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Journal of Steroid Biochemistry and Molecular Biology
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Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity
Yuki Ishikawa ª, Yuto Yamazaki b,*, Yuta Tezuka c,d, Kei Omata ”,”, Yoshikiyo Ono c,d,
Kazuaki Tokodai ª, Fumiyoshi Fujishima b, Shin Kawanabe e,f, Takuyuki Katabami, Akira Ikeya &, Miho Yamashita &, Yutaka Okih, Hiroshi Nanjo1, Fumitoshi Satoh3, Akihiro Ito k,
Michiaki Unnoª, Takashi Kamei ª, Hironobu Sasanob, Takashi Suzuki b
a Department of Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan
b Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan
· Department of Diabetes, Metabolism and Endocrinology, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan
d Division of Nephrology, Rheumatology and Endocrinology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
e Department of Metabolism and Endocrinology, St. Marianna University Yokohama Seibu Hospital, Yokohama, Japan
‘ Department of Metabolism and Endocrinology, St. Marianna University School of Medicine, Kawasaki, Japan
8 Division of Endocrinology & Metabolism, Second Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
h Diabetes & Endocrinology Center, Hamamatsu-Kita Hospital, Hamamatsu, Shizuoka, Japan
i Department of Pathology, Akita University Hospital, Akita, Japan
¿ Division of Clinical Hypertension, Endocrinology and Metabolism, Tohoku University Graduate School of Medicine, Sendai, Japan
k Department of Urology, Tohoku University Graduate School of Medicine, Sendai, Japan
ARTICLE INFO
Keywords:
CYP11B1
CYP17A
Glucocorticoid
Intra-tumoral heterogeneity and disorganized steroidogenesis Tumor-associated macrophages Tumor infiltrating lymphocytes Tumor microenvironment
ABSTRACT
Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocor- ticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH- 1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r= 0.421, p < 0.001), and CD68-positive TAMs (r=0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = - 0.259, p = 0.002) and CD4-positive TILs (r = - 0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and
Abbreviations: ACC, adrenocortical carcinoma; ENSAT, European Network for the Study of Adrenal Tumors; MVD, micro-vessel density; TAMs, Tumor-associated macrophages; TILs, Tumor infiltrating lymphocytes; TME, tumor microenvironment; OS, overall survival; RFS, recurrence-free survival; VASH-1, vasohibin-1.
* Correspondence to: Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail address: y.yamazaki@patholo2.med.tohoku.ac.jp (Y. Yamazaki).
https://doi.org/10.1016/j.jsbmb.2024.106462
Received 19 October 2023; Received in revised form 7 January 2024; Accepted 11 January 2024
CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunore- activity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heteroge- neity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare cancer with an incidence rate of approximately one per million population per year, but its prognosis is poor [1,2]. More than one-third of ACC patients have unresectable advanced disease at the time of diagnosis and even when complete resection could be achieved local recurrence and distant metastasis frequently follow [3,4]. The current standard treatment regimens for advanced-stage ACC patients include administration of mitotane with or without cytotoxic chemotherapy including etoposide, doxorubicin or cisplatin [5,6]. However, the selection and efficacy of the treatment are both rather limited at this juncture [7]. Recently, immune checkpoint inhibitors have been used in various human malignancies and several immunotherapies have been clinically attempted in ACC patients, but their therapeutic effects markedly varied among the studies reported [8-11].
Approximately 90 % of ACC patients are known to harbor increased neoplastic adrenocortical steroid production and among those hormonal symptoms, Cushing’s syndrome due to glucocorticoid excess has been generally considered an adverse clinical prognostic factor [3,12-14]. As described above, several clinical trials of immunotherapy have been reported in ACC patients and despite their variable therapeutic effects in the patients, neoplastic glucocorticoid excess is generally considered an adverse clinical factor in eventual clinical outcome in those patients [9, 15-17]. These results have been interpreted due to the immunosup- pressive effects of glucocorticoids produced and secreted from carci- noma cells, which could be related to modulation of gene transcription, effects on macrophages with suppression of lymphokines production, and promotion of lymphocyte apoptosis [18,19], but their details have remained practically unknown. In addition, steroidogenesis in ACC differs from normal adrenal glands and adrenocortical adenomas in that the intra-tumoral expression pattern of steroidogenic enzymes is mark- edly heterogenous, termed as “disorganized steroidogenesis” [14].
