ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes

Intratumoral formation of tertiary lymphoid structures (TLS) within the tumor microenvironment is considered to be a consequence of antigen challenge during anti-tumor responses. Intracellular adhesion molecule 1 (ICAM1) has been implicated in a variety of immune and inflammatory responses, in addition to associate with triple negative breast cancer (TNBC). In this study, we detected TLS in the aggressive tumor phenotypes TNBC, HER2+ and luminal B, whereas the TLS negative group contained solely tumors of the luminal A subtype. We show that ICAM1 is exclusively expressed in TNBC and HER2 enriched subtypes known to be associated with inflammation and the formation of TLS. Furthermore, cell from normal mammary epithelium and breast cancer cell lines expressed ICAM1 upon stimulation with the proinflammatory cytokines TNFα, IL1β and IFNγ. ICAM1 overexpression was induced in MCF7, MDA-MB-468 and SK-BR-3 cells regardless of hormone receptor status. Taken together, our findings show that ICAM1 is expressed in aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines. ICAM1 may be an attractive molecular target for TNBC, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer.

Inflammation within the tumor microenvironment has been found beneficial for the outcome of breast cancer patients, in particular in cancers with functionally developed tertiary lymphoid structures (TLS) 14 . In TNBC, high levels of inflammation and adjacent TLS has been shown to be a prognostic factor for overall survival 15 . However, the majority of these patients do not achieve complete pathological response and no targeted treatment exists. Recently, it has been postulated that ICAM1 can serve as a therapy target for TNBC 16 . The intercellular adhesion molecule-1 (ICAM1/CD54), a transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily, is expressed by several cell types including leucocytes, fibroblasts, and endothelial cells 17 . ICAM1 plays important roles in adhesion of cells, transendothelial migration of leucocytes to sites of inflammation, and activation of lymphocytes by interacting with lymphocyte function-associated antigen-1 (LFA-1, also known as ITGAL) 18 . Expression of ICAM1 in affected tissues is upregulated in response to a variety of inflammatory mediators and in autoimmune diseases 19,20 . Elevated levels of ICAM1 were reported in several malignancies [21][22][23][24] , where increased ICAM1 expression in breast, gastric, and colorectal cancers is correlated with more favorable prognosis [25][26][27] . Gene expression of ICAM1 differs between breast cancer subtypes, where it is found to be downregulated in luminal subtypes whilst upregulated in basal-like carcinomas 28 . The T cell rich areas of the TLS comprise high endothelial venules (HEVs) that express ICAM1, which represent a major gateway for lymphocyte migration into tumors 29 . The B cell follicle adjacent to the T cell zone, contains a network of CD21 + follicular dendritic cells (FDC) expressing ICAM1. The cell-cell adhesion of B cells to FDC is mediated by interaction between surface expression of ITGAL (LFA-1) and ICAM1, respectively 30 . It has been proposed that ICAM1 has an important function in antitumor immunity by being functionally involved in T cell priming by antigen-presenting cells, trans-endothelial trafficking of effector cells, and facilitating lymphocyte adhesion with tumor cells 31 . Conversely, ICAM1 has also been proposed to be involved in tumor cell invasion and migration into secondary sites [32][33][34][35] . Thus, the biological significance of ICAM1 expression in breast cancers remains controversial.
In this study, we have evaluated the ICAM1 expression in breast cancer. To clarify the expression profile of molecules important for lymphocyte trafficking, we investigated differentially expressed genes by SAGE-sequencing, where RNA was extracted from biopsies in paired samples taken from tumor, with and without TLS formation, and adjacent normal breast tissue. Additionally, we investigated the immunoreactivity of ICAM1 in normal breast tissue and tumor specimens, in addition to samples from a cohort of TNBC patients. Consequently, we evaluated ICAM1 expression in neoplastic cell lines, as well as in normal breast epithelial cell lines, and we surveyed proinflammatory cytokines for their potential to induce ICAM1 expression.

