Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo chemoresponse

Background

Valid prediction of the effectiveness of chemotherapeutic agents in individual head and neck squamous cell carcinoma (HNSCC) is desirable and might be achieved using ex vivo assays.

Methods

Three biopsies from each of 15 HNSCC were taken, minced and digested by collagenase. The digested HNSCC was added to serial dilutions of either cisplatin (CIS) or docetaxel (DTX), which were prepared under flavin-protecting conditions in ECM-coated microtiterplates. After 72-h incubation, cultures were methanol-fixed and Giemsa-stained. The cutoff concentration (COC; concentration completely suppressing colony formation) for epithelial cells (EC) and stromal cells (SC) was evaluated.

Results

12/15 HNSCC (80%) were evaluable. Despite significant correlation of COC of CIS in respect of colony formation of EC or SC, no significant differences in response of individual HNSCC specimens were found in the t test for paired samples (p > 0.16). The same applied to DTX. However, EC and SC showed heterogeneity in chemoresponses leading to COC variability of more than one titration step in 44.1% (CIS) and 20% of HNSCC (DTX). No significant correlation between the COC of both cell populations was found in HNSCC specimens.

Conclusions

The ex vivo chemoresponse of EC and SC of HNSCC must be analyzed separately.     

Inhibition of SRC family kinases reduces myeloid‐derived suppressor cells in head and neck cancer

SRC family kinases (SFKs), a group of nonreceptor tyrosine kinases, modulate multiple cellular functions, such as cell proliferation, differentiation and metabolism. SFKs display aberrant activity in progressive stages of human cancers. However, the precise role of SFKs in the head and neck squamous cell carcinoma (HNSCC) signaling network is far from clear. In this study, we found that the inhibition of SFKs activity by dasatinib effectively reduced the tumor size and population of MDSCs in the HNSCC mouse model. Molecular analysis indicates that phosphorylation of LYN, rather than SRC, was inhibited by dasatinib treatment. Next, we analyzed LYN expression by immunostaining and found that it was overexpressed in the human HNSCC specimens. Moreover, LYN expression in stromal cells positively correlated with myeloid‐derived suppressor cells (MDSCs) makers CD11b and CD33 in human HNSCC. The dual positive expression of LYN in epithelial and stromal cells (EPI⁺ SRT⁺) was associated with unfavorable overall survival of HNSCC patients. These findings indicate that SFKs may be a potential target for an effective immunotherapy of HNSCC by decreasing MDSCs and moreover, LYN will have an impact on such therapeutic strategy.     

Long-term quality of life outcomes in patients with locally advanced prostate cancer after intensity-modulated radiotherapy combined with androgen deprivation

Studies have shown that miR-34c is associated with metastasis and the chemoresponse of several cancers, but its role in osteosarcoma (OS) is unclear. Here, we investigated the role and mechanism of miR-34c in OS metastasis and chemoresponse. In this study, we found that the expression of miR-34c was significantly decreased in specimens from OS patients with a poor chemoresponse or metastasis compared to those with a good chemoresponse and no metastasis. The inhibition of miR-34c significantly stimulated OS cell invasion and chemoresistance in vitro. In contrast, restoring miR-34c significantly inhibited OS cell invasion and chemoresistance. Furthermore, we identified Notch1 and lymphoid enhancer-binding factor 1 (LEF1) as target genes of miR-34c in OS cells and demonstrated that Notch1 and LEF1 have a major role in the effects of miR-34c on OS cell chemosensitivity and metastasis. Taken together, our data indicate that miR-34c suppresses OS metastasis and chemoresistance by targeting Notch1 and LEF1. Restoring miR-34c may have important implications for the development of strategies for inhibiting metastasis and overcoming OS cell resistance to chemotherapy.     

