Study on the correlation between CD80/CD163 and clinical prognosis and the syndrome differentiation in patients with colorectal cancer

Background: To analyze the expression of cluster of differentiation 80 (CD80)/cluster of differentiation 163 (CD163) in colorectal cancer (CRC) patients and their correlation with the syndrome of traditional Chinese medicine, pathology and prognosis. Methods: (1) The correlation between the pathological characteristics of 232 postoperative CRC patients and the deficiency and excess syndromes of traditional Chinese medicine was analyzed using the chi-square test, Spearman’s correlation, and Cox regression methods. (2) Immunohistochemistry and quantitative real-time PCR were used to detect the expression of CD80 and CD163 in cancer and paracancer tissues of CRC patients. (3) The relationships between the changes of CD80 and CD163 and the prognosis of CRC patients with deficiency syndrome. Patient survival were analyzed using cardinality and Cox regression proportional-hazards model regression. Results: (1) The degree of differentiation and tumor node metastasis stage of CRC were statistically different between patients with deficiency and excess syndromes ( P < 0.05); carcinoembryonic antigen and carbohydrate antigen 19-9 were highly expressed in the excess syndrome group, and both were significantly correlated with the distribution of traditional Chinese medicine syndromes ( P < 0.001); deficiency and excess syndromes, and carbohydrate antigen 19-9 were all independent factors affecting the postoperative survival of CRC patients. (2) The distribution of post-operative survival in CRC patients was significantly correlated with the distribution of the disease type ( P < 0.001). (3) The expression levels of CD163 protein and message RNA were significantly higher in CRC cancer tissues than in paraneoplastic tissues ( P < 0.001); whereas the expression of CD80 was significantly higher in paraneoplastic tissues than in cancer tissues ( P < 0.001). (4) The expression levels of CD80/CD163 were significantly different in different parts of the tissues of patients with deficiency and excess syndromes ( P < 0.001). (5) CRC patients with high CD80 expression and low CD163 expression had longer survival cycles ( P < 0.001). Conclusion: The malignant progression of CRC patients with deficiency syndrome is faster than that with excess syndrome. The correlation between deficiency and excess syndromes and the expression levels of CD80 and CD163 could be an independent risk factor for the survival prognosis of patients with CRC.


Background
The pathophysiology of colorectal cancer (CRC), a digestive tract malignant tumor illness with a high incidence and fatality rate, is yet unknown [1]. The "Unity of Heaven and Man" of traditional Chinese medicine (TCM) is congruent with the tumor microenvironment (TME) hypothesis, which states that tumors develop as a result of prolonged and continual internal and external environmental pressure on the body, consistent with the "Unity of Heaven and Man" of TCM [2,3]. In the tumour inflammatory microenvironment, the majority of tumour tissue is surrounded by inflammatory-associated cells that play an integral role in tumourigenesis, progression and metastasis [4,5]. Tumor-associated macrophages (TAMs) are the primary inflammatory cells that infiltrate the tumour mesenchyme. Studies have shown that the imbalance of polarization in TAMs is associated with poor prognosis in CRC [6][7][8]. In the TME, TAMs are polarised into two subtypes regulated by key transcription factors under certain spatial and temporal conditions, which perform biological functions by secreting cytokines. Studies have shown that classically activated macrophages (M1 type), characterised by high expression of protein markers such as cluster of differentiation 64 , cluster of differentiation 80 (CD80) and cluster of differentiation 163 (CD86), play a functional role in antigen presentation and secrete pro-inflammatory cytokines to exert anti-tumour immune responses [9]. Alternatively activated macrophages (M2 type), characterised by high expression of protein markers such as CD11b, CD163 and CD206, are alternatively activated and produce anti-inflammatory cytokines to promote tumour development [10]. Therefore, it is essential to characterize the number of TAMs to reveal their relevance in cancer [8].
The concept of "syndrome" is fundamental in TCM and is used to describe various pathological symptoms that occur during disease progression. Different microenvironments can cause different pathological reactions in the body, reflected in the evolution of syndrome types. The deficiency and excess are the most basic syndrome types, which reflecting the balance of positive and negative forces within the body. TAMs continuously reshape and disrupt the stability of the internal microenvironment during the development of tumor. This process is closely related to the dynamic evolution of TCM deficiency and excess syndrome types [11]. TCM syndrome differentiation, guided by the philosophy of "diagnosis and treatment based on differentiation of syndromes", and diagnosis through the integration of the four diagnostic methods, comprehensively considers the patient's pathological manifestations, physique, external factors, and other aspects of information. This approach improves the accuracy of diagnosis and the efficacy of treatment [12].
However, the relationships between TCM syndrome differentiation, clinical and pathological characteristics of tumor, and TAMs reshaping are unclear. This study aimed to investigate the relationship between CD80 and CD163 expression and CRC patients' clinical characteristics and prognosis. Furthermore, the study compared the reshaping features of TAMs in CRC with deficiency and excess syndrome types, in order to provide a foundation for clinical diagnosis and treatment of CRC.

