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Article

Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities

by
Anne Menz
1,
Julia Zerneke
1,
Florian Viehweger
1,
Seyma Büyücek
1,
David Dum
1,
Ria Schlichter
1,
Andrea Hinsch
1,
Ahmed Abdulwahab Bawahab
2,
Christoph Fraune
1,3,
Christian Bernreuther
1,
Martina Kluth
1,
Claudia Hube-Magg
1,
Katharina Möller
1,
Florian Lutz
1,
Viktor Reiswich
1,
Andreas M. Luebke
1,
Patrick Lebok
1,3,
Sören A. Weidemann
1,
Guido Sauter
1,
Maximilian Lennartz
1,
Frank Jacobsen
1,
Till S. Clauditz
1,
Andreas H. Marx
1,4,
Ronald Simon
1,*,
Stefan Steurer
1,
Eike Burandt
1,
Natalia Gorbokon
1,
Sarah Minner
1 and
Till Krech
1,3add Show full author list remove Hide full author list
1
Institute of Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany
2
Pathology Department, Faculty of Medicine, University of Jeddah, Jeddah 23218, Saudi Arabia
3
Institute of Pathology, Clinical Center Osnabrueck, 49078 Osnabrueck, Germany
4
Department of Pathology, Academic Hospital Fuerth, 90766 Fuerth, Germany
*
Author to whom correspondence should be addressed.
Cancers 2024, 16(13), 2425; https://doi.org/10.3390/cancers16132425
Submission received: 28 May 2024 / Revised: 27 June 2024 / Accepted: 28 June 2024 / Published: 30 June 2024
(This article belongs to the Section Molecular Cancer Biology)
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Abstract

:

Simple Summary

STING is a key element in the cGAS/STING cytosolic sensing pathway and several STING agonists are currently being evaluated as anticancer drugs in the field of cancer immunotherapy. This study provides a unique catalog of STING expressions in tumor cells as well as its clinical relevance and association with PD-L1 expression in tumor and inflammatory cells in more than 130 different tumor entities.

Abstract

Stimulator of interferon genes protein (STING) activates the immune response in inflammatory cells. STING expression in cancer cells is less well characterized, but STING agonists are currently being evaluated as anticancer drugs. A tissue microarray containing 18,001 samples from 139 different tumor types was analyzed for STING by immunohistochemistry. STING-positive tumor cells were found in 130 (93.5%) of 139 tumor entities. The highest STING positivity rates occurred in squamous cell carcinomas (up to 96%); malignant mesothelioma (88.5%–95.7%); adenocarcinoma of the pancreas (94.9%), lung (90.3%), cervix (90.0%), colorectum (75.2%), and gallbladder (68.8%); and serous high-grade ovarian cancer (86.0%). High STING expression was linked to adverse phenotypes in breast cancer, clear cell renal cell carcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, and papillary carcinoma of the thyroid (p < 0.05). In pTa urothelial carcinomas, STING expression was associated with low-grade carcinoma (p = 0.0002). Across all tumors, STING expression paralleled PD-L1 positivity of tumor and inflammatory cells (p < 0.0001 each) but was unrelated to the density of CD8+ lymphocytes. STING expression is variable across tumor types and may be related to aggressive tumor phenotype and PD-L1 positivity. The lack of relationship with tumor-infiltrating CD8+ lymphocytes argues against a significant IFN production by STING positive tumor cells.

1. Introduction

The recognition of pathogens such as viruses, bacteria, and fungi occurs through the perception of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) through a set of specialized receptors. Exogenous DNA derived from pathogens or self-DNA in the cytosol represent highly efficient PAMPs/DAMPs, which can induce strong innate immune responses through the stimulation of downstream signaling cascades including the production of proinflammatory mediators and type I interferons (IFNs). Stimulator of interferon genes (STING) is a key component in this pathogen response system (summarized in [1]). STING “activation” is triggered by cytosolic nucleic acids derived from DNA viruses and bacteria as well as damaged self-DNA in the cyclic GMP-AMP (cGAS)/STING cytosolic DNA-sensing pathway (summarized in [2]). Activated STING acts as an adapter protein to enable, for example, phosphorylation of interferon regulatory factor 3 (IRF3) by serine/threonine protein kinase (TBK1), which leads to type I IFN activation (summarized in [1]). Due to the capability of STING to stimulate immune response, numerous STING agonists are currently being evaluated for their suitability as anticancer drugs in the field of cancer immunotherapy (summarized in [3,4]), and recent evidence indicates that the combination of STING agonists with STAT3 inhibitors can further enhance antitumor immunity [5].
Most studies evaluating the function of STING have focused on inflammatory cells, but STING can also be expressed in cancer cells, in which the role of STING is less well characterized (summarized in [6]). The available data suggest a high complexity of the STING pathway in cancer cells. For example, in vitro STING knockout and STING agonist experiments have revealed reduced, unchanged, or increased cell proliferation and survival in studies employing cell lines from different cancer types (summarized in [6]). Several recent studies have suggested that the STING pathway in cancer cells can be important for regulating the antitumor immune response. For example, programmed cell death 1 ligand 1 (PD-L1) expression in tumor cells could be stimulated by STING agonist in a cancer murine model [7], by elevated STING expression in DNA damage-response-deficient breast cancer cells [8] and by radiation-therapy-induced STING expression in human and mouse liver cancer cells [9]. Furthermore, activation of the cGAS/STING pathway led to natural killer cell (NK) infiltration [10,11], whereas an absence of STING in cancer cells was found to be associated with low numbers of NKs and CD8-positive lymphocytes [12,13]. While these findings could be explained by IFN production in STING positive tumor cells, other studies have questioned the STING-induced secretion of biologically relevant quantities of IFN by tumor cells (summarized in [6]).
Most available data on STING expression in cancer are RNA-based and, therefore, cannot distinguish the specific role of STING expression in cancer cells. Studies evaluating STING expression of tumor cells by immunohistochemistry (IHC) are so far limited in number and the obtained results suggest a rather complex and variable role of STING expression in cancer. For example, STING downregulation as compared to adjacent normal tissue has been reported in colorectal [14] and gastric carcinomas [15], while STING upregulation, as compared to adjacent normal tissue, was described in squamous cell carcinomas (SQCC) of the tongue [16]. Associations of high STING levels with unfavorable tumor features were found in carcinomas of the cervix uteri [17], ovary [18], stomach [19], colon [20], lung [21], larynx [22] tongue [16], and kidney [23], while low STING levels were related to unfavorable tumor features in carcinomas of the breast [24], head and neck [25], the urinary tract [26], lung [27,28], and stomach [15], as well as in T-cell lymphoma [29].
Considering the potential clinical and therapeutic relevance of STING expression in cancer, we aimed at a comprehensive characterization of tumor cell STING expression in human neoplasms. For this purpose, a pre-existing set of tissue microarrays (TMAs) containing more than 18,000 tumor samples from 139 different tumor types and subtypes as well as 76 non-neoplastic tissue categories were analyzed for STING expression by IHC.

