Head and neck tumor cells treated with hypofractionated irradiation die via apoptosis and are better taken up by M1-like macrophages

Abstract Purpose The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2 × 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. Methods Human monocytes were differentiated with the hematopoietic growth factors M‑CSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-γ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2 × 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. Results The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. Conclusion HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.

Download Full-text

Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22179114 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9114

Author(s):

Sebastian Wimmer ◽

Lisa Deloch ◽

Michael Hader ◽

Anja Derer ◽

Fridolin Grottker ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Head And Neck ◽

Tumor Cells ◽

Squamous Cell ◽

Immune Checkpoint ◽

Patient Specific ◽

Immune Checkpoint Molecules ◽

Hpv Status ◽

The Impact

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM.

Download Full-text

CAFs affect the proliferation and treatment response of head and neck cancer spheroids during co-culturing in a unique in vitro model

Cancer Cell International ◽

10.1186/s12935-020-01718-6 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Mustafa Magan ◽

Emilia Wiechec ◽

Karin Roberg

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Head And Neck ◽

Treatment Response ◽

Tumor Cells ◽

In Vitro Model ◽

Tumor Spheroids ◽

Vitro Model ◽

The Impact

Abstract Background Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors for which the overall survival rate worldwide is around 60%. The tumor microenvironment, including cancer-associated fibroblasts (CAFs), is believed to affect the treatment response and migration of HNSCC. The aim of this study was to create a biologically relevant HNSCC in vitro model consisting of both tumor cells and CAFs cultured in 3D to establish predictive biomarkers for treatment response, as well as to investigate the impact of CAFs on phenotype, proliferation and treatment response in HNSCC cells. Methods Three different HNSCC patient-derived tumor cell lines were cultured with and without CAFs in a 3D model. Immunohistochemistry of the proliferation marker Ki67, epidermal growth factor receptor (EGFR) and fibronectin and a TUNEL-assay were performed to analyze the effect of CAFs on both tumor cell proliferation and response to cisplatin and cetuximab treatment in tumor spheroids (3D). mRNA expression of epithelial-mesenchymal transition (EMT) and cancer stem cells markers were analyzed using qRT-PCR. Results The results demonstrated increased cell proliferation within the tumor spheroids in the presence of CAFs, correlating with increased expression of EGFR. In spheroids with increased expression of EGFR, a potentiated response to cetuximab treatment was observed. Surprisingly, an increase in Ki67 expressing tumor cells were observed in spheroids treated with cisplatin for 3 days, correlating with increased expression of EGFR. Furthermore, tumor cells co-cultured with CAFs presented an increased EMT phenotype compared to tumor cells cultured alone in 3D. Conclusion Taken together, our results reveal increased cell proliferation and elevated expression of EGFR in HNSCC tumor spheroids in the presence of CAFs. These results, together with the altered EMT phenotype, may influence the response to cetuximab or cisplatin treatment.

Download Full-text

Survival-Associated Metabolic Genes in Human Papillomavirus-Positive Head and Neck Cancers

Cancers ◽

10.3390/cancers12010253 ◽

2020 ◽

Vol 12 (1) ◽

pp. 253 ◽

Cited By ~ 6

Author(s):

Martin A. Prusinkiewicz ◽

Steven F. Gameiro ◽

Farhad Ghasemi ◽

Mackenzie J. Dodge ◽

Peter Y. F. Zeng ◽

...

Keyword(s):

Human Papillomavirus ◽

Head And Neck ◽

Patient Survival ◽

Tricarboxylic Acid Cycle ◽

Predictive Biomarkers ◽

Head And Neck Cancers ◽

The Cancer Genome Atlas ◽

Metabolic Genes ◽

Hpv Status ◽

The Impact

Human papillomavirus (HPV) causes an increasing number of head and neck squamous cell carcinomas (HNSCCs). Altered metabolism contributes to patient prognosis, but the impact of HPV status on HNSCC metabolism remains relatively uncharacterized. We hypothesize that metabolism-related gene expression differences unique to HPV-positive HNSCC influences patient survival. The Cancer Genome Atlas RNA-seq data from primary HNSCC patient samples were categorized as 73 HPV-positive, 442 HPV-negative, and 43 normal-adjacent control tissues. We analyzed 229 metabolic genes and identified numerous differentially expressed genes between HPV-positive and negative HNSCC patients. HPV-positive carcinomas exhibited lower expression levels of genes involved in glycolysis and higher levels of genes involved in the tricarboxylic acid cycle, oxidative phosphorylation, and β-oxidation than the HPV-negative carcinomas. Importantly, reduced expression of the metabolism-related genes SDHC, COX7A1, COX16, COX17, ELOVL6, GOT2, and SLC16A2 were correlated with improved patient survival only in the HPV-positive group. This work suggests that specific transcriptional alterations in metabolic genes may serve as predictive biomarkers of patient outcome and identifies potential targets for novel therapeutic intervention in HPV-positive head and neck cancers.

