Unravelling heterogeneous effects of cancer­associated fibroblasts on poor prognosis markers in breast cancer EM­G3 cell line: In vitro­targeted treatment (anti­IL-6, anti­VEGF-A, anti­MFGE8) based on transcriptomic profiling.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer­associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co­expression of keratins­8/­14 in the EM­G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin­8, ­14, ­18, ­19) and epithelial­mesenchymal transition­associated markers (SLUG, SNAIL, ZEB1, E­/N­cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGF­A and MFGE8 attenuated the modulatory effect of CAFs on EM­G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM­G3 cells in vitro. CAFs of different origins support the pro­inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.

Utility of fine-needle aspiration biopsy (FNAB) in parotid pleomorphic adenoma diagnosis and management.

PURPOSE: Pleomorphic adenoma (PA), the most common benign tumour of the parotid gland, requires accurate preoperative diagnosis owing to its capacity for malignant transformation. The aim of this study was to evaluate our experience with ultrasound-guided fine-needle aspiration biopsy (FNAB) in the diagnostic algorithm for patients with PA and to assess clinical outcomes for those with different surgical approaches. MATERIAL AND METHODS: We carried out a retrospective analysis of patients treated for parotid gland mass between 2010 and 2016. These had had preoperative FNAB and had undergone subsequent surgery. RESULTS: 165 patients had FNAB with the result of PA and the definitive histology confirmed PA in 159 cases (96.4%). On the other hand, in 179 patients, the definitive histology showed PA and the preoperative FNAB result corresponded in 159 cases (88.9%). The measured sensitivity, specificity and accuracy of ultrasound-guided FNAB in the diagnosis of PA were, respectively, 88.83%, 96.23% and 92.31%. Most of the patients underwent superficial or partial superficial parotidectomy, followed by extracapsular dissection which was associated with statistically lower risk of facial nerve injury (P=0.04). CONCLUSION: Ultrasound-guided FNAB is simple, accurate and valuable in the diagnosis of PA and provides results that can lead to the choice of less invasive operative treatment.

Desmoplastic Crosstalk in Pancreatic Ductal Adenocarcinoma Is Reflected by Different Responses of Panc-1, MIAPaCa-2, PaTu-8902, and CAPAN-2 Cell Lines to Cancer-associated/Normal Fibroblasts.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) still represents one of the most aggressive cancers. Understanding of the epithelial-mesenchymal crosstalk as a crucial part of the tumor microenvironment should pave the way for therapies to improve patient survival rates. Well-established cell lines present a useful and reproducible model to study PDAC biology. However, the tumor-stromal interactions between cancer cells and cancer-associated fibroblasts (CAFs) are still poorly understood. MATERIALS AND METHODS: We studied interactions between four PDAC cell lines (Panc-1, CAPAN-2, MIAPaCa-2, and PaTu-8902) and conditioned media derived from primary cultures of normal fibroblasts/PDAC-derived CAFs (PANFs). RESULTS: When the tested PDAC cell lines were stimulated by PANF-derived conditioned media, the most aggressive behavior was acquired by the Panc-1 cell line (increased number and size of colonies, remaining expression of vimentin and keratin 8 as well as increase of epithelial-to-mesenchymal polarization markers), whereas PaTu-8902 cells were rather inhibited. Of note, administration of the conditioned media to MIAPaCa-2 cells resulted in an inverse effect on the size and number of colonies, whereas CAPAN-2 cells were rather stimulated. To explain the heterogeneous pattern of the observed PDAC crosstalk at the in vitro level, we further compared the phenotype of primary cultures of cells derived from ascitic fluid with that of the tested PDAC cell lines, analyzed tumor samples of PDAC patients, and performed gene expression profiling of PANFs. Immuno-cyto/histo-chemical analysis found specific phenotype differences within the group of examined patients and tested PDAC cell lines, whereas the genomic approach in PANFs found the key molecules (IL6, IL8, MFGE8 and periostin) that may contribute to the cancer aggressive behavior. CONCLUSION: The desmoplastic patient-specific regulation of cancer cells by CAFs (also demonstrated by the heterogeneous response of PDAC cell lines to fibroblasts) precludes simple targeting and development of an effective treatment strategy and rather requires establishment of an individualized tumor-specific treatment protocol.

Molecular Changes Underlying Hypertrophic Scarring Following Burns Involve Specific Deregulations at All Wound Healing Stages (Inflammation, Proliferation and Maturation).

Excessive connective tissue accumulation, a hallmark of hypertrophic scaring, results in progressive deterioration of the structure and function of organs. It can also be seen during tumor growth and other fibroproliferative disorders. These processes result from a wide spectrum of cross-talks between mesenchymal, epithelial and inflammatory/immune cells that have not yet been fully understood. In the present review, we aimed to describe the molecular features of fibroblasts and their interactions with immune and epithelial cells and extracellular matrix. We also compared different types of fibroblasts and their roles in skin repair and regeneration following burn injury. In summary, here we briefly review molecular changes underlying hypertrophic scarring following burns throughout all basic wound healing stages, i.e. during inflammation, proliferation and maturation.

The role of fine-needle aspiration biopsy (FNAB) in the diagnostic management of parotid gland masses with emphasis on potential pitfalls.

