Mouse models for cancer research - current state and the perspective.

Cancer is one of the leading causes of death worldwide. We still do not understand all the details of carcinogenesis, and effective treatment is lacking for many oncological diseases. Animal models provide an irreplaceable tool to observe the growth and spreading of neoplastic cells in an environment of living organisms, to test the efficacy of cancer treatment, side effects, and toxicity, and to study the tumor microenvironment. Mice are the most often used model organisms because of their easy handling, short reproductive period, multiple strains, and complete DNA sequencing. An ideal model should accurately recapitulate each step of tumor development. Recent techniques have established models that enable the study of different aspects of cancer, but choosing a particular model depends on the application of output data. This article aimed to review induced, transplantable, and engineered mice and highlight their significance for recent and future cancer research.

Cystathionine ß-synthase affects organization of cytoskeleton and modulates carcinogenesis in colorectal carcinoma cells.

Background: Cystathionine ß-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins ß-actin and ß-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. Methods: To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. Results: We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal ß-tubulin and, to a lesser extent, also to ß-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. Conclusions: Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC's effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs' changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

In vitro antiproliferative and cytotoxic activities of novel triphenyltin isoselenocyanate in human breast carcinoma cell lines MCF 7 and MDA-MB-231.

Intensive investigation for novel antiproliferative and cytotoxic effective chemical compounds is currently concentrated on structurally modified agents of natural or synthetic source. The selenium derivative of triorganotin compound, triphenyltin isoselenocyanate (TPT-NCSe) caused higher cytotoxicity in hormone sensitive MCF 7 (IC 50-250 nM) in comparison with triple-negative MDA-MB-231 breast carcinoma cell line (IC 50-450 nM) as determined by MTT assay. Measurement of DNA damage showed presence of crosslinks in both cell lines treated by increasing TPT-NCSe concentrations. This compound decreased mitochondrial membrane potential shown by JC-1 staining in a concentration-dependent manner in both cell lines. Activation of caspases-3/7 was observed in MDA-MB-231 cells and was significant only by concentrations causing significant level of crosslinks. On the other hand, migration assay revealed inhibitory effect of viability keeping 100 nM concentration of TPT-NCSe on migration of MDA-MB-231 cells. Our data has shown that this selenium containing triorganotin molecule exerts DNA damage-linked antineoplastic activity in breast carcinoma cell lines studied.

The evaluation of the efficacy and potential genotoxic hazard of combined SAHA and 5-FU treatment in the chemoresistant colorectal cancer cell lines.

5-Fluorouracil (5-FU) is an essential chemotherapeutic drug for colorectal cancer (CRC) treatment. However, the frequent development of drug resistance has dramatically affected its clinical use. Therefore, novel treatment strategies are critical to improving patient outcomes. Herein, we investigated the ability of the epigenetic drug SAHA to increase the sensitivity of chemoresistant CRC cells to 5-FU. In addition, we evaluated the potential genotoxic risk of SAHA+5-FU combination treatment. As a model system, we used three CRC cell lines, HT-29, SW480, and HT-29/EGFP/FUR, differing in their resistance to 5-FU. CRC cell lines were exposed to sub-toxic SAHA concentrations for 24 h, followed by a 48 h treatment with 5-FU. The cytotoxicity of SAHA, 5-FU, and SAHA+5-FU was measured by the MTT test, the genotoxicity by the comet assay, and the micronucleus test. The apoptotic/necrotic activity was assessed using morphological criteria. We found a synergic decrease in the viability of HT-29 and SW480 cells, but not the most resistant HT-29/EGFP/FUR cells after combined SAHA+5-FU exposure compared to 5-FU. Remarkably, SAHA most efficiently induced apoptosis in HT-29/EGFP/FUR cells compared to HT-29 and SW480 cells. Combined SAHA+5-FU treatment resulted in a synergistic increase in apoptotic/necrotic cells in HT-29 cell line, while rather additive/sub-additive effect was determined in the SW480 and HT-29/EGFP/FUR cells. At the same time, however, a synergistic rise in micronuclei was found in CRC cell lines (at least at some concentrations). We have shown that SAHA can sensitize CRC cells to 5-FU; therefore, epigenetic and convential drug combinations could be beneficial for the patients. However, the increase in micronucleus formation after combined SAHA+5-FU treatment indicates a potential health hazard. The clastogenic activity could contribute to cancer heterogeneity, favoring progeny of such aberrant cells to clonal expansion. Therefore, developing new specific epigenetic drugs or nanocarriers for targeted drug delivery might reduce the potential genotoxic risk.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

Development of LC-HRMS methods for evaluation of metabolic conversion of 5-fluorocytosine at GDEPT procedure.

