Patient characteristics
Among the 392 patients in cohort A, radical surgeries were performed in 376 patients, and 16 patients had non-radical surgeries. We divided the 392 patients into two groups according to the weight loss ≥ 5% within six months before the surgery (Table 1). Totally 167 patients in cohort A had weight loss ≥ 5% within the six months before surgery, indicating that at least 42.6% patients had cancer cachexia. There were no significant differences in age, sex and height between the two groups, while patients with weight loss ≥ 5% had significantly lower body weight, BMI, skeletal muscle mass and fat mass. The body weight of patients with weight loss < 5% was on average 4.39 kg higher than those with weight loss ≥ 5%, and particularly the skeletal muscle mass was on average 2.14 kg higher in patients with weight loss < 5%, illustrating that skeletal muscle loss could be a major cause of weight loss. Besides, both in male and female, lower skeletal muscle index (SMI) and handgrip strength were accompanied by weight loss ≥ 5%, suggesting cancer-associated cachexia was a disorder characterized by loss of body weight with particular losses of skeletal muscle mass and function. Particularly, compared to those with weight loss < 5%, women with weight loss ≥ 5% had more reduced SMI (0.21 kg/m2 vs. 0.15 kg/m2) and handgrip strength (2.84 kg vs. 2.67 kg) than men. For pathological parameters, significant differences were found in the TNM stage, depth of invasion and lymph node metastasis between the two groups, in which patients with weight loss ≥ 5% had a higher rate of TNM stage III + IV, T3 + T4 depth of invasion and positive lymph node metastasis. Moreover, the locations of gastric cancer were also different between the two groups, and the main difference was the incidence of entire gastric cancer (7.2% vs. 1.8%). Compared to those with weight loss < 5%, patients with weight loss ≥ 5% had significantly higher scores of NRS2002, indicating that more weight loss in gastric cancer was related to increased risk of malnutrition. As can be seen in Short Form 36 (SF-36) survey, the significant differences between the two groups were physical function, mental health and bodily pain (Table 1), saying that weight loss in gastric cancer may have an impact on the quality of life of patients, especially the physical function and mental health. Additionally, patients with weight loss ≥ 5% had a significantly higher level of preoperative circulating IL-6 and TNF-α.
Table 1
Participants’ characteristics divided by weight loss within 6 months before surgery
Characteristics
|
Weight loss < 5% (n = 225)
|
Weight loss ≥ 5% (n = 167)
|
P
|
Age (years)
|
59.83 ± 10.00a
|
60.25 ± 9.18
|
0.67
|
Sex (Female/ Male)
|
78/147
|
69/98
|
0.18
|
Weight (kg)
|
61.52 ± 10.14
|
57.13 ± 11.97
|
0.001
|
Height (cm)
|
163.66 ± 8.09
|
162.69 ± 8.26
|
0.25
|
BMI (kg/m2)
|
23.24 ± 3.29
|
21.18 ± 3.95
|
< 0.001
|
Body compositionb
|
|
|
|
Skeletal muscle mass (kg)
|
21.58 ± 4.32
|
19.44 ± 5.63
|
< 0.001
|
Fat mass (kg)
|
19.53 ± 2.73
|
18.35 ± 3.23
|
0.001
|
SMI (kg/m2)
|
|
|
|
Female
|
6.01 ± 0.19
|
5.80 ± 0.28
|
< 0.001
|
Male
|
7.38 ± 0.24
|
7.23 ± 0.20
|
< 0.001
|
Handgrip strength (dominant) (kg)
|
|
|
|
Female
|
26.21 ± 4.71
|
23.37 ± 5.64
|
0.001
|
Male
|
44.19 ± 8.52
|
41.52 ± 9.36
|
0.02
|
Location
|
|
|
0.03
|
Esophagogastric junction
|
77 34.2
|
61 36.5
|
|
Body
|
26 11.6
|
12 7.2
|
|
Antrum
|
118 52.4
|
82 49.1
|
|
Entire stomach
|
4 1.8
|
12 7.2
|
|
TNM stage, I + II/III + IV
|
95/130
|
41/126
|
< 0.001
|
Depth of invasion, T1 + T2/T3 + T4
|
67/158
|
31/136
|
0.01
|
Lymph node metastasis, Negative/Positive
