Comparison of primary and passaged tumor cell cultures and their application in personalized medicine.

Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.

Molecular Mechanisms of Tumor Cell Stemness Modulation during Formation of Spheroids.

Cancer stem cells (CSCs), their properties and interaction with microenvironment are of interest in modern medicine and biology. There are many studies on the emergence of CSCs and their involvement in tumor pathogenesis. The most important property inherent to CSCs is their stemness. Stemness combines ability of the cell to maintain its pluripotency, give rise to differentiated cells, and interact with environment to maintain a balance between dormancy, proliferation, and regeneration. While adult stem cells exhibit these properties by participating in tissue homeostasis, CSCs behave as their malignant equivalents. High tumor resistance to therapy, ability to differentiate, activate angiogenesis and metastasis arise precisely due to the stemness of CSCs. These cells can be used as a target for therapy of different types of cancer. Laboratory models are needed to study cancer biology and find new therapeutic strategies. A promising direction is three-dimensional tumor models or spheroids. Such models exhibit properties resembling stemness in a natural tumor. By modifying spheroids, it becomes possible to investigate the effect of therapy on CSCs, thus contributing to the development of anti-tumor drug test systems. The review examines the niche of CSCs, the possibility of their study using three-dimensional spheroids, and existing markers for assessing stemness of CSCs.

Cytochalasin B-Induced Membrane Vesicles from TRAIL-Overexpressing Mesenchymal Stem Cells Induce Extrinsic Pathway of Apoptosis in Breast Cancer Mouse Model.

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

Comparative Analysis of Natural and Cytochalasin B-Induced Membrane Vesicles from Tumor Cells and Mesenchymal Stem Cells.

To date, there are numerous protocols for the isolation of extracellular vesicles (EVs). Depending on the isolation method, it is possible to obtain vesicles with different characteristics, enriched with specific groups of proteins, DNA and RNA, which affect similar types of cells in the opposite way. Therefore, it is important to study and compare methods of vesicle isolation. Moreover, the differences between the EVs derived from tumor and mesenchymal stem cells are still poorly understood. This article compares EVs from human glioblastoma cells and mesenchymal stem cells (MSCs) obtained by two different methods, ultracentrifugation and cytochalasin B-mediated induction. The size of the vesicles, the presence of the main EV markers, the presence of nuclear and mitochondrial components, and the molecular composition of the vesicles were determined. It has been shown that EVs obtained by both ultracentrifugation and cytochalasin B treatment have similar features, contain particles of endogenous and membrane origin and can interact with monolayer cultures of tumor cells.

The Dual Role of Mesenchymal Stromal Cells and Their Extracellular Vesicles in Carcinogenesis.

Mesenchymal stem cells (MSCs) are a major component of the tumor microenvironment (TME) and play an important role in tumor progression. MSCs remodel the extracellular matrix, participate in the epithelial-mesenchymal transition, promote the spread of metastases, and inhibit antitumor immune responses in the TME; however, there are also data pertaining to the antitumor effects of MSCs. MSCs activate the cell death mechanism by modulating the expression of proteins involved in the regulation of the cell cycle, angiogenesis receptors, and proapoptotic proteins. One of the main ways in which MSCs and TME interact is through the production of extracellular vesicles (EVs) by cells. Currently, data on the effects of both MSCs and their EVs on tumor cells are rather contradictory. Various studies have reported that EVs from MSCs can have either antitumor or pro-tumor effects, depending on both the tumor type and developmental stage. In this review, we discuss published data on EV MSCs and their effect on tumor cells. The molecular composition of vesicles obtained from MSCs is also presented in the review. In addition, the use of EV MSCs for the development of new methods for treating oncological diseases is described.

Intrinsic and Extrinsic Factors Impacting Cancer Stemness and Tumor Progression.