Therefore, understanding the tumor microenvironment (TME), including tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and angiogenesis in ACC patients is considered pivotal for establishing immune-based anti-tumor therapy. In particular, the effects of glucocorticoid on tumor immunity are considered to be required, but only two studies were previously reported in this regard in ACC patients. These reported studies include that of Landwehr L-S, et al. using fluorescent immunostaining and of Jordan J Baechle, et al. using The Cancer Genome Atlas analysis, both of which demonstrated that tumor-infiltrating immune cells were decreased in glucocorticoid excess ACC [20,21]. However, the effects of in situ glucocorticoid production as well as the expression profiles of steroidogenic enzymes in tumor cells on the status of tumor immunity itself in ACC have remained unknown. In particular, the effects of intra-tumoral heterogeneity of disorganized steroidogenesis on tumor immunity have not been studied at all. Therefore, in this study, we correlated the status of tumor infiltrating-immune cells (TILs and TAMs) with two key steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity and disorganized steroidogenesis on tumor immunity of ACC. We also examined the correlation among tumor im- munity, angiogenesis, and tumor grade to further evaluate the charac- teristics of tumor microenvironment of ACC patients.
2. Materials and methods
2.1. Patient and tissue
In this study, thirty-four ACC cases were retrieved from pathology files of Department of Pathology, Tohoku University School of Medicine including those submitted for consultation to one of the authors (H.S.). They had been resected in Tohoku University Hospital (N = 19), Hamamatsu University Hospital (N = 9), Sagamihara National Hospital (N = 1), St. Marianna University Hospital (N = 4), and Akita University Hospital (N = 1) from 1993 to 2021. The details of clinicopathological features of the cases studied were summarized in S1. 30 cases were primary, and four were recurrent tumors. The median age, excluding one case that was not available, was 54 years [range: 0-77], and two cases were under 15 years of age and 31 cases were 15 years of age or older. The gender was 12 male, 21 female, and one not available. Glucocorticoid excess was clinically detected in 23 out of 32 patients (72 %), excluding two cases that were not available. Clinically excessive hormone profiles other than glucocorticoid were as follows; androgen in 16, estrogen in four, aldosterone in two cases, non functional in nine cases, and unknown in three cases. The median Weiss score, excluding 11 cases that were not available, was 7 [range: 3-9]. In pT, four cases were pT1, six pT2, five pT3, three pT4, and 16 cases unknown. In pN, 17 cases were pN0, one pN1, and 16 cases unknown. Regarding ENSAT stage, five cases were Stage I, 10 Stage II, six Stage III, eight Stage IV, and five unknown. Glucocorticoid excess was defined as 1 mg dexametha- sone suppression test of cortisol >5 µg/dL in conjunction with sup- pressed adrenocorticotropic hormone. Overall survival (OS) could be evaluated in 16 primary tumors and recurrence-free survival (RFS) available in 11 cases of primary tumors excluding those diagnosed as European Network for the Study of Adrenal Tumors (ENSAT) stage IV. Post operative adjuvant therapy of mitotane was administered to nine cases and that of etoposide + doxorubicin + cisplatin + mitotane to two cases. One recurrent case was treated with mitotane before surgery. Five cases did not receive any adjuvant therapy and unknown in 17 cases. The details of clinicopathological features of the cases examined in this study were summarized in supplemental file 1 (S1). The research pro- tocol of this study was approved by the ethical committee of Tohoku University School of Medicine (accession number 2021-1-482).
