Variable roles of interleukin-17F in different cancers

Background Interleukin (IL)-17 family is a group of six cytokines that plays a central role in inflammatory processes and participates in cancer progression. Interleukin-17A has been shown to have mainly a protumorigenic role, but the other members of the IL-17 family, including IL-17F, have received less attention. Methods We applied systematic review guidelines to study the role of IL-17F, protein and mRNA expression, polymorphisms, and functions, in cancer. We carried out a systematic search in PubMed, Ovid Medline, Scopus, and Cochrane libraries, yielding 79 articles that met the inclusion criteria. Results The findings indicated that IL-17F has both anti- and protumorigenic roles, which depend on cancer type and the molecular form and location of IL-17F. As an example, the presence of IL-17F protein in tumor tissue and patient serum has a protective role in oral and pancreatic cancers, whereas it is protumorigenic in prostate and bladder cancers. These effects are proposed to be based on multiple mechanisms, such as inhibition of angiogenesis, vasculogenic mimicry and cancer cell proliferation, migration and invasion, and aggravating the inflammatory process. No solid evidence emerged for the correlation between IL-17F polymorphisms and cancer incidence or patients' prognosis. Conclusion IL-17F is a multifaceted cytokine. There is a clear demand for more well-designed studies of IL-17F to elucidate its molecular mechanisms in different types of cancer. The studies presented in this article examined a variety of different designs, study populations and primary/secondary outcomes, which unfortunately reduces the value of direct interstudy comparisons.Peer reviewe

Variable roles of interleukin-17F in different cancers

Background Interleukin (IL)-17 family is a group of six cytokines that plays a central role in inflammatory processes and participates in cancer progression. Interleukin-17A has been shown to have mainly a protumorigenic role, but the other members of the IL-17 family, including IL-17F, have received less attention. Methods We applied systematic review guidelines to study the role of IL-17F, protein and mRNA expression, polymorphisms, and functions, in cancer. We carried out a systematic search in PubMed, Ovid Medline, Scopus, and Cochrane libraries, yielding 79 articles that met the inclusion criteria. Results The findings indicated that IL-17F has both anti- and protumorigenic roles, which depend on cancer type and the molecular form and location of IL-17F. As an example, the presence of IL-17F protein in tumor tissue and patient serum has a protective role in oral and pancreatic cancers, whereas it is protumorigenic in prostate and bladder cancers. These effects are proposed to be based on multiple mechanisms, such as inhibition of angiogenesis, vasculogenic mimicry and cancer cell proliferation, migration and invasion, and aggravating the inflammatory process. No solid evidence emerged for the correlation between IL-17F polymorphisms and cancer incidence or patients' prognosis. Conclusion IL-17F is a multifaceted cytokine. There is a clear demand for more well-designed studies of IL-17F to elucidate its molecular mechanisms in different types of cancer. The studies presented in this article examined a variety of different designs, study populations and primary/secondary outcomes, which unfortunately reduces the value of direct interstudy comparisons.Peer reviewe

Vasculogenic Mimicry: A Promising Prognosticator in Head and Neck Squamous Cell Carcinoma and Esophageal Cancer? A Systematic Review and Meta-Analysis

Vasculogenic mimicry (VM) is an intratumoral microcirculation pattern formed by aggressive cancer cells, which mediates tumor growth. In this study, we compiled the evidence from studies evaluating whether positive VM status can serve as a prognostic factor to patients with squamous cell carcinoma of the head and neck (HNSCC) or esophagus (ESCC). Comprehensive systematic searches were conducted using Cochrane Library, Ovid Medline, PubMed, and Scopus databases. We appraised the quality of studies and the potential for bias, and performed random-effect meta-analysis to assess the prognostic impact of VM on the overall survival (OS). Seven studies with 990 patients were eligible, where VM was detected in 34.24% of patients. Positive-VM was strongly associated with poor OS (hazard ratio = 0.50; 95% confidence interval: 0.38–0.64), which remained consistent following the subgroup analysis of the studies. Furthermore, VM was associated with more metastasis to local lymph nodes and more advanced stages of HNSCC and ESCC. In conclusion, this study provides clear evidence showing that VM could serve as a promising prognosticator for patients with either HNSCC or ESCC. Further studies are warranted to assess how VM can be implemented as a reliable staging element in clinical practice and whether it could provide a new target for therapeutic intervention

Human β-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus

Human β-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2–histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP

