CEACAM expression in an in-vitro prostatitis model

Background Prostatitis is an inflammatory disease of the prostate gland, which affects 2-16% of men worldwide and thought to be a cause for prostate cancer (PCa) development. Carcinoembryogenic antigen-related cell adhesion molecules (CEACAMs) are deregulated in inflammation and in PCa. The role of CEACAMs in prostate inflammation and their possible contribution to the malignant transformation of prostate epithelial cells is still elusive. In this study, we investigated the expression of CEACAMs in an in-vitro prostatitis model and their potential role in malignant transformation of prostate epithelial cells. Methods Normal prostate epithelial RWPE-1 cells were treated with pro-inflammatory cytokines to achieve an inflammatory state of the cells. The expression of CEACAMs and their related isoforms were analyzed. Additionally, the expression levels of selected CEACAMs were correlated with the expression of malignancy markers and the migratory properties of the cells. Results This study demonstrates that the pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interferon-gamma (IFNγ), induce synergistically an up-regulation of CEACAM1 expression in RWPE-1 cells, specifically favoring the CEACAM1-L isoform. Furthermore, overexpressed CEACAM1-L is associated with the deregulated expression of JAK/STAT, NFκB, and epithelial-mesenchymal transition (EMT) genes, as well as an increased cell migration. Conclusion We postulate that CEACAM1 isoform CEACAM1-4L may synergistically contribute to inflammation-induced oncogenesis in the prostate.


Introduction
Prostatitis refers to an inflammation of the prostate gland, which affects 2-16% of men worldwide, with a higher incidence observed in middle-aged individuals (1-5).Prostatitis is clinically heterogeneous and comprises acute bacterial prostatitis, chronic bacterial prostatitis, chronic pelvic pain syndrome, and asymptomatic inflammatory prostatitis, according to the US National Institutes of Health (NIH) (6).The inflammatory response within the prostate can be induced by systemic disseminated or an organ-restricted infection (7).Potential sources of prostatitis include sexually transmitted infections, physical trauma, and chemical exposure induced by urine reflux, diet, estrogens, or a combination of two or more of these factors (8)(9)(10).
About 20% of all human cancers are caused by chronic infection or chronic inflammation (11) and evidence arised that prostatitis may lead to PCa initiation and progression to a metastatic disease (8,12,13).In particular, acute prostatitis has been correlated with an increased risk of developing PCa (14).However, the underlying mechanisms are not fully understood so far.
CEACAMs belong to the immunoglobulin superfamily (15) and can be expressed in epithelial (16,17), endothelial (18), and immune cells (19,20).CEACAMs are engaged in cell-cell communication that influences a variety of signaling events, including those involved in mitogenesis, survival/apoptosis, differentiation, migration, invasion, arrangement of threedimensional tissue structure, angiogenesis, and modulation of immune responses (21,22).The most extensively characterized member of the CEACAM family is CEACAM1.It consists of an Nterminal ectodomain (N-domain), extracellular Ig-like domains (AB-domains), a conserved transmembrane domain, and either a short (CEACAM1-S) or long cytoplasmic domain (CEACAM1-L) generated due to the alternative splicing of CEACAM1 premRNA (23).CEACAM1 has been described as a tumor suppressor being down-regulated in several tumor entities including colon carcinoma, hepatocellular carcinoma, a proportion of breast cancers, bladder cancer, and PCa (24)(25)(26).Nonetheless, CEACAM1 appeared overexpressed or neo-expressed in thyroid cancer, gastric cancer, and malignant melanoma (27,28).One reason for the contradictory expression pattern and functional role of CEACAM1 could be altered CEACAM1-S/CEACAM1-L ratios (22).
In normal adult prostate tissue, CEACAM1 is abundantly expressed on the apical side of glandular prostatic epithelial cells, and its expression is downregulated in PCa (29)(30)(31).Additionally, the co-expression of CEACAM20 has been observed and found to be repressed in PCa, indicating its potential involvement in disease progression (32).Furthermore, CEACAM5 has been identified as a validated cell-surface antigen in neuroendocrine PCa, which presents intriguing prospects for its utilization as a therapeutic target (33,34).
In addition to their potential roles in tumor formation and progression, CEACAMs have been implicated in the modulation of immune response mechanisms as they are expressed on immune cells (22) and serve as pathogen receptors (35-37).Few reports have documented that the stimulation of specific epithelial cells with proinflammatory cytokines leads to the up-regulation of several CEACAMs (38, 39), indicating their potential involvement in bridging the gap between inflammation and the establishment of cancer.
To comprehensively investigate the involvement of CEACAMs in prostate inflammation and their potential contribution to the malignant transformation of prostate epithelial cells, we conducted a characterization of CEACAM expression levels in RWPE-1 cells under different culture conditions.Additionally, we correlated these expression levels with the presence of malignancy markers and the migratory properties of the cells.Our study demonstrates that the pro-inflammatory cytokines, tumor necrosis factor alpha (TNFa) and interferon-gamma (IFNg), induce an up-regulation of CEACAM1 expression in RWPE-1 cells, specifically favoring the CEACAM1-L isoform.This up-regulation is associated with the deregulated expression of JAK/STAT, NFkB, and EMT genes, as well as an increase in cell migration.