Cytokeratins 6 and 16 are frequently expressed in head and neck squamous cell carcinoma cell lines and fresh biopsies

BACKGROUND: Cytokeratins (CK) are members of intermediate filaments, which are predominantly found in epithelial cells. Different types of epithelia are characterized by a distinct composition of CK. Recently immunohistochemical investigations demonstrated that, among others, CKs 6, 14, 16 and 17 are regularly expressed in benign stratified squamous epithelium of the head and neck as well as in squamous cell carcinoma of the head and neck (HNSCC) in contrast to CKs 1, 10 and 11, that were only rarely expressed in these tissues. MATERIALS AND METHODS: Total RNA was isolated from 15 primary cell lines derived from HNSCC and from 15 tissue samples of oro- and hypopharyngeal carcinomas obtained from surgery specimens. CK expression was evaluated by RT-PCR, Western blot analysis and immunohistochemistry. RESULTS: CK6 and 16 were found to be expressed in both groups at almost 100%. The expression level of CK14 remained constant (73%) in both groups, at the RNA and protein level. CK17 was more frequently present in tumour specimens than in HNSCC cell lines. The immunohistochemical results of the surgical tumour specimens confirmed the results of Western blot analysis. CONCLUSION: The presented results show high and stable expression rates for CK6 and CK16 in HNSCC. These results will serve as a basis for further investigations concerning the search for circulating tumour cells and micrometastases. In addition, we found that cytokeratin expression in HNSCC is different on the RNA level compared to the protein level.     

Chemotherapeutic drug sensitivity testing of human leukemia cells in vitro using a semiautomated fluorometric assay

A simple and reproducible semiautomated fluorometric method for drug sensitivity testing of leukemic cells in microculture is described. The assay is based on hydrolysis of nonfluorescent fluorescein diacetate (FDA) to a strongly fluorescent product (fluorescein) by cells with intact plasma membranes after 72 hr of culture and was in the present study applied to acute lymphocytic leukemia (ALL) cell lines and specimens from patients with lymphocytic and myelocytic leukemia. FDA fluorescence was linearly related to viable cell number within a wide range of cell densities (3-4 logs) as well as in the presence of different added proportions of dead cells. The assay reliably detects high and low grade resistance to vincristine (vcr) and daunorubicin, respectively, as well as the subsequent reversal of vcr resistance by cyclosporin A and the calcium channel blocker verapamil. Using ALL cell lines, drug sensitivity was in good correspondence with data obtained by the microculture tetrazolium assay. Furthermore, drug sensitivity data of fresh leukemia cells from patients with leukemia were readily obtained. The results indicate that the presently described method is applicable for simple and reliable chemosensitivity testing of leukemia cell lines as well as tumor specimens from patients with leukemia.     

Frequent alterations of Smad signaling in human head and neck squamous cell carcinomas: a tissue microarray analysis

Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most frequent cancer worldwide. HNSCC cell lines are typically refractory to transforming growth factor-beta (TGF-beta)-mediated cell cycle arrest. A number of these cell lines carry inactivating mutations of the TGF-beta type II (TbetaR-II) receptor, and fail to phosphorylate receptor-associated Smads, Smad2 and Smad3. In addition, we identified several intragenic mutations of the TbetaR-I gene in a small series of metastatic HNSCC specimens, suggesting that disruptions of TGF-beta signaling might contribute to the development and progression of HNSCC. To test this idea, we have now embarked on a larger scale analysis of the patterns of expression and activation of Smads in 170 HNSCC specimens assembled in tissue microarrays. Smad2 protein was expressed by 99% (95% CI: 96-100%) of tumors. The activated form of Smad2, pSmad2, was expressed in 86% (95% CI: 80-91%) of HNSCC, indicating their ability to survive and proliferate in spite of the presence of bioactive TGF-beta within the tissue microenvironment. In the 24 remaining cases (14%; 95% CI: 9-20%), pSmad2 was not detected in the tumor cells, although it was expressed by surrounding stromal cells and capillaries. In addition, 38 tumors (22%; 95% CI: 16-29%) failed to express Smad4 protein. Thus, we found evidence of loss of TGF-beta/Smad signaling in approximately 15-20% of HNSCC specimens, which is consistent with the phenotype of established human SCC lines. Moreover, we found that these Smad signaling defects were associated with a greater tendency for metastatic spread and regional or distant recurrence of HNSCC. These results indicate that inactivation of TGF-beta/Smad signaling occurs frequently in HNSCC and might have an adverse effect on patient outcome.     