Human β-Defensin 2 Expression in Oral Epithelium: Potential Therapeutic Targets in Oral Lichen Planus

Human β-defensin 2 (hBD-2) is a potent antimicrobial peptide that participates in defense against invading bacteria. We recently showed that bacterial components and histamine, through histamine H4 receptor (H4R), are involved in the pathogenesis of the potentially malignant lesion, oral lichen planus (OLP). However, the underlying mechanisms remain unknown. We, therefore, investigated the role of hBD2–histamine crosstalk signaling in promoting OLP pathology. Biopsies from OLP and oral tongue squamous cell carcinoma (OTSCC) patients, and healthy controls were used. Two OTSCC cell lines and normal human oral keratinocytes (HOKs) were used. HBD-2 and other targets were mapped by immunostaining and analyzed by ImageJ2 software. The highly sensitive droplet-digital PCR technology and qRT-PCR were utilized to study the clinically derived and in vitro samples, respectively. H4R was challenged with the specific agonist HST-10 and inverse agonist ST-1007. HBD-2 was highly induced in OLP lesions. In contrast, hBD2 expression was attenuated in OTSCC tissues, while very low levels of hBD-2 messenger RNA (mRNA) were observed in OTSCC cells. Together with tumor necrosis factor-α (TNF-α), histamine upregulated hBD-2 mRNA expression in HOKs. Activation of H4R seems to modulate the expression of epithelial hBD-2. These findings suggest the involvement of hBD-2 in the pathogenesis of OLP and may, thus, be harnessed for therapeutic interventions in OLP

Interleukin-17F Has Anti-Tumor Effects in Oral Tongue Cancer

We recently showed that extracellular interleukin-17F (IL-17F) correlates with better disease-specific survival in oral tongue squamous cell carcinoma (OTSCC) patients. However, the underlying mechanisms of such effect remain obscure. Here, we used qRT-PCR to assess the expression of IL-17F and its receptors (IL-17RA and IL-17RC) in two OTSCC cell lines (HSC-3 and SCC-25) and in normal human oral keratinocytes (HOKs). IL-17F effects on cancer cell proliferation, migration, and invasion were studied using a live-imaging IncuCyte system, and a Caspase-3/7 reagent was used for testing apoptosis. 3D tumor spheroids were utilized to assess the impact of IL-17F on invasion with or without cancer-associated fibroblasts (CAFs). Tube-formation assays were used to examine the effects of IL-17F on angiogenesis using human umbilical vein endothelial cells (HUVEC). OTSCC cells express low levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with HOKs. IL-17F inhibited cell proliferation and random migration of highly invasive HSC-3 cells. CAFs promoted OTSCC invasion in tumor spheroids, whereas IL-17F eliminated such effect. IL-17F suppressed HUVEC tube formation in a dose-dependent manner. Collectively, we suggest that IL-17F counteracts the pro-tumorigenic activity in OTSCC. Due to its downregulation in tumor cells and inhibitory activity in in vitro cancer models, targeting IL-17F or its regulatory pathways could lead to promising immunotherapeutic strategies against OTSCC.Peer reviewe

Evaluation Challenges in the validation of B7-H3 as oral tongue cancer prognosticator

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers

Evaluation Challenges in the Validation of B7-H3 as Oral Tongue Cancer Prognosticator

B7-H3 was the only molecule identified with prognostic potential from a recent systematic review of the prognostic value of immune checkpoints in oral cancer. We aimed to validate this finding in a multicenter international cohort. We retrospectively retrieved 323 oral tongue squamous cell carcinoma (OTSCC) samples from three different countries (Brazil, Finland, and Norway) for immunostaining and scoring for B7-H3. We evaluated tumor immunogenicity by analyzing the amount of tumor-infiltrating lymphocytes and divided the tumors into immune hot and cold. To increase the reliability of the results, both digital and manual visual scoring were used. Survival curves were constructed based on the Kaplan-Meier method, and the Cox proportional hazard model was utilized for univariate and multivariate survival analysis. B7-H3 expression was not significantly associated with overall or disease-specific survival in the whole OTSCC cohort. When divided into immune hot and cold tumors, high B7-H3 expression was significantly associated with poor disease-specific and overall survival in the immune hot group, depending on the scoring method and the country of the cohort. This was achieved only in the univariate analysis. In conclusion, B7-H3 was a negative prognosticator for OTSCC patient survival in the subgroup of immune hot tumors, and was not validated as a prognosticator in the full cohort. Our findings suggest that the immune activity of the tumor should be considered when testing immune checkpoints as biomarkers.Peer reviewe