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.

Overexpression of TNFα induces senescence, autophagy and mitochondrial dysfunctions in melanoma cells.

BACKGROUND: Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with both anti-tumorigenic and pro-tumorigenic activity, affecting tumor cell biology, the balance between cell survival and death. The final effect of TNFα is dependent on the type of malignant cells, with the potential to arrest cancer progression. METHODS: In order to explain the diverse cellular response to TNFα, we engineered melanoma and colorectal carcinoma cell lines stably overexpressing this cytokine. RESULTS: Under the TNFα overexpression, significant upregulation of two genes was observed: proinflammatory cytokine IL6 gene in melanoma cells A375 and gene for pro-apoptotic ligand TRAIL in colorectal carcinoma cells HT29, both mediated by TNFα/TNFR1 signaling. Malignant melanoma line A375 displayed also increased autophagy on day 3, followed by premature senescence on day 6. Both processes seem to be interconnected, following earlier apoptosis induction and deregulation of mitochondrial functions. We documented altered mitochondrial status, lowered ATP production, lowered mitochondrial mass, and changes in mitochondrial morphology (shortened and condensed mitochondria) both in melanoma and colorectal carcinoma cells. Overexpression of TNFα was not linked with significant affection of the subpopulation of cancer stem-like cells in vitro. However, we could demonstrate a decrease in aldehyde dehydrogenase (ALDH) activity up to 50%, which is associated with to the stemness phenotype. CONCLUSIONS: Our in vitro study of direct TNFα influence demonstrates two distinct outcomes in tumor cells of different origin, in non-epithelial malignant melanoma cells of neural crest origin, and in colorectal carcinoma cells derived from the epithelium.

Targeting of Deregulated Wnt/ß-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines.

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/ß-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of ß-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of ß-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/ß-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/ß-catenin pathway inhibition in GCTs is therefore warranted.

Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy.

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

Molecular features and gene expression signature of metastatic colorectal cancer (Review).

Uncontrollable metastatic outgrowth process is the leading cause of mortality worldwide, even in the case of colorectal cancer. Colorectal cancer (CRC) accounts for approximately 10% of all annually diagnosed cancers and 50% of CRC patients will develop metastases in the course of disease. Most patients with metastatic CRC have incurable disease. Even if patients undergo resection of liver metastases, the 5­year survival rate ranges from 25 to 58%. Next­generation sequencing of tumour specimens from large colorectal cancer patient cohorts has led to major advances in elucidating the genomic landscape of these tumours and paired metastases. The expression profiles of primary CRC and their metastatic lesions at both the gene and pathway levels were compared and led to the selection of early driver genes responsible for carcinogenesis and metastasis­specific genes that increased the metastatic process. The genetic, transcriptional and epigenetic alteration encoded by these genes and their combination influence many pivotal signalling pathways, enabling the dissemination and outgrowth in distant organs. Therapeutic regimens affecting several different active pathways may have important implications for therapeutic efficacy.

Calcium signaling affects migration and proliferation differently in individual cancer cells due to nifedipine treatment.

Several papers have reported that calcium channel blocking drugs were associated with increased breast cancer risk and worsened prognosis. One of the most common signs of breast tumors is the presence of small deposits of calcium, known as microcalcifications. Therefore, we studied the effect of dihydropyridine nifedipine on selected calcium transport systems in MDA-MB-231 cells, originating from triple negative breast tumor and JIMT1 cells that represent a model of HER2-positive breast cancer, which possesses amplification of HER2 receptor, but cells do not response to HER2 inhibition treatment with trastuzumab. Also, we compared the effect of nifedipine on colorectal DLD1 and ovarian A2780 cancer cells. Both, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and type 1 sodium calcium exchanger (NCX1) were upregulated due to nifedipine in DLD1 and A2780 cells, but not in breast cancer MDA-MB-231 and JIMT1 cells. On contrary to MDA-MB-231 and JIMT1 cells, in DLD1 and A2780 cells nifedipine induced apoptosis in a concentration-dependent manner. After NCX1 silencing and subsequent treatment with nifedipine, proliferation was decreased in MDA-MB-231, increased in DLD1 cells, and not changed in JIMT1 cells. Silencing of IP3R1 revealed increase in proliferation in DLD1 and JIMT1 cells, but caused decrease in proliferation in MDA-MB-231 cell line after nifedipine treatment. Interestingly, after nifedipine treatment migration was not significantly affected in any of tested cell lines after NCX1 silencing. Due to IP3R1 silencing, significant decrease in migration occurred in MDA-MB-231 cells after nifedipine treatment, but not in other tested cells. These results support different function of the NCX1 and IP3R1 in the invasiveness of various cancer cells due to nifedipine treatment.