Cell culture
The human prostate epithelial cell line RWPE-1 was purchased from the American Type Culture Collection (ATCC/LGC Standards GmbH, Germany).The cells were maintained in keratinocyte serumfree medium (K-SFM) containing 50 μg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Thermo Fisher Scientific, Germany) at 37°C in a humidified 5% CO 2 atmosphere.For the experiments, serum-starved post-confluent cells were treated with 100 ng/ml TNFa (ImmunoTools, Germany), 100ng/ml IFNg (ImmunoTools, Germany) and combination for 24 h in K-SFM without supplements.Non-treated cells were used as a control.All experiments were performed with mycoplasma-free cells.

Real-time PCR
Total RNA extraction was performed with RNAmagic (Bio-Budget, Germany) in accordance with manufacturer's instructions.cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Germany).
qRT-PCR was performed using qTOWER³ (Analytik Jena, Germany), specific primers and 5× EvaGreen ® QPCR-Mix II (ROX) (Bio-Budget, Germany).The thermal cycling conditions were set as followed: 95°C for 15 min followed by 45 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec.Melting curve analysis was performed for quality control.Evaluation of relative mRNA expression was determined by DDCt method using GAPDH and ACTB as housekeeping genes.The oligonucleotides sequences are shown in Supplementary Table S2.

Tissue collection
Postoperative material from patients treated at the Sumy Regional Clinical Hospital between 2020 and 2022 (Departments of Urology; Sumy, Ukraine) was used.This study included six cases of prostatitis and six controls obtained from patients after transrectal prostate needle biopsy or transurethral resection of the prostate.Prostatitis was confirmed by at least two pathologists.The patients received written study information from their treating physician and provided written informed consent for tissue investigation.The written informed consent was kept in the patient's file (Inpatient Health Record).The Institutional Review Board of the Academic and Research Medical Institute of Sumy State University (Sumy, Ukraine) approved the study design (№ 05/ 3-2022), which adhered to ethical guidelines for experimental and clinical research.

DAB staining
Serial sections of 4 μm were prepared from paraffin-embedded tissue previously fixed in neutrally buffered formalin and mounted on 3-aminopropyltriethoxysilane-coated slides.The tissue slices were deparaffinized with xylene and rehydrated in a descending alcohol series (100%, 96% and 70%).Heat-mediated antigen retrieval was performed in 0.01 M sodium citrate buffer for 30 min at 97°C.Endogenous peroxidase activity was blocked by treating the samples with 3% H2O2 for 5 min.Background staining was inhibited by incubation with 1% BSA/PBS for one hour at RT.The tissue slices were incubated with 10 μg/ml anti-CEACAM1 (C5-1x8), anti-CEACAM5 (5C8C4), anti-CEACAM6 (1H7-4B) and anti-CEACAM20 (1-11A) mAbs in 0.5% BSA/PBS overnight at 4°C.Isotype-matched Ig mAb (10 μg/ml) was used as a negative control.A biotinylated secondary rabbit anti-mouse Ab (Dako, Germany) was applied 1:200 v/v in 0.5% BSA/PBS and incubated for one hour at RT.After washing, the tissue slices were incubated with VECTASTAIN ABC reagent (Vector Laboratories, USA) for 30 min according to the manufacturer's instructions.The staining was visualized using diaminobenzidine (DAB) substrate, and the color intensity was monitored using light microscopy.The DAB reaction was stopped with distilled H2O as soon as the desired color intensity was achieved.DAB-negative structures were identified by additional counterstaining with hematoxylin.Finally, slices were dehydrated in ascending alcohol series/xylene and mounted with Xylene Substitute Mountant (Thermo Fisher Scientific, Germany).For signal detection Nikon Eclipse Ni-E microscope (Nikon, Germany), Ri2 camera (Nikon, Germany) and NIS-Elements version 5.30.02 (Nikon, Germany) were used.