2. Materials and Methods

2.1. Tissue Microarrays (TMAs)

Two sets of TMAs made from formalin-fixed, paraffin-embedded tissue samples were employed in our study. These included a normal TMA with a total of 608 samples that was built from 8 samples (from 8 different donors) from each of 76 different normal tissue types, as well as cancer TMAs. The cancer TMAs included a total of 18,001 primary tumors from 139 different tumor types and subtypes. The TMAs contained one 0.6 mm spot per tumor and were manufactured as described earlier [30,31]. A histopathological and molecular database was attached to the TMAs containing data of cancers of the breast (n = 1208), kidney (n = 1534), liver (n = 231), bladder (n = 1073), thyroid (n = 518), colon (n = 2351), pancreas (n = 598), stomach (n = 327), endometrium (n = 182), and ovary (n = 369). Clinical follow-up data were available from 877 patients with invasive breast carcinomas of no special type with a median follow-up time of 43 months. From an earlier study, data on PD-L1 expression were available for 11,938 cancers and on the density of CD8-positive lymphocytes for 6,897 cancers [32]. Details on the normal tissues and cancers represented in the TMAs are given in Section 3. All samples were obtained from the routine archives of the Institute of Pathology, University Hospital of Hamburg, Germany; the Institute of Pathology, Clinical Center Osnabrueck, Germany; and the Department of Pathology, Academic Hospital Fuerth, Germany. The use of archived remnants of diagnostic tissues for TMA manufacturing, their analysis for research purposes, and the use of patient data complied with local laws (HmbKHG, §12) and analysis had been approved by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Declaration of Helsinki.

2.2. Immunohistochemistry (IHC)

All experiments were carried out on the same day and with the same batch of reagents. For best immunostaining results, all TMA blocks were freshly cut one day in advance of immunostaining. Slides were deparaffinized with xylol, rehydrated through a graded alcohol series, and exposed to heat-induced antigen retrieval for 5 minutes in an autoclave at 121 °C in pH 7.8 Tris-EDTA-Citrate (TEC) buffer. Endogenous peroxidase activity was blocked with Dako REAL Peroxidase-Blocking Solution (Agilent Technologies, Santa Clara, CA, USA; #S2023) for 10 minutes. Primary antibody specific for STING (mouse monoclonal, MSVA-515M, MS Validated Antibodies, Hamburg, Germany; #5473-515M) was applied at 37 °C for 60 minutes at a dilution of 1:150. For the purpose of antibody validation, the normal tissue TMA was also analyzed by the rabbit recombinant monoclonal STING antibody clone D2P2F (Cell Signaling Technologies®, Danvers, MA, USA; #13647) at a dilution of 1:600 and an otherwise identical protocol. Bound antibody was visualized using the Dako REAL EnVision Detection System Peroxidase/DAB+ Rabbit/Mouse kit (Agilent Technologies, Santa Clara, CA, USA; #K5007), according to the manufacturer’s directions. The sections were counterstained with hemalaun. Scoring of tumor samples was performed as described before [33]. In brief, the percentage of positive neoplastic cells was estimated, and the staining intensity was semi-quantitatively recorded (0, 1+, 2+, or 3+). For statistical analyses, the staining results were categorized into four groups (see Supplementary Table S1). Tumors without any staining were considered negative. Tumors with 1+ staining intensity in ≤70% of tumor cells and 2+ intensity in ≤30% of tumor cells were considered weakly positive. Tumors with 1+ staining intensity in >70% of tumor cells, 2+ intensity in 31–70%, or 3+ intensity in ≤30% of tumor cells were considered moderately positive. Tumors with 2+ intensity in >70% or 3+ intensity in >30% of tumor cells were considered strongly positive. We used this scoring system in many earlier TMA studies and found it suitable for the identification of numerous known and novel prognostic molecular features in various tumor types [33].

2.3. Statistics

The JMP17® software package (SAS®, Cary, NC, USA) was used for statistical analyses including the chi2-test to search for associations between STING immunostaining and tumor phenotype and PD-L1 immunostaining in tumor and immune cells, analysis of variance (ANOVA) to search for associations between STING immunostaining and the density of CD8-positive lymphocytes, and the Log-rank test along with Kaplan–Meier plots for survival analysis.

3. Results

3.1. Technical Issues

In the tumor TMAs, a total of 15,345 (85.2%) of 18,001 tumor samples were interpretable in our analysis. In the normal tissue TMA, at least four samples were evaluable of each normal tissue type. Non-interpretable samples demonstrated an absence of specific cell types, absence of unequivocal tumor cells, or a complete lack of individual tissue spots.