|
89/136
|
47/120
|
0.02
|
NRS2002
|
1.63 ± 0.14
|
3.65 ± 0.09
|
< 0.001
|
SF-36
|
|
|
|
Physical function
|
80.18 ± 12.79
|
75.87 ± 14.05
|
0.002
|
Role-physical
|
80.78 ± 16.53
|
79.19 ± 18.28
|
0.39
|
Bodily pain
|
80.67 ± 13.89
|
77.66 ± 16.26
|
0.04
|
General health
|
64.11 ± 23.23
|
61.05 ± 22.40
|
0.19
|
Vitality
|
73.51 ± 17.06
|
71.08 ± 19.54
|
0.19
|
Social functioning
|
83.61 ± 13.23
|
81.59 ± 14.73
|
0.14
|
Role-emotional
|
74.96 ± 22.67
|
74.45 ± 24.73
|
0.83
|
Mental health
|
79.45 ± 12.37
|
74.49 ± 13.75
|
0.0002
|
Circulating IL-6 (pg/mL)
|
3.43 ± 4.43
|
6.16 ± 6.57
|
< 0.0001
|
Circulating TNF-α (pg/mL)
|
6.71 ± 1.84
|
11.21 ± 5.93
|
< 0.0001
|
BMI, Body Mass Index; IL-6, interleukin-6; NRS 2002, Nutritional Risk Screening 2002; SF-36, Short Form 36; SMI, skeletal muscle index; TNF-α, tumor necrosis factor alpha; TNM, tumor, node, and metastasis. |
aMean ± standard deviation. |
bMeasurement results of human body composition analysis (InBody770) |
Circulating TNF-α, an independent important factors with weight loss
We further conducted a univariate analysis, which revealed that circulating IL-6, circulating TNF-α, TNM stage, location of tumors, depth of invasion and lymph node metastasis were significantly associated with weight loss ≥ 5%. However, in multivariate analysis, only circulating TNF-α and TNM stage had significant associations with weight loss ≥ 5% (Table 2). According to the laboratory examination reports, circulating TNF-α < 8.0 pg/ml was regarded as normal level. Thus we divide the circulating TNF-αinto four groups according to the multiple of normal TNF-α. When circulating TNF-α was more than two times of the normal level, the patients with weight loss ≥ 5% outnumbered those with weight loss < 5% (Fig. 1A). The rate of weight loss ≥ 5% was also extremly increasing when circulating TNF-α was two to three times of the normal (Fig. 1B). Next, we used the ROC curve to test the sensitivity and specificity of TNF-α associated with weight loss ≥ 5% (Fig. 1C). Circulating TNF-α was a sensitive indicator of weight loss ≥ 5% (AUC = 0.66, P < 0.001). Through the ROC curve, the cut-off point value was calculated as 9.96 pg/ml, about 1.25 times higher than normal value. This is a reminder that circulating TNF-α, 1.25 times higher than normal was an important warning of weight loss in patient with gastric cancer. Then, the values of circulating TNF-α in different TNM stages were compared to clarify the relationship between TNF-α and TNM stage. The data showed that circulating TNF-α in stage III was significantly higher than that in stage I (P = 0.01). Although circulating TNF-α in stage IV was the highest and that in stage I was the lowest, the difference was not significant (Fig. 1D). This could be due to too small number of patients with stage I and stage IV. When patients were divided into two groups (I + II vs. III + IV), the difference of TNF-α was significant (Fig. 1E). To explore the effect of circulating TNF-α and TNM stage on weight loss, we divided circulating TNF-α into two groups according to the cut-off value of 9.96 pg/ml. In stage I and II, most patients with weight loss ≥ 5% were affected by high circulating TNF-α (≥ 9.96 pg/ml), while in stage III and IV, nearly half of patients with weight loss ≥ 5% had low circulating TNF-α (Fig. 1F,G,H,I). It meant that TNF-α and TNM stage were two independent factors causing weight loss, and the mechanisms of how TNF-α and TNM stage promoting weight loss might be different. Thus, we conjectured that the characteristics of weight loss induced by TNF-α and TNM stage should be variant.
