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.

Tyrosine kinase inhibitor SU11274 increased tumorigenicity and enriched for melanoma-initiating cells by bioenergetic modulation.

BACKGROUND: Small molecule inhibitor of tyrosine kinase activity, compound SU11274, was reported to have antitumorigenic and antimetastatic effect in melanoma. In this study, we evaluated, whether similar effect could be achieved also in other melanoma cells including highly tumorigenic and hypermetastatic variant. METHODS: The effect of SU11274 was evaluated in adherent and non-adherent melanosphere cultures of human melanoma cells M14, M4Beu, A375 and EGFP-A375/Rel3. Tumorigenicity of SU11274-treated cells was tested by limiting dilution assay in xenograft model in vivo. RESULTS: Here we show that SU11274 enriched for melanoma-initiating cells in vivo. SU11274 substantially decreased number of cells in adherent and spheroid cultures, but increased their tumorigenic potential as determined by higher frequency of tumor-initiating cells in vivo. SU11274 treatment was not associated with any significant alteration in the expression of stem cell markers, but the inhibitor stimulated higher level of pluripotent markers. SU11274-treated melanoma cells exhibited higher ATP content and lactate release indicative of increased glycolysis. Our data suggest that the SU11274 altered bioenergetic state of the cells. Indeed, pharmacological intervention with a glycolytic inhibitor dichloroacetate significantly reduced SU11274-promoted increase in melanoma-initiating cells and decreased their tumorigenicity. CONCLUSIONS: Our data suggest critical role of glycolysis regulation in melanoma-initiating cells. Moreover, these data unravel substantial plasticity of melanoma cells and their adoptive mechanisms, which result in ambivalent response to therapeutic targeting.

Genetically engineered mesenchymal stromal cells producing TNFα have tumour suppressing effect on human melanoma xenograft.

BACKGROUND: Mesenchymal stromal cells (MSC) are a promising tool for targeted cancer therapy due to their tumour-homing ability. Intrinsic resistance enables the MSC to longer tolerate therapeutic factors, such as prodrug converting enzymes, cytokines and pro-apoptotic proteins. Tumour necrosis factor alpha (TNFα) is known to be cytotoxic to a variety of cancer cells and exert a tumour-destructive capacity. METHODS: MSC were retrovirally transduced to stable express an exogenous gene encoding the desired therapeutic agent hTNFα. The effect of a TNFα-producing adipose tissue-derived MSC (AT-MSC/hTNFα) was tested on the tumour cell lines of different origins: melanoma (A375), breast carcinoma (SKBR3, MDA-MB-231), colon carcinoma (HT29), ovarian carcinoma (SKOV3) and glioblastoma (U87-MG) cells. The tumour suppressing effect of AT-MSC/hTNFα on A375 melanoma xenografts was monitored in an immunodeficient mouse model in vivo. RESULTS: Engineered AT-MSC are able to constitutively secrete human TNFα protein, induce apoptosis of tumour cell lines via caspase 3/7 activation and inhibit the tumour cell proliferation in vitro. Melanoma A375 and breast carcinoma SKBR3 cells were the most sensitive, and their proliferation in vitro was reduced by conditioned media produced by AT-MSC/hTNFα to 60% and 40%, respectively. The previously reported tumour supportive effect of AT-MSC on subcutaneous A375 melanoma xenograft growth was neutralised and suppressed by engineered AT-MSC stably producing hTNFα. When AT-MSC/hTNFα were coinjected with A375 melanoma cells, the tumour mass inhibition was up to 97.5%. CONCLUSIONS: The results of the present study demonstrate that tumour cells respond to hTNFα-based treatment mediated by genetically engineered AT-MSC/hTNFα both in vitro and in vivo.

Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice.

BACKGROUND: Previously, we validated capability of human adipose tissue-derived mesenchymal stem cells (AT-MSC) to serve as cellular vehicles for gene-directed enzyme prodrug molecular chemotherapy. Yeast fusion cytosine deaminase : uracil phosphoribosyltransferase expressing AT-MSC (CD y-AT-MSC) combined with systemic 5-fluorocytosine (5FC) significantly inhibited growth of human colon cancer xenografts. We aimed to determine the cytotoxic efficiency to other tumour cells both in vitro and in vivo. METHODS: CD y-AT-MSC/5FC-mediated proliferation inhibition against a panel of human tumour cells lines was evaluated in direct and indirect cocultures in vitro. Antitumour effect was tested on immunodeficient mouse model in vivo. RESULTS: Although culture expansion of CD y-AT-MSC sensitized these cells to 5FC mediated suicide effect, expanded CD y-AT-MSC/5FC still exhibited strong bystander cytotoxic effect towards human melanoma, glioblastoma, colon, breast and bladder carcinoma in vitro. Most efficient inhibition (91%) was observed in melanoma A375 cell line when directly cocultured with 2% of therapeutic cells CD y-AT-MSC/5FC. The therapeutic paradigm of the CD y -AT-MSC/5FC system was further evaluated on melanoma A375 xenografts on nude mice in vivo. Complete regression in 89% of tumours was achieved when 20% CD y-AT-MSC/5FC were co-injected along with tumour cells. More importantly, systemic CD y-AT-MSC administration resulted in therapeutic cell homing into subcutaneous melanoma and mediated tumour growth inhibition. CONCLUSIONS: CD y-AT-MSC capability of targeting subcutaneous melanoma offers a possibility to selectively produce cytotoxic agent in situ. Our data further demonstrate beneficial biological properties of AT-MSC as a cellular vehicle for enzyme/prodrug therapy approach to molecular chemotherapy.

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.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

BACKGROUND: Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. METHODS: Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. RESULTS: We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. CONCLUSIONS: Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine.

In our work, we have evaluated efficiency of gene-directed enzyme/prodrug therapy (GDEPT) based on combination of fusion yeast cytosine deaminase (yCD) and 5-fluorocytosine (5FC) on model human medullary thyroid carcinoma (MTC) cell line TT. We determined the efficiency of this GDEPT approach in suicide and bystander cytotoxicity induction. We have shown significant bystander effect in vitro and 5FC administration resulted in potent antitumor effect in vivo. Furthermore, we have unraveled high efficiency of cell-mediated GDEPT, when human mesenchymal stromal cells (MSC) were used as delivery vehicles in direct cocultures in vitro. Nevertheless, effector MSC exhibited inhibitory effect on TT cell proliferation and abrogated TT xenotransplant growth in vivo. We suggest that yCD/5FC combination represents another experimental treatment modality to be tested in MTC and our data further support the exploration of MSC antitumor potential for future use in metastatic MTC therapy.