3.2. STING in Normal Tissues

STING immunostaining of cells was always cytoplasmic. Particularly strong STING staining was observed in endothelial cells of vessels of all sizes; macrophages/dendritic cells; subsets of lymphocytes and of bone marrow cells, respiratory, fallopian tube, and endocervical epithelium; as well as in basal cells of the prostate. The few cell types that were always negative in our screening of up to eight samples per tissue type included heart and skeletal muscle cells; syncytiotrophoblast and cytotrophoblast cells; amnion and chorion cells of the placenta; epithelial cells of the parathyroid, thyroid, and adrenal glands; acinar cells of the pancreas and of the prostate; hepatocytes; principal cells of the caput epididymis; and Sertoli cells and germ cells of the testis. Representative images of these tissues are shown in Figure 1. In many other cell types, the STING staining pattern varied between samples. For example, STING staining ranged from negative to strong in the gastrointestinal epithelium, gallbladder, collecting ducts of the kidney, urothelium, epithelial cells of the cauda epididymis and the seminal vesicle, and epithelial and stromal cells of the endometrium. STING staining varied from negative to moderate intensity in the salivary glands, Brunner glands, breast epithelium, and in subsets of cells in the adeno- and neurohypophysis. In squamous epithelium, STING staining was usually limited to the basal cell layer, although STING staining was more intense in tonsil crypts. All these normal tissue findings were obtained by using the mouse monoclonal antibody MSVA-515M and the rabbit recombinant monoclonal antibody D2P2F and were, therefore, considered to be specific. Representative images of tissue staining are given in Supplementary Figure S1.

3.3. STING Expression in Cancer

In tumor samples, STING immunostaining was seen in macrophages, lymphocytic cells, endothelial cells, other stroma cells, and (often) also in tumor cells. STING positivity of tumor cells was detectable in 8908 (58.1%) of the 15,345 analyzable tumors, including 4169 (27.2%) with weak, 2005 (13.1%) with moderate, and 2734 (17.8%) with strong immunostaining. Overall, 130 of 139 tumor categories showed detectable STING staining, while 96 tumor categories included at least one case with strong positivity (Table 1). Representative images of STING-positive tumors are shown in Figure 2. Particularly high rates of STING positivity occurred in SQCCs of different sites of origin (up to 96%), malignant mesothelioma (88.5–95.7%), ductal adenocarcinoma of the pancreas (94.9%), pulmonary adenocarcinoma (90.3%), cervical adenocarcinoma (90.0%), serous high-grade ovarian cancer (86.0%), anaplastic thyroid carcinoma (82.9%) colorectal adenocarcinoma (75.2%), adenocarcinoma of the gallbladder (68.8%), and in breast carcinoma (up to 66%). The prevalence of STING positivity was intermediate for urothelial neoplasms (51.3–79.6%), adenocarcinoma of the esophagus (57.4%), and several important sarcoma categories such as liposarcoma (50.9%) and osteosarcoma (48.3%). Particularly low rates of STING positivity were observed for prostatic adenocarcinomas (up to 11%), different subtypes of renal cell carcinomas (RCC; 18.8–24.5%), neuroendocrine tumors (NETs) of various sites (17.0–28.6%), neuroendocrine carcinomas (NECs; 12.5–38.5%), and Merkel cell carcinoma of the skin (5%) and small cell neuroendocrine carcinomas of the bladder (31.6%) and the prostate (17.6%). A graphical representation of a ranking order of STING-positive and strongly positive cancers is given in Supplementary Figure S2.

3.4. STING Expression, Tumor Phenotype, and Prognosis

The relationship between STING expression in tumor cells and clinically important histopathological and molecular tumor features in carcinomas from different sites is shown in Table 2. In clear cell renal cell carcinoma (ccRCC), high STING expression was linked to a poor histologic grade (p < 0.005), high pT category (p < 0.0001), and high UICC stage (p = 0.0060). In colorectal adenocarcinoma, high STING expression was linked to right-side tumor location (p = 0.0008), microsatellite instability (p = 0.0016), and RAS mutations (p < 0.0001). High STING expression was also related to nodal metastases in hepatocellular carcinoma (HCC; p = 0.0435) and in papillary carcinoma of the thyroid (p = 0.0074). In invasive breast cancer of no special type, high STING expression was linked to estrogen receptor (ER) and progesterone receptor (PR) expression (p < 0.0001 each), non-triple-negative status (p = 0.0028), and poor overall survival (p = 0.0196; Figure 3). However, in non-invasive urothelial carcinoma of the urinary bladder, high STING expression was associated with a low grade (p = 0.0002). A combined analysis of 480 SQCC from nine different sites showed a significant link between STING positivity and HPV infection (p = 0.0212; Supplementary Table S2). This statistical relationship was also retained in a subgroup of 50 pharyngeal SQCC (p = 0.0390). STING expression was unrelated to tumor phenotype in gastric adenocarcinoma, high-grade serous and endometrioid ovarian cancer, endometrioid endometrial carcinoma, pancreatic adenocarcinoma, and papillary RCC (Supplementary Table S3).

3.5. STING Expression, PD-L1 Status, and Tumor Microenvironment

Data on PD-L1 status were available from 10,579 and on the density of CD8 positive lymphocytes from 5880 tumors for which data on STING expression on tumor cells were collected in our project. Across all tumor entities, there was a significant relationship between high STING expression in tumor cells and PD-L1 positivity of tumor cells and tumor-associated inflammatory cells (p < 0.0001 each), while STING expression was unrelated to the density of CD8 positive cells (p = 0.4253, Table 3). Examples of PD-L1 and CD8 positive and negative immunostainings are shown in Supplementary Figure S3.