Table 2
Univariate and multivariate analysis for the weight loss ≥ 5% before surgery
Variable
|
Univariate
|
Multivariate
|
HR
|
95% CI
|
P
|
HR
|
95% CI
|
P
|
Age (years)
|
0.98
|
0.96–1.01
|
0.29
|
0.98
|
0.95–1.02
|
0.29
|
Sex (Female/Male)
|
0.84
|
0.48–1.49
|
0.20
|
1.15
|
0.55–2.39
|
0.71
|
Circulating IL-6 (> median)
|
2.66
|
1.50–4.71
|
0.001
|
1.28
|
0.57–2.90
|
0.55
|
Circulating TNF-α (> median)
|
3.19
|
1.89–5.37
|
< 0.001
|
2.84
|
1.46–5.55
|
0.001
|
TNM stage (IV + III/II + I)
|
11.21
|
5.80-21.66
|
< 0.001
|
8.03
|
3.60-17.92
|
0.002
|
Location
|
2.07
|
1.18–3.64
|
0.01
|
1.44
|
0.63–3.28
|
0.45
|
Depth of invasion (T3 + T4/T1 + T2)
|
2.07
|
1.18–4.64
|
0.01
|
1.50
|
0.72–3.12
|
0.31
|
Lymph node metastasis (Yes/No)
|
3.45
|
1.93–6.18
|
< 0.001
|
1.97
|
0.99–3.89
|
0.28
|
95% CI, 95% confidence intervals; HR, hazard ratios; IL-6, interleukin-6; TNF-α, tumor necrosis factor alpha; TNM, tumor, node, and metastasis. |
Clinic characteristics of patients in different TNM stage and circulating TNF-α
Next, through comparing the clinic parameters of patients in different TNM stage (stage I + II vs. III + IV) and circulating TNF-α (TNF-αL vs. TNF-αH), many interesting different characteristics related to metabolism, nutrition and immune indexes were found, showing in Table 3 and Table 4. Among the 392 patients with gastric cancer, totally 104 patients (26.5%) had high level of circulating TNF-α (TNF-αH ≥ 9.96 pg/ml), and those with TNF-αH had significantly lower albumin (ALB), total protein (TP), transferrin (TRF) and prealbumin (PAB), which were important indicators of nutrition (Table 3). In stage III and IV, the results were as same as those with TNF-αH. It was consistent that circulating TNF-α and TNM stage could have an adverse impact on nunutrition. However, It was the opposite in globulin (GLB), a herd of the major proteins associated with immunity. Glucose (GLU), one of the important metabolic indexes, in TNF-αH group was significant higher than it in TNF-αL group, while it was lower in stage III + IV than stage I + II. And lactate dehydrogenase (LDH) was as same as GLU between TNF-αH group and TNF-αL group or between stage III + IV and stage I + II. But other metabolic indexes such as creatinine (CREA), triglyceride (TG), and cholesterol (CHOL), in TNF-αH group and stage III + IV were respectively lower than that in TNF-αL group and stage III + IV. In nutrition and metabolism, the clinic characteristics of circulating TNF-α and TNM stage were slightly different. However, in immunity, the divergences of circulating TNF-α and TNM stage were considerable. Even though, the number of lymphocyte (LYMPH) and percentage of CD4 were significant lowerer in TNF-αH group compared to those in TNF-αL group, and white blood cell (WBC), complement 3 (C3), CD3, CD8, immunoglobulin G (IgG) and IL-6 were all higher in TNF-αH group. From Table 4, in TNM stage, immune indexes were decreased in III + IV stage. By the data from clinic characteristics, we found that although both TNF-α and TNM stage could result in weight loss, the mechanisms should be diversity. Body weight was mainly composed of skeletal muscle mass and fat mass. And skeletal muscle mass loss has recently been identified as a risk factor for poor outcomes in various malignancies. Since clinic characteristics between circulating TNF-α and TNM stage were different, we speculated that the changes of body compositions were also different, despite identical weight loss.