Immunofluorescence staining
The 3 μm sections of paraffin-embedded tissue were deparaffinized and rehydrated using graded alcohol and xylene.To prevent autofluorescence from endogenous fluorophores, MaxBlock ® Autofluorescence Reducing Reagent Kit (Dianova, Germany) was employed, followed by washing with 60% ethanol and distilled water.Heat-mediated antigen retrieval, background staining prevention, incubation with primary antibodies, and biotinylated secondary antibody incubation were performed as previously described.The double staining procedure was carried out sequentially, with the first staining using rabbit polyclonal anti-CEACAM1 antibody.Subsequently, Cy3-labeled Streptavidin was applied, followed by overnight incubation at 4°C with mouse monoclonal anti-CD45 antibody.Mouse and rabbit IgG control antibodies were used as negative controls, respectively.For visualization of receptor-positive green signals and general tissue structure, Alexa 488 with DAPI, diluted in 0.5% BSA/PBS (1:200), was applied.Before final mounting using Fluoromount-G ® (SouthernBiotech, USA), a MaxBlock ® Post-Detection Conditioner Kit (Dianova, Germany) was utilized.It was crucial to employ primary antibodies from different species (mouse and rabbit) to avoid cross-reactivity between them.

Gap closure migration assay
RWPE-1 cells (56.000 cells/well) were seeded in Ibidi chambers (Culture-Inserts 2 well for self-insertion, Cat.No. 80209) on a 24 well plate (Greiner Bio-One, Germany) and incubated overnight.The following day, inserts were removed to create a gap and cell patches were washed with 1x PBS, before adding 2 ml of the respective medium (without cytokines, +TNFa, +IFNg, +TNFa+IFNg) per well.Three fields of view per well in three replicate wells were monitored.Time-lapse imaging with ImageXpress ® Pico (Molecular Devices, USA) and Cell Reporter Xpress 2.9 software was set up for acquisition every hour for 24 h in total.Images were analyzed using ImageJ 1.53t software.In total three fields of view per well from three replicate well for each experiment were analyzed.

Statistics
All data are shown as mean ± SEM with n = 3. Statistical analysis was performed using GraphPad Prism (Vers.9,Statcon GmbH, Germany).One-way ANOVA followed by Bonferroni post hoc analysis was used.Expression data of qRT-PCR were analyzed using anti-logarithmic data.Values of *p< 0.05, ** p< 0.01, *** p< 0.001 and # p< 0.0001 were considered statistically significant.

TNFa and IFNg increase CEACAM1 expression in RWPE-1 cells
In order to mimic an inflammation of prostate epithelial cells, RWPE-1 cells were subjected to various treatments, including lipopolysaccharide (LPS), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), TNFa, and IFNg.The individual and combined effects of these cytokines were evaluated (Supplementary Figure 1).Among all the treatments, only TNFa and IFNg showed significant alterations in the expression of CEACAMs in RWPE-1 cells.Consequently, a treatment with TNFa and IFNg at a concentration of 100 ng/ml for a duration of 24 h was selected for further experiments due to their predisposition to induce changes in CEACAM expression.
To assess the inflammatory state of the cells following cytokine treatment, gene expression analysis of endogenous proinflammatory cytokines was performed using quantitative realtime polymerase chain reaction (qRT-PCR).Specifically, the expression levels of IL-1b, IL-6, IL-8, and IL-18 were analyzed (Figure 1A).Treatment with TNFa results in an increased expression of IL-8, while IFNg treatment leads to elevated expression of IL-1b and IL-6.Notably, the most significant effect is observed for IL-1b (p<0.05) and IL-6 (p<0.0001) when TNFa and IFNg is used in combination, indicating a synergistic effect.No change in IL-18 expression is detected after stimulation with TNFa and IFNg.Cell morphology (Figure 1B), cell size (Figure 1C), and cell granularity (Figure 1D) remains unaffected by the treatment with TNFa and IFNg.
CEACAM protein expression in RWPE-1 cells was analyzed using flow cytometry, immunocytochemistry, and western blot.The initial flow cytometry analysis using the polyspecific 6G5j mAb reveals an expression of CEACAMs in non-treated cells.Upon treatment of RWPE-1 cells with TNFa and IFNg a synergistic increase of CEACAM expression is observed (Figure 2A).In order to identify the specific CEACAMs contributing to the signal, further mAbs were used.Notably, an equal signal is detected with Sab mAb, which detects the N-domain of CEACAM1, suggesting that the signal detected by 6G5j mAb is likely attributed exclusively to CEACAM1 (Figure 2A).Interestingly, when B3-17 mAb, which recognizes the A1B1-domains of CEACAM1, was used in flow cytometry, a lower signal compared to the Sab mAb was observed (Figure 2A).Furthermore, immunocytochemistry (Figure 2B) and western blot analysis (Figure 2C) demonstrates elevated CEACAM1 The CEACAM1-3S/L and CEACAM1-4S/L isoform ratio is decreased after TNFa, IFNg and combined treatment compared to the control.CEACAM1 isoform ratio was calculated from band intensity using ImageJ software.Data are represented as mean ± SEM, n=3, one-way ANOVA followed by Bonferroni post hoc analysis, # p < 0.0001.expression in response to TNFa and IFNg treatment in an additive manner.In contrast, CEACAM5, CEACAM6, and CEACAM20 are not detected in RWPE-1 cells (data not shown).