4. Discussion

The main goal of our study was to provide a comprehensive overview of the prevalence of STING expression in tumor cells across a broad range of different tumor entities. Our data from 15,345 tumors from 139 tumor entities demonstrate substantial heterogeneity of tumoral STING expression between cancer types and individual patients. A total of >90% of the analyzed tumor entities had at least one STING positive case, >65% tumor entities had at least one case with strong STING positivity, >50% of all tumors were STING-positive, and 18% of all tumors were strongly STING-positive, demonstrating that STING expression is a common feature of cancer cells. It is still conspicuous that some cancer entities such as SQCCs of various sites and colorectal, pancreatic, gallbladder, pulmonary, or cervical adenocarcinomas did often show high STING expression levels while other entities such as prostatic adenocarcinomas, renal cell carcinomas, or neuroendocrine neoplasm were less frequently STING-positive. Considering that STING expression can be stimulated by the accumulation of cytosolic DAMPs, which are often found in cancer cells with a high degree of genomic instability (summarized in [34]), it could be hypothesized that cancer entities with a lesser degree of genomic instability such as prostatic adenocarcinomas [35], renal cell carcinomas [36], or neuroendocrine neoplasm [37] may be less prone to STING expression than cancers with high levels of genomic alterations such as SQCCs [38] or pulmonary [39] and pancreatic adenocarcinomas [40].
The high variability of STING expression in different samples from identical normal tissue types found in this study demonstrates that the level of STING expression fluctuates in non-neoplastic cell types. Although the reason for this variability remains elusive, it is conspicuous that many of the tissues and cell types that never showed any STING expression in our study, such as the placenta, testicular tubules, or endocrine organs, are particularly well protected from infection by viruses or bacteria as well as other causes of inflammation. The obvious inconsistency of STING expression levels in normal cell types makes it difficult to distinguish whether cancers do up- or downregulate STING, as compared to normal tissue. Most functional studies on STING in cancer have suggested that upregulation may be the prevalent mechanism (summarized in [6]). However, 42% of our tumor samples were completely STING-negative, with numerous STING-positive cells observed in the adjacent tumor stroma. This suggests that suppression of STING expression may occur in a subset of cancers.
The significant associations between high STING expression and poor overall survival in breast cancer, poor histological grade, and advanced stage in ccRCC as well as nodal metastases in HCC and papillary thyroid carcinoma suggests that STING upregulation rather than downregulation in tumor cells tends to be a feature of aggressive cancers. This observation fits well with reports of a possible tumor-promoting role of STING in cancer cells, although the molecular basis is not yet understood [41]. For example, Bakhoum et al. [42] reported that genetically instable tumors can activate STING-dependent noncanonical NF-κB signaling that facilitates metastasis. Alternatively, it is also possible that the upregulation of STING in cancer cells may represent a consequence of progressive dedifferentiation. The fact that loss of STING expression correlated with grade progression in non-invasive urothelial carcinoma shows that—depending on tumor type—the role of STING in determining the aggressiveness of tumor cells may vary. The absence of associations between STING immunostaining and parameters of cancer aggressiveness in gastric adenocarcinomas, high-grade serous and endometrioid ovarian cancer, endometrioid endometrial carcinoma, pancreatic adenocarcinoma, and papillary RCC further demonstrates that the level of STING expression in tumor cells is not likely to represent a pivotal general feature of cancer aggressiveness. Earlier studies analyzing the prognostic impact of STING expression in tumor cells have found inconsistent results. A relationship between high STING expression and unfavorable tumor phenotype or poor prognosis has been found in SQCC of the tongue [16] and—in agreement with our data—in ccRCC [23] and adenocarcinoma of the colon [20], while associations between low STING expression and poor prognosis were described in non-small cell lung carcinoma [27], gastric cancer [15], small cell lung carcinoma [28], and head and neck carcinomas [25]. Other authors could not find significant associations with patient prognosis or tumor phenotype in colorectal cancer [14], SQCC of the head and neck [43], or early gastric neoplastic lesions [44].
STING expression levels were also related to several critical molecular features of tumors. The particularly high rate of STING positivity in HPV-positive SQCC is consistent with the role of STING as a sensor for the presence of cytosolic PAMPs. In addition, data from The Cancer Genome Atlas (TCGA) also showed higher STING expression on RNA and protein levels in HPV-positive versus HPV-negative SQCC of the head and neck [45]. However, studies have shown that this correlation is maybe HPV-type specific as HPV18 E7 and HPV16 E7 lead to inhibition of the cGAS/STING pathway and reduced IFN production [46,47]. The significant link of high STING expression to MSI and RAS mutations in this study is also consistent with previous studies examining STING expression in colorectal and lung carcinomas [27,48,49], although some authors have found more RAS mutations in colorectal cancers with STING expression loss [48]. The relationship between STING positivity and ER/PR loss in breast cancer is consistent with recent studies showing elevated expression and activation of STING by DNA damage in ER/PR-negative breast cancer cell lines [50,51]. Another study found that higher STING expression in ER-positive breast cancers was associated with favorable prognosis [52].
The continuous increase in PD-L1 expression in both tumor cells and tumor-associated inflammatory cells with increasing levels of STING expression in tumor cells represents a strong confirmation of studies showing a functional relationship between STING activation and PD-L1 upregulation in cancer cells. For example, PD-L1 upregulation by STING-dependent activation of TBK1 was observed in HCC [9] and in HPV-positive cervical cancer cells [53]. Vasiyani et al. showed a STING-mediated, NF-kB-induced upregulation of PD-L1 in triple-negative breast cancer cells [50], and Grabosch et al. found PD-L1 upregulation by STING activation in an ovarian cancer mouse model [54]. Based on the assumption that STING-expressing tumor cells can produce type I IFN, a more intense population of these tumors by tumor-infiltrating lymphocytes was to be expected. The fact that we could not find a significant relationship between the level of STING expression and the density of CD8-positive, tumor-infiltrating immune cells, although data were available from more than 5500 tumors, strongly argues against functionally relevant type I IFN production in these tumors. This is consistent with data from studies exposing STING-expressing tumor cells to the STING agonist cGAMP or dsDNA and finding only low or even absent IFN-β production in mice with chronic lymphatic leukemia and colon cancer cell lines [55,56]. However, others have found that IFN-ß production is independent of cGAS-STING signaling in mismatch-repair-deficient colon cancer cells [43].
Given the high number of tumors analyzed in our study, we focused on thoroughly validating our STING IHC assay. The International Working Group for Antibody Validation (IWGAV) recommends that acceptable antibody validation for IHC on formalin-fixed tissues should involve either a comparison of results from two different independent antibodies or a comparison with expression data from another independent method [57]. Due to the high variability and cell-type specificity of STING expression, comparison with a method based on disaggregated tissue is not ideal for validating the STING antibody. Crucial evidence for the validity of our assay comes from the confirmation of all STING-positive cell types, including staining variabilities within individual tissue samples observed by MSVA-515M and by the independent second antibody D2P2F. It is noteworthy that using a broad selection of normal tissues (n = 76) for antibody validation increases the likelihood of detecting cross-reactivities, since virtually all proteins found in normal human adult cells are included in the validation process.