ICAM1 is upregulated in breast carcinomas and associated with aggressive tumor phenotypes with TLS formation.
We previously demonstrated that intratumoral TLS formation is associated with higher levels of immune cell infiltration, hormone receptor status, and histological grade 3 tumors 36 . In the present study, we evaluated 23 surgically resected tumor biopsies from primary operable breast carcinomas with pair-matched normal tissue samples based on the organization of infiltrating lymphocytes, TLS formation, and ICAM1 expression. Patient clinicopathological parameters are presented in Table 1. Tumor samples were classified into TLS positive and TLS negative groups (Table 1) based on the detection of FDC in germinal centers (GC) within the B cell follicle and HEVs found in the T cell zone (Fig. 1). The T cell rich area consisted of clusters of CD3+ T cells (Fig. 1b-d) surrounded by PNAd+ HEVs (Fig. 1e). The B cell follicle comprised CD20 + B cells (Fig. 1f) and CD21 + FDC (Fig. 1g), with GC Bcl6 + B cells (Fig. 1h). The group of tumors with TLS formation were associated with a more aggressive tumor phenotype, and included subtypes such as TNBC, HER2 enriched, and luminal B, whereas the TLS negative group contained solely tumors of luminal A subtype (Table 1). We further assessed ICAM1 gene expression levels in the TLS positive and TLS negative groups of tumors by quantitative PCR (qPCR). The qPCR results from the 23 patient samples showed a significant upregulation of ICAM1 mRNA expression in tumors compared to normal tissue specimens (Fig. 2a, p < 0.01). Analyses of the ICAM1 immunoreactivity in these tumor specimens revealed positive staining in endothelial cells, lymphocytes in stroma, and macrophages. Interestingly, we detected ICAM1 positive tumor cells exclusively in triple negative and HER2 enriched subtypes ( Table 1). The ICAM1 expression in these samples correlated to the individual mRNA expression in tumor and normal samples with the exception of one patient sample with HER2 enriched tumor (Fig. 2b). Interestingly, all tumors with ICAM1 positive tumor cells (n = 4) had high amount of immune cell infiltration and all were TLS positive ( Fig. 2b and Table 1). In addition, we analyzed ICAM1 expression in tumor metastasis (n = 6) within dissected lymph nodes (LN) of these 23 patients. No positive ICAM1 staining of the tumor cells could be detected within the LNs (data not shown). ICAM1 is overexpressed in triple negative breast cancer. As previously stated, ICAM1 overexpression has been associated with TNBC, and ICAM1 was suggested as a possible target for treatment in this patient group 16 . We therefore evaluated the ICAM1 protein expression by immunohistochemistry in a larger population of patients by including all 54 TNBCs diagnosed at UNN, Tromsø, Norway, from 2012 to 2015. Among the TNBC tumors tested, 63% of the samples showed positive ICAM1 staining in tumor cells (Table 2). Notably, the remaining triple negative tumors did not stain positively for ICAM1 in tumor cells, but all showed positive staining for ICAM1 expression in infiltrating immune cells. Among the positive cases of ICAM1 in tumor cells, the expression was predominantly detected in the invasive front of the tumor, whereas around one third showed prominent staining in the central tumor areas (Table 2). In all ICAM1 positive tumors, the protein was expressed in infiltrating immune cells in the periphery ( Table 2).
ICAM1 immunoreactivity was mainly localized in the cell membrane of the tumor cells ( Fig. 2c and d), but different staining patterns of ICAM1 were observed. Some tumors showed pronounced staining throughout the tissue (Fig. 2c), whereas others had focally distributed staining for positive and negative ICAM1 (Fig. 2d). Tumor infiltrating lymphocytes stained always positively for ICAM1, both in the center of the tumor and the periphery ScieNTiFic RepoRts | (2018) 8:11720 | DOI:10.1038/s41598-018-29604-2 ( Fig. 2e). TLS within the tumors showed strong ICAM1 staining of FDC within GC, and HEVs located in areas surrounding the GC (Fig. 2f). ITGAL, the counterreceptor-ligand of ICAM1, was expressed in immune cells surrounding the tumor cells, both in the periphery and central tumor, as well as in the tumor-associated TLS (Supplementary Figure S1).