PURPOSE: The treatment strategy of parotid gland tumours depends mainly on the histopathological type of the lesion. Fine-needle aspiration biopsy (FNAB) is recommended in preoperative diagnostics. The aim of the study was to evaluate the FNAB standing in the diagnostic algorithm of parotid gland lesions and to correlate FNAB results in relation to the definitive histopathological diagnosis. MATERIAL AND METHODS: The retrospective analyses of 651 examined and consequently surgically treated parotid gland lesions at the Department of Otorhinolaryngology and Head and Neck Surgery, 1st Faculty of Medicine, Charles University in Prague between 2006 and 2016 were used. Preoperative cytological results were consequently evaluated in relation to the definitive histopathological diagnosis. RESULTS: The cohort consisted of 367 women and 284 men (average age 58 years). FNAB was diagnostic in 604 (92.8%) patients and non-diagnostic in 47 (7.2%) patients. The result of FNAB was positive (suspicious for malignant tumour) in 89 (14.7%) patients and negative (benign) in 515 (85.3%) patients. Sensitivity of the examination was 80.00%, specificity was 93.82%, PPV 62.92%, NPV 97.28%, and LR + and LR- were 12.95 and 0.21, respectively, with an accuracy of 92.22%. CONCLUSION: Our results confirm the significant role of FNAB in the diagnostic algorithm of parotid gland lesions.

The role of fine-needle aspiration biopsy (FNAB) in Warthin tumour diagnosis and management.

PURPOSE: Warthin tumour (WT) is the second most common benign tumour of the parotid gland. The aim of this study was to assess the value of the FNAB in the diagnosis and treatment decision in patients with WT. MATERIALS AND METHODS: We performed a retrospective study of patients treated for parotid gland mass between 2006 and 2016. Patients who underwent the surgery with preoperative FNAB were considered. The first group was comprised of patients with preoperative FNAB showing WT and the second group was formed by patients with definitive histology of WT. RESULTS: 216 patients had FNAB with the result of WT and underwent surgery (98 women-45.4% and 118 men-54.6%). The definitive histology corresponded with the preoperative diagnosis in 201 cases (93.1%). The other way round, 222 patients were operated with definitive histology showing WT and we correlated this finding with preoperative FNAB. The result of FNAB corresponded with definitive histology of WT in 201 cases (90.5%). Counted sensitivity and specificity of the ultrasound-guided FNAB for the diagnosis of WT were, respectively: 96.63% (CI 93.19-98.64%) and 96.21 (CI 93.83-97.86%). The accuracy of this method was 96.36% (CI 94.54-97.70%). CONCLUSION: Ultrasound-guided FNAB is a safe, accurate and important method in WT diagnosis. The therapeutic approach can be chosen based on FNAB results correlated with other clinical findings. We propose that when WT is suspected, follow-up or enucleation of the tumour are appropriate treatments. Patient preferences should be also considered.

Detection of Distinct Changes in Gene-expression Profiles in Specimens of Tumors and Transition Zones of Tenascin-positive/-negative Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Having previously initiated genome-wide expression profiling in head and neck squamous cell carcinoma (HNSCC) for regions of the tumor, the margin of surgical resecate (MSR) and normal mucosa (NM), we here proceed with respective analysis of cases after stratification according to the expression status of tenascin (Ten). MATERIALS AND METHODS: Tissue specimens of each anatomical site were analyzed by immunofluorescent detection of Ten, fibronectin (Fn) and galectin-1 (Gal-1) as well as by microarrays. RESULTS: Histopathological examination demonstrated that Ten+Fn+Gal-1+ co-expression occurs more frequently in samples of HNSCC (55%) than in NM (9%; p<0.01). Contrary, the Ten-Fn+Gal-1- (45%) and Ten-Fn-Gal-1- (39%) status occurred with significantly (p<0.01) higher frequency than in HNSCC (3% and 4%, respectively). In MSRs, different immunophenotypes were distributed rather equally (Ten+Fn+Gal-1+=24%; Ten-Fn+Gal-1-=36%; Ten-Fn-Gal-1-=33%), differing to the results in tumors (p<0.05). Absence/presence of Ten was used for stratification of patients into cohorts without a difference in prognosis, to comparatively examine gene-activity signatures. Microarray analysis revealed i) expression of several tumor progression-associated genes in Ten+ HNSCC tumors and ii) a strong up-regulation of gene expression assigned to lipid metabolism in MSRs of Ten- tumors, while NM profiles remained similar. CONCLUSION: The presented data reveal marked and specific changes in tumors and MSR specimens of HNSCC without a separation based on prognosis.

How Signaling Molecules Regulate Tumor Microenvironment: Parallels to Wound Repair.

It is now suggested that the inhibition of biological programs that are associated with the tumor microenvironment may be critical to the diagnostics, prevention and treatment of cancer. On the other hand, a suitable wound microenvironment would accelerate tissue repair and prevent extensive scar formation. In the present review paper, we define key signaling molecules (growth factors, cytokines, chemokines, and galectins) involved in the formation of the tumor microenvironment that decrease overall survival and increase drug resistance in cancer suffering patients. Additional attention will also be given to show whether targeted modulation of these regulators promote tissue regeneration and wound management. Whole-genome transcriptome profiling, in vitro and animal experiments revealed that interleukin 6, interleukin 8, chemokine (C-X-C motif) ligand 1, galectin-1, and selected proteins of the extracellular matrix (e.g., fibronectin) do have similar regulation during wound healing and tumor growth. Published data demonstrate remarkable similarities between the tumor and wound microenvironments. Therefore, tailor made manipulation of cancer stroma can have important therapeutic consequences. Moreover, better understanding of cancer cell-stroma interaction can help to improve wound healing by supporting granulation tissue formation and process of reepithelization of extensive and chronic wounds as well as prevention of hypertrophic scars and formation of keloids.