5. Conclusions

Our data provide an overview on the prevalence of STING expression in cancer cells across 139 different tumor types. The highest STING positivity rates occurred in squamous cell carcinomas, malignant mesothelioma, and adenocarcinomas of various origins. Comparison with the tumor phenotype demonstrates that high STING expression rather than STING deficiency tends to be linked to a more aggressive cancer phenotype. High STING expression was linked to adverse phenotypes in breast cancer, clear cell renal cell carcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, and papillary carcinoma of the thyroid. Moreover, our data demonstrate that STING expression in tumor cells is tightly linked to PD-L1 expression, but it may not exert a measurable impact on the quantity of tumor-infiltrating inflammatory cells.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers16132425/s1. Supplementary Figure S1: Immunohistochemistry validation by comparison of two antibodies. The panels show a concordance of immunostaining results obtained by two independent STING antibodies with respect to both stained cell types and intensity distribution within tissues. Using MSVA-515M, cytoplasmic staining is seen in basal cells in the prostate (A); a fraction of epithelial cells of the seminal vesicle (B); basal, suprabasal, and superficial epithelial cells of the urothelium (C); basal squamous epithelial cells of the ectocervix (D); a fraction of epithelial cells of the rectal mucosa (E); endothelial cells of the testis (F); and variable subsets of cells in a normal (G) and a focally inflamed and atrophic kidney (H). Using clone D2P2F, comparable staining was seen in the prostate (I), seminal vesicle (J), urothelium (K), ectocervix (L), rectal mucosa (M), testis (N), and the kidney (O, P). The images A–H and I–P are from consecutive tissue sections. Supplementary Figure S2: Ranking order of STING immunostaining in tumors. Both the percentage of positive cases (blue dots) and the percentage of strongly positive cases (orange dots) are shown. Supplementary Figure S3: Examples of PD-L1 and CD8 immunostaining. Strong PD-L1 staining in a muscle-invasive urinary bladder cancer (a) and in a clear cell carcinoma of the ovary (b); a PD-L1-negative, muscle-invasive urinary bladder cancer (c); high intratumoral density of CD8+ lymphocytes in a squamous cell carcinoma of the cervix (d); low intratumoral density of CD8+ lymphocytes in a serous carcinoma of the ovary (e); and a CD8-negative endometrioid carcinoma of the ovary (f). Supplementary Table S1: IHC scoring criteria. Supplementary Table S2: STING immunostaining and HPV in squamous cell carcinomas. Supplementary Table S3: STING immunostaining and tumor phenotype (non-significant).

Author Contributions

A.M., J.Z., T.K., M.K., R.S. (Ronald Simon) and G.S.: contributed to conception, design, data collection, data analysis, and manuscript writing. A.M., F.V., J.Z., S.B., D.D., R.S. (Ria Schlichter), A.H., A.A.B., C.F., C.B., K.M., F.L., V.R., A.M.L., P.L., S.A.W., M.L., F.J., T.S.C., A.H.M., S.S., E.B., N.G. and S.M.: participated in pathology data analysis, data interpretation, and collection of samples. R.S. (Ria Schlichter), M.K. and C.H.-M.: data analysis. T.K., R.S. (Ronald Simon) and G.S.: study supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The usage of archived, diagnostic, left-over tissues for manufacturing TMAs, their analysis for research purposes, and patient data analysis complied by local laws (HmbKHG, §12,1) and were approved by the local ethics committee (Ethics commission Hamburg, WF-049/09). All work has been carried out in compliance with the Declaration of Helsinki.

Informed Consent Statement

Patient consent was waived due to local laws (HmbKHG, §12,1) that permit research with anonymized, diagnostic, left-over tissue samples.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

We are grateful to Melanie Steurer, Laura Behm, Inge Brandt, and Sünje Seekamp for excellent technical assistance.

Conflicts of Interest

The mouse monoclonal antibody, clone MSVA-515M, was provided from MS Validated Antibodies GmbH, Hamburg, Germany (owned by a family member of G.S.).