Download Full-text

Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization

Cells ◽

10.3390/cells9051303 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1303 ◽

Cited By ~ 2

Author(s):

Alexandra Pritchard ◽

Sultan Tousif ◽

Yong Wang ◽

Kenneth Hough ◽

Saad Khan ◽

...

Keyword(s):

Bone Marrow ◽

Tumor Cells ◽

Bone Marrow Cells ◽

Lung Tumor ◽

Macrophage Polarization ◽

M2 Macrophage ◽

M2 Macrophages ◽

M2 Macrophage Polarization ◽

The Impact ◽

M2 Phenotype

Cellular cross-talk within the tumor microenvironment (TME) by exosomes is known to promote tumor progression. Tumor promoting macrophages with an M2 phenotype are suppressors of anti-tumor immunity. However, the impact of tumor-derived exosomes in modulating macrophage polarization in the lung TME is largely unknown. Herein, we investigated if lung tumor-derived exosomes alter transcriptional and bioenergetic signatures of M0 macrophages and polarize them to an M2 phenotype. The concentration of exosomes produced by p53 null H358 lung tumor cells was significantly reduced compared to A549 (p53 wild-type) lung tumor cells, consistent with p53-mediated regulation of exosome production. In co-culture studies, M0 macrophages internalized tumor-derived exosomes, and differentiated into M2 phenotype. Importantly, we demonstrate that tumor-derived exosomes enhance the oxygen consumption rate of macrophages, altering their bioenergetic state consistent with that of M2 macrophages. In vitro co-cultures of M0 macrophages with H358 exosomes demonstrated that exosome-induced M2 polarization may be p53 independent. Murine bone marrow cells and bone marrow-derived myeloid-derived suppressor cells (MDSCs) co-cultured with lewis lung carcinoma (LLC)-derived exosomes differentiated to M2 macrophages. Collectively, these studies provide evidence for a novel role for lung tumor-exosomes in M2 macrophage polarization, which then offers new therapeutic targets for immunotherapy of lung cancer.

Download Full-text

Patient reported outcomes (PROs) in patients with human papilloma virus (HPV) positive versus negative head and neck cancer (HNC).

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.26_suppl.87 ◽

2016 ◽

Vol 34 (26_suppl) ◽

pp. 87-87

Author(s):

Jessica Ruth Bauman ◽

Areej El-Jawahri ◽

Karen Quinn ◽

Lisa Arcikowski ◽

Gina Chan ◽

...

Keyword(s):

Depressive Symptoms ◽

Head And Neck ◽

Regression Models ◽

Illness Perception ◽

Depression Scale ◽

Symptom Burden ◽

Hpv Status ◽

Patient Reported ◽

Prospective Longitudinal ◽

The Impact

87 Background: HNC caused by HPV has become an epidemic. Treatment for HNC results in a tremendous symptom burden. The impact of HPV-status on quality of life (QOL) and how this effects illness perception and mood has not been described. We sought to explore differences in PROs in patients with HPV + vs. - HNC. Methods: This is a secondary analysis of data from a prospective, longitudinal intervention study of 60 patients with newly diagnosed HNC undergoing concurrent chemoradiation. 30 control patients received standard care followed by 30 intervention patients who received an educational intervention. Satisfaction (information satisfaction questionnaire (ISQ)), mood (Hospital Anxiety and Depression Scale (HADS)), illness perception (Brief Illness Perception Questionnaire (IPQ)), and QOL (MD Anderson Symptom Inventory- Head and Neck (MDASI-HN)) were evaluated at baseline and 3 weeks. Data were analyzed using linear regression models controlling for the effect of the intervention to assess the association between HPV status and changes in PROs. Results: From 8/2014 to 10/2015, we enrolled 60 patients (2 excluded for hospitalization or care elsewhere). 29/58 (50%) had HPV + HNC. 40 (69%) were men. 54 (93%) had stage III/IV disease. Compared to patients with HPV - HNC, patients with HPV + HNC reported an increase in symptom burden, symptom interference, and depressive symptoms, as well as a more threatening illness perception from baseline to 3 weeks. There were no differences in anxiety or satisfaction. Table 1 details the regression models. Conclusions: Patients with HPV + HNC have a larger decrement in QOL during treatment than patients with HPV - HNC, which corresponds to a more threatening illness perception and more depressive symptoms. Interventions tailored to symptom management and mood should be developed for this unique population. [Table: see text]

Download Full-text

The impact of HPV infection on survival of patients with non-oropharyngeal head and neck cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.6048 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. 6048-6048 ◽

