[Mesenchymal stromal cells]. / Mezenchymalne komórki zrebu.

Mesenchymal stromal cells (MSCs) have been among the most intensively studied cells in recent years. Lack of specific unique markers for these cells makes it difficult to distinguish MSCs from other types of cells, such as fibroblasts or pericytes. MSCs are a mixture of morphologically different cells with expression of various cellular markers, with varying degrees of differentiation, as well as varying proliferation capacities. The majority of phenotypic features of these cells have been identified through cell culture. One of their basic features is the capacity to differentiate into three cell lines: osteoblasts, adipocytes and chondroblasts. Under in vivo conditions, MSCs form an important functional element of the hematopoietic stem cell niche. Residing within the blood vessel wall, MSCs assist in its formation and functioning. MSCs release anti­apoptotic and pro­angiogenic factors, as well as agents that stimulate cell proliferation and also immunostimulating factors. In this study, we focused in particular on therapeutic strategies employing MSCs to improve the performance of the infarcted heart as well as on their involvement in the repair of hard-to-heal wounds. Thanks to the released anti-inflammatory agents, MSCs can inhibit inflammatory reactions. Owing to cytokines and growth factors they can also stimulate regeneration of damaged tissues and organs. The therapeutic effect that follows MSCs administration is linked to their paracrine activity.

[From precursor to reprogrammed cells: evolution of cardiomyoplasty]. / Od komórek prekursorowych do komórek przeprogramowanych: ewolucja kardiomioplastyki.

Myocardial infarction is underoxygenation-driven limited necrosis of heart tissues which results in elimination of ca. 0.5 to 1 billion spontaneously contracting cardiomyocytes (CM). Since the ability of human heart to regenerate is limited, efforts have been undertaken to increase the number of cardiomyocytes in post-infarction myocardium. Theoretically, such proposals might involve transplantation of 1) skeletal myoblasts and cardiomyocytes, or 2) progenitor/stem cells, theoretically capable of differentiating into cardiomyocytes, or 3) pluripotent cells such as embryonal stem cells (ESC) and induced pluripotent stem cells (iPSC) differentiating into cardiomyocytes. The efforts to increase CM could also involve 4) in situ reprogramming of fibroblasts into active cardiomyocyte-like cells, or 5) stimulating in situ proliferation of cardiomyocytes using pharmacological agents. Only three proposals merit closer scrutiny (2, 4 and 5). However, preclinical and clinical data have demonstrated weak ability of progenitor cells to differentiate (proposal 2). Nevertheless, transplanted cell-induced paracrine effects accompanying such therapy do improve functioning of the damaged heart muscle. The proposals that would permit the number of CM to be increased include in situ reprogramming of fibroblasts into active cardiomyocytes (proposal 4), as well as in situ stimulation of quiescent cardiomyocytes' proliferation (proposal 5). It appears that an optimized therapeutic solution (increasing left ventricular ejection fraction and decreasing the post-infarct scar) might combine agents stimulating paracrine effects and reprogramming of fibroblasts.

[Endoglin as a target of antitumor therapy]. / Endoglina jako cel terapii przeciwnowotworowej.

Blood vascular supply significantly affects progression of tumor growth. Inhibition of endothelial cell proliferation by antiangiogenic drugs should lead to growth arrest of both primary tumors and metastases. During the course of lengthy therapy, endothelial cells may, however, become refractory to the action of antiangiogenic agents. Novel approaches to anticancer treatment should explore the issue of drug resistance shown by endothelial cells. One possible therapeutic solution might be tumor immunotherapy directed against antigens expressed on the surface of endothelial cells which co-form tumor blood vasculature. Such therapy is supposed to break immune tolerance to own antigens and to eliminate tumor blood vessel endothelial cells by activating cytotoxic T lymphocytes. This kind of response can be obtained against endoglin (CD105). Endoglin is overexpressed in proliferating endothelial cells which line tumor blood vessels. Presence of endoglin in solid tumor blood vessels has prognostic value in cancer treatment. CD105 is also expressed by certain cancer cells (prostate, melanoma and Ewing sarcoma). It appears that therapeutic strategies directed against endoglin allow several mechanisms of resistance to antiangiogenic drugs to be omitted. The therapeutic approach that we propose, i.e. a tumor blood vessel-destroying strategy combined with immunotherapy, may become an effective therapeutic tool. 