2.2. Immunohistochemistry
The resected specimens had been fixed and paraffin-embedded and the serial tissue sections (thickness: 3-4 um) were prepared. Deparaffi- nization was performed in xylene, and dehydration in graded ethanol solutions. In this study, CD3, CD4, CD8, and FOXP3 were immunolo- calized to evaluate the status of TILs, CD68 (PG-M1) and CD163 to evaluate TAMs, CYP17A and CYP11B1 to study key steroidogenic en- zymes of glucocorticoid, CD31 (PECAM-1) and vasohibin-1 (VASH-1) to examine the status of angiogenesis, and Ki-67 (MIB-1) to evaluate tumor cell proliferation or tumor grade. The primary antibodies and immu- nohistochemistry protocols used in this study were summarized in Table 1.
2.3. Evaluation of immunoreactivity
All immunohistochemically stained slides were digitally scanned by Nanozoomer S360 (C13220-01, Hamamatsu Photonics, Shizuoka, Japan). Digital image analysis was performed using HALO® Membrane v1.7 (Indica Laboratories, Corrales, NM, USA) [22]. The representative images of immunohistochemistry were illustrated in Fig. 1.
CD3, CD4, CD8 and FOXP3 immunoreactivity were evaluated by nuclear segmentation and quantification algorithms [23]. CD68, CD163, CYP17A and CYP11B1 immunoreactivity were evaluated by cytoplasm segmentation and quantification algorithms. CD3, CD4, CD8, FoxP3, CD64, CD163, CYP17A and CYP11B1 were firstly evaluated in the whole tumor area and then in each of four randomly selected areas (8 mm2 at x 200) matched in each stain (Fig. 2). The value represented the number of CD3-, CD4-, CD8- and FoxP3-positive TILs and CD64- and CD163-positive TAMs divided by the analysis area (mm2) [24]. CYP17A and CYP11B1 immunoreactivity were evaluated using H-score. H-score (range: 0-300) is defined as the sum of the product of the intensity score (0: negative; 1: weak; 2: moderate; 3: strong positive) and the corre- sponding percentage of positive cells (range: 0-100 %) [25].
According to the previous study, CD31- and VASH-1-positive vessels were quantified using a combination of digital image analysis and manual methods [22]. All of the immunohistochemically stained endothelial cells or clusters isolated from adjacent vessels were counted as single vessel, even if the lumen was absent [26]. CD31 immunore- activity was evaluated in each five hot spot areas (1 mm2 at x 200) after careful evaluation of the whole tumor area. The number of the vessels with endothelial cells positive for CD31 was subsequently determined, and the area with the highest value was defined as micro-vessel density (MVD) -CD31. VASH-1 immunoreactivity was determined by counting the number of the vessels with vascular endothelial cells positive for VASH-1 in the MVD-CD31 area, which was defined as MVD-VASH-1. The ratio of MVD-VASH-1 to MVD-CD31 was defined as VASH-1 expression [22,27].
Ki-67 proliferative index was evaluated by nuclear segmentation and quantification algorithms in the whole tumor area. The value repre- sented the number of positive per total number of carcinoma cells.
2.4. Statistical analysis
Statistical analyses were performed using JMP Pro software (Ver 16.0.0, SAS Institute, Cary, NC, USA). In the whole tumor area analyses, the results of immunoreactivity of individual markers and Weiss score were divided into “Low” and “High” groups based on the median as cut- off value. OS was defined as the period from the date of surgery to that of death or the last follow-up. RFS was defined as the period from the date of surgery to that of the first recurrence or the last follow-up. Wilcoxon signed-rank test was applied to comparative analysis between two groups. Correlations between CYP17A and CYP11B1 immunoreactivity, and between CYP17A and CYP11B1 immunoreactivity and TILs and TMAs in randomly selected areas were analyzed using Spearman’s rank
correlation coefficient. Survival analysis was using Cox proportional hazards regression modeling. We tentatively defined the significance as p < 0.05 for all statistical analyses in this study.
3. Results
3.1. Differences of tumor-infiltrating immune cell profiles between ACCs with and without glucocorticoid excess
The results of immune-profiles of TILs and TAMs in the whole tumor area were summarized in S2. The number of TILs and TAMs and their subset ratios were compared between those with and without gluco- corticoid excess and results were summarized in S3. In ACC associated with glucocorticoid excess, CD8-positive TILs and CD8/CD3 ratio were significantly lower (CD8: p = 0.019, CD8/CD3: p = 0.008) than those not (Fig. 3).
