Lung cancer is currently the most common malignancy with the highest mortality rate worldwide [1]. In China, lung cancer ranks first among all malignancies in terms of incidence and mortality rate [2]. Lung adenocarcinoma (LUAD) is the most common pathological type of lung cancer, accounting for approximately 45% of all cases, and has a significantly higher incidence than other pathological subtypes such as squamous cell, small cell, and large cell carcinomas [3]. Surgical resection is the mainstay of treatment for early to intermediate stage (stage I–IIIA) nonsmall cell lung cancer (NSCLC), but the risk of tumor recurrence and death after radical surgery remains high [4]. Therefore, the search for molecular markers associated with tumor progression or prognosis in lung cancer is of great significance for elucidating tumor pathogenesis and screening patients with poor prognosis [5, 6].

Tumor growth is accompanied by angiogenesis, and antiangiogenic therapy has always been a crucial target in antitumor treatment [7]. The relationship between tumor angiogenesis and the prognosis of tumor patients has also been widely studied and is currently a hot topic of discussion. Microvascular density (MVD) is an indicator used to assess the number of blood vessels within a tumor [8] and plays an important role in predicting the prognosis of patients with tumors [9–11].

CD34 is a pan-endothelial cell marker commonly used to assess tumor vascularity. Although researchers have used CD34 antibodies to label the tumor microvasculature by performing immunohistochemical (IHC) staining of lung cancer tumor tissues and to assess the relationship between MVD, tumor progression, and patient prognosis, the results of different studies have been controversial. A study by Tanaka et al. [12] that included 236 NSCLC patients found that intratumoral CD34-MVD was not associated with tumor stage or five-year survival rate. Similarly, Donnem et al. found that among 335 NSCLC patients, high vs. low CD34-MVD was not associated with a five-year survival rate [13]. In contrast, Kadota et al. found that among 147 NSCLC patients, those with high CD34-MVD had lower survival rates [14]. In a study of 81 patients with NSCLC, Bing et al. showed that an increase in CD34-MVD was associated with tumor progression but not lymph node metastasis; unfortunately, the study did not include a survival analysis [15]. The results of the study by Pomme et al., which included 371 NSCLC patients, were contrary to all the studies above and instead showed that the increase in CD34-MVD was associated with lower TNM stage and better prognosis [16]. Furthermore, all the above studies included NSCLC patients, and none analyzed patients with lung adenocarcinoma separately. Therefore, the relationship between CD34-MVD and tumor progression and prognosis in patients with lung adenocarcinoma remains inconclusive and warrants further investigation.

In this study, IHC staining was performed using CD34 antibodies on tumor tissue sections from 238 LUAD patients who underwent radical surgery to determine intratumoral MVD and to analyze the relationship between CD34-MVD and patient clinicopathological characteristics and survival. Based on the above, we aimed to explore the role of CD34-MVD in tumor development and its prognostic value.

Intratumoral blood vessels provide oxygen and nutrients to tumor cells; hence, angiogenesis is thought to play an important role in tumor development. However, the processes and mechanisms underlying tumor neovascularization are complex. They may include sprouting angiogenesis, intussusceptive angiogenesis, vasculogenesis, recruitment of endothelial progenitor cells, vascular mimicry, and transdifferentiation of cancer stem cells [18]. Studies identifying various aspects of tumor angiogenesis, especially biomarkers targeting tumor angiogenesis, can help us better understand its molecular mechanisms and discover targets for designing effective anti-tumor therapies [19]. The selection of appropriate tumor vascular markers for MVD counting enables quantitative morphological analysis of the tumor vasculature and reflects the extent of tumor angiogenesis [8]. CD34, as a powerful pan-endothelial cell marker, is widely expressed in mature vascular endothelial cells and has garnered extensive interest among clinicians in clinical practice, especially in the fields of cardiovascular and cerebrovascular diseases and cancer [20]. This was the starting point of our study, in which the abundance of microvessels within LUAD tumors was quantified using CD34-MVD, and the relationship between CD34-MVD, clinicopathological characteristics, and patient prognosis was analyzed.