Evolving models of tumor origin and progression.

History of cancer disease models clearly illustrates the evolving nature of these concepts. Since such models undergo continual revisions and additions as a result of underlying medical research, they also tend to reorganize knowledge and allow perceiving previously unseen relationships. Growth of medical thought has been influenced for many centuries by an ancient Hippocratic concept of disease seen as a disturbance in bodily "humors." True mechanisms of cell and tissue injury started to be elucidated only with the advent of postmortem pathological findings. Concerning cancer, when first disease-producing bacteria were identified in the nineteenth century, also neoplasms were treated as infectious diseases. Foreign organisms were thought to be present inside tumors. However, this hypothesis could not be confirmed by microscopic or histochemical studies. The latter suggested, instead, that tumors were rather formed by abnormal cells. Cancer was then started to be regarded as a disease of cells. This interpretation was radically altered by later developments in genetics which suggested that neoplasms can be treated as genetic diseases as pathologic cellular lesions are caused by mutations in specific genes. More recent models have compared carcinogenesis to evolutionary processes. Due to genetic instability, successive mutations, appearing in cells, lead to selection of cancer cells which feature specific phenotypic traits. The newest data indicate that there may be also a link between cancer and mutated stem cells. The review discusses main concepts of tumor origin forwarded since the beginnings of the nineteenth century.

["Vicious circles" of glioblastoma tumors: vascularization and invasiveness]. / "Bledne kola" glejaków: unaczynienie i inwazyjnosc.

Glioblastoma multiforme is the most common and a particularly aggressive form of glial primary brain tumors. This malignancy accounts for ca. 70% of all diagnosed cases. Unfortunately, average survival of glioma patients does not exceed one year from diagnosis. Specific vascularization pattern (presence of numerous microvessels and glomerular vessels) and exceptional invasiveness are characteristic features of glioblastoma tumors. Both of these features reflect complex underlying processes forming two vicious circles. Common to both of these circles is the state of tumor underoxygenation. Hypoxia that occurs in the vicinity of abnormal tumor blood vessels stimulates formation of novel microvessels and invasiveness of tumor cells. In their essence, both of the vicious circles are processes allowing tumor cells to adapt to an underoxygenated tumor milieu. These processes play an important role in tumor progression, which reflects a specific type of evolution of cancer cells. Late effects of this evolution include appearance of highly aggressive, chemo- and radiotherapy resistant neoplastic cells. Increased adaptation capabilities of such cancer cells have a negative influence on the therapeutic process. Effective therapeutic strategies should not be directed against single cancer cell markers; instead, they should be targeted so as to break both vicious cycles. Herein we discuss several such strategies. In our opinion, effective therapeutic approaches must include a combination of several agents that recognize and simultaneously break both vicious cycles, i.e. vascularization and invasiveness. Also, agents that decrease hypoxia in cancer cells, for example drugs inhibiting activity of HIF-1α, might also prove therapeutically effective in such approaches. 

[HMGB1--its role in tumor progression and anticancer therapy]. / HMGB1--rola w progresji i terapii przeciwnowotworowej.

HMGB1 is an evolutionarily conserved protein with a wide spectrum of action. Its main receptors are RAGE and TLR found on the surface of immune system cells as well as endothelial cells. Although signaling pathways for both receptor groups are different, ultimately they both activate NFκB transcription factor which, in turn, activates genes encoding adhesion proteins, proinflammatory cytokines and proangiogenic factors. Inside cells, HMGB1 is found mainly in the cell nucleus, where it participates in replication, recombination, transcription and DNA repair processes. Following release into the extracellular space, HMGB1 becomes a proinflammatory cytokine which stimulates formation of new blood microvessels, enhances cell migration, activates the inflammatory condition and affects cell proliferation. HMGB1 protein also takes part in regeneration of damaged tissues and stimulates autophagy. HMGB1 plays a potential role in anticancer therapy. Increased amounts of HMGB1 in cancer cells and elevated levels in the bloodstream are noted among patients afflicted with various cancers. HMGB1 protects cells from apoptosis, as it affects telomere stability. HMGB1 also stimulates a number of proteins involved in proliferation of cancer cells and inhibits signals that control cell growth. Ability to arrest HMGB1 release from cells or to inhibit its activity appears to be a promising therapeutic approach. At present, several inhibitors of HMGB1 are known and can be used in anticancer therapy.  