Metastatic Lung Disease to the Central Nervous System: in vitro Response to Chemotherapeutic Agents

BACKGROUND: Metastatic lung disease to the central nervous system (CNS) comprises a significant percentage of cranial metastases. For those cases where chemotherapy may be of palliative or therapeutic benefit, in vitro chemoresponse testing may identify the agent(s) most likely to be effective clinically. METHODS: Tumor-derived cell cultures were established from 14 surgically excised lung lesions metastatic to the CNS. In vitro chemoresponse testing was performed with a variety of anticancer agents on the tumor cells that grew out of the cultured tissue specimens. Drug concentrations and exposure times were adjusted to bracket approximate average peak plasma levels that are observed typically in vivo. For each tumor-derived cell culture, a complete dose-response curve was established for each chemotherapeutic agent tested. RESULTS: Approximately 80% of the 14 tumor cell cultures had a definitive response to one or more chemotherapeutic agents in vitro, with approximately one-third of these cultures displaying a response to at least three of the drugs tested. There was considerable heterogeneity in the response of individual tumor cell cultures to the chemotherapeutic drugs. The agents that showed the highest cytotoxic response rate against the individual tumor cell cultures included lomustine, carboplatin, cisplatin and etoposide. CONCLUSIONS: The tumor cells isolated from individuals with lung lesions metastatic to the brain demonstrated differential chemoresponses to the agents tested. No single agent was effective against every tumor cell culture. These data suggest that in vitro chemoresponse testing of cultured tumor cells may be useful to identify biologically effective chemotherapeutic agents for individual patients, thereby addressing at least one factor in this complex therapeutic challenge.     

S19-mRNA expression in squamous cell carcinomas of the upper aerodigestive tract

BACKGROUND: The differential display method showed altered expression of ribosomal protein S19 gene in human head and neck squamous cell carcinoma (HNSCC) cell lines. MATERIALS AND METHODS: To verify these results, RT-PCR analysis was carried out in 18 HNSCC and 17 benign epithelial cell lines as well as 30 HNSCC and 8 reference tissue samples. In the HNSCC cells S19 mRNA expression was significantly reduced as compared to benign epithelial cells. RESULTS: Change of the S19 gene expression in surgical samples was detectable but not significant although the histopathological grading of the HNSCC biopsies correlated significantly with the S19 mRNA expression levels. The expression of ribosomal protein S6 and S14 genes were additionally analyzed using the same methods. CONCLUSION: High correlation was found between the expression of S6/S14 and S19 suggesting that changes in S19 gene expression might be the result of loss of ribosomes in HNSCC cells.     

NM23-H1 expression of head and neck squamous cell carcinoma in association with the response to cisplatin treatment

We recently reported that low NM23-H1 expression of head and neck squamous cell carcinoma (HNSCC) correlated with poor patients'' prognosis. Growing evidence has indicated that high tumor NM23-H1 expression contributes to a good response to chemotherapy. Therefore, we investigated the role of NM23-H1 in susceptibility of HNSCC cells to cisplatin and its clinical significance, as well as the in vitro study for validation was performed. Using immunohistochemistry, we analyzed NM23-H1 expression in surgical specimens from 46 HNSCC patients with cervical metastases receiving surgery and adjuvant chemoradiotherapy. Low tumor NM23-H1 expression correlated with locoregional recurrence of HNSCC following postoperative cisplatin-basedtherapy (p = 0.056) and poor patient prognosis (p = 0.001). To validate the clinical observation and the effect of NM23-H1 on cisplatin cytotoxicity, we established several stable clones derived from a human HNSCC cell line (SAS) by knockdown and overexpression. Knockdown of NM23-H1 attenuated the chemosensitivity of SAS cells to cisplatin, which was associated with reduced cisplatin-induced S-phase accumulation and downregulation of cyclin E1 and A. Overexpression of NM23-H1 reversed these results, indicating the essential role of NM23-H1 in treatment response to cisplatin. NM23-H1 may participate in HNSCC cell responses to cisplatin and be considered a potential therapeutic target.     