Tumor heterogeneity represents an important limitation to the development of effective cancer therapies. The presence of cancer stem cells (CSCs) and their differentiation hierarchies contribute to cancer complexity and confer tumors the ability to grow, resist treatment, survive unfavorable conditions, and invade neighboring and distant tissues. A large body of research is currently focusing on understanding the properties of CSCs, including their cellular and molecular origin, as well as their biological behavior in different tumor types. In turn, this knowledge informs strategies for targeting these tumor initiating cells and related cancer stemness. Cancer stemness is modulated by the tumor microenvironment, which influences CSC function and survival. Several advanced in vitro models are currently being developed to study cancer stemness in order to advance new knowledge of the key molecular pathways involved in CSC self-renewal and dormancy, as well as to mimic the complexity of patients' tumors in pre-clinical drug testing. In this review, we discuss CSCs and the modulation of cancer stemness by the tumor microenvironment, stemness factors and signaling pathways. In addition, we introduce current models that allow the study of CSCs for the development of new targeted therapies.

Cytochalasin B-induced membrane vesicles from human mesenchymal stem cells overexpressing TRAIL, PTEN and IFN-ß1 can kill carcinoma cancer cells.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are of interest as a new vector for the delivery of therapeutic agents into the tumor microenvironment. Cell-free EV-based therapy has a number of advantages over cell-based therapy, since the use of EVs allows avoiding potential undesirable transformation associated with MSCs. MSC-derived EVs can transfer natural proteins with immunomodulatory or antitumor properties. The aim of this study was to produce vesicles from mesenchymal stem cells with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 and analyze its antitumor and immunomodulatory properties. In this work, a stable line of human adipose tissue-derived mesenchymal stem cells (hADSCs) with simultaneous overexpression of TRAIL, PTEN and IFN-ß1 was produced. To obtain this cell line hADSCs were genetically modified with a genetic multicistronic cassette encoding TRAIL, PTEN, and IFN-ß1 genes separated with a self-cleaving P2A peptide nucleotide sequence. Membrane vesicles (CIMVs) were obtained from genetically modified hADSCs using cytochalasin B treatment. Antitumor and immunomodulatory properties of the CIMVs were analyzed in vitro. It was shown that CIMVs isolated from genetically modified hADSCs overexpressing TRAIL, PTEN and IFN-ß1 genes are able to activate human immune cells and induce apoptosis in various types of carcinomas in vitro. Thus, the immunomodulatory and antitumor properties of CIMVs were shown. However, further studies on animal models in vivo are required.

Cytochalasin B-Induced Membrane Vesicles from Human Mesenchymal Stem Cells Overexpressing IL2 Are Able to Stimulate CD8+ T-Killers to Kill Human Triple Negative Breast Cancer Cells.

Interleukin 2 (IL2) was one of the first cytokines used for cancer treatment due to its ability to stimulate anti-cancer immunity. However, recombinant IL2-based therapy is associated with high systemic toxicity and activation of regulatory T-cells, which are associated with the pro-tumor immune response. One of the current trends for the delivery of anticancer agents is the use of extracellular vesicles (EVs), which can carry and transfer biologically active cargos into cells. The use of EVs can increase the efficacy of IL2-based anti-tumor therapy whilst reducing systemic toxicity. In this study, human adipose tissue-derived mesenchymal stem cells (hADSCs) were transduced with lentivirus encoding IL2 (hADSCs-IL2). Membrane vesicles were isolated from hADSCs-IL2 using cytochalasin B (CIMVs-IL2). The effect of hADSCs-IL2 and CIMVs-IL2 on the activation and proliferation of human peripheral blood mononuclear cells (PBMCs) as well as the cytotoxicity of activated PBMCs against human triple negative cancer MDA-MB-231 and MDA-MB-436 cells were evaluated. The effect of CIMVs-IL2 on murine PBMCs was also evaluated in vivo. CIMVs-IL2 failed to suppress the proliferation of human PBMCs as opposed to hADSCs-IL2. However, CIMVs-IL2 were able to activate human CD8+ T-killers, which in turn, killed MDA-MB-231 cells more effectively than hADSCs-IL2-activated CD8+ T-killers. This immunomodulating effect of CIMVs-IL2 appears specific to human CD8+ T-killer cells, as the same effect was not observed on murine CD8+ T-cells. In conclusion, the use of CIMVs-IL2 has the potential to provide a more effective anti-cancer therapy. This compelling evidence supports further studies to evaluate CIMVs-IL2 effectiveness, using cancer mouse models with a reconstituted human immune system.