[Tumor blood vessels]. / Nowotworowe naczynia krwionosne.

Growth of tumors usually depends on the development of the tumor's own vasculature. Small avascular tumors (1­2 mm3) cannot continue growth provided an equilibrium between pro-angiogenic and anti-angiogenic factors is maintained within the tumor environment. Angiogenesis is not the only factor responsible for tumor blood vessels forming, as vasculogenic mimicry plays an equally substantial role in this process. Vessel-like structures formed during this process are made up from cancer cells, macrophages and mast cells. Certain neoplasms are capable of growing without developing their own vasculature; instead they secure growth via normal blood vessels of the host. Slowed-down blood flow through an abnormally built tumor vascular network is the main reason for cancer cells' underoxygenation (hypoxia). Defective blood vessels, with hypoxia resulting, play a major role in tumor progression. Underoxygenation induces formation of novel vessels and these new defective vessels are in turn the principal reason for hypoxia. The latter increases cancer cells' malignancy and invasiveness. A particular process, called transdifferentiation, takes place in tumor vasculature when hypoxia is present and involves neoplastic cells transforming into endothelial cells. Since growth of a tumor is dependent on its own blood supply, inhibition of such vascular network growth and/or damage to this network should exert a strong impact on tumor growth. Long-term administration of anti-angiogenic drugs, however, encounters unexpected problems. Anti-angiogenic drug resistance, together with paradoxical stimulation of invasiveness and metastasis by these drugs, has lately become a dominant issue in anticancer therapy.

Mesenchymal stromal cells as carriers of IL-12 reduce primary and metastatic tumors of murine melanoma.

Due to immunosuppressive properties and confirmed tropism towards cancer cells mesenchymal stromal cells (MSC) have been used in many trials. In our study we used these cells as carriers of IL-12 in the treatment of mice with primary and metastatic B16-F10 melanomas. IL-12 has confirmed anti-cancer activity, induces a strong immune response against cancer cells and acts as an anti-angiogenic agent. A major limitation of the use of IL-12 in therapy is its systemic toxicity. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study MSC were used as carriers of the IL-12. We confirmed antitumor effectiveness of the cells secreting IL-12 (MSC/IL-12) in primary and metastatic murine melanoma models. We observed inhibition of tumor growth and a significant reduction in the number of metastases in mice after MSC/IL-12 administration. MSC/IL-12 decreased vascular density and increased the number of anticancer M1 macrophages and CD8+ cytotoxic T lymphocytes in tumors of treated mice. To summarize, we showed that MSC are an effective, safe carrier of IL-12 cytokine. Administered systemically they exert therapeutic properties of IL-12 cytokine without toxicity. Therapeutic effect may be a result of pleiotropic (proinflammatory and anti-angiogenic) properties of IL-12 released by modified MSC.

Anticancer effects of CAMEL peptide.

This study analyzed whether therapy with CAMEL, an antimicrobial peptide (KWKLFKKIGAVLKVL), possess anticancer benefits. Although the peptide was cytotoxic for all the cell lines tested, it did not cause hemolysis, which suggests that CAMEL does not damage cell membranes. After cellular internalization, CAMEL localized to mitochondria and lowered the mitochondrial potential, resulting in the organelles' swelling, a decrease in cellular ATP level and, finally, cellular breakdown. High mobility group box 1 (HMGB1) protein, a necrotic death marker, was shown to be released from cells treated with CAMEL. Growth of B16-F10 melanoma tumors was clearly restrained after injections with CAMEL and could be kept in check throughout the period of peptide administration. However, if therapy was stopped, tumors started to grow again 3-4 days later. To reduce tumor volume and block tumor relapse, a combined therapy was required involving CAMEL and plasmid DNA carrying the interleukin-12 (IL-12) gene. The two therapeutic agents used in combination (a series of CAMEL injections first, followed by daily administration of plasmid DNA) delayed tumor growth and extended survival of treated animals in a statistically significant manner. Complete tumor regression was found in 60% of cases.

Can inhibition of angiogenesis and stimulation of immune response be combined into a more effective antitumor therapy?