Subjects of study
A retrospective analysis was conducted by screening and completing a case observation form on 232 CRC patients who were admitted to the second Department of Surgery at Gaozhou Hospital of Traditional Chinese Medicine between January 1, 2015, and December 31, 2020. Immunohistochemistry and clinical/prognostic analysis were performed on 45 CRC patients, while 27 CRC surgical specimens were tested using quantitative real-time PCR. All CRC patients were diagnosed according to the Colorectal Cancer Diagnosis Guidelines and Clinicopathological Staging Criteria of The (AJCC)/(UICC) Tumor Node Metastasis (TNM) Staging Criteria (8th edition) [13,14]. Syndrome differentiation of TCM were conducted in line with the "Guidelines for the Diagnosis and Treatment of Tumors in Chinese Medicine" [15]. The Ethics Committee of Gaozhou City Hospital of Traditional Chinese Medicine approved the study, grant No. Y(2021)0013. All patients signed informed consent and were informed that their privacy would be properly protected.

Inclusion and exclusion criteria Incorporation criteria.
(1) Patients with a confirmed pathological diagnosis of primary CRC after surgery; (2) successful completion of radical CRC surgery (including open or laparoscopic surgery with intraoperative resection of intact tumor tissue); (3) availability of completed clinical history information and follow-up data; (4) patients who provided signed informed consent and voluntarily agreed to participate; (5) availability of completed information of the four diagnostic methods of TCM. Exclusion criteria. (1) Patients without definitive pathological diagnosis of CRC; (2) patients with a history of prior radiotherapy treatment; (3) patients who have undergone palliative resection of tumor; (4) patients with vital organ failure, such as heart, liver etc. ; (5) patients with familial polyposis carcinoma, Crohn's disease, or non-polyposis CRC; (6) patients with insufficient information of the Four Diagnostic Methods of TCM, or difficulty in distinguishing between the deficiency and excess syndromes; (7) patients who died during the initial hospitalization. Data collection and follow-up. Medical history of all CRC patients was retrieved from the hospital information system, including tumor infiltration depth, differentiation, TNM stage, lymph node metastasis, and tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA199) and alpha-fetoprotein (AFP) etc. CEA > 5 ng/mL, CA199 > 37 U/mL, and AFP > 20 ng/mL were defined as positive. The final follow-up was conducted by face-to-face interview or by telephone, which continued until December 31, 2021.