Cited By ~ 1

Author(s):

Samer Alsidawi ◽

Gustavo Figueiredo Marcondes Westin ◽

Ashish V. Chintakuntlawar ◽

Scott H. Okuno ◽

Katharine Andress Rowe Price

Keyword(s):

Multivariate Analysis ◽

Head And Neck Cancer ◽

Head And Neck ◽

Neck Cancer ◽

Tumor Grade ◽

Hpv Infection ◽

Smoking History ◽

Hpv Status ◽

Number Of Patients ◽

The Impact

6048 Background: The role of Human Papilloma Virus (HPV) infection in non-oropharyngeal squamous cell carcinoma (non-OPSCC) of the head and neck is unknown. Current available studies have yielded conflicting results due to limited number of patients. We present a large analysis from the National Cancer Database (NCDB) evaluating HPV-positive non-OPSCC. Methods: Using the NCDB registry, we included adults diagnosed with non-OPSCC from 2004-2012 with available HPV status. A cohort of patients with OPSCC was analyzed for HPV prevalence comparison. Survival analysis was performed using Kaplan-Meier method and stratified using HPV-status. The prognostic effect of variables was studied using Cox proportional hazards models. The JMP software was used for statistical analysis. Results: A total of 8726 non-OPSCC patients were identified and primary sites included the oral cavity (50%), larynx (41%) and hypopharynx (9%). 11% of non-OPSCC patients had evidence of infection with high-risk HPV strains compared to 61% of OPSCC patients. HPV-positive non-OPSCC patients presented at slightly younger age, had more advanced stage and higher tumor grade compared to HPV-negative patients (P < 0.01). HPV-positive non-OPSCC patients had better survival than HPV-negative patients (HR 0.82, 95% CI 0.72-0.93, P < 0.01) and this was most pronounced in patients with locally advanced disease (5-year survival 50% versus 40%, HR 0.69, 95% CI 0.6-0.8, P < 0.01). A univariate and multivariate analysis were performed adjusting for age, sex, race, stage, primary site, Charlson/Deyo comorbidity score, financial income, tumor grade, surgery, radiation and chemotherapy administration. Smoking history was unavailable. HPV positivity was an independent predictor of better survival in non-OPSCC in multivariate analysis (HR 0.69, 95% CI 0.59-0.8, P < 0.01). Conclusions: HPV infection is seen in a subset of patients with non-OPSCC head and neck cancer and these present with more advanced tumors. The survival of patients with HPV-positive non-OPSCC is significantly better than HPV-negative tumors. Routine HPV testing and enrollment in treatment de-intensification clinical trials similar to OPSCC might be appropriate for this patient population.

Download Full-text

High Level Expression of MHC-II in HPV+ Head and Neck Cancers Suggests that Tumor Epithelial Cells Serve an Important Role as Accessory Antigen Presenting Cells

Cancers ◽

10.3390/cancers11081129 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1129 ◽

Cited By ~ 7

Author(s):

Steven F. Gameiro ◽

Farhad Ghasemi ◽

John W. Barrett ◽

Anthony C. Nichols ◽

Joe S. Mymryk

Keyword(s):

T Cell ◽

Epithelial Cells ◽

Antigen Presentation ◽

Head And Neck ◽

Antigen Presenting Cells ◽

Human Papillomaviruses ◽

Mhc Ii ◽

Hpv Status ◽

Antigen Presenting ◽

The Impact

High-risk human papillomaviruses (HPVs) are responsible for a subset of head and neck squamous cell carcinomas (HNSCC). Expression of class II major histocompatibility complex (MHC-II) is associated with antigen presenting cells (APCs). During inflammation, epithelial cells can be induced to express MHC-II and function as accessory APCs. Utilizing RNA-seq data from over 500 HNSCC patients from The Cancer Genome Atlas, we determined the impact of HPV-status on the expression of MHC-II genes and related genes involved in their regulation, antigen presentation, and T-cell co-stimulation. Expression of virtually all MHC-II genes was significantly upregulated in HPV+ carcinomas compared to HPV− or normal control tissue. Similarly, genes that encode products involved in antigen presentation were also significantly upregulated in the HPV+ cohort. In addition, the expression of CIITA and RFX5—regulators of MHC-II—were significantly upregulated in HPV+ tumors. This coordinated upregulation of MHC-II genes was correlated with higher intratumoral levels of interferon-gamma in HPV+ carcinomas. Furthermore, genes that encode various co-stimulatory molecules involved in T-cell activation and survival were also significantly upregulated in HPV+ tumors. Collectively, these results suggest a previously unappreciated role for epithelial cells in antigen presentation that functionally contributes to the highly immunogenic tumor microenvironment observed in HPV+ HNSCC.