Role of Interleukin-17F in Oral Tongue Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a group of common and aggressive malignant tumours that arise in the oral cavity, oropharynx, hypopharynx and larynx. The majority of HNSCC tumours are encountered in the oral cavity, where the tongue is the most frequently affected site intraorally (oral tongue SCC; OTSCC). The multimodality treatment includes surgery and radiotherapy with or without chemotherapy. In addition, some immunotherapeutic agents have been recently approved to treat HNSCC patients. However, patients with HNSCC in general, and those with OTSCC in particular, have a relatively unfavourable prognosis. Unfortunately, five-year overall survival rates in the Nordic countries including Finland remain between 50-60%. Therefore, there is an urgent need to identify new prognostic and therapeutic targets to improve the survival outcomes of OTSCC patients. Interleukin‐17F (IL‐17F) is the latest identified member of the IL‐17 cytokine family. IL‐17F was recently shown to induce antitumorigenic effects in several cancer types through different mechanisms. The serum level of IL-17F was lower in OSCC patients compared with leukoplakia patients and healthy individuals. Furthermore, IL‐17F inhibited tumour angiogenesis and the level of IL-17F was significantly lower in tumour tissues such as in colon and hepatocellular carcinoma (HCC) compared with the healthy control samples. Overall, these reports suggest an inverse relationship between IL‐17F levels and tumorigenesis. The aims of the present doctoral thesis were set as follows: 1) to investigate the expression levels and cellular sources of IL‐17F in OTSCC tissues and its potential association with patients' prognosis and mortality outcomes; 2) to assess the effects of IL-17F on several tumorigenic features including cell proliferation, migration, invasion and angiogenesis; 3) to test the possible effect of IL-17F on the formation of tumour-derived de novo vessel-like structures (vasculogenic mimicry; VM); 4) based on the antiangiogenic effect of IL-17F, we also aimed to systematically review the prognostic value of blood microvessel density (MVD) and lymphatic vessel density (LVD) in patients with tongue SCC (TSCC). The studies included clinical samples obtained from OTSCC patients who were treated surgically at Oulu and Tampere University Hospitals during the period from 1990‐2016. The study was approved by the relevant Ethics Committees. In addition, different OTSCC cell lines, human umbilical vein endothelial cells (HUVEC), cancer-associated fibroblasts (CAF), and human oral keratinocytes (HOKs) were used for the in vitro assays. Gene analysis assays were performed using RT-PCR and droplet-digital PCR. Immunostaining and blinded-scoring were used to map IL-17F expression in patient samples. The IncuCyte system, tumour spheroids, FACS analysis, and tube-formation assays were also used. Survival curves were constructed according to the Kaplan-Meier method. We also tested different patterns of VM structures in patient samples and in an orthotopic mouse model of OTSCC. The systematic review search was conducted in the following databases: Ovid Medline, Scopus, and Cochrane libraries. Briefly, we first showed that mast cells are the main source of IL-17F in OTSCC. Using multivariate analysis, the extracellular mast cell-derived IL-17F at the tumour invasion front was associated with better disease-specific survival in all-stages and early-stages OTSCC patients. We also showed that OTSCC cells had lower levels of IL-17F, IL-17RA, and IL-17RC mRNA compared with normal HOKs. Importantly, IL-17F inhibited tumour cell proliferation, random migration, and CAF-mediated tumour cell invasion. The tube formation by HUVEC was inhibited by IL-17F in a dose-dependent manner. Additionally, we showed that OTSCC cells contained CD31 at both mRNA and protein levels, and formed tube-like VM on Matrigel similar to those formed by HUVEC. These VM structures were also identified in a mouse orthotopic model of human OTSCC. Furthermore, IL-17F supressed the formation of VM in vitro, which mimics the effect produced by the anti-angiogenic drug Sorafenib. Finally, our systematic review results revealed that higher expression of MVD/LVD markers were associated with worse survival outcomes in TSCC patients in most of the relevant studies. Taken together, our findings support an anticancerous role of IL-17F in OTSCC. Based on levels of extracellular mast cell-derived IL-17F at the tumour invasive front, patients with OTSCC were categorized into high- and low-risk groups. The in vitro assays revealed inhibitory effects of IL-17F on tumour cell proliferation, migration, invasion and VM formation, and thus further preclinical studies are warranted. We also concluded that more clinical studies with larger patient cohorts are needed before the MVD/LVD markers can be recommended for clinical prognostication. Development of IL-17F-based drugs, or targeting one of its regulatory pathways, may have prognostic implications as well as serve as a promising therapeutic approach in patients with