Methods
Immunohistochemistry. Following dewaxing, antigen repair, and closure, the sections were routinely incubated overnight at 4°C with a drop of primary antibody (CD80 or CD163). The following day, the secondary antibody was incubated at room temperature for 1 hour, a horseradish enzyme-labeled streptavidin working solution was incubated for 20 minutes, a DAB chromogenic solution was stained for 3-5 minutes, hematoxylin stained nuclei for 30 seconds, and the samples were transparent, air-dried, and sealed with neutral resin before being examined under a light microscope. Image J image analysis software calculated the average optical density value. The integrated optical density sum (IOD sum)/positive area sum was calculated. The average optical density of three fields of view in each section was selected as the optical density value of this sample [16]. Multiplex fluorescence immunohistochemistry. The steps are as follows, according to the basin reagent method instructions: In a 60°C oven, incubate the tissue sections of the collected clinical samples for 1 hour, then dewax with xylene and all levels of ethanol regularly; after rinsing with tris buffered saline with tween 20 (TBST), place the sections in antigen repair solution and microwave for 8 minutes on high and 15 minutes on low for antigen repair; wait for the sections to return to room temperature, TBST rinsing, dropwise addition of The sections were incubated for 10 min at room temperature and rinsed with TBST; standard sheep serum working solution was added dropwise and blocked for 30 minutes at 37°C; primary antibody (CD80/CD163) was added dropwise and incubated overnight at 4°C. The following day, the samples are incubated for 10 minutes with a drop of secondary antibody working solution (Ebenezer) before being rinsed with TBST; the samples are incubated with a fluorescent staining amplification signal (Ebenezer) at a dilution of 1:100. Fluorescence microscopy is used to confirm the staining after each round of staining, and then additional staining is performed. After further labeling, the slides are thermally repaired to remove the tissue-bound primary and secondary antibody complexes. Repeat these steps until the clinical tissue slide has been diluted 1:100 with all markers and spectral DAPI (Ebenezer). Finally, an anti-fluorescence quenching sealer is applied dropwise, and the slide is sealed with a coverslip. Each case was photographed using an Olympus slide panoramic scanner.

Detection of message RNA (mRNA) expression by real-time PCR.
Total RNA was extracted from CRC tissues using TRIzol solution, and 0.8 μg of total RNA was reverse transcribed to obtain cDNA. qPCR was performed using SYBR Premix Ex Taq Ⅱ (2X). 50 μL of the reaction system was pre-denatured at 95°C for 30 s, denatured at 95°C for 5 s, and annealed at 60°C for 30 s. A total of 40 cycles were completed. Data were calculated using the PCR instrument's software and analyzed for relative quantification using the 2-ΔΔCT method. (Table  1 lists the primer sequences)

Statistical analysis
All statistical analyses of this study were conducted with SPSS software (version 26.0). All participants with available relevant variables were included in the analysis, and there were no missing data. Normally distributed continuous data were expressed as mean and mean SD (± s), while the non-normal distribution continuous data were expressed as the median and interquartile range (M [P25, P75]). Categorical variables were presented by frequency and percentages (%). The critical value was determined to be the median (average optical density value) of the quantitative analysis of CD80 and CD163. For normal distributed continuous data with equal variance, the Student's t test was applied in the comparison between two groups. And the Mann-Whitney U test was used for non-normally distributed data. Chi-square test or Fisher's exact test were used for comparison between categorical variables. The Kaplan-Meier method was conducted for survival analysis, and the log-rank test was used to compare the overall survival curves. The Cox regression was used to identify potential independent predictors of survival prognosis. All hypothesis tests were two-sided, and P ≤ 0.05 was considered statistically significant.

Demographic and clinicopathological characteristics of CRC patients with deficiency and excess syndromes of TCM
As shown in Table 1, no statistical differences were found in gender, age, tumor diameter, depth of tumor infiltration, and metastasis between CRC patients with deficiency and excess syndromes of TCM (P > 0.05). Statistical differences were found in the degree of differentiation and TNM stage of tumor. Specifically, patients in the hypofractionated deficiency syndrome group had a higher degree of tumor differentiation and lower TNM stage than patients in the hypofractionated excess syndrome, while patients in the deficiency syndrome group had a lower degree of tumor differentiation and higher TNM stage than those in the excess syndrome group in the early stage (stage I & II). In the late stage (stage III & IV), CRC patients with deficiency syndrome were more likely to exhibit a higher degree of malignant progression of the tumor. These findings suggest that there are significant differences between the deficiency and excess syndrome of CRC in terms of the degree of tumor malignancy, and that CRC patients in the late stage are more likely to exhibit deficiency syndrome (P < 0.05, Table 2).

Relationship between deficiency and excess syndromes and tumor markers in CRC patients
By comparing the expression of CEA, CA199, and AFP between the deficiency and excess syndromes, we discovered that all of the serum tumor markers were highly expressed in the excess group, with the most significant variability in CEA and CA199 (as shown in Figure 1A, Figure 1B).

