The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

Multimodal evaluation of 19F-BPA internalization in pancreatic cancer cells for boron capture and proton therapy potential applications.

PURPOSE: One of the obstacles to the application of Boron Neutron Capture Therapy (BNCT) and Proton Boron Fusion Therapy (PBFT) concerns the measurement of borated carriers' biodistribution. The objective of the present study was to evaluate the in vitro internalization of the 19F-labelled p-boronophenylalanine (19F-BPA) in the human cancer pancreatic cell line (PANC-1) for the potential application of BNCT and PBFT in pancreatic cancer. The 19F-BPA carrier has the advantage that its bio-distribution may be monitored in vivo using 19F-Nuclear Magnetic Resonance (19F NMR). MATERIALS AND METHODS: The 19F-BPA internalization in PANC-1 cells was evaluated using three independent techniques on cellular samples left in contact with growing medium enriched with 13.6 mM 19F-BPA corresponding to a 11B concentration of 120 ppm: neutron autoradiography, which quantifies boron; liquid chromatography hyphenated to tandem mass spectrometry and UV-Diode Array Detection (UV-DAD), which quantifies 19F-BPA molecule; and 19F NMR spectroscopy, which detects fluorine nuclei. RESULTS: Our studies suggested that 19F-BPA is internalized by PANC-1 cells. The three methods provided consistent results of about 50% internalization fraction at 120 ppm of 11B. Small variations (less than 15%) in internalization fraction are mainly dependent on the proliferation state of the cells. CONCLUSIONS: The ability of 19F NMR spectroscopy to study 19F-BPA internalization was validated by well-established independent techniques. The multimodal approach we used suggests 19F-BPA as a promising BNCT/PBFT carrier for the treatment of pancreatic cancer. Since the quantification is performed at doses useful for BNCT/PBFT, 19F NMR can be envisaged to monitor 19F-BPA bio-distribution during the therapy.

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype.

PURPOSE: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). METHODS: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. RESULTS: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. CONCLUSION: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Wharton's jelly mesenchymal stromal cells have contrasting effects on proliferation and phenotype of cancer stem cells from different subtypes of lung cancer.

Studies on the role of multipotent mesenchymal stromal cells (MSC) on tumor growth have reported both a tumor promoting and a suppressive effect. The aim of the present study was to determine the effect of MSC isolated from Wharton's jelly of umbilical cord (WJMSC) on lung cancer stem cells (LCSC) derived from human lung tumors: two adenocarcinomas (AC) and two squamous cell carcinomas (SCC). LCSC derived from SCC and AC expressed, to varying extents, the more relevant stem cell markers. The effect of WJMSC on LCSC was investigated in vitro using conditioned medium (WJ-CM): a proliferation increase in AC-LCSC was observed, with an increase in the ALDH+ and in the CD133+ cell population. By contrast, WJ-CM hampered the growth of SCC-LCSC, with an increase in the pre-G1 phase indicating the induction of apoptosis. Furthermore, the ALDH+ and CD133+ population was also reduced. In vivo, subcutaneous co-transplantation of AC-LCSC/WJMSC generated larger tumors than AC-LCSC alone, characterized by an increased percentage of CD133+ and CD166+ cells. By contrast, co-transplantation of WJMSC and SCC-LCSC did not affect the tumor size. Our results strongly suggest that WJMSC exert, both in vitro and in vivo, contrasting effects on LCSC derived from different lung tumor subtypes.

Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population.

BACKGROUND: The identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD). METHODS: Rhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium. RESULTS: MEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells. CONCLUSIONS: Our results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance. Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.

Cord blood CD34+ cells expanded on Wharton's jelly multipotent mesenchymal stromal cells improve the hematopoietic engraftment in NOD/SCID mice.

OBJECTIVE: This study aims to investigate the capability of Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSC) to support the in vitro expansion of hematopoietic stem/progenitor cells (HSPC) derived from cord blood (CB) in the absence of exogenous cytokines, and the effect on engraftment of the expanded cells in a mouse model. METHODS: CB-CD34+ cells were seeded on WJ-MSC layer and cultured in HP01 serum-free medium. Day-7 and day-13 expanded cells were transplanted in NOD/SCID mice. After 8 wk, engraftment was evaluated in mouse bone marrow as percentage of human CD45+ cells. RESULTS: CD34+ population was expanded without increasing the differentiation rate. Co-culture increased the expansion of the CD34+ cells by 2.0 and 7.3 times after 7 and 13 d, respectively, and maintained the CD34+ cells up to day 20. In particular, earlier CD34+/CD90+ and CD34+/CD33- subtypes were increased. An advantage of the day-7 co-cultured HSPC in respect of HSPC at day 0 in the engraftment of NOD/SCID mice was obtained both as percentage of mice engrafted (100% vs. 75%) and as percentage of chimerism. CONCLUSIONS: Although the increase in hematopoietic progenitors is not dramatic as in the presence of added cytokines, this study demonstrates the effectiveness of the WJ-MSC not only to preserve the CD34+ population but also to improve the repopulating efficacy of the amplified HSPC, also in the absence of added cytokines and growth factors.