Table 3
Participants’ characteristics divided by level of TNF-α in peripheral blood
Characteristics
|
TNF-αL (n = 288)
|
TNF-αH (n = 104)
|
P
|
ALB (g/L)
|
42.08 ± 4.00
|
38.11 ± 5.25
|
< 0.001
|
TP (g/L)
|
67.01 ± 5.69
|
65.85 ± 7.52
|
0.10
|
GLB (g/L)
|
24.91 ± 4.01
|
26.71 ± 9.15
|
0.007
|
PAB (mg/L)
|
212.33 ± 55.30
|
201.33 ± 48.97
|
0.07
|
TRF (g/L)
|
2.32 ± 0.50
|
2.25 ± 0.46
|
0.21
|
GLU (mmol/L)
|
5.16 ± 1.29
|
5.66 ± 2.30
|
0.007
|
CREA (µmol/L)
|
66.98 ± 16.34
|
58.61 ± 11.76
|
< 0.001
|
TG (mmol/L)
|
1.25 ± 0.63
|
1.11 ± 0.44
|
0.04
|
CHOL (mmol/L)
|
4.33 ± 0.92
|
4.06 ± 0.98
|
0.01
|
LDH (IU/L)
|
154.01 ± 26.67
|
169.67 ± 51.24
|
< 0.001
|
RBC (× 1012/L)
|
4.30 ± 0.68
|
4.26 ± 0.80
|
0.62
|
HGB (g/L)
|
126.33 ± 25.75
|
115.39 ± 29.25
|
< 0.001
|
WBC (× 109/L)
|
5.44 ± 1.42
|
6.19 ± 2.34
|
< 0.001
|
LYMPH# (× 109/L)
|
1.60 ± 0.54
|
1.49 ± 0.43
|
0.06
|
C3 (g/L)
|
0.81 ± 0.15
|
0.88 ± 0.20
|
< 0.001
|
CD3 (%)
|
68.52 ± 10.57
|
69.40 ± 10.83
|
0.47
|
CD4 (%)
|
41.05 ± 9.56
|
39.95 ± 9.14
|
0.31
|
CD8 (%)
|
22.79 ± 7.74
|
25.04 ± 10.39
|
0.02
|
CD4/CD8
|
2.08 ± 1.04
|
1.71 ± 0.86
|
0.001
|
IgG (g/L)
|
10.80 ± 2.33
|
13.36 ± 9.28
|
< 0.001
|
IL-6 (pg/ml)
|
5.64 ± 13.56
|
16.51 ± 29.76
|
< 0.001a
|
ALB, albumin; CD, cluster of differentiation; CHOL, cholesterol; CREA, Creatinine; C3, complement 3; GLB, globulin; GLU, glucose; HGB, hemoglobin; IgG, immunoglobulin G; IL-6, interleukin-6; LDH, lactate dehydrogenase; LYMPH, lymphocyte; PAB, prealbumin; RBC, red blood cell; TG, triglyceride; TP, total protein; TRF, transferrin; WBC, white blood cell. |
aThe Mann-Whitney Test was used. |
Table 4
Participants’ characteristics divided by TNM stage
Characteristics
|
I + II (n = 136)
|
III + IV (n = 256)
|
P
|
ALB (g/L)
|
42.53 ± 3.98
|
41.32 ± 4.44
|
0.008
|
TP (g/L)
|
67.57 ± 5.82
|
65.24 ± 6.30
|
< 0.001
|
GLB (g/L)
|
26.04 ± 3.96
|
24.92 ± 5.19
|
0.03
|
PAB (mg/L)
|
222.68 ± 49.06
|
201.49 ± 56.08
|
< 0.001
|
TRF (g/L)
|
2.44 ± 0.54
|
2.21 ± 0.48
|
< 0.001
|
GLU (mmol/L)
|
5.70 ± 1.80
|
5.01 ± 1.06
|
< 0.001
|
CREA (µmol/L)
|
70.31 ± 17.38
|
64.16 ± 14.20
|
< 0.001
|
TG (mmol/L)
|
1.41 ± 0.53
|
1.26 ± 0.63
|
0.02
|
CHOL (mmol/L)
|
4.48 ± 0.93
|
4.24 ± 0.93
|
0.02
|
LDH (IU/L)
|
161.74 ± 25.63
|
152.43 ± 32.43
|
0.004
|
RBC (× 1012/L)
|
4.49 ± 0.63
|
4.23 ± 0.73
|
< 0.001
|
HGB (g/L)
|
131.76 ± 23.08
|
122.27 ± 26.21
|
< 0.001
|
WBC (× 109/L)
|
5.71 ± 1.91
|
5.37 ± 1.26
|
0.04
|
LYMPH# (× 109/L)
|
1.70 ± 0.60
|
1.54 ± 0.48
|
0.004
|
C3 (g/L)
|
0.85 ± 0.14
|
0.81 ± 0.18
|
0.025
|
CD3 (%)
|
70.20 ± 11.24
|
66.33 ± 8.94
|
< 0.001
|
CD4 (%)
|
44.53 ± 9.34
|
38.71 ± 9.27
|
< 0.001
|
CD8 (%)
|
23.34 ± 8.33
|
19.27 ± 7.26
|
< 0.001
|
CD4/CD8
|
1.90 ± 1.21
|
2.00 ± 0.93
|
0.36
|
IgG (g/L)
|
11.07 ± 2.43
|
9.16 ± 7.47
|
0.004
|
IL-6 (pg/ml)
|
0.85 ± 0.14
|
0.81 ± 0.18
|
0.025
|
ALB, albumin; CD, cluster of differentiation; CHOL, cholesterol; CREA, Creatinine; C3, complement 3; GLB, globulin; GLU, glucose; HGB, hemoglobin; IgG, immunoglobulin G; IL-6, interleukin-6; LDH, lactate dehydrogenase; LYMPH, lymphocyte; PAB, prealbumin; RBC, red blood cell; TG, triglyceride; TP, total protein; TRF, transferrin; WBC, white blood cell. |