3.2. Correlation between CYP17A/CYP11B1 and glucocorticoid excess in ACC
The results of CYP17A and CYP11B1 immunoreactivity in the whole tumor areas were summarized in S2. There were no significant differ- ences in CYP17A and CYP11B1 immunoreactivity in the whole tumor area between those with and without glucocorticoid excess (Fig. 4A). There was a significantly positive correlation between CAP17A and CYP11B1 in both whole tumor (r = 0.613, p = 0.001) and randomly selected areas (r = 0.357, p < 0.001) (Fig. 4B).
3.3. Correlation between CYP17A/CYP11B1 and tumor-infiltrating immune cells in ACC
The number of TILs and TAMs and their subset ratios were compared between high and low CYP17A and CYP11B1 groups in the whole tumor areas and results were summarized in S4. In high CYP17A group, FOXP3-positive TILs were significantly higher, but CD4/CD3 ratio was significantly lower (FOXP3: p = 0.021, CD4/CD3: p = 0.001) than low group. In high CYP11B1 group, CD8/CD3 and CD163/CD68 ratios were both significantly higher, but CD4-positive TILs and CD4/CD3 ratio were significantly lower (CD4: p = 0.036, CD4/CD3: p = 0.001, CD8/ CD3: p = 0.039, CD163/CD68: p = 0.006) than low group (Fig. 5A). The correlations between the tumor-infiltrating immune cells (TILs and TAMs) and CYP17A/CYP11B1 immunoreactivity in randomly selected areas were examined and the results were summarized in S5. There were significantly positive correlations between CYP17A and CD8 and FOXP3-positive TILs, CD8/CD3 ratio, and CD68-positive TAMs (CD8: r = 0.334, p < 0.001, FOXP3: r =0.414, p<0.001, CD8/CD3: r = 0.421, p < 0.001, CD68: r = 0.298, p < 0.001). There was signifi- cantly positive correlation between CYP11B1 and CD8/CD3 ratio, while significantly inverse correlations detected between CYP11B1 and CD3- and CD4-positive TILs (CD3: r = - 0.259, p = 0.002, CD4: r = - 0.312, p < 0.001, CD8/CD3: r = 0.276, p = 0.001) (Fig. 5B).
| Antibody | Source | Species | Mono/poly | Antigen retrieval (Buffer pH) | Dilution | Secondary antibody |
|---|---|---|---|---|---|---|
| CD3 | DAKO | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (9.0) | 1:500 | Histofine SAB-PO (M) Kit |
| CD4 | Abcam | Rabbit | Monoclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:400 | Histofine SAB-PO (R) Kit |
| CD8 | DAKO | Mouse | Monoclonal | Autoclave, 121 °℃, 5 min (6.0) | 1:50 | Histofine SAB-PO (M) Kit |
| FOXP3 | Abcam | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:100 | Histofine SAB-PO (M) Kit |
| CD68 (PG-M1) | DAKO | Mouse | Monoclonal | Trypsin, 37 ℃, 30 min (7.6) | 1:100 | Histofine SAB-PO (M) Kit |
| CD163 | Lecia biosystems | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:600 | Histofine SAB-PO (M) Kit |
| CYP17A | BEX | Rabbit | Polyclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:500 | Histofine SAB-PO (R) Kit |
| CYP11B1 | Provided | Rat | Monoclonal | Autoclave, 121 ℃, 5 min (9.0) | 1:70 | ImmPRESS® Goat Anti-Rat IgG Polymer Kit |
| CD31 (PECAM-1) | DAKO | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:100 | Histofine SAB-PO (M) Kit |
| vasohibin-1 | Tohoku University | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (9.0) | 1:400 | Histofine SAB-PO (M) Kit |
| Ki-67 (MIB-1) | DAKO | Mouse | Monoclonal | Autoclave, 121 ℃, 5 min (6.0) | 1:100 | Histofine SAB-PO (M) Kit |
a
b
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CD4
CD8
FOXP3
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CD68
CD163
CYP17A
CYP11B1
120
1
p=0.019*
p=0.008*
100
0.8
80
CD8
CD8/CD3
60
0.6
40
0.4
20
0.2
0
-
+
+
Glucocorticoid excess
Glucocorticoid excess
A
150
p=0.476
p=0.933
100
CYP17A
CYP11B1
100
50
50
0
0
+
+
Glucocorticoid excess
Glucocorticoid excess
B
Whole tumor area
Randamly selected areas
7=0.613
7=0.357
p=0.001*
300
p<0.001*
100
CYP17A
CYP17A
200
50
100
0
0
0
50
100
150
0
50
100
150
200
CYP11B1
CYP11B1
3.4. Association between clinical glucocorticoid excess/CYP17A/ CYP11B1 and angiogenesis or Ki-67 proliferative index
There were no significant differences of the MVD-CD31, MVD-VASH- 1, VASH-1 and/or the Ki-67 proliferative index between those with and without glucocorticoid excess (S6). In addition, there were no signifi- cant differences of the MVD-CD31, MVD-VASH-1 and VASH-1 and/or the Ki-67 proliferative index between high and low CYP17A and CYP11B1 groups in the cases examined in this study (S7).
