Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma.

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene.

Efficient and simple approach to in vitro culture of primary epithelial cancer cells.

Primary cancer cells constitute a favourable testing platform for in vitro research in oncology field as they reflect tumour state more accurately than the most commonly employed stable cell lines. Unfortunately, due to limited availability of material and difficulties with protocols validation, primary models are rarely implemented into laboratory practice.We have compared protocols for primary cultures, differing in media components and plate coatings. In terms of culture establishment, application of Geltrex® coating demonstrated equal efficiency to feeder layer (83% compared with 72% successfully established breast and 80% compared with 80% prostate tumour specimens), yet it was substantially less complicated and easier to validate. Both Geltrex® coating and tissue-specific primary cell medium were permanently required to successfully maintain primary epithelial prostate cancer cells (PEPCs) in culture. In case of primary epithelial breast cancer cells (PEBCs), collagen I coating enabled to obtain comparable number of passages to Geltrex® coating (P=0.438). Commercial primary cell media demonstrated lower efficiency than tissue-specific ones (PEPCs-5 compared with 8 and PEBCs-6 compared with 9 passages). Interestingly, both analysed tumour types were unsusceptible to induction of culture lifespan extension when transduced with SV40LT, BMI-1 or hEST2 genes, commonly applied as potential immortalizing agents.In conclusion, the approach based on extracellular matrix reconstitution and tissue-specific primary cell media is easy to validate and provides in vitro expansion sufficient for analytical purposes (approximately 8 passages). Therefore, it may facilitate implementation of hardly available experimental models for a variety of analyses.

EGFR Activation Leads to Cell Death Independent of PI3K/AKT/mTOR in an AD293 Cell Line.

The Epidermal Growth Factor Receptor (EGFR) and its mutations contribute in various ways to tumorigenesis and biology of human cancers. They are associated with tumor proliferation, progression, drug resistance and the process of apoptosis. There are also reports that overexpression and activation of wild-type EGFR may lead to cell apoptosis. To study this phenomenon, we overexpressed in an AD293 cell line two most frequently observed forms of the EGFR receptor: wild-type and the constitutively active mutant-EGFR variant III (EGFRvIII). Then, we compared the effect of EGF stimulation on cell viability and downstream EGFR signaling. AD293 cells overexpressing wild-type EGFR, despite a significant proliferation increase in serum supplemented medium, underwent apoptosis after EGF stimulation in serum free conditions. EGFRvIII expressing cells, however, were unaffected by either serum starvation or EGF treatment. The effect of EGF was completely neutralized by tyrosine kinase inhibitors (TKIs), indicating the specificity of this observation. Moreover, apoptosis was not prevented by inhibiting EGFR downstream proteins (PI3K, AKT and mTOR). Here we showed another EGFR function, dependent on environmental factors, which could be employed in therapy and drug design. We also proposed a new tool for EGFR inhibitor analysis.

Mesenchymal phenotype of CTC-enriched blood fraction and lymph node metastasis formation potential.

INTRODUCTION: Circulating tumor cells (CTCs) that present mesenchymal phenotypes can escape standard methods of isolation, thus limiting possibilities for their characterization. Whereas mesenchymal CTCs are considered to be more malignant than epithelial CTCs, factors responsible for this aggressiveness have not been thoroughly defined. This study analyzed the molecular profile related to metastasis formation potential of CTC-enriched blood fractions obtained by marker unbiased isolation from breast cancer patients without (N-) and with lymph nodes metastases (N+). MATERIALS AND METHODS: Blood samples drawn from 117 patients with early-stage breast cancer were enriched for CTCs using density gradient centrifugation and negative selection with anti-CD45 covered magnetic particles. In the resulting CTC-enriched blood fractions, expression of CK19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2, CXCR4 and uPAR was analyzed with qPCR. Results were correlated with patients' clinicopathological data. RESULTS: CTCs (defined as expression of either CK19, MGB1 or HER2) were detected in 41% (20/49) of N- and 69% (34/49) of N+ patients (P = 0.004). CTC-enriched blood fractions of N+ patients were more frequently VIM (P = 0.02), SNAIL (P = 0.059) and uPAR-positive (P = 0.03). Positive VIM, CXCR4 and uPAR status correlated with >3 lymph nodes involved (P = 0.003, P = 0.01 and P = 0.045, respectively). In the multivariate logistic regression MGB1 and VIM-positivity were independently related to lymph node involvement with corresponding overall risk of 3.2 and 4.2. Moreover, mesenchymal CTC-enriched blood fractions (CK19-/VIM+ and MGB1+ or HER2+) had 4.88 and 7.85-times elevated expression of CXCR4 and uPAR, respectively, compared with epithelial CTC-enriched blood fractions (CK19+/VIM- and MGB1+ or HER2+). CONCLUSIONS: Tumors of N+ patients have superior CTC-seeding and metastatic potential compared with N- patients. These differences can be attributed to VIM, uPAR and CXCR4 expression, which endow tumor cells with particularly malignant phenotypes.

Heterogeneity of mesenchymal markers expression-molecular profiles of cancer cells disseminated by lymphatic and hematogenous routes in breast cancer.