The diversity of body composition and handgrip strength associated with circulating TNF-α and TNM stage
Our data showed that circulating TNF-α and TNM stage had significant associations with weight loss ≥ 5% before surgery, therefore, we firstly analyzed the preoperative body composition and handgrip strength of patients in cohort A with different circulating TNF-α and TNM stage. The body weight (P = 0.02), skeletal muscle mass (P < 0.001) and handgrip strength (P < 0.001) but not fat mass (P = 0.06) of patients in TNF-αL group were significantly higher than those in TNF-αH group, respectively (Fig. 2A, B, C, D). Meanwhile, patients of TNM stage I + II had significantly higher body weight (P < 0.001), skeletal muscle mass (P = 0.007), fat mass (P < 0.001) and handgrip strength (P < 0.001) than those of TNM stage III + IV (Fig. 2E, F, G, H). Except for fat mass, the trends of changes between circulating TNF-α and TNM stage were similar. We further analyzed the differences between TNF-αH and TNF-αL groups and found that the skeletal muscle mass accounted for 76.4% of the total body weight difference, while between TNM stage I + II and III + IV groups, this result was only 23.1%. Moreover, the average difference of handgrip strength between different TNF-α groups was higher than that between different TNM stage groups (3.80 kg vs. 1.96 kg). That was to say, circulating TNF-α seemed to have a greater effect on preoperative skeletal muscle than TNM stage.
Current studies suggested that TNF-α and IL-6, the important inflammatory cytokines, were related to cancer cachexia [8], thus we investigated the postoperative changes of circulating TNF-α and IL-6. Compared to those in TNF-αL group before surgery, patients in TNF-αH group tended to have higher postoperative circulating TNF-α (Fig. 3A). For patients with resected gastric cancer, the circulating TNF-α was elevated within the postoperative 3 days and then decreased slowly (Fig. 3B), and the circulating IL-6 was just increased within the first day after surgery and then decreased rapidly (Fig. 3C). Moreover, In patients with non-radical surgery, the changes of circulating IL-6 were similar to those with radical surgery (Fig. 3C), while the circulating TNF-α was elevated within the postoperative 3 days and still higher one month later (Fig. 3B), indicating that the existence of tumor after non-radical surgery might affect the circulating TNF-α continuously. Based on the findings mentioned above, we supposed that circulating TNF-α but not IL-6 might be associated with postoperative changes in body weight and composition in gastric cancer. This also reminded us that circulating TNF-α recovered to normal slowly after operation, and remained at a high level for a period of time. However, for patients with resected gastric cancer, tumor would have a weak effect on weight loss. This also showed that high circulating TNF-α should be caused by tumor but not all released by a tumor.