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Figure 1. STING immunostaining of normal tissues. (A) Strong cytoplasmic staining of many different cell types in the lymph node, (B) respiratory epithelial cells of the bronchus, (C) a large subset of epithelial cells of the fallopian tube, (D) basal cells of the prostate, (E) endothelial cells of the testis, and (F) stroma cells in the first trimester placenta. (G) STING staining is less intense and limited to the basal cell layer in the squamous epithelium of the ectocervix. (H) Focal staining in epithelial cells of the rectum.
Figure 1. STING immunostaining of normal tissues. (A) Strong cytoplasmic staining of many different cell types in the lymph node, (B) respiratory epithelial cells of the bronchus, (C) a large subset of epithelial cells of the fallopian tube, (D) basal cells of the prostate, (E) endothelial cells of the testis, and (F) stroma cells in the first trimester placenta. (G) STING staining is less intense and limited to the basal cell layer in the squamous epithelium of the ectocervix. (H) Focal staining in epithelial cells of the rectum.
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Figure 2. STING immunostaining in cancer. In all tumor samples, cytoplasmic STING staining was seen in stroma cells, including endothelial and inflammatory cells. STING expression was more variable in tumor cells. Strong, cytoplasmic STING positivity for all tumor cells in (A) squamous cell carcinoma of the penis, (B) epithelioid malignant mesothelioma, (C) ductal adenocarcinoma of the pancreas, and (D) adenocarcinoma of the lung. STING staining was absent in tumor cells of (E) invasive urothelial carcinoma of the urinary bladder, (F) serous high-grade carcinoma of the ovary, (G) neuroendocrine tumor of the pancreas, and (H) adenocarcinoma of the prostate.
Figure 2. STING immunostaining in cancer. In all tumor samples, cytoplasmic STING staining was seen in stroma cells, including endothelial and inflammatory cells. STING expression was more variable in tumor cells. Strong, cytoplasmic STING positivity for all tumor cells in (A) squamous cell carcinoma of the penis, (B) epithelioid malignant mesothelioma, (C) ductal adenocarcinoma of the pancreas, and (D) adenocarcinoma of the lung. STING staining was absent in tumor cells of (E) invasive urothelial carcinoma of the urinary bladder, (F) serous high-grade carcinoma of the ovary, (G) neuroendocrine tumor of the pancreas, and (H) adenocarcinoma of the prostate.
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Figure 3. STING immunostaining and prognosis in invasive breast cancer of no special type.
Figure 3. STING immunostaining and prognosis in invasive breast cancer of no special type.
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Table 1. STING immunostaining in human tumors.
Table 1. STING immunostaining in human tumors.
STING Immunostaining
Tumor CategoryTumor Entityon TMA (n)Analyzable (n)Negative (%)Weak (%)Moderate (%)Strong (%)
Tumors of the skinPilomatricoma352343.521.717.417.4
Basal cell carcinoma of the skin89588.653.420.717.2
Benign nevus29254.044.044.08.0
Squamous cell carcinoma of the skin14512928.745.716.39.3
Malignant melanoma656028.318.328.325.0
Malignant mel. lymph node metastasis866927.530.421.720.3
Merkel cell carcinoma484095.02.50.02.5
Tumors of the head and neckSquamous cell carcinoma of the larynx1099424.539.419.117.0
Squamous cell carcinoma of the pharynx60523.840.423.132.7
Oral squamous cell carcinoma13011219.635.723.221.4
Pleomorphic adenoma (parotid gland)50424.89.521.464.3
Warthin tumor of the parotid gland494634.858.76.50.0
Basal cell adenoma of the salivary gland151414.357.114.314.3
Tumors of the lung, pleura, and thymusAdenocarcinoma of the lung1961659.718.220.052.1
Squamous cell carcinoma of the lung805825.927.615.531.0
Small cell carcinoma of the lung161275.00.025.00.0
Mesothelioma, epithelioid402611.538.534.615.4
Mesothelioma, biphasic77464.319.630.445.7
Thymoma29277.437.022.233.3
Lung, neuroendocrine tumor (NET)292871.410.77.110.7
Tumors of the female genital tractSquamous cell carcinoma of the vagina786312.742.919.025.4
Squamous cell carcinoma of the vulva15713523.047.416.313.3
Squamous cell carcinoma of the cervix1361239.834.122.034.1
Adenocarcinoma of the cervix232010.025.020.045.0
Endometrioid endometrial carcinoma33827822.331.719.826.3
Endometrial serous carcinoma866627.334.812.125.8
Carcinosarcoma of the uterus574822.945.818.812.5
Endometrial carcinoma, high grade, G3131154.527.39.19.1
Endometrial clear cell carcinoma9757.142.90.00.0
Endometrioid carcinoma of the ovary13011520.925.226.127.8
Serous carcinoma of the ovary58052014.029.416.540.0
Mucinous carcinoma of the ovary1018145.727.213.613.6
Clear cell carcinoma of the ovary514759.621.36.412.8
Carcinosarcoma of the ovary474511.168.96.713.3
Granulosa cell tumor of the ovary444146.339.014.60.0
Leydig cell tumor of the ovary4450.025.025.00.0
Sertoli cell tumor of the ovary110.0100.00.00.0
Sertoli–Leydig cell tumor of the ovary3366.733.30.00.0