OTSCC .Pään ja kaulan alueen syöpä (HNSCC) pitää sisällään ryhmän yleisiä ja aggressiivisia pahanlaatuisia kasvaimia, joita esiintyy suuontelossa, nielussa ja kurkunpään alueella. Suurin osa HNSCC-kasvaimista esiintyy suuontelossa, ja erityisesti kielen alueella (liikkuvan kielen syöpä, OTSCC). Multimodaalinen hoito sisältää leikkauksen ja sädehoidon, johon voidaan yhdistää kemoterapia. Lisäksi joitakin immunoterapeuttisia aineita on äskettäin hyväksytty HNSCC -potilaiden hoitoon. HNSCC-potilailla, erityisesti kielisyöpäpotilailla, on suhteellisen huono ennuste. Valitettavasti viiden vuoden eloonjäämisaste on Pohjoismaissa, myös Suomessa, vain 50-60%. Siksi uusia ennustavia ja terapeuttisia menetelmiä tarvitaan kiireisesti kielisyöpäpotilaiden eloonjäämisennusteen parantamiseksi. Interleukiini 17F (IL-17F) on viimeisin tunnistettu IL-17 sytokiiniperheen jäsen. IL-17F:n on äskettäin osoitettu indusoivan kasvaimia estäviä vaikutuksia useissa syöpätyypeissä eri mekanismien kautta. Lisäksi seerumin IL-17F-tasot ovat olleet pienempiä kielisyöpäpotilailla verrattuna leukoplakiapotilaisiin ja terveisiin yksilöihin. Lisäksi IL-17F:n on osoitettu estävän kasvaimen angiogeneesiä ja IL-17F:n tasot ovat olleet merkittävästi pienempiä kasvainkudoksissa, muun muassa paksusuolen ja maksasolusyövässä (HCC), verrattuna terveisiin kontrollinäytteisiin. Nämä aikaisemmat tutkimukset viittaavat käänteiseen suhteeseen IL-17F-tasojen ja tuumorigeneesin välillä. Tämän väitöskirjan tavoitteena oli: 1) tutkia IL-17F:n ilmentymistasoja ja solulähteitä kielisyöpäkudoksissa ja sen mahdollista yhteyttä potilaiden ennusteeseen ja kuolleisuuteen; 2) arvioida IL-17F:n vaikutuksia useisiin tuumorigeenisiin ominaisuuksiin, kuten solujen jakautumiseen, migraatioon invasoitumiseen ja angiogeneesiin; 3) testata IL-17F:n mahdollista vaikutusta tuumorista peräisin olevien de novo suonen kaltaisten rakenteiden muodostumiseen (vaskulogeeninen matkiminen; VM); 4) IL-17F:n antiangiogeenisen vaikutuksen huomioiden, tarkastella systemaattisesti veren mikroverisuonitiheyden (MVD) ja imusuonten tiheyden (LVD) ennustearvoa kielisyöpäpotilailla. Tutkimukset sisälsivät kliinisiä näytteitä, jotka saatiin OTSCC-potilailta, joita hoidettiin kirurgisesti Oulun ja Tampereen yliopistollisissa sairaaloissa vuosina 1990–2016. Asianomaiset tutkimuseettiset toimikunnat hyväksyivät tutkimuksen. Lisäksi eri OTSCC-solulinjoja, ihmisen napanuoran endoteelisoluja (HUVEC), syöpään liittyviä fibroblasteja (CAF) ja ihmisen suun keratinosyyttejä (HOK) käytettiin in vitro -määrityksissä. Geenianalyysit suoritettiin käyttäen PCR-pohjaisia menetelmiä (RT-PCR ja digitaalinen pisara-PCR). Vasta-ainevärjäystä ja sokkopisteytystä käytettiin IL-17F:n ilmentymisen kartoittamiseen potilasnäytteistä. Lisäksi tutkimuksissa käytettiin IncuCyte-järjestelmää, tuumori palloja, FACS-analyysiä ja putken muodostus määrityksiä. Selviytymiskäyrät tuotettiin Kaplan-Meier-menetelmällä. Testasimme myös erilaisia VM -rakenteiden malleja potilasnäytteillä ja ortotooppisessa kielisyöpähiirimallissa. Systemaattinen tiedonhaku suoritettiin seuraavissa tietokannoissa: Ovid Medline-, Scopus- ja Cochrane. Lyhyesti, osoitimme ensin, että syöttösolut ovat tärkein IL-17F:n lähde OTSCC:ssä. Käyttämällä monimuuttuja-analyysiä selvitimme, että kasvaimen invaasiorintaman solunulkoinen syöttösolujen erittämä IL-17F oli yhteydessä parempaan tapauskohtaiseen eloonjäämiseen kaikissa OTSCC-potilaiden vaiheissa ja alkuvaiheessa. Osoitimme myös, että OTSCC-soluilla oli alhaisemmat IL-17F-, IL-17RA- ja IL-17RC-mRNA-tasot verrattuna normaaleihin HOK-soluihin. Huomattavaa oli, että IL-17F esti kasvainsolujen lisääntymistä, satunnaista migraatiota ja CAF-välitteistä kasvainsolujen invaasiota. IL-17F esti HUVEC-solujen putken muodostumista annoksesta riippuvaisella tavalla. Lisäksi osoitimme, että OTSCC-solut sisälsivät CD31: tä sekä mRNA- että proteiinitasolla ja muodostivat samalaisia putkimaisia VM -rakenteita matrigeessä kuin HUVEC-solut. Nämä VM -rakenteet tunnistettiin myös ortotooppisessa kielisyöpähiirimallissa. Lisäksi IL-17F vähensi VM -rakenteiden muodostusta in vitro samalla tavoin kuin antiangiogeeninen lääke sorafenib. Systemaattiset tarkastelutuloksemme paljastivat, että MVD/LVD -merkkiaineiden korkeampi ilmentyminen liittyi huonompaan eloonjäämistulokseen OTSCC -potilailla useimmissa tutkimuksissa. Tuloksemme tukevat IL-17F:n syövänvastaista roolia kielisyövässä. OTSCC-potilaat luokiteltiin korkean ja matalan riskin ryhmiin kasvaimen invasiivisen rintaman syöttösolujen erittämän solunulkoisten IL-17F:n perusteella. In vitro -määritykset osoittivat, että IL-17F vaikuttaa kasvainsolujen lisääntymiseen, migraatioon, invasoitumiseen ja VM:n muodostumiseen, ja siksi prekliinisiä lisätutkimuksia tarvitaan. Lisäksi laajempia kliinisiä tutkimuksia suuremmilla potilaskohorteilla tarvitaan, ennen kuin MVD/LVD -markkereita voidaan suositella kliiniseen ennustamiseen. IL-17F-pohjaisten lääkkeiden kehittämisellä tai IL-17F säätelyreitteihin kohdentamisella voi olla ennustavia vaikutuksia ja voivat toimia lupaavana terapeuttisina lähestymistapoina kielisyöpäpotilaille