Valproic acid affects the engraftment of TPO-expanded cord blood cells in NOD/SCID mice.

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34+ cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro, VPA+TPO inhibited HSPC differentiation and preserved the CD34+ cell fraction; the self-renewal of the CD34+ TPO+VPA-treated cells was suggested by the increased replating efficiency. In vivo, short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO+VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO+VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO+VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO+VPA-treated cells in vitro.

Long-term platelet production assessed in NOD/SCID mice injected with cord blood CD34+ cells, thrombopoietin-amplified in clinical grade serum-free culture.

OBJECTIVE: Delayed platelet recovery post-cord blood (CB) transplantation might be due to CB characteristics: low maturity of stem cell compartment, poor production of CD34+/CD41+ cells when induced to differentiate along the megakaryocytic (MK) lineage, retention of a low ploidy in the expanded MKs. Ex vivo expansion of CB hematopoietic progenitor cells for reconstitution of different human hematopoietic lineages has already been developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. However, optimal conditions for MK-progenitor engraftment to reduce hemorrhaging risk still to be developed. This study assesses the hypothesis that CB-CD34+ amplification with thrombopoietin (TPO) can be applied to a portion of a CB transplant unit to stimulate recovery along MK differentiation program. MATERIALS AND METHODS: Human CB-CD34+ cells were amplified in a serum-free, clinical grade medium with 100 ng/mL TPO alone and in addition to other cytokines (Kit ligand, interleukin-6, and Flt-3 ligand). Seven-day cultured cells were transplanted into irradiated NOD/SCID mice and engraftment, megakaryocytopoiesis, and platelet production were assessed. RESULTS: Platelet release was successful and continuously present for at least 8 weeks in NOD/SCID mice transplanted with CB cells stimulated by TPO. Thrombocytopoiesis was more effective with transplanted TPO-amplified cells than with the cytokine cocktails. CONCLUSION: Platelet number obtained is within the minimum level considered sufficient for hemostasis. Furthermore, amplified cells maintain their self-renewal capacity and multilineage potential differentiation. Thus, transplantation of TPO-expanded CB cells has the potential favoring both platelet recovery and human engraftment.

Factor-V expression in platelets from human megakaryocytic culture.

The origin of platelet-factor-V has long been discussed. To elucidate whether and when human platelet-factor-V is synthesized by megakaryocytes, we utilized in vitro-generated megakaryocytes capable of producing platelets. Factor-V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor-V protein was expressed in mature proplatelet-bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor-V.

Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release.

Ploidy could be the key to understanding megakaryocyte (MK) biology and platelet production. Human CD34+ cells purified from umbilical cord blood (CB) and peripheral blood (PB) were investigated on their capability to give rise, in a serum-free medium containing thrombopoietin, to MKs and platelets. CB-MKs showed reduced polyploidization and platelet number compared with PB-MKs, but a similar membrane phenotype. Most CB-MKs showed a 2N content of DNA (approximately 80%) and only 2.6% had 8N, whereas 40% of the PB cells had 8N or more. Platelets were substantially released in PB culture from day 12; at day 14 the CB-derived MKs were able to release platelets although at a reduced level (approximately 35%), correlating with their reduced size. A direct correlation was demonstrated by sorting polyploid cells from PB-MKs and evaluating the platelets released in the supernatant. Furthermore, the study analyzed the expression and distribution of cyclin D3 and cyclin B1. Cyclin D3 protein was increased in PB in comparison to CB-MKs; in PB culture most cells rapidly became positive, whereas in CB-derived cells cyclin D3 expression was evident only from day 9 and in a reduced percentage. Cyclin B1 was essentially localized at the nuclear level in the CB and was expressed during the whole culture. In PB-MKs, at day 9, a reduction was observed, correlating with an advanced ploidy state. The data indicate the inability of the CB-MKs to progress in the endomitotic process and a direct correlation between DNA content and platelet production.