Decreased CEACAM1-S/CEACAM1-L ratio upon inflammation stimulus
CEACAM1 has a number of membrane anchored or soluble isoforms.The membrane anchored CEACAM1 splice variants include 1-4 ectodomains with either short (S) or long (L) cytoplasmic domain.As the optimal CEACAM1-S/CEACAM1-L ratio is important for maintaining normal cellular function, the gene expression of the most abundant membrane anchored CEACAM1 splice variants CEACAM1-3S, CEACAM1-3L, CEACAM1-4S, and CEACAM1-4L was analyzed.The mRNA expression was investigated using reverse transcriptionpolymerase chain reaction (RT-PCR), while the gene products were separated and visualized using agarose gel electrophoresis (Figure 2D).TNFa and IFNg treatment induces the mRNA expression of CEACAM1-3S, CEACAM1-3L, and CEACAM1-4L.Notably, TNFa+IFNg causes even a reversal of the CEACAM1-S/ CEACAM1-L ratio specifically for the CEACAM1-4 variant (3.79 to 0.77) (Figure 2E).Soluble CEACAM1 isoforms are not detected in cell culture supernatants (data not shown).

Intracellular localization of CEACAM1 in prostatitis tissue
The expression of CEACAMs was examined in prostate tissue samples (n=12) collected from patients who underwent transrectal prostate needle biopsy or transurethral resection of the prostate.The prostatitis tissue sections (n=6) contained regions with leukocytes infiltrates as well as non-infiltrated tissue areas, which were used as an internal control.Normal prostate tissue (n=6) was used as an external control (Supplementary Figure 2).Alterations in the subcellular localization of CEACAM1 depending on the side of inflammation are notable.Specifically, a redistribution of CEACAM1 from the plasma membrane to the cytoplasm in the areas infiltrated by leukocytes (Figures 3A, B; box 1; leukocytes indicated with black arrows) is observed, whereas apical CEACAM1 expression is observed in non-infiltrated areas (Figures 3A, B; box 2).Similar findings are obtained using DAB (3,3'-Diaminobenzidine) and Immunofluorescence (IF).CEACAM20 is also detected on the apical side of prostate epithelial cells.However, while CEACAM1 exhibits a stronger signal and is continuously present in every gland, CEACAM20 displays a relatively weak signal and is not present in every gland (Supplementary Figure 3).CEACAM5 and CEACAM6 are not detectable in neither DAB nor IF staining (data not shown).

A B
Intracellular staining of CEACAM1 in inflamed human prostatic tissue.

Increased cell migration and expression of malignancy markers are associated with CEACAM1-L overexpression during inflammation
Pro-inflammatory cytokines and CEACAM1 have been implicated in the modulation of cellular functions and signaling pathways, contributing to the expression of critical mediators in cancer and inflammation.The findings of this study demonstrate that TNFa and IFNg treatment results in a deregulated expression of JAK/STAT (Figure 4B) and NFkB (Figure 4C) pathway genes, as well of genes involved in EMT (Figure 4D).Moreover, the protein expression as well as phosphorylation of the NFkB pathway member RelA are increased when treated with TNFa and IFNg in a synergistic manner (Supplementary Figure 4).Additionally, gap closure assay reveals an increased migration after IFNg treatment (gap closure: 18h vs. 24h), which is further enhanced by TNFa (gap closure: 15h vs 24) (Figure 4A).To test the hypothesis that overexpressed CEACAM1-L isoform upon an inflammation stimulus may lead to an increased cell migration and deregulation of the expression of the malignancy markers in RWPE-1 cells we transfected the cells with human CEACAM1-4L and analyzed the parameters, respectively.The transfection leads to a deregulation of STAT1, STAT2, REL B, E-CADH, and VIM in CEACAM1-4L transfected cells compared to the controls (Figure 4E).Furthermore, the cell migration is enhanced in CEACAM1-4L transfected cells compared to the controls (gap closure: 19h vs 24h).The effect is comparable to the IFNg treatment (Figure 4A).

