Isolation and characterization of head and neck cancer-derived peritumoral and cancer-associated fibroblasts.

Introduction: Head and neck squamous cell carcinomas (HNSCC) are characterized by strong cellular and molecular heterogeneity and treatment resistance entailing poor survival. Besides cell-intrinsic properties, carcinoma cells receive important cues from non-malignant cells within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a major component of the TME that impact on the molecular make-up of malignant cells and have a decisive function in tumor progression. However, the potential functionality of fibroblasts within tumor-adjacent, macroscopically normal tissue remains poorly explored. Methods: Here, we isolated primary peritumoral fibroblasts (PtFs) from macroscopically normal tissue in vicinity of primary human papillomavirus-negative and -positive oropharyngeal HNSCC and compared their phenotype and functionality with matched CAFs (n = 5 pairs) and with human oral fibroblasts (hOFs). Results: Expression patterns of CD90, CD73, CD105, smooth muscle actin, Vimentin, and S100A4 were comparable in PtFs, CAFs, and hOFs. Cell proliferation and doubling times of CAFs and PtFs were heterogeneous across patients (n =2 PtF>CAF; n = 1 CAF>PtF; n = 2 CAF=PtF) and reflected inferior growth than hOFs. Furthermore, PtFs displayed an reduced heterogeneity in cell size compared to matched CAFs, which were characterized by the presence of single large cells. Overall, conditioned supernatants from CAFs had more frequently growth-promoting effects on a panel of carcinoma cell lines of the upper aerodigestive tract carcinoma cell lines (Cal27, Cal33, FaDu, and Kyse30), whereas significant differences in migration-inducing effects demonstrated a higher potential of PtFs. Except for Kyse30, CAFs were significantly superior to hOFs in promoting proliferation, while PtFs induced stronger migration than hOFs in all carcinoma lines tested. Analysis of soluble factors demonstrated significantly increased VEGF-A production in CAFs (except in pat.8), and significantly increased PDGF-BB production in PtFs of two patients. Tube formation assays confirmed a significantly enhanced angiogenic potential of conditioned supernatants from CAFs compared to hOFs on human umbilical vascular endothelial cells (HUVECs) in vitro. Discussion: Hence, matched CAFs and PtFs present in HNSCC patients are heterogeneous in their proliferation-, migration-, and angiogenesis-promoting capacity. Despite this heterogeneity, CAFs induced stronger carcinoma cell proliferation and HUVEC tube formation overall, whereas PtFs promoted migration of tumor cells more strongly.

Interactome analysis reveals endocytosis and membrane recycling of EpCAM during differentiation of embryonic stem cells and carcinoma cells.

Transmembrane epithelial cell adhesion molecule (EpCAM) is expressed in epithelia, carcinoma, teratoma, and embryonic stem cells (ESCs). EpCAM displays spatiotemporal patterning during embryogenesis, tissue morphogenesis, cell differentiation, and epithelial-to-mesenchymal transition (EMT) in carcinomas. Potential interactors of EpCAM were identified in murine F9 teratoma cells using a stable isotope labeling with amino acids in cell culture-based proteomic approach (n = 77, enrichment factor >3, p value ≤ 0.05). Kyoto Encyclopedia of Genes and Genomes and gene ontology terms revealed interactions with regulators of endosomal trafficking and membrane recycling, which were further validated for Rab5, Rab7, and Rab11. Endocytosis and membrane recycling of EpCAM were confirmed in mF9 cells, E14TG2α ESC, and Kyse30 carcinoma cells. Reduction of EpCAM during mesodermal differentiation and TGFß-induced EMT correlated with enhanced endocytosis and block or reduction of recycling in ESCs and esophageal carcinoma cells. Hence, endocytosis and membrane recycling are means of regulation of EpCAM protein levels during differentiation of ESC and EMT induction in carcinoma cells.

Molecular Characterization of Lipoaspirates Used in Regenerative Head and Neck Surgery.