Disulfiram Overcomes Cisplatin Resistance in Human Embryonal Carcinoma Cells.

Cisplatin resistance in testicular germ cell tumors (TGCTs) is a clinical challenge. We investigated the underlying mechanisms associated with cancer stem cell (CSC) markers and modalities circumventing the chemoresistance. Chemoresistant models (designated as CisR) of human embryonal carcinoma cell lines NTERA-2 and NCCIT were derived and characterized using flow cytometry, gene expression, functional and protein arrays. Tumorigenicity was determined on immunodeficient mouse model. Disulfiram was used to examine chemosensitization of resistant cells. ALDH1A3 isoform expression was evaluated by immunohistochemistry in 216 patients' tissue samples. Chemoresistant cells were significantly more resistant to cisplatin, carboplatin and oxaliplatin compared to parental cells. NTERA-2 CisR cells exhibited altered morphology and increased tumorigenicity. High ALDH1A3 expression and increased ALDH activity were detected in both refractory cell lines. Disulfiram in combination with cisplatin showed synergy for NTERA-2 CisR and NCCIT CisR cells and inhibited growth of NTERA-2 CisR xenografts. Significantly higher ALDH1A3 expression was detected in TGCTs patients' tissue samples compared to normal testicular tissue. We characterized novel clinically relevant model of chemoresistant TGCTs, for the first time identified the ALDH1A3 as a therapeutic target in TGCTs and more importantly, showed that disulfiram represents a viable treatment option for refractory TGCTs.

Recent advances in understanding tumor stroma-mediated chemoresistance in breast cancer.

Although solid tumors comprise malignant cells, they also contain many different non-malignant cell types in their micro-environment. The cellular components of the tumor stroma consist of immune and endothelial cells combined with a heterogeneous population of stromal cells which include cancer-associated fibroblasts. The bi-directional interactions between tumor and stromal cells therefore substantially affect tumor cell biology.Herein, we discuss current available information on these interactions in breast cancer chemo-resistance. It is acknowledged that stromal cells extrinsically alter tumor cell drug responses with profound consequences for therapy efficiency, and it is therefore essential to understand the molecular mechanisms which contribute to these substantial alterations because they provide potential targets for improved cancer therapy. Although breast cancer patient survival has improved over the last decades, chemo-resistance still remains a significant obstacle to successful treatment.Appreciating the important experimental evidence of mesenchymal stromal cells and cancer-associated fibroblast involvement in breast cancer clinical practice can therefore have important therapeutic implications.

Sodium/calcium exchanger is involved in apoptosis induced by H2S in tumor cells through decreased levels of intracellular pH.

We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S-induced apoptosis in tumor cells.

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Although the involvement of type 1 (IP3R1) and type 2 (IP3R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP3R (IP3R3) is still elusive. Therefore, in this work we focused on the role of IP3R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP3R3 compared to IP3R1 and IP3R2 in tumor cells. Silencing of IP3R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP3R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP3R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP3R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP3R3_del cells than in DLD1 cells. These results clearly show that IP3R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP3R1.

ALDH1A3 upregulation and spontaneous metastasis formation is associated with acquired chemoresistance in colorectal cancer cells.

BACKGROUND: Efficiency of colorectal carcinoma treatment by chemotherapy is diminished as the resistance develops over time in patients. The same holds true for 5-fluorouracil, the drug used in first line chemotherapy of colorectal carcinoma. METHODS: Chemoresistant derivative of HT-29 cells was prepared by long-term culturing in increasing concentration of 5-fluorouracil. Cells were characterized by viability assays, flow cytometry, gene expression arrays and kinetic imaging. Immunomagnetic separation was used for isolation of subpopulations positive for cancer stem cells-related surface markers. Aldehyde dehydrogenase expression was attenuated by siRNA. In vivo studies were performed on SCID/bg mice. RESULTS: The prepared chemoresistant cell line labeled as HT-29/EGFP/FUR is assigned with different morphology, decreased proliferation rate and 135-fold increased IC50 value for 5-fluorouracil in comparison to parental counterparts HT-29/EGFP. The capability of chemoresistant cells to form tumor xenografts, when injected subcutaneously into SCID/bg mice, was strongly compromised, however, they formed distant metastases in mouse lungs spontaneously. Derived cells preserved their resistance in vitro and in vivo even without the 5-fluorouracil selection pressure. More importantly, they were resistant to cisplatin, oxaliplatin and cyclophosphamide exhibiting high cross-resistance along with alterations in expression of cancer-stem cell markers such as CD133, CD166, CD24, CD26, CXCR4, CD271 and CD274. We also detected increased aldehyde dehydrogenase (ALDH) activity associated with overexpression of specific ALDH isoform 1A3. Its inhibition by siRNA approach partially sensitized cells to various agents, thus linking for the first time the ALDH1A3 and chemoresistance in colorectal cancer. CONCLUSION: Our study demonstrated that acquired chemoresistance goes along with metastatic and migratory phenotype and can be accompanied with increased activity of aldehyde dehydrogenase. We describe here the valuable model to study molecular link between resistance to chemotherapy and metastatic dissemination.