Importantly, neither tumors of positive hormone receptor subtype nor the normal epithelium of the breast showed any positive ICAM1 staining. No positive correlation could be found between lymph node status, tumor grade, and ICAM1 positive tumors ( Table 2).

Proinflammatory cytokines are potent inducers of ICAM1 expression in normal breast epithelial cells and breast cancer cell lines.
In the tumor microenvironment, proinflammatory cytokines can induce expression of other genes that are important for homing of immune cells and promoting inflammation. Since we observed a selective expression pattern of ICAM1 in our study, we wanted to investigate whether expression of ICAM1 in breast cancer cell lines could be induced by cytokines. First, we analyzed the basal expression profile of ICAM1 in different cell lines, both of cancerous origin: MDA-MB-231 ( , and non-neoplastic cells: HMLE, Hs 578Bst, and MCF10A (Fig. 3a). Both TNBC cell lines, MDA-MB-231 and MDA-MB-468, showed the highest expression of ICAM1, with a 26-and 37-fold difference compared to MCF10A, respectively (p < 0.01) (Fig. 3a). In contrast, the expression of ICAM1 in MCF7 and SK-BR-3 cell lines was similar compared to MCF10A (Fig. 3a). Of the additional non-tumorigenic cell lines, Hs 578Bst and HMLE, only Hs 578Bst showed a significantly increased expression of ICAM1 compared to MCF10A (fc 10.6, p < 0.01). MCF7 cells were chosen for further dose dependent stimulation experiments based on the low basal expression level of ICAM1 and hormone receptor status. Both IFNγ and TNFα cytokines significantly induced ICAM1 mRNA expression, with peak levels reached after 24 hours (Fig. 3b). Subsequently, we examined all the neoplastic cell lines for ICAM1 expression after exposure to several cytokines ( Fig. 3c-f). TNFα, IFNγ, and IL1β significantly increased the production of ICAM1 in both SK-BR-3, MCF7, and MDA-MB-468 cell lines after 24 hours in comparison to unstimulated basal conditions (Fig. 3c-e). Notably for IL1β, there was a pulse induction of ICAM1 expression after 3 hours followed by a second peak at 24 hours (Fig. 3e). In contrast, no significant induction of ICAM1 expression was observed upon IFNα stimulation for MCF7 and MDA-MB-468 cells over the 48-hour period examined, whereas an 8-fold induction was observed for SK-BR-3 cells (Fig. 3f). ICAM1 mRNA expression was induced with IFNγ in HMLE cells (Fig. 3d). TNFα and IL1β did also induce the ICAM1 mRNA expression, but to a lesser extent ( Fig. 3c and e). At the protein level, the production of ICAM1 was detected in HMLE cells exposed to IFNγ after 24 and 48 hours, and a weak band was observed after IL1β stimulation at both time points (Fig. 3g). ICAM1 protein expression was also evident at 24 and 48 hours after IFNγ stimulation in SKBR3 and MDA-MB-468 cells, whereas in MCF7 cells only a weak band could be detected at 24 hour (Fig. 3g). Protein expression of ICAM1 after IL1β stimulations was detected in SK-BR-3, MCF7, and MDA-MB-468 cells (Fig. 3g). TNFα induced expression of ICAM1 in MCF7 and MDA-MB-468 cells after 24 and 48 hours, whereas SK-BR-3 cells showed only weak ICAM1 expression (Fig. 3g). Unstimulated MDA-MB-468 cells showed weak expression of ICAM1 at 0 hour that increased during the 24 and 48 hours incubation time (Fig. 3g). No increase in ICAM1 protein expression could be observed after IFNα induction in any of the cell lines. Altogether, these results show that each stimulant is a potent inducer of ICAM1, regardless of hormone receptor status and metastatic potential of the cells.

