228	2.765	0.035	1.368	0.769～1.842

No competing interests reported.

The expression and clinical significance of tumor stem cell markers CD34 and CD40 in osteosarcoma tissue

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Abstract

Figures

Introduction

Method

Results

Discussion

Declarations

References

Tables

Additional Declarations

Status:

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