IMPORTANCE: Adipose-derived mesenchymal stem cells (ASCs) have been used commonly in regenerative medicine and increasingly for head and neck surgical procedures. Lipoaspiration with centrifugation is purported to be a mild method for the extraction of ASCs used for autologous transplants to restore tissue defects or induce wound healing. The content of ASCs, their paracrine potential, and cellular potential in wound healing have not been explored for this method to our knowledge. OBJECTIVE: To evaluate the characteristics of lipoaspirates used in reconstructive head and neck surgical procedures with respect to wound healing. DESIGN, SETTING, AND PARTICIPANTS: This case series study included 15 patients who received autologous fat injections in the head and neck during surgical procedures at a tertiary referral center. The study was performed from October 2017 to November 2018, and data were analyzed from October 2017 to February 2019. MAIN OUTCOMES AND MEASURES: Excessive material of lipoaspirates from subcutaneous abdominal fatty tissue was examined. Cellular composition was analyzed using immunohistochemistry (IHC) and flow cytometry, and functionality was assessed through adipose, osteous, and chondral differentiation in vitro. Supernatants were tested for paracrine ASC functions in fibroblast wound-healing assays. Enzyme-linked immunosorbent assay measurement of tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), stromal-derived factor 1α (SDF-1α), and transforming growth factor ß3 (TGF-ß3) was performed. RESULTS: Among the 15 study patients (8 [53.3%] male; mean [SD] age at the time of surgery, 63.0 [2.8] years), the stromal vascular fraction (mean [SE], 53.3% [4.2%]) represented the largest fraction within the native lipoaspirates. The cultivated cells were positive for CD73 (mean [SE], 99.90% [0.07%]), CD90 (99.40% [0.32%]), and CD105 (88.54% [2.74%]); negative for CD34 (2.70% [0.45%]) and CD45 (1.74% [0.28%]) in flow cytometry; and negative for CD14 (10.56 [2.81] per 300 IHC score) and HLA-DR (6.89 [2.97] per 300 IHC score) in IHC staining; they differentiated into osteoblasts, adipocytes, and chondrocytes. The cultivated cells showed high expression of CD44 (mean [SE], 99.78% [0.08%]) and CD273 (82.56% [5.83%]). The supernatants were negative for TNF (not detectable) and SDF-1α (not detectable) and were positive for VEGF (mean [SE], 526.74 [149.84] pg/mL for explant supernatants; 528.26 [131.79] pg/106 per day for cell culture supernatants) and TGF-ß3 (mean [SE], 22.79 [3.49] pg/mL for explant supernatants; 7.97 [3.15] pg/106 per day for cell culture supernatants). Compared with control (25% or 50% mesenchymal stem cell medium), fibroblasts treated with ASC supernatant healed the scratch-induced wound faster (mean [SE]: control, 1.000 [0.160]; explant supernatant, 1.369 [0.070]; and passage 6 supernatant, 1.492 [0.094]). CONCLUSIONS AND RELEVANCE: The cells fulfilled the international accepted criteria for mesenchymal stem cells. The lipoaspirates contained ASCs that had the potential to multidifferentiate with proliferative and immune-modulating properties. The cytokine profile of the isolated ASCs had wound healing-promoting features. Lipoaspirates may have a regenerative potential and an application in head and neck surgery. LEVEL OF EVIDENCE: NA.

Radiation-induced sensitivity of tissue-resident mesenchymal stem cells in the head and neck region.

BACKGROUND: Tissue-resident mesenchymal stem cells (MSCs) possess the ability to migrate to areas of inflammation and promote the regeneration of damaged tissue. However, it remains unclear how radiation influences this capacity of MSC in the head and neck region. METHODS: Two types of MSCs of the head and neck region (mucosa [mMSC] and parotid gland [pMSC]) were isolated, cultured and exposed to single radiation dosages of 2 Gy/day up to 10 days. Effects on morphology, colony forming ability, apoptosis, chemokine receptor expression, cytokine secretion, and cell migration were analyzed. RESULTS: Although MSC preserved MSC-specific regenerative abilities and immunomodulatory properties following irradiation in our in vitro model, we found a deleterious impact on colony forming ability, especially in pMSC. CONCLUSIONS: MSC exhibited robustness and activation upon radiation for the support of tissue regeneration, but lost their potential to replicate, thus possibly leading to depletion of the local MSC-pool after irradiation.

Membrane-associated epithelial cell adhesion molecule is slowly cleaved by γ-secretase prior to efficient proteasomal degradation of its intracellular domain.

Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and ß-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aß-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen.

BACKGROUND: The search for intrinsic factors, which account for a protein's capability to act as an allergen, is ongoing. Fold stability has been identified as a molecular feature that affects processing and presentation, thereby influencing an antigen's immunologic properties. OBJECTIVE: We assessed how changes in fold stability modulate the immunogenicity and sensitization capacity of the major birch pollen allergen Bet v 1. METHODS: By exploiting an exhaustive virtual mutation screening, we generated mutants of the prototype allergen Bet v 1 with enhanced thermal and chemical stability and rigidity. Structural changes were analyzed by means of x-ray crystallography, nuclear magnetic resonance, and molecular dynamics simulations. Stability was monitored by using differential scanning calorimetry, circular dichroism, and Fourier transform infrared spectroscopy. Endolysosomal degradation was simulated in vitro by using the microsomal fraction of JAWS II cells, followed by liquid chromatography coupled to mass spectrometry. Immunologic properties were characterized in vitro by using a human T-cell line specific for the immunodominant epitope of Bet v 1 and in vivo in an adjuvant-free BALB/c mouse model. RESULTS: Fold stabilization of Bet v 1 was pH dependent and resulted in resistance to endosomal degradation at a pH of 5 or greater, affecting presentation of the immunodominant T-cell epitope in vitro. These properties translated in vivo into a strong allergy-promoting TH2-type immune response. Efficient TH2 cell activation required both an increased stability at the pH of the early endosome and efficient degradation at lower pH in the late endosomal/lysosomal compartment. CONCLUSIONS: Our data indicate that differential pH-dependent fold stability along endosomal maturation is an essential protein-inherent determinant of allergenicity.

IRAK1 is a therapeutic target that drives breast cancer metastasis and resistance to paclitaxel.

Metastatic tumour recurrence due to failed treatments remains a major challenge of breast cancer clinical management. Here we report that interleukin-1 receptor-associated kinase 1 (IRAK1) is overexpressed in a subset of breast cancers, in particular triple-negative breast cancer (TNBC), where it acts to drive aggressive growth, metastasis and acquired resistance to paclitaxel treatment. We show that IRAK1 overexpression confers TNBC growth advantage through NF-κB-related cytokine secretion and metastatic TNBC cells exhibit gain of IRAK1 dependency, resulting in high susceptibility to genetic and pharmacologic inhibition of IRAK1. Importantly, paclitaxel treatment induces strong IRAK1 phosphorylation, an increase in inflammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1 is able to reverse paclitaxel resistance by triggering massive apoptosis at least in part through inhibiting p38-MCL1 pro-survival pathway. Our study thus demonstrates IRAK1 as a promising therapeutic target for TNBC metastasis and paclitaxel resistance.