3.5. Differences of tumor-infiltrating immune cell profiles, angiogenesis, and Ki-67 proliferative index between ACCs with and without androgen excess
There were no significant differences in the number of TILs and TAMs and the ratios of their subsets, the MVD-CD31, MVD-VASH-1 and VASH-1, and the Ki-67 proliferative index between those with and without androgen excess (S8).
3.6. Association between angiogenesis and tumor-infiltrating immune cells in TME of ACC
The results of MVD-CD31, MVD-VASH-1, and VASH-1 were sum- marized in S2. The number of TILs and TAMs and their subset ratios were compared between high and low MVD-CD31, MVD-VASH-1 and VASH-1 groups and the results were summarized in S9. There were no significant differences in the number of TILs and TAMs and their subset ratios between high and low MVD-CD31 groups (S6). In high MVD- VASH-1 group, CD68- and CD163-positive TAMs, and CD163/CD68 ratio were significantly higher (CD68: p = 0.015, CD168: p = 0.009,
CD163/CD68: p = 0.042) than low group. In high VASH-1 expression group, CD163-positive TAMs was significantly higher (CD163: p = 0.042) than low group (Fig. 6).
3.7. Association between Ki-67 proliferative index/Weiss scores and TME in ACC
The results of Ki-67 proliferative index were summarized in S2. The number of TILs and TAMs and their subset ratios, MVD-CD31, MVD- VASH-1, and VASH-1 were compared between high and low Ki-67 proliferative index and Weiss score groups, and results were summa- rized in S10. In high Ki-67 proliferative index group, MAD-VASH-1 and VASH-1 were significantly higher (MVD-VASH-1: p = 0.006, VASH-1 expression: p = 0.006) than low one (Fig. 7). There were no signifi- cant differences in the number of TILs and TAMs and their subset ratios, MVD-CD31, MVD-VASH-1, and VASH-1 immunoreactivities between high and low Weiss score groups (S7).
3.8. Survival analysis according to the TME factors examined in ACC patients
The results of univariate analysis of OS and RFS were illustrated in S11. In the analysis of OS and RFS, there were no prognostic factors in those examined in this study (S11).