Breast cancers can metastasize via hematogenous and lymphatic routes, however in some patients only one type of metastases are detected, suggesting a certain proclivity in metastatic patterns. Since epithelial-mesenchymal transition (EMT) plays an important role in cancer dissemination it would be worthwhile to find if a specific profile of EMT gene expression exists that is related to either lymphatic or hematogenous dissemination. Our study aimed at evaluating gene expression profile of EMT-related markers in primary tumors (PT) and correlated them with the pattern of metastatic spread. From 99 early breast cancer patients peripheral blood samples (N = 99), matched PT (N = 47) and lymph node metastases (LNM; N = 22) were collected. Expression of TWIST1, SNAI1, SNAI2 and VIM was analyzed in those samples. Additionally expression of CK19, MGB1 and HER2 was measured in CTCs-enriched blood fractions (CTCs-EBF). Results were correlated with each other and with clinico-pathological data of the patients. Results show that the mesenchymal phenotype of CTCs-EBF correlated with poor clinico-pathological characteristics of the patients. Additionally, PT shared more similarities with LNM than with CTCs-EBF. Nevertheless, LNM showed increased expression of EMT-related markers than PT; and EMT itself in PT did not seem to be necessary for lymphatic dissemination.

Epithelial-mesenchymal transition: a hallmark in metastasis formation linking circulating tumor cells and cancer stem cells.

The occurrence of either regional or distant metastases is an indicator of poor prognosis for cancer patients. The mechanism of their formation has not yet been fully uncovered, which limits the possibility of developing new therapeutic strategies. Nevertheless, the discovery of circulating tumor cells (CTCs), which are responsible for tumor dissemination, and cancer stem cells (CSCs), required for tumor growth maintenance, shed light on the metastatic cascade. It seems that CTCs and CSCs are not necessarily separate populations of cancer cells, as CTCs generated in the process of epithelial-mesenchymal transition (EMT) can bear features characteristic of CSCs. This article describes the mechanisms of CTC and CSC formation and characterizes their molecular hallmarks. Moreover, we present different types of EMT occurring in physiological and pathological conditions, and we demonstrate its crucial role in providing CTCs with a CSC phenotype. The article delineates molecular changes acquired by cancer cells undergoing EMT that facilitate metastasis formation. Deeper understanding of those processes is of fundamental importance for the development of new strategies of early cancer detection and effective cancer treatment approaches that will be translated into clinical practice.

Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release.

In solid malignant tumors, lactate has been identified as a prognostic parameter for metastasis and overall survival of patients. To investigate the effects of lactate on tumor cell migration, Boyden chamber assays were applied. We could show here that lactate enhances tumor cell motility of head and neck carcinoma cell lines significantly in a dose-dependent manner. The changes in tumor cell migration could be attributed to L-lactate or a conversion of lactate to pyruvate, as only these two substances were able to increase migration. Addition of D-lactate or changes in osmolarity or intracellular pH did not alter the migratory potential of the cells investigated. Because lactate was shown earlier to impair the penetration of dendritic cells in a tumor spheroid model, which is contrary to the response of the malignant cell population in the present study, we included blood monocytes in our assay as a highly motile immune cell type and precursor of tumor-associated macrophages. Interestingly, high levels of L-lactate (20 mM) at a pH of 7.4 inhibited monocyte migration in the Boyden chamber system. In addition, cytokine release of TNF and IL-6 was inhibited. The obtained data suggest that high lactate content promotes tumor progression by contributing to the phenomenon of tumor immune escape and by enhancing the migratory potential of the malignant cell population which may directly be coupled to a higher incidence of metastasis.

Importance of CCL2-CCR2A/2B signaling for monocyte migration into spheroids of breast cancer-derived fibroblasts.

A considerable fraction of tumor-associated macrophages (TAM) is located in the fibroblast-rich stromal compartment of desmoplastic breast carcinoma. We analyzed the migratory activity of blood monocytes (MO), the precursor cells of TAM, into 3-D cultures of carcinoma cells and fibroblasts from breast tumor origin. MO migration into breast tumor spheroids was highly variable: Hs578T spheroids showed high MO infiltration rates, T47D cultures were intermediate, whereas BT549, BT474 and MCF-7 spheroids were poorly infiltrated. MO infiltration was also high in tumor-derived fibroblast spheroids; however, no MO subpopulation with specific infiltrative potential was identified by CD14/CD16 expression profile. The infiltration of MO could be inhibited by pre-exposure to pertussis and cholera toxins, but only pertussis toxin, which blocks G(i) protein function, entirely inhibited MO migration. The G(i) coupled CCL2 receptor CCR2A/2B was expressed on roughly all MO. Furthermore, highly infiltrated tumor-derived fibroblast and Hs578T spheroids secreted considerable amounts of CCL2. In line with this, the infiltration of MO into fibroblast spheroids was suppressed by either addition of recombinant CCL2 to disturb the CCL2 gradient or by pre-incubation of MO with a CCR2A/2B blocking antibody. MO infiltration of Hs578T spheroids, however, could not be inhibited by CCL2 receptor blockade. Our study clearly shows that the CCL2-CCR2A/2B pathway is crucial for the recruitment of blood MO into tumor fibroblastic areas, whereas additional factors may be relevant for the migration of MO into tumor cell sites.