Promising Applications of Tumor Spheroids and Organoids for Personalized Medicine.

One of the promising directions in personalized medicine is the use of three-dimensional (3D) tumor models such as spheroids and organoids. Spheroids and organoids are three-dimensional cultures of tumor cells that can be obtained from patient tissue and, using high-throughput personalized medicine methods, provide a suitable therapy for that patient. These 3D models can be obtained from most types of tumors, which provides opportunities for the creation of biobanks with appropriate patient materials that can be used to screen drugs and facilitate the development of therapeutic agents. It should be noted that the use of spheroids and organoids would expand the understanding of tumor biology and its microenvironment, help develop new in vitro platforms for drug testing and create new therapeutic strategies. In this review, we discuss 3D tumor spheroid and organoid models, their advantages and disadvantages, and evaluate their promising use in personalized medicine.

Therapeutic Potential of Pharmacological Targeting NLRP3 Inflammasome Complex in Cancer.

Introduction: Dysregulation of NLRP3 inflammasome complex formation can promote chronic inflammation by increased release of IL-1ß. However, the effect of NLRP3 complex formation on tumor progression remains controversial. Therefore, we sought to determine the effect of NLRP3 modulation on the growth of the different types of cancer cells, derived from lung, breast, and prostate cancers as well as neuroblastoma and glioblastoma in-vitro. Method: The effect of Caspase 1 inhibitor (VX765) and combination of LPS/Nigericin on NLRP3 inflammasome activity was analyzed in A549 (lung cancer), MCF-7 (breast cancer), PC3 (prostate cancer), SH-SY5Y (neuroblastoma), and U138MG (glioblastoma) cells. Human fibroblasts were used as control cells. The effect of VX765 and LPS/Nigericin on NLRP3 expression was analyzed using western blot, while IL-1ß and IL-18 secretion was detected by ELISA. Tumor cell viability and progression were determined using Annexin V, cell proliferation assay, LDH assay, sphere formation assay, transmission electron microscopy, and a multiplex cytokine assay. Also, angiogenesis was investigated by a tube formation assay. VEGF and MMPs secretion were detected by ELISA and a multiplex assay, respectively. Statistical analysis was done using one-way ANOVA with Tukey's analyses and Kruskal-Wallis one-way analysis of variance. Results: LPS/Nigericin increased NRLP3 protein expression as well as IL-1ß and IL-18 secretion in PC3 and U138MG cells compared to A549, MCF7, SH-SY5Y cells, and fibroblasts. In contrast, MIF expression was commonly found upregulated in A549, PC3, SH-SY5Y, and U138MG cells and fibroblasts after Nigericin treatment. Nigericin and a combination of LPS/Nigericin decreased the cell viability and proliferation. Also, LPS/Nigericin significantly increased tumorsphere size in PC3 and U138MG cells. In contrast, the sphere size was reduced in MCF7 and SH-SY5Y cells treated with LPS/Nigericin, while no effect was detected in A549 cells. VX765 increased secretion of CCL24 in A549, MCF7, PC3, and fibroblasts as well as CCL11 and CCL26 in SH-SY5Y cells. Also, VX765 significantly increased the production of VEGF and MMPs and stimulated angiogenesis in all tumor cell lines. Discussion: Our data suggest that NLRP3 activation using Nigericin could be a novel therapeutic approach to control the growth of tumors producing a low level of IL-1ß and IL-18.

MicroRNA Post-transcriptional Regulation of the NLRP3 Inflammasome in Immunopathologies.