4. Discussion
This is the first study to evaluate the effects of in situ neoplastic glucocorticoid production and intra-tumoral heterogeneity of steroido- genesis or disorganized steroidogenesis on tumor immunity of ACC. In
A
1
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p=0.001*
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p=0.006
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:
CD4
CD4/CD3
CD8/CD3
CD163/CD68
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:00
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0.6
:
50
0.4
:
0.4
0.4
:
0
0.2
0.2
0.2
Low
CYP11B1
High
Low
High
Low
High
Low
High
CYP11B1
CYP11B1
CYP11B1
B
250
10
7=0.334
7=0.414
8
7=0.421
1=0.298
200
p<0.001*
S
p<0.001*
p<0.001*
1000
p<0.001*
6
CD8
150
FOXP3
6
CD8/CD
4
CD68
100
4
3
500
50
2
2
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0
0
0
0
100
200
300
0
100
200
300
0
100
200
300
0
100
200
300
CYP17A
CYP17A
CYP17A
CYP17A
400
1 =- 0.259
400
7 =- 0.312
8
1=0.276
p=0.002*
p<0.001*
p=0.001
300
300
6
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CD3
200
CD4
CD8/CD3
200
4
100
100
2
0
0
0
0
50
100
150
200
0
50
100
150
200
0
50
100
150
200
CYP11B1
CYP11B1
CYP11B1
terms of intra-tumoral immune-profiling, the presence of CD4- and CD8- positive TILs has been reported to be generally correlated with favorable prognosis of the patients with various malignancies [20,28-30]. On the other hand, FOXP3-positive TILs and M2 phenotype CD163-positive TAMs have generally been reported to contribute to tumor progression or aggressive clinical course in those tumor patients [31-34]. In our present study of ACC, higher CYP17A immunoreactivity increased CD8-positive TILs as well as FOXP3-positive TILs, while higher CYP11B1 decreased CD4-positive TILs but increased the ratio of CD163-positive TAMs. Therefore, these steroidogenic enzymes involved in glucocorti- coid production could modulate the tumor immunity in a different manner possibly because of their unique characteristics of “disorganized
steroidogenesis” in dedifferentiation of tumor cells. However, further investigations are warranted for clarification due to the relatively small number of the cases examined in this study.
In ACC with glucocorticoid excess, Landwehr et al. reported decreased CD4-positive TILs and Baechle et al. reported decreased CD8- positive TILs [20,21], both of which were consistent with those in our present study. Glucocorticoid has been reported to generally suppress CD4-positive lymphocytes activation and CD8-positive lymphocytes effector differentiation, while promoting FOXP3-positive lymphocytes activation and differentiation [18,35,36]. It is also well known that glucocorticoid could also contribute to monocyte maturation or devel- opment into M2 phenotypes CD163-positive macrophages [37].
1
p=0.015*
400
500
p=0.009*
p=0.042*
400
300
CD163/CD68
0.8
CD68
$ :
:
300
:
:
CD163
200
0.6
200
8
100
:
0.4
:
100
8
0.2
0
0
Low
MVD-VASH-1
High
Low
High
MVD-VASH-1
Low
MVD-VASH-1
High
400
p=0.042*
300
CD163
200
100
0
Low
High
VASH-1 expression
80
p=0.006*
0.7
VASH-1 expression
p=0.006*
0.6
MVD-VASH-1
60
0.5
0.4
40
0.3
20
0.2
0.1
0
0
Low
High
Low
High
Ki-67 proliferative index
Ki-67 proliferative index
Therefore, not only serum glucocorticoid excess but also in situ gluco- corticoid excess, as demonstrated by increased CYP17A and CYP11B1 levels in this study, are considered to suppress tumor immunity. How- ever, it is also true that the localization of CYP17A and CYP11B1 was not necessarily concordant with those detected in normal adrenal cortex because of the disorganized steroidogenesis and deviation of tumor cell differentiation, which made it rather difficult to interpret the unique pathophysiology of ACC.
We used CYP17A and CYP11B1 immunoreactivity, which was re- ported to be correlated with the extent of glucocorticoid excess in adrenocortical adenoma [38,39] and to be generally regarded to represent in situ glucocorticoid production. However, it is also true that in our present study, there were no significant differences in CYP17A and CYP11B1 immunoreactivity between ACCs with and without
glucocorticoid excess. In addition, the correlation between CYP17A and CYP11B1, both of which were involved in glucocorticoid production, was generally less pronounced in the randomly selected areas than in the whole tumor area. These results above were all consistent with intra-tumoral heterogeneity of steroidogenesis enzymes expression in ACC. This intra-tumoral heterogeneity results in disorganized steroido- genesis, which could account for the discrepancy of CYP17A and CYP11B1 with glucocorticoid secretion, especially in randomly selected tumor areas of ACC. These findings also indicated that ACC was poorly differentiated compared to adrenocortical adenoma and had a func- tionally deviant differentiation tendency called disorganized steroido- genesis. Therefore, CYP17A immunoreactivity could indicate the deviation from normal adrenal cortex and the tissue microenvironment related to abnormal steroidogenesis. The immune system was
considered to be activated in TME of ACC in contrast to normal adrenal cortex with infiltration of CD8-positive TILs. In addition, CYP11B1 is involved in the final stage of cortisol production, which could more directly influence the levels of in situ glucocorticoid. Therefore, this CYP11B1 expression might result in immunosuppressive effects of glucocorticoid with decreased CD4-positive TILs and increased CD163-positive TAMs. However, the heterogenous patterns of neoplastic steroidogenic enzymes expression further complicated glucocorticoid production, making it difficult to determine exactly how the expression status of CYP17A and CYP11B1 influence the TME, including tumor immune cells.