Steroid cell tumor of the ovary3366.733.30.00.0
Brenner tumor413743.245.92.78.1
Tumors of the breastInvasive breast ca. of no special type1764156434.032.417.516.0
Lobular carcinoma of the breast36327435.833.619.311.3
Medullary carcinoma of the breast342835.732.110.721.4
Tubular carcinoma of the breast291947.442.110.50.0
Mucinous carcinoma of the breast655257.726.95.89.6
Phyllodes tumor of the breast503366.721.212.10.0
Tumors of the digestive systemAdenomatous polyp, low-grade dysplasia50372.724.324.348.6
Adenomatous polyp, high-grade dyspl.50405.015.030.050.0
Adenocarcinoma of the colon2483224524.834.415.525.3
Gastric adenocarcinoma, diffuse type21515143.035.113.28.6
Gastric adenocarcinoma, intestinal type21518839.932.410.617.0
Gastric adenocarcinoma, mixed type624930.642.916.310.2
Adenocarcinoma of the esophagus836842.636.813.27.4
Squamous cell carcinoma of the esophagus765827.634.515.522.4
Squamous cell carcinoma of the anal canal916811.836.827.923.5
Cholangiocarcinoma585860.317.26.915.5
Gallbladder adenocarcinoma514831.333.312.522.9
Carcinoma of the extrahepatic bile duct423122.619.419.438.7
Hepatocellular carcinoma31227684.47.23.64.7
Ductal adenocarcinoma of the pancreas6595945.117.026.451.5
Pancreatic/ampullary adenocarcinoma989413.823.424.538.3
Acinar cell carcinoma of the pancreas181877.816.75.60.0
Gastrointestinal stromal tumor (GIST)626232.316.122.629.0
Appendix, neuroendocrine tumor (NET)251376.915.40.07.7
Colorectal, neuroendocrine tumor (NET)12977.80.022.20.0
Ileum, neuroendocrine tumor (NET)5349100.00.00.00.0
Pancreas, neuroendocrine tumor (NET)1018883.05.76.84.5
Colorectal, neuroendocrine carcinoma (NEC)141376.915.47.70.0
Ileum, neuroendocrine carcinoma (NEC)8887.50.012.50.0
Gallbladder, neuroendocrine carcinoma (NEC)4475.025.00.00.0
Pancreas, neuroendocrine carcinoma (NEC)141361.530.80.07.7
Tumors of the urinary systemNon-invasive papillary urothelial carcinoma, pTa G2 low grade17711320.469.08.02.7
Non-invasive papillary urothelial carcinoma, pTa G2 high grade14110037.050.06.07.0
Non-invasive papillary urothelial carcinoma, pTa G321915248.740.18.62.6
Urothelial carcinoma, pT2-4 G373551742.630.412.614.5
Squamous cell carcinoma of the bladder221921.163.25.310.5
Small cell neuroendocrine carcinoma of the bladder231968.421.15.35.3
Sarcomatoid urothelial carcinoma251729.447.123.50.0
Urothelial carcinoma of the kidney pelvis625244.246.23.85.8
Clear cell renal cell carcinoma1287106575.515.73.65.3
Papillary renal cell carcinoma36832081.313.44.11.3
Clear cell tubulopapillary renal cell carcinoma262176.29.514.30.0
Chromophobe renal cell carcinoma17014979.218.12.70.0
Oncocytoma of the kidney25721959.434.25.01.4
Tumors of the male genital organsAdenocarcinoma of the prostate, Gleason 3+3837397.32.70.00.0
Adenocarcinoma of the prostate, Gleason 4+4806288.79.71.60.0
Adenocarcinoma of the prostate, Gleason 5+5857792.26.50.01.3
Adenocarcinoma of the prostate (recurrence)25822491.56.31.80.4
Small cell neuroendocrine carcinoma of the prostate191782.411.85.90.0
Seminoma68265996.23.60.20.0
Embryonal carcinoma of the testis5447100.00.00.00.0
Leydig cell tumor of the testis312751.937.011.10.0
Sertoli cell tumor of the testis22100.00.00.00.0
Sex cord stromal tumor of the testis110.0100.00.00.0
Spermatocytic tumor of the testis11100.00.00.00.0
Yolk sac tumor534395.34.70.00.0
Teratoma534443.245.59.12.3
Squamous cell carcinoma of the penis92738.243.828.819.2
Tumors of endocrine organsAdenoma of the thyroid gland11311188.39.91.80.0
Papillary thyroid carcinoma39134922.117.815.844.4
Follicular thyroid carcinoma15414278.913.41.46.3
Medullary thyroid carcinoma11110257.827.514.70.0
Parathyroid gland adenoma432996.63.40.00.0
Anaplastic thyroid carcinoma454117.126.824.431.7
Adrenal cortical adenoma4845100.00.00.00.0
Adrenal cortical carcinoma2720100.00.00.00.0
Pheochromocytoma5151100.00.00.00.0
Tumors of hematopoietic and lymphoid tissuesHodgkin’s lymphoma103951.114.742.142.1
Small lymphocytic lymphoma, B-cell type504413.684.10.02.3
Diffuse large B cell lymphoma (DLBCL)11310028.057.08.07.0
Follicular lymphoma88746.877.013.52.7
T-cell non-Hodgkin’s lymphoma25214.838.14.852.4
Mantle cell lymphoma18130.092.30.07.7
Marginal zone lymphoma161414.378.67.10.0
Diffuse large B-cell lymphoma (DLBCL) in the testis161625.068.86.30.0
Burkitt lymphoma51100.00.00.00.0
Tumors of soft tissue and boneTenosynovial giant cell tumor45313.23.212.980.6
Granular cell tumor5331100.00.00.00.0
Leiomyoma504783.014.92.10.0
Leiomyosarcoma948638.430.215.116.3
Liposarcoma14511449.121.914.014.9
Malignant peripheral nerve sheath tumor (MPNST)151323.130.823.123.1
Myofibrosarcoma262429.225.016.729.2
Angiosarcoma74557.325.520.047.3
Angiomyolipoma91755.312.022.760.0
Dermatofibrosarcoma protuberans21156.726.76.760.0
Ganglioneuroma14147.171.414.37.1
Kaposi sarcoma850.020.020.060.0
Neurofibroma11711238.449.112.50.0
Sarcoma, not otherwise specified (NOS)746431.329.712.526.6
Paraganglioma413997.42.60.00.0
Ewing sarcoma231136.49.136.418.2
Rhabdomyosarcoma7650.016.70.033.3
Schwannoma12211323.050.415.011.5
Synovial sarcoma12988.911.10.00.0