Normal and malignant breast gene expression profiles.
To investigate global gene expression patterns in tumor versus adjacent normal tissue, we performed a comparative analysis of differentially expressed genes from clinical biopsies of breast tumor and pair-matched normal tissue specimens (Table 1 and  Supplementary Tables S1 and S2). SAGE data generated in total close to 1.3 billion reads (1,290,594,733 raw SAGE-seq reads), out of which approximately 67% were mapped to the human genome (Supplementary Table S3). In order to identify the gene expression differences between normal and tumor specimens, we employed a statistically stringent analysis. This analysis revealed 1,323 differentially expressed genes between normal and tumor specimens. Among these genes, 613 genes were upregulated, and 710 genes were downregulated in tumors (see Supplementary Table S4 for complete gene list).

Comparison of differentially expressed genes in TLS positive and TLS negative breast tumors.
In order to identify the most commonly occurring gene expression changes in the two tumor groups (TLS positive or TLS negative, Table 1), the tumors were profiled independently from the patient-matched normal tissue samples. We found 1,423 differentially expressed genes when comparing TLS positive tumor samples with paired normal tissue (N) samples. In the TLS positive group, we noted 604 upregulated and 819 downregulated genes (p < 0.05; see Supplementary Table S5 for complete gene list). In the TLS negative group, 1,807 transcripts were found to be differentially expressed, 790 genes were upregulated and 1,017 genes were downregulated between TLS negative samples and paired normal samples (N) (p < 0.05; see Supplementary Table S6 for complete gene list). The relationships between significantly upregulated genes among the pairwise comparisons of Tumor versus Normal samples, TLS positive versus paired normal samples (TLS positive versus Normal) and TLS negative versus paired normal samples (TLS negative versus Normal) are presented in a Venn diagram in Fig. 4a. The comparison revealed 215 upregulated genes in common that were found to be functionally enriched in different biological processes, e.g. mitotic cell cycle (GO:0000278), extracellular matrix disassembly (GO:0022617) and negative regulation of macromolecule metabolic process (GO:0010605) among others (Supplementary  Table S9). Surprisingly, in the TLS positive versus Normal comparison, only a few genes were involved in immune   responses. We therefore compared the differentially expressed genes of the normal samples taken adjacent to tumors with and without TLS. The comparative analysis revealed 155 genes, of which 60 were upregulated and 95 were downregulated in normal samples taken from paired TLS positive tumors compared to normal samples  Table S10).
Since the differentially expressed genes in normal samples taken from TLS positive and TLS negative tumors are known to be involved in immune responses, we performed a comparative analysis of genes expressed only in TLS positive and TLS negative tumors (Table 1 and Supplementary Table S2). Our comparative analysis revealed 163 genes that were differently expressed in tumors with and without TLS formation (see Supplementary Table S8 for complete gene list). Of these, 70 genes were upregulated and 93 were downregulated among the TLS positive tumors. Selected genes related to leukocyte migration that were upregulated in TLS positive tumors included CTLA4, ITGAL, CXCL9, CXCL10, CXCL11, CCR7, XCL1, and XCL2. Downregulated genes of interest in tumors with TLS formation were PGR, PIK3R1, and HMGB1 (Supplementary Table S8 Table S11). We could not detect ICAM1 among the differentially expressed genes found in the different comparisons. We therefore used STRING to select the 20 nearest interaction partners (Fig. 5a). When comparing the genes from the Tumor versus Normal, TLS positive versus TLS negative and TLS positive versus Normal with the interaction partners of ICAM1, four genes were identified ( Fig. 5b and Supplementary Table S12). ITGAL, GBP1, GBP2 and ITGB2 were all upregulated in TLS positive tumors compared to TLS negative, but no changes could be observed in Tumor versus Normal and TLS positive versus Normal (Fig. 5b). We also identified an upregulation of ITGAL in normal samples from TLS positive tumors compared to normal samples from TLS negative tumors (Supplementary Table S7).