Survival analysis
The findings suggest that deficiency syndrome is correlated with the malignant progression of CRC and tumor marker levels. Additionally, clinicopathological characteristics of CRC patients have a particular assessment value on survival prognosis. Therefore, the relationship between clinicopathological characteristics and survival time of the 232 CRC patients was analyzed. The results showed that gender, depth of infiltration, size of tumor, degree of differentiation, lymph node metastasis, TNM stage, deficiency and excess syndrome, and CA199 were all factors influencing survival prognosis. Notably, in the   investigation of deficiency and excess syndrome types, patients with deficiency syndrome (n = 118) had a median survival of 24 months, while patients with excess syndrome (n = 114) had a median survival of 34 months (P < 0.001, hazard ratio (HR) = 2.574), suggesting that CRC patients with deficiency syndrome had worse prognosis ( Figure  2).  Table 3).

The expression of CD80 and CD163 in tissues of CRC patients with eficiency syndrome and excess syndromes
The expression of CD80 in paracancer tissues was significantly higher in CRC patients with deficiency syndrome than excess syndrome. And it was significantly lower in cancer tissues of patients with deficiency syndrome, when compared with patients with excess syndrome. Whereas the expression of CD163 was significantly higher in patients with excess syndrome (Figure 3A-3D). In addition, we performed multiple immunostainings on tissue sections from CRC patients. The intestinal tissue samples were labeled with CD80 and CD163  macrophage markers, with CD80 in green, CD163 in red, and DAPI in blue ( Figure 3E). CD80 was found to be highly expressed in para-cancerous tissues and was distributed in the intestinal mucosal glands and the lamina propria, whereas it was significantly weaker in cancerous tissues and was only found in the lamina propria. CD163 was rarely expressed in para-cancerous tissues but significantly more expressed in cancerous tissues, and the expression areas were dominated by the lamina propria surrounding the glands. The distribution of CD80 and CD163 in cancer tissues partially overlapped, implying that M1 and M2 polarization patterns in tumor tissues could be interconverted.
CD80/CD163 mRNA levels in CRC tissues mRNA CD80 and CD163 in CRC cancer and paraneoplastic tissues were detected by quantitative real-time PCR. CD80 was down-regulated in intestinal cancer tissues compared to paraneoplastic tissues. However, CD163 was up-regulated in intestinal cancer tissues. ( *** P < 0.001, Figure 3F, Figure 3G)

Correlation of CD80/CD163 expression levels with clinical characteristics
According to the median value of CD80 and CD163 expression, patients were divided into two groups: those with high expression (> median value) and those with low expression (< median value). The relationships between CD80 and CD163 expression in CRC tissues and clinical features were probed. The level of CD80 expression was significantly correlated with pathological type, degree of differentiation, and TCM syndrome (P < 0.05, Table 4); whereas the  CD163 was only significantly correlated with TCM syndrome (P < 0.05, Table 5). Therefore, the result suggested that the level of CD80 expression was more likely related to clinical characteristics.

Prognostic value of CD80 and CD163 expression in CRC patients
According to the Kaplan-Meier survival analysis, patients with high CD80 expression versus low CD163 expression had a more prolonged survival and a better prognosis (P < 0.001, Figure 4). TNM stage, CA199, and TCM evidence were independent predictors of overall survival in a Cox regression analysis. Patients with early-stage (TNM I& II) CRC had a lower risk than intermediate and late-stage (TNM III&IV) patients, as well as a lower risk for low serum CA199 expression (< 37 U/mL); patients with deficiency syndrome had a higher risk than patients with excess syndrome (P < 0.01, Table 6).