Download Full-text

837 Preliminary insights into the impact of tumor microbiome in head and neck squamous cell carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.837 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A878-A878

Author(s):

Vidhya Karivedu ◽

Rebecca Hoyd ◽

Caroline Wheeler ◽

Sachin Jhawar ◽

Priyanka Bhateja ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Head And Neck ◽

Clinical Outcomes ◽

Squamous Cell ◽

Neck Squamous Cell Carcinoma ◽

Smoking History ◽

Predictive Tool ◽

Hpv Status ◽

The Impact

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a heterogeneous set of distinct malignancies. Recognized prognostic factors rely on clinical and biological features, consisting mainly of stage, site of disease, performance status, comorbidities, smoking history and human papilloma virus (HPV) status. However, patients clustered by these parameters still differ in their clinical behavior and therapy response. The impact of the tumor microbiome on human disease has been explored and discussed extensively. Evaluating the tumor microbiome is a promising new approach that could be used as a prognostic and predictive tool in HNSCC, with the potential for improved treatment options and better clinical outcomes.MethodsWe utilized The Cancer Genome Atlas (TCGA) database to obtain RNA sequencing (RNAseq) data to identify microbes in HNSCC samples. We utilized ExoTIC, ”Exogenous sequences in Tumors and Immune cells,” a tool recently developed by Spakowicz et al. ExoTIC takes raw RNAseq reads and carefully aligns them to both human and non-human reference genomes to identify low-abundance microbes. We performed Cox proportional hazards regression to identify the microbes associated with overall survival (OS), controlling for age, stage, and smoking status.ResultsWe evaluated 498 RNAseq samples from TCGA (table 1). ExoTIC identified 5838 microbes including bacteria, viruses and fungi, of which 330 were statistically associated with OS. Interestingly, 20% (n=100) of samples had HPV virus which was significantly associated with improved OS (HR 0.59, CI 0.4–0.9, p<0.01). There were also several other viruses and bacteria associated with significantly improved OS.Abstract 837 Table 1Patient characteristics of TCGA datasetConclusionsWe found the presence of certain microbes in tumor biopsies statistically correlated with OS in HNSCC patients. This supports further study into the presence and correlation of specific microbes with tumor subsite and outcomes. Assessing individual characteristics of a HNSCC subtype with its particular microenvironment (e.g., microbes) can lead to personalized treatment insights and improved outcomes. Our future research will validate and correlate the microbial profile of HNSCC subtypes with clinical outcomes retrospectively and prospectively.

Download Full-text

Personalizing Postoperative Treatment of Head and Neck Cancers

American Society of Clinical Oncology Educational Book ◽

10.1200/edbk_201087 ◽

2018 ◽

pp. 515-522 ◽

Cited By ~ 3

Author(s):

Ellie Maghami ◽

Shlomo A. Koyfman ◽

Jared Weiss

Keyword(s):

Adjuvant Therapy ◽

Head And Neck ◽

Locally Advanced ◽

Tumor Biology ◽

Postoperative Treatment ◽

Trimodality Therapy ◽

Pathologic Features ◽

Quality Life ◽

Hpv Status ◽

The Impact

Head and neck cancer (HNC) treatment is a complex multidisciplinary undertaking. Although overtreatment can result in functional and cosmetic defects, undertreatment can result in cancer recurrence. Surgery and chemoradiotherapy are both accepted standards for the curative intent treatment of locally advanced mucosal squamous cell carcinoma of the head and neck, but are often prioritized differently depending on the site of tumor origin (e.g., oral cavity/sinonasal vs. oropharynx/larynx), tumor burden, tumor biology, quality-life considerations, and patient preference. Regardless of modalities chosen, failure to cure remains a considerable problem in locally advanced disease. For patients treated with primary surgery, high-risk pathologic features portend higher recurrence rates, and adjuvant therapy can reduce these rates and improve outcomes. This report details which tumor- and nodal-related factors are indications for adjuvant therapy, examines the impact of tumor HPV status on adjuvant treatment paradigms, and considers which systemic therapies should be used for which patients when trimodality therapy is indicated.

Download Full-text

Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns

Cancers ◽

10.3390/cancers13205221 ◽

2021 ◽

Vol 13 (20) ◽

pp. 5221

Author(s):

Senem Uzun ◽

Yüksel Korkmaz ◽

Nora Wuerdemann ◽

Christoph Arolt ◽

Behrus Puladi ◽

...

Keyword(s):

Blood Vessels ◽

Tumor Cells ◽

Expression Patterns ◽

Growth Factor Receptor ◽

Decisive Role ◽

Hpv Infection ◽

Stem Cell Markers ◽

Autocrine Loop ◽

Hpv Status ◽

The Impact

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

Download Full-text