Osteosarcoma442951.727.610.310.3
Chondrosarcoma402470.812.54.212.5
Rhabdoid tumor5560.00.020.020.0
Solitary fibrous tumor171612.537.537.512.5
Table 2. STING immunostaining and tumor phenotype.
Table 2. STING immunostaining and tumor phenotype.
Tumor EntityPathological and Molecular Parameters STING Immunostaining
nNegative (%)Weak (%)Moderate (%)Strong (%)p
Invasive breast carcinoma of no special typepT154335.530.620.313.60.2402
pT241933.934.617.414.1
pT3-48432.128.615.523.8
G116130.434.821.113.70.3571
G254135.129.419.416.1
G338436.533.915.614.1
pN033733.129.020.917.00.1367
pN+43534.435.017.812.7
pM016838.725.020.815.50.4778
pM19734.034.017.514.4
HER2 negative83134.532.118.514.80.0746
HER2 positive11437.740.413.28.8
ER negative19646.432.79.211.7<0.0001
ER positive70931.034.320.014.7
PR negative38441.932.812.512.8<0.0001
PR positive56129.932.821.216.0
Non-triple negative74831.734.819.414.20.0028
Triple negative13047.730.010.811.5
Clear cell renal cell carcinomaISUP 123281.914.21.32.60.0002
ISUP 235279.012.24.34.5
ISUP 323166.219.54.310.0
ISUP 46663.621.27.67.6
Fuhrman 15685.78.93.61.80.0002
Fuhrman 260379.813.33.33.6
Fuhrman 325968.719.33.58.5
Fuhrman 48158.024.77.49.9
Thoenes 130682.413.12.02.60.0017
Thoenes 242472.616.33.87.3
Thoenes 38763.220.75.710.3
UICC 127079.314.12.64.10.0060
UICC 23167.725.80.06.5
UICC 38265.915.92.415.9
UICC 45865.513.88.612.1
pT159381.612.33.22.9<0.0001
pT211471.919.33.55.3
pT3-429765.719.54.710.1
pN014770.715.64.88.80.5488
pN+2360.913.08.717.4
pM08670.918.64.75.80.7933
pM+7969.615.26.38.9
Hepatocellular carcinomapT16783.66.04.56.00.3890
pT27474.314.92.78.1
pT3-45682.15.47.15.4
G13688.95.60.05.60.2932
G211376.19.75.38.8
G34684.88.74.32.2
pN06675.87.67.69.10.0435
pN+3548.622.911.417.1
Papillary carcinoma of the thyroidpT113519.319.323.038.50.0961
pT27331.515.112.341.1
pT3-49420.218.111.750.0
pN08223.219.522.035.40.0074
pN+1199.216.816.857.1
Urothelial bladder carcinomapTa G2 low11320.469.08.02.70.0002
pTa G2 high10037.050.06.07.0
pTa G313147.341.29.22.3
pT210341.726.215.516.50.4343
pT318937.631.713.816.9
pT49146.234.19.99.9
G21833.333.327.85.60.2402 *
G337341.330.812.315.6
pN022439.730.412.517.40.3742 *
pN+14644.531.513.011.0
Adenocarcinoma of the colonpT18218.328.023.230.50.1202
pT242724.433.718.323.7
pT3120025.734.513.626.3
pT442525.435.516.522.6
pN0112525.732.515.126.70.1591
pN+99924.236.316.023.4
V0153525.632.915.725.80.2577
V155722.637.315.324.8
L069125.631.515.227.60.1674
L1141124.535.715.724.0
Right side44521.130.618.030.30.0008
Left side119627.135.614.622.7
MMR proficient112425.035.716.123.20.0016
MMR deficient8316.922.919.341.0
RAS wildtype44533.033.315.718.0<0.0001
RAS mutation33117.231.418.732.6
BRAF wildtype12526.432.016.824.80.5262
BRAF V600E mutation1915.836.810.536.8
* only in pT2-4 urothelial bladder carcinomas. Abbreviations: pT: pathological tumor stage, G: grade, pN: pathological lymph node status, pM: pathological status of distant metastasis, V: venous invasion, L: lymphatic invasion, PR: progesterone receptor, MMR: mismatch repair, ER: estrogen receptor, ISUP: International Society of Urological Pathology, UICC: Union for International Cancer Control.
Table 3. STING immunostaining versus PD-L1 immunostaining and density of CD8-positive lymphocytes.
Table 3. STING immunostaining versus PD-L1 immunostaining and density of CD8-positive lymphocytes.
PD-L1-Positive (% of Tumors)CD8+ Density (Cells/mm2)
STING ImmunostainingnTumor CellsnImmune CellsnMean ± SE
negative41428.0413627.21893287.6 ± 11.6
weak276714.2276333.81615270.9 ± 12.5
moderate131819.5131632.7862266.1 ± 17.2
strong179923.21796371222298.4 ± 14.1
p <0.0001 <0.0001 0.4253
Abbreviations: SE: standard error.
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Menz, A.; Zerneke, J.; Viehweger, F.; Büyücek, S.; Dum, D.; Schlichter, R.; Hinsch, A.; Bawahab, A.A.; Fraune, C.; Bernreuther, C.; et al. Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities. Cancers 2024, 16, 2425. https://doi.org/10.3390/cancers16132425

AMA Style

Menz A, Zerneke J, Viehweger F, Büyücek S, Dum D, Schlichter R, Hinsch A, Bawahab AA, Fraune C, Bernreuther C, et al. Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities. Cancers. 2024; 16(13):2425. https://doi.org/10.3390/cancers16132425

Chicago/Turabian Style

Menz, Anne, Julia Zerneke, Florian Viehweger, Seyma Büyücek, David Dum, Ria Schlichter, Andrea Hinsch, Ahmed Abdulwahab Bawahab, Christoph Fraune, Christian Bernreuther, and et al. 2024. "Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities" Cancers 16, no. 13: 2425. https://doi.org/10.3390/cancers16132425

APA Style

Menz, A., Zerneke, J., Viehweger, F., Büyücek, S., Dum, D., Schlichter, R., Hinsch, A., Bawahab, A. A., Fraune, C., Bernreuther, C., Kluth, M., Hube-Magg, C., Möller, K., Lutz, F., Reiswich, V., Luebke, A. M., Lebok, P., Weidemann, S. A., Sauter, G., ... Krech, T. (2024). Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities. Cancers, 16(13), 2425. https://doi.org/10.3390/cancers16132425

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