Involvement of ABC transporters in chemosensitivity of human renal cell carcinoma, and regulation of MRP2 expression by conjugated bilirubin

In this study, the involvement of ATP-binding cassette (ABC) transporters in in vitro chemosensitivity of surgically removed human renal cell carcinomas was investigated. The relative expression levels of transporter mRNAs in the renal tumors from 13 patients were similar to those in the surrounding normal kidney tissues. Five renal cell carcinomas cultured successfully in vitro for 14 days showed significantly decreased expression of multi-drug resistance-associated proteins 2 and 6 (MRP2 and MRP6) mRNAs. In vitro chemosensitivity testing of the same specimens using the collagen-gel matrix assay indicated that some anticancer drugs were effective, especially cisplatin, which is an MRP2 substrate. MRP2 mRNA expression in renal carcinoma was significantly increased when cells were cultured in the presence of conjugated bilirubin. In an established renal proximal tubule epithelial cell line (RPTEC), conjugated bilirubin increased MRP2 expression at the mRNA and protein levels, and decreased the cisplatin sensitivity of the cells. These results indicate that MRP2 expression in renal cell carcinoma may be regulated by conjugated bilirubin in the body and decreased during in vitro culture. Thus, the effectiveness of anticancer drugs selected on the basis of in vitro chemosensitivity testing of clinical cancers may be overestimated.     

Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5-fluorouracil (5-FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5-FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5-FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC.     

长链非编码RNA HOTAIR抑制膀胱尿路上皮癌对阿霉素的敏感性

目的:研究长链非编码RNA HOTAIR在膀胱尿路上皮癌中的表达,明确其对膀胱尿路上皮癌对阿霉素敏感性的调控作用.方法:HOTAIR基因在膀胱尿路上皮癌及癌旁组织中的表达由实时定量PCR法检测.将HOTAIR基因的表达载体和沉默载体分别转染膀胱尿路上皮癌J82细胞,实时定量PCR验证转染效果.应用MTT法检测HOTAIR基因表达改变对于J82细胞的增殖能力及对阿霉素敏感性的调控作用.结果:HO-TAIR基因在膀胱尿路上皮癌表达显著上调.HOTAIR基因的表达载体和沉默载体能够显著上调或沉默HO-TAIR基因的表达.HOTAIR高表达能够促进J82细胞增殖,抑制J82细胞对阿霉素的敏感性;而HOTAIR表达沉默的J82细胞增殖能力下调明显,对阿霉素的敏感性明显增加.结论:长链非编码RNA HOTAIR在膀胱尿路上皮癌中高表达,HOTAIR能够作为癌基因促进膀胱尿路上皮癌的细胞增殖能力并抑制膀胱尿路上皮癌对阿霉素敏感性.     

Microculture-based chemosensitivity testing: a feasibility study comparing freshly explanted human melanoma cells with human melanoma cell lines.