To further determine the different effects of circulating TNF-α and TNM on postoperative human body compositions, we followed up the patients with radical surgery (n = 376) in cohort A for half a year. From the analysis of postoperative body composition, we observed decreased body weight, skeletal muscle mass and fat mass at the first month after surgery, and all of them got a bit recovered in the sixth month after surgery (Fig. 3D). To determine whether preoperative circulating TNF-α was associated with postoperative changes in body composition, we next compared the patients between TNF-αH (n = 93) and TNF-αL (n = 283). We found that patients in TNF-αH group had significantly more loss of body weight (-3.37 kg vs. -2.02 kg, P < 0.001), skeletal muscle mass (-1.83 kg vs. -0.86 kg, P < 0.001) and fat mass (-0.85 kg vs. -0.25 kg, P < 0.001) than those in TNF-αL group at first month after surgery (Fig. 3E), while at sixth month after surgery, patients in TNF-αH group also had significantly more loss of body weight (-1.01 kg vs. -0.21 kg, P < 0.001) and skeletal muscle mass (-0.76 kg vs. -0.18 kg, P < 0.001) but not fat mass (-0.21 kg vs. -0.21 kg, P > 0.9) (Fig. 3F). Additionally, we also investigated the associations between TNM stage and postoperative changes of body composition. Interestingly, in the first month after surgery, patients of advanced stage (III + IV, n = 240) had significantly more loss of body weight, skeletal muscle mass and fat mass than those of early stage (I + II, n = 136) (Fig. 3G), whereas there was no significant difference in change of body composition between patients in early and advanced stage at sixth month after surgery (Fig. 3H). In summary, for postoperative changes of body composition, high circulating TNF-α that was mainly associated with skeletal muscle loss was further clarified. From the analysis, we surmised that circulating TNF-α mainly affected skeletal muscle loss to bring about weight loss, and TNM stage could influence both skeletal muscle and fat mass.
Low skeletal muscle mass and handgrip strength are associated with increased surgical adverse events
Given that current studies suggested that weight loss might be associated with increased risk of postoperative complications, we also evaluated whether the body composition and handgrip strength had associations with postoperative adverse events in patients of cohort A with radical surgeries. Based on the Common Terminology Criteria for Adverse Events (CTCAE) version 4, we found that low skeletal muscle mass and handgrip strength, but not body weight and fat mass, were associated with a higher risk of in-hospital grade 3–4 non-hematological surgical adverse events after radical surgery (Table 5). Thus patients with weight loss caused by high circulating TNF-α should deserves our due attention, especially that in advanced stage with high circulating TNF-α.
Table 5
In-hospital grade 3–4 non-hematological surgical adverse events for resection
CTCAE v4.0
|
Weight
|
Skeletal muscle mass
|
Fat mass
|
Handgrip strength
|
High (n = 196)
|
Low (n = 196)
|
High (n = 196)
|
Low (n = 196)
|
High (n = 196)
|
Low (n = 196)
|
High (n = 196)
|
Low (n = 196)
|
Overall
|
7
|
12
|
4
|
15**
|
9
|
10
|
2
|
17***
|
Pancreatic fistula
|
0
|
2
|
0
|
2
|
0
|
2
|
0
|
2
|
Anastomotic leak
|
0
|
1
|
0
|
1
|
0
|
1
|
0
|
1
|
Abdominal infection
|
2
|
3
|
1
|
4
|
2
|
3
|
1
|
4
|
Postoperative hemorrhage
|
0
|
1
|
0
|
1
|
1
|
0
|
0
|
1
|
Anastomotic stenosis
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Cholecystitis
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Dumping syndrome
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Delayed gastric emptying
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Gastroesophageal regurgitation
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Bowel obstruction
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Ileus
|
1
|
0
|
1
|
0
|
1
|
0
|
0
|
1
|
Thromboembolic event