Our findings revealed that CD34-MVD was higher in patients with tumor size ≤3 cm than in those with tumor size >3 cm, and higher in patients without lymph node metastasis than in those with lymph node metastasis. This result reflects the fact that in LUAD, CD34 positive microvessels are more abundant in the early tumor microenvironment, which then decreases in abundance as the tumor progresses (tumor enlargement and lymph node metastasis). Our results are similar to those of Carlini et al. [21] who found that intratumoral CD34-MVD was lower in patients with stage T2 NSCLC and higher in those with stage T1 NSCLC. We analyzed two possible reasons for this result. First, CD34 positive microvessels in lung cancer tumor tissues as a differentiated microvessels tpye, which was more likely to display intratumoral mature blood vessels and peritumoral normal blood vessels [22]. Second, CD34-MVD was also shown to be associated with intratumoral necrosis, resulting in uneven oxygen distribution [16]. Thus, we hypothesized that, as the tumor increased in size, there was a decrease in mature differentiated blood vessels, an increase in intratumoral necrosis, and the occurrence of tumor hypoxia, which in turn promoted the metastasis of lung cancer to the lymph nodes. Therefore, a reduction in CD34 positive microvessels may be associated with tumor development in LUAD.

According to the survival analysis conducted in this study, patients with high CD34-MVD showed better prognosis for both DFS and OS. High CD34-MVD reduced the risk of tumor recurrence in a multivariate Cox proportional hazards regression model of postoperative tumor recurrence. Since we previously found that the decrease in CD34-MVD was associated with tumor invasion events (tumor enlargement and lymph node metastasis), thus explaining the favorable prognosis of patients with high CD34-MVD and the poor prognosis of those with low CD34-MVD. This detrimental effect of reduced CD34-MVD on prognosis has been found not only in NSCLC [16] but also in other malignancies, such as kidney cancer [23], bladder cancer [24], ovarian cancer [25], intrahepatic cholangiocarcinoma [26], and hepatocellular carcinoma [27]. Therefore, we suggest that CD34-MVD should be routinely detected in patients with lung adenocarcinoma after operation, and the patients with low CD34-MVD should be examined and evaluated for tumor recurrence more frequently during the follow-up period.

Antiangiogenic therapy, such as bevacizumab, can inhibit tumor angiogenesis and normalize tumor blood vessels. Hence, this is currently an important approach for antitumor drug therapy [28]. Some studies have found that antiangiogenic therapy, such as chemotherapy plus bevacizumab [22]or anlotinib alone [29], is more effective in patients with more undifferentiated (CD31+/CD34-) MVD in advanced NSCLC. CD34 is a marker of mature microvessels in differentiated tumors, and we found that a high abundance of CD34 positive microvessels reduced tumor invasion and improved patient prognosis. This suggests that increasing CD34 positive microvessels may be a novel approach to antitumor therapy.

Previous studies on the correlation between CD34-MVD and lung cancer have yielded mixed results, which may have been due to the following reasons. First, different MVD evaluation and counting methods were employed. Tanaka et al. [12], Kyuichi et al. [14] and Bing et al. [15] used the hotspot method in their studies, in which several areas with the densest concentration of microvessels were selected at low power and counted at high power. The average value was used as the final MVD value. In contrast, Donnem et al. [13] and Pomme et al. [16] used the microarray construction method in their studies, where MVD was defined as the number of microvessels found within a microarray core (0.6 mm in diameter). Second, different grouping criteria were used for MVD. Tanaka et al. [12] defined the high and low MVD groups using the median as the cutoff. The MVD values in the study by Donnem et al. [13] were based on IHC scoring. Kadota et al. [14] defined the high and low MVD groups by referring to the results of other previous studies. Pomme et al. [16] directly defined 65/mm² as the cutoff for high and low MVD (the authors did not justify this definition). Bing et al. [15] did not divide MVD into high and low groups. Since the Weidner correction method (i.e., the hot spot method) was first proposed, it has gradually gained popularity as the most classic and widely adopted method for MVD measurement owing to its simplicity and rapidity. However, with advances in artificial intelligence and algorithms, MVD measurements assisted by computer imaging technology are expected to become a reliable indicator for evaluating therapeutic efficacy in the future [30, 31]. Based on the above, we can see that there is as yet no unified standard for MVD counting and grouping protocols, and the standardization and automation of its application should be the direction of future research.

In conclusion, our study demonstrated that a high abundance of intratumoral CD34 positive microvessels in LUAD may reduce tumor invasion, whereas a decrease in CD34 positive microvessels may be associated with tumor progression. CD34-MVD is an independent risk factor for postoperative tumor recurrence in patients with LUAD and can serve as a prognostic indicator for such patients.