Discussion
Non-resolving inflammation is one of the defining features of the tumor microenvironment, in addition to involvement of proinflammatory mediators in cancer progression, such as adhesion molecules, chemokines, and cytokines. TLS are thought to act as a powerhouse of anti-tumor immunity by bringing the whole immune response machinery at the tumor site. We have previously been studying the intriguing observation that TLS forms within the tumor microenvironment in human breast cancer patients 36 . Our initial work focused on tumor infiltrating lymphocytes, how they were organized, and the association with clinicopathological features. We observed that breast carcinomas frequently contained TLS and that the presence of these structures was associated with a more aggressive tumor phenotype 36 . However, it is still unclear whether TLS are either a consequence of an immune response per se or sites of an active immune reaction against the components within the tumor microenvironment. Here we present findings that TLS positive tumors included the more aggressive subtypes, such as TNBC, HER2 enriched and luminal B, while tumors in TLS negative groups were all of luminal A subtype. In addition, we identified genes involved in important migratory and signaling events related to tumor-associated TLS formation to be expressed in TLS positive tumors. ICAM1 is an important molecule for lymphoid trafficking and has been shown to be upregulated in several types of cancers, including breast cancer 24 . Our verification studies showed that ICAM1 mRNA was expressed at higher level in tumor compared to the adjacent normal breast tissue. Furthermore, immunohistochemical analyses revealed ICAM1 immunoreaction in tumor cells of more aggressive subtypes, including HER-2 enriched and triple negative tumors. Supporting this, Guo and colleagues detected ICAM1 expression solely in triple negative tumors and therefore proposed ICAM1 as a molecular target for TNBC 16 . TNBC is characterized by high influx of tumor-associated lymphocytes, an aggressive phenotype, and high metastatic potential of tumor cells. Tumor-localized TLS are associated with a more aggressive tumor phenotype and are frequently found in TNBC. Challenging this, in the cohort of triple negative patients approximately 60% of the samples had ICAM1 positive tumor cells. It is important to mention that the percentage of tumor cells that expressed ICAM1 ranged from less than 10 to 100% with intertumoral differences in staining patterns. Questioning the proposed TNBC-specific expression profile of ICAM1, we investigated whether a proinflammatory stimuli could mimic the milieu found in the tumor microenvironment and result in overexpression of ICAM1 also in hormone sensitive subtypes. We showed that ICAM1 expression could be induced by proinflammatory cytokines, both in hormone receptor positive cells and normal breast epithelial cells, in addition to the basal-like and HER2 positive cell lines.
A series of lines of evidences suggests that ICAM1 can potentially be involved in invasion of tumor cells and metastasis of human breast cancer 24 . Supporting our findings, Rosette and colleagues observed that the level of ICAM1 expression was positively correlated with the metastatic potential of cell lines, and they reported a trend towards elevated expression in tumor specimens compared to normal control tissues 37 . Furthermore, ICAM1 might be involved in cancer metastasis, as shown by silencing of ICAM1 in MCF7 breast cancer cells. This can attenuate the metastatic ability in vitro and result in decreased migration and invasion accompanied by a lower MMP14 expression 38 . It has also been demonstrated by exploiting ICAM1 overexpression in breast cancer cells, that ICAM1 may serve as an effective nanomedicine target by delivering siRNA to TNBC cells and inhibiting cancer progression 39 .