Inflammation has a crucial role in protection against various pathogens. The inflammasome is an intracellular multiprotein signaling complex that is linked to pathogen sensing and initiation of the inflammatory response in physiological and pathological conditions. The most characterized inflammasome is the NLRP3 inflammasome, which is a known sensor of cell stress and is tightly regulated in resting cells. However, altered regulation of the NLRP3 inflammasome is found in several pathological conditions, including autoimmune disease and cancer. NLRP3 expression was shown to be post-transcriptionally regulated and multiple miRNA have been implicated in post-transcriptional regulation of the inflammasome. Therefore, in recent years, miRNA based post-transcriptional control of NLRP3 has become a focus of much research, especially as a potential therapeutic approach. In this review, we provide a summary of the recent investigations on the role of miRNA in the post-transcriptional control of the NLRP3 inflammasome, a key regulator of pro-inflammatory IL-1ß and IL-18 cytokine production. Current approaches to targeting the inflammasome product were shown to be an effective treatment for diseases linked to NLRP3 overexpression. Although utilizing NLRP3 targeting miRNAs was shown to be a successful therapeutic approach in several animal models, their therapeutic application in patients remains to be determined.

In vitro assessment of electrospun polyamide-6 scaffolds for esophageal tissue engineering.

Artificial tissue-engineered grafts offer a potential alternative to autologous tissue grafts for patients, which can be traumatic. After decellularizing Papio hamadryas esophagus and studying the morphology and physical properties of the extracellular matrix (ECM), we generated electrospun polyamide-6 based scaffolds to mimic it. The scaffolds supported a greater mechanical load than the native ECM and demonstrated similar 3D microstructure, with randomly aligned fibers, 90% porosity, 29 µm maximal pore size, and average fiber diameter of 2.87 ± 0.95 µm. Biocompatibility studies showed that human adipose- and bone marrow-derived mesenchymal stromal cells (AD-MSC and BMD-MSC) adhered to the scaffold surface and showed some proliferation: scaffold cell coverage was 25% after 72 h of incubation when seeded with 1000 cells/mm2 ; cells elongated processes along the polyamide-6, although they flattened 1.67-4 times less than on cell culture plastic. Human umbilical vein endothelial cells, however, showed poor adherence and proliferation. We thus provide in vitro evidence that polyamide-6 scaffolds approximating the esophageal biomechanics and 3D topography of nonhuman primates may provide a biocompatible substrate for both AD-MSC and BMD-MSCs, supporting their adhesion and survival to some degree. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 253-268, 2019.

Allogenic Adipose Derived Stem Cells Transplantation Improved Sciatic Nerve Regeneration in Rats: Autologous Nerve Graft Model.

We examined the effect of transplantation of allogenic adipose-derived stem cells (ADSCs) with properties of mesenchymal stem cells (MSCs) on posttraumatic sciatic nerve regeneration in rats. We suggested an approach to rat sciatic nerve reconstruction using the nerve from the other leg as a graft. The comparison was that of a critical 10 mm nerve defect repaired by means of autologous nerve grafting versus an identical lesion on the contralateral side. In this experimental model, the same animal acts simultaneously as a test model, and control. Regeneration of the left nerve was enhanced by the use of ADSCs, whereas the right nerve healed under natural conditions. Thus the effects of individual differences were excluded and a result closer to clinical practice obtained. We observed significant destructive changes in the sciatic nerve tissue after surgery which resulted in the formation of combined contractures in knee and ankle joints of both limbs and neurotrophic ulcers only on the right limb. The stimulation of regeneration by ADSCs increased the survival of spinal L5 ganglia neurons by 26.4%, improved sciatic nerve vascularization by 35.68% and increased the number of myelin fibers in the distal nerve by 41.87%. Moreover, we have demonstrated that S100, PMP2, and PMP22 gene expression levels are suppressed in response to trauma as compared to intact animals. We have shown that ADSC-based therapy contributes to significant improvement in the regeneration.