Discussion
CRC is a systemic disease caused by the interplay of several pathogenic factors, resulting an imbalance of the body's internal Yin and Yang (A pair of categories in ancient Chinese philosophy, is a generalisation of the properties of the opposing sides of certain interrelated things or phenomena in nature.), and dysfunction of the Qi (Qi is the most basic substance that makes up the human body and sustains its vital activities.) and blood transportation in internal organs. However, the various clinical symptoms that arise are merely macroscopic manifestations of syndrome types, which is essentially an imbalance in the composition and structure of the organism's microenvironment [17]. The intrinsic properties of the microenvironment differ between deficiency and excess syndromes. TAMs are important microenvironmental components, and their remodeling characteristics influence the composition and function of the TME, which are critical predictors of tumor development [18]. TAMs constantly undergo evolving and remodeling during tumor development, leading to instability in the in vivo microenvironment. The TME is constantly reconstructed, similar to the dynamic evolution of the body's internal environment caused by the "Yin and Yang imbalance" in Chinese medicine [11]. The TCM syndrome is a generalization of various pathological symptoms in the disease process that can reveal the essence of the disease more thoroughly and thus assess the disease process's progress [19]. Several studies have linked Submit a manuscript: https://www.tmrjournals.com/tmr   TCM evidence transformation to tumor development and metastasis [20,21]. Tumor-associated protein expression differs in CRC patients with deficiency and excess syndrome [22]. Early-stage CRC symptoms are not obvious, and most patients have metastases to lymph nodes, liver, and other organs by the time they are clinically diagnosed. The differentiation between deficiency and excess syndrome of TCM provides clinicians with a foundation for understanding the patient's disease and aid in determining the prognosis and treatment of CRC patients. Therefore, investigating the relationship between pathological features of CRC and TCM syndromes is crucial for CRC clinical diagnosis and treatment strategies, ultimately improving the CRC prognosis.
Our results showed that males have poorer survival rate than females after CRC resection surgy, which is consistent with national reports [23]. This was discovered through Kaplan-Meier and Cox regression survival analysis performed on postoperative CRC patients [24]. Possible explanations for this finding include: (1) Males may have diets that are higher in fats, proteins, and carbohydrates; (2) the pace of life is faster, and exercise intensity is inadequate for Males; (3) males are more likely to engage in unhealthy lifestyle, such as smoke and alcohol use. Previous research has shown that the size of tumor is negatively correlated with clinical outcome [25]. CRC tumors measuring less than 5 cm in diameter have a longer survival after surgery compared to those with tumors measuring 5 cm or greater, indicating that the tumor diameter size defined of 5 cm can be used as an evaluation index of prognosis. Furthermore, tumor differentiation, which reflects the aggressiveness of the tumor, is another important factor. Tumors with lower degrees of differentiation exhibit higher rates of cell proliferation, division, and change, and are more aggressive and metastatic, resulting in poorer patient's prognosis.
Hashemi et al. have demonstrated a strong relationship between CRC differentiation and malignancy, with higher degrees of differentiation associated with lower malignancy levels [26].
The results of this study indicate that CRC patients with high or medium differentiation had better postoperative survival than those with low differentiation, highlighting a potential link between differentiation and postoperative survival. Adenocarcinoma was the most common type of CRC, with mucinous adenocarcinoma and indolent cell carcinoma being more malignant and invasive, resulting in significantly lower postoperative survival rates than adenocarcinoma patients, as reported by Sun et al [27]. Patients with stage I & II CRC showed considerably better postoperative survival than those with stage III & IV CRC. Several studies have also identified TNM staging as an independent prognostic factor for CRC patients [28,29]. Additionally, CRC patients with substantial evidence of deficiency had remarkable better postoperative survival than patients in the deficiency syndrome. In a multivariate Cox regression analysis, it was found that deficiency and excess syndrome were independent variables significantly affecting CRC survival prognosis. Previous studies have shown that the combined effect of deficiency of healthy Qi and dysregulation of visceral Qi is a key factor in the formation and development of malignant tumours, which can be further aggravated by treatments such as surgery, radiotherapy and chemotherapy [30,31]. On one hand, patients with advanced CRC with deficiency syndrome have poorer prognosis, similar to the results of this study [32]. On the other hand, tumor blockage of the vascular tracts, stagnation of Qi and blood, malfunction of the spleen and stomach, and lack of biochemical sources can result in deficiency of Qi and blood. The deficiency of Qi is then unable to restrain the cancer toxin, which eventually leads to critical symptoms of Yin and Yang leaving the body [33].