Cytotoxic response of 5-fluorouracil-resistant cells to gene- and cell-directed enzyme/prodrug treatment.

Gene-directed enzyme/prodrug therapy (GDEPT) mediated by mesenchymal stromal cells (MSC) was already approved for clinical study on a progressive disease refractory to standard therapy. In this work, we examined the effect of several GDEPT approaches on chemoresistant cells. First, we derived 5-fluorouracil (5-FU)-resistant variant of human colorectal adenocarcinoma cells HT-29 designated HT-29/EGFP/FUR. Our data show that the upregulation of thymidylate synthase (TS) and downregulation of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) contributed to the 5-FU resistance in cancer cells. Next, we combined the MSC expressing either yeast cytosine deaminase (CD-MSC) or fusion yeast CD::uracil phosphoribosyl transferase (CD::UPRT-MSC) and prodrug 5-fluorocytosine (5-FC) in a cell-mediated GDEPT approach. Bystander cytotoxic effect in the direct co-cultures of the tumor and therapeutic cells mixed in a 5:1 ratio resulted in 55% and 70% inhibition of proliferation, respectively. However, the acquired chemoresistance to 5-FU can be overcome by introducing the prodrug-converting transgene into the tumor cells. When the transgene CD::UPRT was expressed in the chemoresistant cells (CD::UPRT-FUR), substantial suicide effect and a 90% decrease in viability was observed using non-toxic concentration of 62.5 µg/ml 5-FC. In summary, we demonstrate here that the transgene introduction circumvented 5-FU resistance in the tumor cells.

Targeted antitumor therapy mediated by prodrug-activating mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) were introduced as tumor-targeted vehicles suitable for delivery of the gene-directed enzyme/prodrug therapy more than 10 years ago. Over these years key properties of tumor cells and MSCs, which are crucial for the treatment efficiency, were examined; and there are some critical issues to be considered for the maximum antitumor effect. Moreover, engineered MSCs expressing enzymes capable of activating non-toxic prodrugs achieved long-term curative effect even in metastatic and hard-to-treat tumor types in pre-clinical scenario(s). These gene-modified MSCs are termed prodrug-activating MSCs throughout the text and represent promising approach for further clinical application. This review summarizes major determinants to be considered for the application of the prodrug-activating MSCs in antitumor therapy in order to maximize therapeutic efficiency.

Metastatic Ovarian Cancer Can Be Efficiently Treated by Genetically Modified Mesenchymal Stromal Cells.

Due to late diagnosis, often recurrence, formation of metastases and resistance to commonly used chemotherapeutics human ovarian carcinoma represents a serious disease with high mortality. Adipose tissue-derived mesenchymal stromal cells (AT-MSC) can serve as vehicles for therapeutic genes and we engineered AT-MSC to express either Herpes simplex virus thymidine kinase (HSVtk-MSC), which phosphorylates ganciclovir (GCV) to its toxic metabolites or yeast fused cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT-MSC), which converts 5-fluorocytosine (5-FC) to highly toxic 5-fluorouracil (5-FU). Here, we reported different responses of cytotoxicity mediated by CD::UPRT-MSC/5-FC treatment on human ovarian carcinoma cell lines-SKOV-3 and A2780 used in adherent or three-dimensional (3D) cell culture and we proved high potential of 3D model to predict results in our in vivo experiments. Both tumor cell lines showed similarly high chemosensitivity to the used treatment in adherent culture, but 3D model revealed severe discrepancy-only 36% of SKOV-3 cells but even 90% of A2780 cells were eliminated. This result served as a prognostic marker-we were able to achieve significantly decreased tumor volumes of subcutaneous xenografts of A2780 cells in nude mice and we prolonged tumor-free survival in 33% of animals bearing highly metastatic ovarian carcinoma after CD::UPRT-MSC/5-FC treatment.