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Pneumonia
|
3
|
4
|
2
|
5
|
3
|
4
|
1
|
6
|
Chyle leakage
|
0
|
1
|
0
|
1
|
1
|
0
|
0
|
1
|
Wound infection
|
1
|
0
|
0
|
1
|
1
|
0
|
0
|
1
|
Wound dehiscence
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
CTCAE, Common Terminology Criteria for Adverse Events. |
**P < 0.01 |
***P < 0.001 |
High TNF-α leads to myofiber change, interfered glycometabolism of skeletal muscle and increased myoblast apoptosis
Based on clinical data from cohort A, we found that high circulating TNF-α was associated with skeletal muscle loss. To further investigate the effect of TNF-α on skeletal muscle, we next collected 60 skeletal muscle samples from patients in cohort B, in which 16 patients had high circulating TNF-α (> 9.96 pg/ml) and 21 patients were in TNM stage I + II. Immumohistochemical analysis of ATPase staining showed that the skeletal muscle of patients in the TNF-αH group had a significantly decreased ratio of type II muscle fibers (P < 0.001) (Fig. 4A), which was a typical pathogenic characteristic of sarcopenia muscle [11]. Besides, immumohistochemical analysis also showed that skeletal muscles in TNF-αH group had significantly lower expression of AMPK than those in TNF-αL group (P < 0.001) (Fig. 4B). For validating this result, we cultured the HSMMs with different concentrations of TNF-α in vitro. Western blot showed that the expressions of AMPK and phosphate-AMPK (p-AMPK) in HSMMs were inversely associated with concentrations of TNF-α (Fig. 4C). Moreover, flow cytometry revealed a positive correlation between the apoptosis ratio of HSMMs and the concentration of TNF-α (Fig. 4D). These findings indicated that high concentrations of TNF-α could decrease the glycometabolism in skeletal muscle and induce HSMMs cells apoptosis, which might lead to the skeletal muscle wasting.
The relationships between TNF-α and CD8+ T cells in vitro and in vivo
From data in cohort A, we found that patients with TNF-αH had significantly higher CD3 and CD8 in peripheral blood. However, the relationships between TNF-α and CD8+ T cells in peripheral blood were unclear. We collected the peripheral blood of patients in cohort B before surgery. PBMCs were isolated and then used for testing the expression of co-inhibitory receptors on CD8+ T cells. Flow cytometry showed that the expressions of PD-1, LAG-3, TIM-3 and TIGIT on CD8+ T cells had no significant differences between patients in TNF-αH group (n = 16) and TNF-αL group (n = 44) (Fig. 5A). However, patients of TNM stage I + II (n = 21) had significantly lower expressions of PD-1, LAG-3, TIM-3 but not TIGIT on CD8+ T cells than those of TNM stage III + IV (n = 39) (Fig. 5B). In vitro, we cultured the PBMCs with different concentrations of TNF-α, whereas the expressions of PD-1, LAG-3, TIM-3 and TIGIT on CD8+ T cells were not associated with the concentrations of TNF-α (Fig. 5C). Additionally, be consistent with the result in Fig. 1E, we also found that patients in cohort B with advanced TNM stage (III + IV) had significantly higher circulating TNF-α (Fig. 5D). Moreover, we also extracted the CD8+ T cells from PBMCs. After being stimulated with soluble anti-CD3 plus anti-CD28 and cultured with different concentrations of TNF-α, flow cytometry showed that the apoptosis ratio and proportions of S phase of CD8+ T cells were positively correlated with concentrations of TNF-α (Fig. 5E and 5F). Besides, CD8+ T cells cultured with 10 pg/ml TNF-α had higher proliferation than those without TNF-α (P = 0.04). While no significant difference was found between CD8+ T cells cultured with 20 pg/ml and 10 pg/ml TNF-α, as well as between 0 pg/ml and 20 pg/ml (Fig. 5G). In summary, TNM stage instead of circulating TNF-α was associated with expressions of PD-1, LAG-3, TIM-3 and TIGIT on peripheral CD8+ T cells, and vitro experiments showed that TNF-α promoted the proliferation of CD8+ T cells but significantly increased their apoptosis ratio at higher concentration.