The remodelling characteristics of TAMs in the TME may be a microscopic manifestation of the macroscopic subtype classification of CRC. TAMs are polarised into two subtypes under different influences of the tumour microenvironment, each playing a different biological function; M1 TAMs promote inflammatory responses and anti-tumour activity, while M2 TAMs play a "bad role" in the anti-inflammatory response and promote malignant tumour growth [10]. Chen et al. analyzed the immunohistochemistry results of CRC tissues and found that high-density CD68+ /CD163+ TAMs in tumor tissues were significantly correlated with late TNM staging and poor prognosis of CRC patients. These TAMs were identified as independent prognostic factors for survival prognosis of CRC [34]. Several studies have shown that the development and metastasis of tumors are related to the transformation of TCM syndromes [20,21]. In this study, relationship between TAMs infiltration and clinical-pathological information in patients with CRC was analyzed. The results showed that CRC patients with high expression of CD80 and low expression of CD163 had a longer survival period. Patients with the deficiency syndrome had a significant increase in CD163 infiltration and a higher mortality rate, which was consistent with the results of Zou et al. on late-stage CRC patients with deficiency syndrome [35]. According to TCM theory, Healthy Qi is the foundation for regulating balance, resisting pathogenic factors, and maintaining normal physiological functions. When Healthy Qi is deficient and fail to fight pathogenic factors, various diseases may occur. In clinical practice, if the patient with tumors has a weak constitution and insufficient Healthy Qi to promote the circulation of Qi, blood, and body fluids, the accumulation of pathogenic factors may worsen. The invasion of pathogenic factors may further consume Healthy Qi, accelerating the invasion and metastasis of the tumor [36]. Therefore, for CRC patients with the deficiency syndrome, clinicians should pay more attention to the prevention and treatment of metastasis or recurrence [37].
In this study, we evaluated the macrophage spectrum in a series of 45 cases of CRC by quantitatively assessing the expression of CD80 and CD163 corresponding to pro-inflammatory macrophages. Immunohistochemistry was used to analyze macrophages in CRC cases, with CD80 as a marker for pro-inflammatory macrophages and CD163 as a marker for anti-inflammatory macrophages. Quantitative analysis was performed in the tumor and matched adjacent normal mucosa regions by computer-assisted analysis. The CD80 was mainly present in the mucosal glands and cytoplasmic layers, indicating the main anti-inflammatory polarization of TAMs. Conversely, the CD163 was rarely expressed in the para-cancerous tissue but was significantly more expressed in the cancer tissue, and the expression areas were mainly around the glandular layer, which suggests that M2 macrophages may promote tumor growth and metastasis in that area.
Wei et al. found that the increase in CD163+ TAM infiltration at the tumor invasive front is significantly associated with poor prognosis in CRC patients, and may play a role in promoting the spread and invasion of CRC [38]. Other studies have also shown that CD163 can distinguish M2-type macrophages, which are considered "bad" cells, as they participate in Th2 immune responses, release anti-inflammatory cytokines that promote tumor development, and are associated with poor prognosis in CRC [39]. Our study found that high expression of CD80 and low expression of CD163 in colon tissues were associated with longer survival in CRC patients. However, the relationship between high expression of CD163 and poor prognosis in CRC remains uncertain, and further studies are needed to provide more definitive evidence on its association with M2 cells.
Although our study provides new insights into the search for novel prognostic biomarkers for CRC, our study still has some limitations. First of all, we reviewed the relevant literatures and found that CD80 and CD163 had an impact on the development, metastasis and prognosis of CRC. Therefore, we mainly focused on the changes of CD80 and CD163 in the microenvironment of CRC. However, macrophages can be divided into two classical subtypes, and each has a variety of protein markers. Unfortunately, we did not carry out experimental verification of macrophage markers. Besides, invitro cell experiments for in-depth analysis of the mechanism is needed to support our findings.
In summary, this study investigated the relationship between clinical pathological features and TCM syndrome differentiation and found that TCM syndrome differentiation is related to CRC survival prognosis. By comparing the macrophage remodeling features between deficiency and excess syndrome differentiation and the prognostic significance of this feature for CRC, we further elucidated that the differences in TAMs remodeling features may be a potential factor leading to differences in the malignant progression degree of deficiency and excess syndrome differentiation in CRC. These findings suggest that a more comprehensive diagnosis and treatment strategy may be provided for CRC patients by considering TCM syndrome differentiation along with macrophage remodeling features.