Discussion
Several studies reported deregulated CEACAMs in PCa (29)(30)(31)(32)(33)(34) and the there is an evidence of deregulated CEACAMs in inflammation (38, 39), indicating their potential involvement in bridging the gap between inflammation and the tumorigenesis.In this study, we investigated the expression of CEACAMs in an invitro prostatitis model and the potential role of CEACAMs in malignant transformation of prostate epithelial cells.
In accordance with previous findings, we have successfully validated the expression of CEACAM1 in RWPE-1 cells and prostate tissue.Interestingly, the expression of CEACAM20 was detected in prostate tissue, but not in RWPE-1 cells.In contrast to the observations reported by Zhang et al. (32), we did not detect a concurrent co-expression of CEACAM1 and CEACAM20 in prostate tissue.While CEACAM1 and CEACAM20 are restricted to the apical membrane of glandular prostate epithelial cells, the expression of CEACAM20 is not observed in every gland as it is the Increased cell migration and malignancy marker expression after TNFa and IFNg treatment.case for CEACAM1.This discrepancy may account for the absence of CEACAM20 in RWPE-1 cells, as the cells used in this study might lack the expression of CEACAM20.Interestingly, immune infiltrated areas of prostate tissue revealed also an intracellular staining of CEACAM1 indicating enhanced CEACAM1 synthesis and storage within the cells.Intracellular CEACAM1 may possess additional functions beyond its involvement in cell-cell communication and signaling, potentially exerting effects on the cell itself through intracellular signaling.
Our findings demonstrate an up-regulation of CEACAM1 in response to a treatment with TNFa and IFNg in RWPE-1 cells associated with induced IRF-1 expression.However, we observed no up-regulation of CEACAMs upon treatment with LPS, IL-6, IL-8, and IL-17 (data not shown).TNFa and IFNg are pleiotropic Th1type cytokines that play crucial roles in modulating immune and inflammatory responses, as well as contributing to pathogenesis at aberrant expression levels (40,41).Both cytokines induce IRF-1 (40,42), which binds to Interferon-Stimulated Response Element (ISRE) in IFNg-inducible gene promotors thereby activating the expression of genes involved in immune response, cell growth, apoptosis, tumor suppression, or tumorigenesis (43,44).Previous studies have shown that CEACAM1 possesses an ISRE in its promoter region and is synergistically induced by TNFa and IFNg in endothelial cells, colorectal carcinoma cells, cervix carcinoma cells, and breast carcinoma cells (45)(46)(47)(48).In this study, we provide confirmation of these findings for the first time using non-malignant epithelial cells.
Twelve CEACAM1 splice variants with different features can be expressed in human cells, including membrane anchored or soluble isoforms.The membrane anchored CEACAM1 splice variants include 1-3 or 1-4 extracellular Ig-like domains with either short (S) or long (L) cytoplasmic domains (49).Different CEACAM1-isoforms are most frequently co-expressed by the same cell and their ratio determine the outcome of cellular signaling (50, 51).While RWPE-1 cells predominantly express the CEACAM1-S isoforms as already reported for non-malignant prostate tissue by Gaur et al. (23), the treatment with TNFa and IFNg increases the expression of splice variants CEACAM1-3S, CEACAM1-3L, CEACAM1-4S, and CEACAM1-4L with a shift to the CEACAM1-L isoform.The most prominent effect was observed for CEACAM1-4 with an even reversed CEACAM1-S/CEACAM1-L ratio.Similar results of alternative splicing towards the CEACAM1-L isoform upon IFNg treatment were reported by Dery et al. for breast cancer cells (40).It is assumed that during inflammation the induction of IRF-1 leads to the expression of CEACAM1-L isoform, which if chronically expressed may promote malignant transformation of epithelial cells.Indeed, overexpression of CEACAM1-L variant is linked to melanoma progression and metastasis (52) and tumorigenesis in breast tissue (23).Due to the high heterogeneity of PCa, the CEACAM1-S/CEACAM1-L ratio varies among different subsets of cancer cells (23).However, a transfection of PCa cell line DU145 with CEACAM1-L decreased the tumorigenic potential in a xenograft animal model, suggesting that CEACAM1 is a tumor suppressor in PCa (24).Therefore, the widely accepted tumor suppressor activity of CEACAM1-L in PCa should be reevaluated by analyzing different PCa cell lines, considering factors such as androgen sensitivity, neuroendocrine characteristics, and the given CEACAM1-S/CEACAM1-L ratio.Nevertheless, increased expression of the CEACAM1-L isoform in normal prostate epithelial cells upon inflammation stimulus as shown in this study may lead to an early onset of malignant transformation.