Comparative Analysis of Vascular Mimicry in Head and Neck Squamous Cell Carcinoma: In Vitro and In Vivo Approaches

Tissue vasculature provides the main conduit for metastasis in solid tumours including head and neck squamous cell carcinoma (HNSCC). Vascular mimicry (VM) is an endothelial cell (EC)-independent neovascularization pattern, whereby tumour cells generate a perfusable vessel-like meshwork. Yet, despite its promising clinical utility, there are limited approaches to better identify VM in HNSCC and what factors may influence such a phenomenon in vitro. Therefore, we employed different staining procedures to assess their utility in identifying VM in tumour sections, wherein mosaic vessels may also be adopted to further assess the VM-competent cell phenotype. Using 13 primary and metastatic HNSCC cell lines in addition to murine- and human-derived matrices, we elucidated the impact of the extracellular matrix, tumour cell type, and density on the formation and morphology of cell-derived tubulogenesis in HNSCC. We then delineated the optimal cell numbers needed to obtain a VM meshwork in vitro, which revealed cell-specific variations and yet consistent expression of the EC marker CD31. Finally, we proposed the zebrafish larvae as a simple and cost-effective model to evaluate VM development in vivo. Taken together, our findings offer a valuable resource for designing future studies that may facilitate the therapeutic exploitation of VM in HNSCC and other tumours.Peer reviewe