ACC produces hormones other than glucocorticoid, such as androgen, estrogen and aldosterone but also precursor steroids including those hormonally less inactive, which could also affect TME. ACCs with aldosterone or estrogen hypersecretion are extremely rare [40] and only a few cases were available for examination in our present study. Therefore, we attempted to evaluate the possible influence of androgens on TME of ACC cases examined. Results of previously re- ported studies on the association between androgen-deprivation therapy and tumor immunity in prostate cancer suggested that androgens sup- pressed various subsets of infiltrating lymphocytes [41,42]. In our pre- sent study, tumor derived androgens had extremely limited effects on tumor microenvironment of ACC. In addition, Sada et al. reported that the status of androgen secretion was by no means related to clinical outcome including survival rate in the patients of ACC [40]. Therefore, it awaits further investigations to clarify the effects of androgen on the TME in ACC.
The status of neovascularization is also an important component of the TME, and increased vascular density and endothelial areas of each vessel were reported in ACC compared to adrenocortical adenomas [43, 44]. To further evaluate the characteristics of TME in ACC, in this study, we examined angiogenesis by immunolocalization of CD31, which is a vascular endothelial cell marker, and VASH-1, which was reported to reflect angiogenesis in several human malignancies[45,46], and studied their association with the status of tumor-infiltrating immune cells. Results did demonstrate that CD163-positive TAMs increased with the increased VASH-1 positive vessels, and similar results were also reported in mixed neuroendocrine non-neuroendocrine neoplasms [22]. These results above are considered to reflect promoting angiogenic effects of M2 phenotypes macrophages [33,34].
We also examined the association of tumor-infiltrating immune cells and angiogenesis with Ki-67 proliferative index, which represented not only tumor cell proliferative activity but also tumor grade of ACC pa- tients [47], and Weiss score, which has been used to diagnose ACC [48], to explore how tumor grade could influence TME of the patients. Results did demonstrate that both Ki-67 proliferative index and Weiss score were not significantly associated with TILs and TAMs. The correlation between tumor grade and tumor-infiltrating immune cells has been re- ported to be markedly different among different types of human ma- lignancies [49-51]. The results of our present study did demonstrate that TILs were by no means related to tumor grade in ACC. On the other hand, the results of our present study showed the correlation between Ki-67 proliferative index and VASH-1 expressing vessels as reported in hepatocellular carcinomas [45]. Those results suggested that increased angiogenesis was also associated with tumor growth in ACC as in other malignant tumors, but further investigations are required for clarification.
We evaluated the TME of ACC from various aspects including glucocorticoid production, tumor-infiltrating immune cells, angiogen- esis, and tumor grade, suggesting that neoplastic steroidogenesis, especially glucocorticoid production markedly influenced the status of TME in ACC patients. In addition, not only systemic glucocorticoid excess, but also in situ glucocorticoid excess was demonstrated to be associated with the status of tumor immunity, which could be very important in evaluating the tumor prior to immune based therapy including immune check point inhibitors in ACC patients.