BACKGROUND: The culture of cancer cells has many applications in chemosensitivity testing and new drug development. PURPOSE: Our goal was to adapt simple semiautomated microculture methods for testing the chemosensitivity of melanoma cells freshly recovered from patients' tumors. METHODS: Cells were cultured on a substrate of agarose and exposed continuously to cytotoxic drugs, the effects of which were measured by determining the uptake of [3H]thymidine 4-7 days later. RESULTS: Immunocytochemical staining of cells cultured with 5-bromo-2'-deoxyuridine demonstrated that tumor cells were responsible for the measured thymidine incorporation. The effects of cytotoxic drugs were calculated as logarithmic 50% inhibitory concentrations and expressed as divergences from the mean in a log-mean graph. The inhibitory effects of amsacrine, etoposide, doxorubicin, cisplatin, mitomycin C, and fluorouracil were tested. Tumors differed widely in their sensitivity to these drugs, although sensitivity to the three topoisomerase-II-directed agents was highly correlated. Cells from two non-neoplastic hematopoietic progenitor cell lines (FT and 32D) showed chemosensitivity patterns distinct from those in the melanoma cells, indicating tissue selectivity. Two established melanoma cell lines, MM-96 and FME, were tested under the same conditions and showed sensitivity typical of at least some fresh specimens. CONCLUSIONS: These results support the validity of melanoma cell lines as models of freshly resected melanoma cells. If successfully applied to other tumor types, such semiautomated approaches could find wide application in routine hospital laboratories for the chemosensitivity testing of patients' tumor cells.     

Histological and immunohistochemical prediction for local control of cervical squamous cell carcinoma treated with radiotherapy alone

Predictability of local control following radiotherapy was evaluated with morphological methods such as histology and immunohistochemistry for 36 cervical squamous cell carcinomas. All these patients showed viable cancer cell predominance on the specimens excised with drill biopsy after radiation therapy. These specimens were stained with routine haematoxylin and eosin staining as well as with antibodies against epithelial membrane antigen (EMA), carcinoembryonic antigen, and S-100 protein. Five histological features, the number of the cancer nests per 22.5mm2 section, stromal reaction, space formation in the cancer nests, foamy cell or foreign body giant cell (FBG) clusters, and epithelial membrane antigen reactivity on the specimens excised after radiation therapy, were significantly related to the local control probability. Namel, less than 20 cancer nests, granulomatous stroma, presence of the space formation, absence of the foamy cell or foreign body giant cell clusters, and epithelial membrane antigen negativity were favorable for local control of the cervical cancer with radiation therapy. These data suggested that radiation sensitivity of cancer and stromal reaction for drainage of the degenerated cancer debris were important for local control of the tumors.     

Paracrine stimulation of polarized secretion from monolayers of a neoplastic prostatic epithelial cell line by prostatic stromal cell proteins

The paracrine influence of prostatic stromal cell proteins on a neoplastic prostate cell line (PA-III) was investigated. We have utilized an in vitro experimental model whereby confluent epithelial sheets of PA-III cells are grown on Matrigel-coated filters in bicameral chambers (Millicell-HA). Confluence of the epithelial sheet was confirmed morphologically by electrical resistance measurements and by impedence of [3H]inulin permeability across paracellular channels. Stromal cells were isolated from the ventral prostate of 50-day-old rats by isopyknic Percoll centrifugation. Purity (92%) of the isolated stromal cells was confirmed by indirect immunofluorescence of vimentin intermediate filaments. Prostatic epithelial cells were negative for vimentin immunofluorescence. Prostatic stromal cell secretory proteins with molecular weights greater than 10,000 were placed in the basal reservoir of the bicameral chambers underneath the confluent epithelial sheets of PA-III in a manner that mimics the relationship between stroma and epithelia in vivo. After 24 h incubation the stromal cell proteins increased the [35S]methionine-labeled protein secretion from the epithelial sheet of cells. Trypsinization of the stromal cell secretory proteins eliminated the stimulatory effect on epithelial protein secretion. In addition, conditioned media from Swiss 3T3 fibroblasts, A431 cells, or bovine serum albumin did not stimulate epithelial protein secretion. Two-dimensional gel electrophoresis of the [35S]methionine-labeled epithelial protein secretion showed that the stromal cell proteins induced the secretion of a novel peptide (SE-1) from the basal domain of the epithelial sheet of cells within the first hour of metabolic labeling. These results indicate that stromal cell secretory proteins contain a stimulatory protein that can induce overall protein secretion as well as the vectorial secretion of a novel peptide from the basal domain of PA-III epithelial cells. These results are consistent with a paracrine interaction between epithelial and stromal cells in the regulation of prostatic secretion.     