To investigate the potential role of CEACAM1-L in inflammation as a potential initiator of malignant transformation, transfected RWPE-1 cells were transfected with human CEACAM1-4L and the expression of malignancy markers compared to the non-transfected, cytokine-treated cells were analyzed.The overexpression of CEACAM1-L in glioblastomainitiating cells has been associated with increased tumorigenesis through the activation of the STAT3 signaling pathway (53).Additionally, it has been reported that CEACAM1 relates to the activation of the non-canonical NFkB pathway (54) and controls the EMT switch in murine mammary carcinoma (55).Thus, we analyzed the gene expression of JAK/STAT pathway genes, NFkB pathway genes, as well as genes involved in EMT.On the one hand, the treatment of RWPE-1 cells with TNFa and IFNg results in an increase of the expression of not only CEACAM1 but also of JAK2, STAT1, STAT2, REL A, REL B, NFkB1, NFkB2, and VIM.Additionally, a decrease in the expression of E-CAD is observed.The deregulated expression of malignancy markers after cytokine treatment could be verified at the protein level by examining exemplary REL A and its phosphorylated form.On the other hand, RWPE-1 cells transfected with human CEACAM1-4L exhibit an increased expression of STAT1, STAT2, REL B and a decreased expression of E-CAD and VIM, indicating that the deregulation of genes associated with malignancy can be attributed to the overexpression of CEACAM1-4L.However, one functional principle of CEACAM1 is that it barely does anything on its own but influences many other molecules in their function (22) and represent only a part of a complex mechanism contributing to carcinogenesis.As malignant transformation of the cells is associated with increased migration, the migration properties of RWPE-1 cells after cytokine treatment versus CEACAM1-4L transfected cells were analyzed in this study.Increased migration is observed after IFNg treatment, which is enhanced by TNFa.CEACAM1-4L transfected RWPE-1 cells show also increased migration compared to the untreated controls.These findings support and further emphasize the significance of CEACAM1-4L in the context of inflammation-related oncogenesis.
Furthermore, we could observe a neo-expression of the soluble CEACAM1 isoform CEACAM1-4C1, and CEACAM5 upon IFNg treatment of RWPE-1 cells.Due to homophilic and heterophilic dimerization of CEACAM1, the soluble CEACAM1 can serve as a ligand for CEACAM1, CEACAM5, CEACAM6 or CEACAM8 on epithelial cells, immune cells, and endothelia promoting different functions, e. g. angiogenesis, activation/inactivation of immune cells, proliferation and migration (56-63).CEACAM5 leads to angiogenesis, tumor formation, metastasis, modulation of immune cells, and is a wide accepted tumor marker for different tumor entities including neuroendocrine PCa (33, 64).Thus, elevated levels of soluble CEACAM1 as well as membraneanchored or soluble CEACAM5 proteins can modify immune response, angiogenesis and properties of epithelial cells favoring tumor-appropriated environment.However, no corresponding protein expression was observed in the RWPE-1 cells.Thus, soluble CEACAM1 and CEACAM5 most likely do not participate in PCa initiation and progression.

Conclusion
This study provides groundbreaking insights into the involvement of CEACAMs, specifically CEACAM1 and its isoforms, in the inflammatory response of prostate epithelial cells and their potential contribution to malignant transformation.We demonstrate that TNFa and IFNg stimulation leads to an upregulation of CEACAM1 expression, predominantly favoring the CEACAM1-L isoform.This isoform shift correlates with deregulated expression of genes associated with the JAK/STAT and NFkB signaling pathways, as well as genes involved in EMT, and enhanced cell migration.Our findings provide supporting evidence for the hypothesis that CEACAM1-4L may synergistically contribute to inflammation-induced oncogenesis in the prostate.