Why 3D in vitro cancer models are the future of cancer research?

Tumors are three-dimensional (3D) entities characterized by complex structural architecture which is necessary for adequate intercellular, intracellular and cell-to-matrix interactions among the aberrant cells in cancer. In the field of cancer research, 2D cell cultures are traditionally used for decades in the majority of experiments. The reasons for this are the vast benefits these models provide, including simplicity and cost effectiveness. However, it is now known that these models are exposed to much higher stiffness, they lose physiological extracellular matrix (ECM) on artificial plastic surfaces as well as differentiation, polarization and cell-cell communication. This leads to the loss of crucial cellular signaling pathways and changes in cell responses to stimuli when compared to in vivo conditions. Moreover, they cannot adequately mimic the complexity and dynamic interactions of the tumor microenvironment (TME) which is of great importance in anticancer drug treatments. 3D models seem more biomimetic compared to 2D cell monolayers because they offer the opportunity to model the cancer mass together with its environment which seems the key factor in promoting and directing cancer invasion. 3D cell culture with its additional dimensionality makes the difference in cellular responses because it influences the spatial and physical aspects of the cells in 3D culture. This affects the signal transduction and makes the behavior of 3D-cultured cells more physiologically relevant and reflective of in vivo cellular responses. This review focuses on major differences between 2D and 3D cell cultures, highlighting the importance of considering bioengineering humanized 3D cancer models as the future in cancer research. Additionally, it presents diverse 3D models currently used in cancer research, outlining their benefits and limitations. Precisely, this review highlights the differences between the 3D models with the focus on tumor stroma interactions, cell population and extracellular matrix composition providing methods and examples for each model from the studies done so far

Combination of cyclopamine and tamoxifen promotes survival and migration of MCF-7 breast cancer cells – interaction of Hedgehog-Gli and Estrogen receptor signaling pathways

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estroge

Promoter methylation in the PTCH gene in cervical epithelial cancer and ovarian cancer tissue as studied by eight novel Pyrosequencing assays

DNA methylation status in the CpG sites of promoter regions in cancer-related genes, such as PTCH, has traditionally been investigated using either dye-terminator sequencing or methylation-specific PCR. We aimed to study the PTCH gene promoter methylation in gynecological cancers, with a method that gives a quantitative measure of the methylation status of the promoter region of the studied gene, and for this purpose, we designed novel Pyrosequencing based assays. Bisulfite-treated genomic DNA (bsDNA)was amplified by standard PCR and applied to novel Pyrosequencing assays, in order to measure the methylated fraction(%) at each CpG site of the PTCH gene promoter. We analyzed 22 squamous cell cervical cancer tissue specimens (11 with good and 11 with poor outcomes after radiotherapy)and 5 ovarian cancer tissue specimens matched with 5 normal ovarian tissue specimens. Six optimized PCR protocols which generated 8 Pyrosequencing assays covering 63 CpG sites in the promoter regions 1 and 2 as well as the previously unanalyzed promoter region 3 in the PTCH gene were developed. The 27 tumor tissue specimens and 5 normal tissues did not show any methylation within any of the 63 CpG sites. Our data suggest that methylation of the PTCH promoter is not a high-prevalence feature of squamous cell cervical cancer or ovarian cancer, but Pyrosequencing assays are a good method for studying promoter methylation

Potential hot spot for de novo mutations in PTCH1 gene in Gorlin syndrome patients: a case report of twins from Croatia

We describe a case of twins with sporadic Gorlin syndrome. Both twins had common Gorlin syndrome features including calcification of the falx cerebri, multiple jaw keratocysts, and multiple basal cell carcinomas, but with different expressivity. One brother also had benign testicular mesothelioma. We propose this tumor type as a possible new feature of Gorlin syndrome. Gorlin syndrome is a rare autosomal dominant disorder characterized by both developmental abnormalities and cancer predisposition, with variable expression of various developmental abnormalities and different types of tumors. The syndrome is primarily caused by mutations in the Patched 1 (PTCH1) gene, although rare mutations of Patched 2 (PTCH2) or Suppressor of Fused (SUFU) genes have also been found. Neither founder mutations nor hot spot locations have been described for PTCH1 in Gorlin syndrome patients. Although de novo mutations of the PTCH1 gene occur in almost 50% of Gorlin syndrome cases, there are a few recurrent mutations. Our twin patients were carriers of a de novo mutation in the PTCH1 gene, c.3364_3365delAT (p.Met1122ValfsX22). This is, to our knowledge, the first Gorlin syndrome-causing mutation that has been reported four independent times in distant geographical locations. Therefore, we propose the location of the described mutation as a potential hot spot for mutations in PTCH1