Increased serum concentrations of tumor necrosis factor α (TNFα) and transforming growth factor β-1 (TGFβ-1) in the blood of patients with pancreatic cancer (PC) have previously been demonstrated. In addition, exogenous exposure to these cytokines promotes various cancer cell invasive and cancer stem cell (CSC) phenotypes. However, their importance in pancreatic CSCs remains elusive. In the present study, the effects of TNFα and TGFβ-1 on the human PC cell line MiaPaCa-2 were examined. Using flow cytometry, it was revealed that TNFα and TGFβ-1 synergistically increase cluster of differentiation (CD) 44v6, CD133 and ATP-binding cassette transporter G2 (ABCG2) expressing populations in adherent tumor cell culture conditions. Furthermore, a similar trend was observed in cells pretreated with these cytokines grown in sphere forming culture conditions. Similar to previous studies, TNFα treatment increased the proportion of epidermal growth factor receptor (EGFR) expressing cells in adherent culture, and this data was further supported by the results of the sphere formation assay, in which the subculture with a high proportion of EGFR expressing cells exhibited the most efficient sphere forming ability. However, the proportion of vascular endothelial growth factor receptor 1 (VEGFR1) expressing cells did not increase upon treatment with these cytokines individually or in combination. This data was subsequently supported by the results of the wound healing assay in which cytokine treatment did not increase the migration of cells. The MTT cell proliferation and cytotoxicity assay revealed that TNFα + TGFβ-1 treatment significantly increased cell proliferation and daunorubicin resistance, but not gemcitabine resistance. In conclusion, the data of the current study provide a mechanistic association between TNFα, TGFβ-1 and the CSC properties of MiaPaCa-2 cells. In addition, it suggests that targeting TNFα and TGFβ-1 is beneficial for improving the therapeutic efficacy of treatments for patients with PC.

Failure of antitumor immunity in cancer was shown to be mediated by myeloid-derived suppressor cells (MDSCs), which are considered to be one of the key factors contributing to the development of malignant diseases. Therefore, the development of pharmacological approaches to effectively eliminate MDSCs in organisms carrying growing tumors is a promising pathway for potential treatment. For this purpose we propose alpha-fetoprotein (AFP) conjugated with a cytotoxic agent as a vector molecule, specifically recognizing MDSCs. The present study was aimed at examination of this suggestion using both in vitro and in vivo approaches. MDSCs, obtained from the spleen of Ehrlich carcinoma bearing mice, selectively bound AFP labeled with fluorescein isothiocyanate. AFP conjugated to daunorubicin (AFP-DR) and DR alone showed similar in vitro cytotoxicity against the granulocytic MDSC subpopulation. The monocytic MDSC subpopulation was resistant to treatment with DR, whereas it was completely depleted in the presence of AFP-DR. Treatment of mice bearing Ehrlich carcinoma with AFP-DR resulted in reduced numbers of splenic MDSCs, normalization of NK cell levels, and inhibition of tumor growth. The obtained results demonstrate that cytotoxic conjugates based on AFP are promising anticancer drugs, which, in addition to the direct effect on tumor cells expressing receptors to AFP, may contribute to elimination of MDSCs.

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