Immunotherapy was generally reported to be less effective in ACC with glucocorticoid excess [9,15-17]. However, in situ glucocorticoid pro- duction also affected the effectiveness of immunotherapy, which should be considered when administering those therapies. On the other hand, there were some limitations in this study. First, ACC is a rare tumor and only 34 cases were available in this study and some of the cases sub- mitted for histopathology diagnosis consultation did not have sufficient clinical findings available. Second, the definition of systemic or clinical glucocorticoid excess did differ among hospitals involved in this study and was by no means standardized. Third, the actual amount of in situ levels of glucocorticoid could not be evaluated. This was retrospective study evaluating archival or 10 % formalin-fixed and paraffin embedded histology materials, which made biochemical analysis of steroids including evaluation of steroid contents impossible because all steroids were extracted in the process of tissue specimen preparation. Therefore, in this study, we could not help employing CYP17A and CYP11B1 as indicators of in situ glucocorticoid production. Considering those limi- tations above, further investigations such as evaluation of a larger number of fresh frozen ACC cases are warranted.
In summary, in this study, we firstly demonstrated that in situ glucocorticoid production of ACC, especially intra-tumoral heteroge- neity of glucocorticoid biosynthesis could markedly influence tumor immunity. The findings and hypothetical mechanism based on the re- sults of our present study regarding the influence of intra-tumoral het- erogeneity of glucocorticoid biosynthesis on in situ tumor immunity of ACC are illustrated in Fig. 8. The results of our present study should provide important insights when considering new immune based ther- apies in ACC patients.
In ACC, CYP17A and CYP11B1 immunoreactivity did not always coincide, indicating intra-tumoral heterogeneity of steroidogenesis en- zymes expression. This intra-tumoral heterogeneity results in disorga- nized steroidogenesis. In our present study, higher CYP17A immunoreactivity increased CD8-positive TILs and their ratio as well as FOXP3-positive TILs, while higher CYP11B1 immunoreactivity decreased CD4-positive TILs and their ratio and also increased ratio of CD163-positive TAMs. The inconsistent results for CYP17A and CYP11B1 were thought to be due to disorganized steroidogenesis. ACC, adrenocortical carcinoma; TILs, Tumor infiltrating lymphocytes; TAMs, Tumor-associated macrophages.
Funding
This study was funded by grants from the Ministry of Health, Labour, and Welfare, Japan (Nos. 23FC1041 and No. 22K16406).
CRediT authorship contribution statement
Unno Michiaki: Writing - review & editing. Ono Yoshikiyo: Writing - review & editing. Ito Akihiro: Writing - review & editing. Yamashita Miho: Resources, Writing - review & editing. Ikeya Akira: Resources, Writing - review & editing. Nanjo Hiroshi: Resources, Writing - review & editing. Yamazaki Yuto: Conceptualization, Project administration, Writing - review & editing. Oki Yutaka: Resources, Writing - review & editing. Ishikawa Yuki: Data curation, Formal analysis, Investigation, Methodology, Writing - original draft. Sasano Hironobu: Conceptualization, Supervision, Writing - review & editing. Fujishima Fumiyoshi: Writing - review & editing. Kamei Takashi: Writing - review & editing. Tokodai Kazuaki: Writing - review & editing. Katabami Takuyuki: Resources, Writing - review & editing. Suzuki Takashi: Conceptualization, Supervision, Writing - review & editing. Kawanabe Shin: Resources, Writing - review & editing. Omata Kei: Writing - review & editing. Satoh Fumitoshi: Writing - review & editing. Tezuka Yuta: Writing - review & editing.
Intra-tumoral heterogeneity of steroidogenic enzymes
CD4-positive tumor infiltrating lymphocytes
CYP17A
CYP11B1
CD4
CD8-positive tumor infiltrating lymphocytes
CD8
Disorganized steroidogenesis
Cortisol
FOXP3-positive tumor infiltrating lymphocytes
CYP17A
CYP11B1
FOXP3
CD163
CD163-positive tumor- associated macrophages
CD8
CD163
FOXP3
CD4
Conflict of interest
The authors declare that they have no conflicts of interest.
Data Availability
Data will be made available on request.
Acknowledgment
We thank Prof. Gomez-Sanchez for providing the antibody of CYP11B1.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jsbmb.2024.106462.
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