The migration of lymphocytes from blood vessels and TLS associated HEVs in into tissue is well characterized at a molecular level, in contrast to the emigration of tumor cells 40 . However, ICAM1 is hypothesized to facilitate the attachment of carcinoma cells to the lymphatic endothelium and therefore promote the micro-metastatic movement in regional lymph nodes. Furthermore, it has been shown that tumor cells utilize leukocytes as linker-cells to endothelium for their extravasation. Strell and colleagues demonstrated that tumor cells expressing ICAM1 interact with neutrophil granulocytes that facilitates the contact to the endothelium by β2-integrins 41 . The same interaction has been shown for IL-8 producing melanoma cells 42,43 . We demonstrated that ICAM1 expression was inducible in tumor cells upon pro-inflammatory stimuli which might be important for the extravasation process from the blood vessels. We speculate that ICAM1 expression can be upregulated by cytokines in the tumor microenvironment prior to intravasation into the circulation. The blood vessels that arise in the tumor are often immature and vulnerable that can result in leakage through the vessel wall and this allows the tumor cells to leave the tissue more easily. On the other hand, expression of ICAM1 by the tumor cells might lead to specific T cell recognition and enhancement of effector CTL adhesion that might not be in favor for the tumor cells. ICAM1/LFA-1 interactions have been proposed to play critical roles in enhancing antitumor immune responses 31,44 . Moreover, induced overexpression of ICAM1 in tumor cells have been shown to result in reduced tumor growth correlated with increased lysis by tumor infiltrating lymphocytes 45,46 . It is intriguing to speculate that it might be the cytokine production within the tumor microenvironment that causes the expression of ICAM1 in the tumor cells, since TNFα and IFNγ are known to induce synthesis of ICAM1 47 . However, the absence of ICAM1 expression in hormone receptor positive tumors remains to be elucidated and warrant more attention to this interesting phenomenon. Taken together, our findings show that ICAM1 is upregulated in more aggressive subtypes of breast cancer and its expression is inducible by well-known proinflammatory cytokines, regardless of hormone receptor status of the tumor cells. ICAM1 may still be an attractive molecular target for TNBCs, but further investigations elucidating the role of ICAM1 in targeted therapies have to take into consideration selective subtypes of breast cancer.

Methods
Patients. The study was approved by the Regional Committees for Medical and Health Research Ethics (REC; Norway, 2010/1523). Tumor and normal tissue specimen for SAGE-sequencing were collected from 23 patients that were operated at the University hospital of North Norway (UNN) in Tromsø in 2012. A written informed consent was obtained from all subjects and all methods were carried out in accordance with relevant guidelines and regulations. The main clinical and pathological parameters of the included patients are presented in Table 1. Full-faced hematoxylin and eosin (HE) stained sections were used to assess the degree of infiltrating immune cells in the breast tumors as described previously 36 . Histological tumor grade was assessed by the Nottingham Grading System 48 . The cut off values for Estrogen (ER) and Progesterone (PR) were 10%. Tumors demonstrating HER2 protein overexpression or amplified HER2 gene (IHC 3+ or FISH HER2 gene ratio ≥2) were considered to be positive. None of the included patients received adjuvant therapy before surgery, nor did they have any other known malignancies.
Immunohistochemistry. 54 triple negative breast carcinomas (TNBC), 30 tumors with different hormone receptor status, and 30 normal breast tissue samples were scored for ICAM1 staining. We used archived (2012-2015) formalin-fixed paraffin embedded (FFPE) specimen obtained from the Department of Clinical Pathology (UNN, Tromsø). ICAM1 expression was defined as follows: negative, <10% of tumor cells showing membranous staining; positive >10% of tumor cells showing membranous staining, both in the center of tumor and in the periphery. In addition, tumor and normal breast tissue sections from biopsies (n = 23) used for SAGE-sequencing and dissected lymph nodes (n = 6) from the same patients were evaluated for ICAM1 expression. Immunohistochemistry was performed on FFPE sections using Envision+ System-HRP(DAB+) detection kit (K4011, Dako), Polink-2 Plus HRP Goat with DAB kit (D43-18, GBI Labs), or platform-specific assays on BenchMark XT (Ventana Medical systems Inc., USA) as described previously 36