Gene expression profiling of advanced head and neck squamous cell carcinomas and two squamous cell carcinoma cell lines under radio/chemotherapy using cDNA arrays.

BACKGROUND AND PURPOSE: The response of squamous cell carcinomas of the head and neck (HNSCC) to radio/chemotherapy is accompanied by complex changes in patterns of gene expression. It is highly probable that a better understanding of molecular and genetic changes can help to optimize the treatment of HNSCC. cDNA arrays provide a powerful tool for high-throughput monitoring of gene expression in small clinical specimens. MATERIALS AND METHODS: We used tumour biopsies from four patients with HNSCC which have been taken prior to and during radio/chemotherapy. The patterns of gene expression obtained from clinical samples were compared with gene expression profiles of two squamous cell carcinoma cell lines (FaDU and UD-7A). RESULTS: The experimental data analysis revealed changes in expression levels of several genes during radio/chemotherapy. Despite treatment, independent samples taken from the same cell line or tumour in situ were more similar to each other than either was to other specimens. The data indicate a high gene heterogeneity of HNSCC that is preserved during treatment. CONCLUSIONS: From our preliminary results we conclude that the cDNA array experimental approach can detect differences in gene expression between treated and untreated small tumour biopsies, as well as inter-individual differences in expression profiles between HNSCC tumours. The examination of a greater sample size will be needed to make this preliminary evaluation useful to elucidate the functional significance of individual genes which exhibit altered levels of expression under radiation therapy.     

Microvessel density in advanced head and neck squamous cell carcinoma before and after chemotherapy

OBJECTIVE: Studies of tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC) in regard to correlation with prognostic significance have yielded inconclusive results. To determine whether the microvessel density (MVD) within the tumor of advanced (Stages III and IV) HNSCC has any impact on tumor response to 2-3 courses of paclitaxel (Taxol) and carboplatin, we prospectively studied pre-chemotherapy specimens from patients with previously untreated, advanced stage HNSCC. We also attempted to study residual tumors after chemotherapy to determine if the MVD within the tumor had changed. STUDY DESIGN: The MVD within the tumor was obtained by immunohistochemical staining of the tumors with Q-Bend 10 (CD34). The "hot-spot" areas of each tumor (ie., areas with most intense blood neovascularization) were considered for evaluation. Results were expressed as the Average number of microvessels identified in 5-400x microscope fields (ie., the number of microvessels counted in 5-400x microscope fields divided by 5). SETTING: Tertiary University Medical Center. INTERVENTION: Two to 3 courses of chemotherapy with paclitaxel and carboplatin. MAIN OUTCOME MEASURES: Progression-free and overall survival with 5 years follow-up, RESULTS: The tumoral MVD in 32 HNSCC specimens before chemotherapy ranged from 4.0-39.0 (mean, 14.8; median, 14.2). Eight out of 32 patients achieved pathologically complete remission; their tumoral MVD revealed a mean of 10.9. The 24 remaining patients had pathologically-confirmed residual tumor post-chemotherapy; their tumoral MVD revealed a mean of 16.5. CONCLUSION: Statistical analyses revealed no evidence of a relationship between remission and a MVD > or < 10 or > or < 16.5. There was no correlation of tumoral MVD with overall or progression-free survival. In 15 patients, tumoral MVD results were also available on the post-chemotherapy specimens. The greater the difference of the tumoral MVD between the pre- and post-chemotherapy specimens, the shorter the patients overall and progression-free survival (p = 0.042).