Cancer initiation and progression is strongly influenced by the tumor microenvironment consisting of various types of host cells (inflammatory cells, vascular cells and fibroblasts), extracellular matrix and non-matrix molecules. Host cells play a defining role in two major processes crucial for tumor growth: angiogenesis and escape from immune surveillance. The interdependence of these processes resemble the principles of Yin and Yang, as the stimulation of tumor angiogenesis inhibits effective immune responses, while angiogenesis inhibition may have the opposite effect. These considerations may be useful in developing anticancer strategies based on the potentially synergistic combinations of antiangiogenic and immunostimulatory drugs.

Author Correction: Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combination of combretastatin A4 phosphate and doxorubicin-containing liposomes affects growth of B16-F10 tumors.

The study aimed to check the effectiveness of anticancer therapy combining a vascular-disruptive drug (combretastatin phosphate, CA4P) and a liposomal formulation of a chemotherapeutic (doxorubicin). CA4P was synthesized in our laboratory according to a previously described procedure. The antivascular drug and long-circulating doxorubicin-loaded liposomes were used to treat B16-F10 murine melanoma experimental tumors. Seventy-four hours after drug administration, a decrease in the number of tumor blood vessels was apparent and necrotic areas within tumors were visible. Combination therapy consisting of alternate administrations of CA4P and liposomal doxorubicin yielded greater inhibition of tumor growth than monotherapies alone. The best therapeutic results were obtained with the antivascular drug administered intratumorally every second day at 50 mg/kg body mass. In the case of combined therapy, the best results were obtained when the vascular-disruptive agent (CA4P) and the antineoplastic agent (liposomal doxorubicin) were administered in alternation.

Chimeric protein ABRaA-VEGF121 is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth.

It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.

Oxidation of carbidopa by tyrosinase and its effect on murine melanoma.

Oxidation of the anti-Parkinsonian agent carbidopa by tyrosinase was investigated. The products of this reaction were identified as 3-(3,4-dihydroxyphenyl)-2-methylpropanoic acid and 6,7-dihydroxy-3-methylcinnoline. These results demonstrate that after oxidation of the catechol moiety to an o-quinone either a redox exchange with the hydrazine group or a cyclization reaction occur. The cyclization product underwent additional oxidation reactions leading to aromatization. The cyclization reaction is undesired in the case of hydrazine-containing anti-melanoma prodrugs and will have to be taken into account in designing such compounds. Carbidopa was tested against B16(F10) melanoma cells in culture and showed cytotoxicity significantly higher than either of its oxidation products and l-dopa. This effect, however, was not specific to this cell line.

[Angiogenesis and immune suppression: yin and yang of tumor progression?]. / Angiogeneza i immunosupresja: jin i jang progresji nowotworów?

Specialized variants of neoplastic cells that appear in tumors during cancer disease progression possess the ability to recruit certain kinds of hematopoietic and mesenchymal cells from the bone marrow or bloodstream. These tumor-recruited hematopoietic cells include monocytes, macrophages, granulocytes, mast and dendritic cells, as well as myeloblastic suppressor cells. Fibroblasts derived from undifferentiated mesenchymal cells are also recruited. Some of these cells (especially macrophages and fibroblasts) then undergo "education-like" phenotype reprogramming under the influence of the neoplastic cell population, resulting in the appearance of tumor-associated macrophages (TAM) and fibroblasts (CAF). Together with the extracellular matrix (ECM) as well with the remaining types of recruited cells, they contribute to the formation of a specific tumor microenvironment. Both the cells forming the tumor microenvironment and neoplastic cells engage in the two intimately linked processes of angiogenesis and immune suppression. The network of defective blood vessels formed during tumor angiogenesis and the resulting fluctuations in blood flow lead to under-oxygenation of the surrounding neoplastic cells and have substantial impact on their metabolic profile. A number of processes triggered in these under-oxygenated neoplastic cells appear to strongly favor further tumor progression. Such processes result in lower oxygen demand, enhanced angiogenesis, and epithelial-mesenchymal transition, owing to which the neoplastic cells acquire the ability to translocate. Under-oxygenation also leads to augmented genetic instability of the neoplastic cells. The tumor environment-forming cells also have their share in the establishment of an immunosuppressive environment which enables the neoplastic cells to escape immune surveillance. By providing a sophisticated milieu for the selection of increasingly malignant neoplastic cells (i.e. with proangiogenic and immunosuppressive phenotypes), the tumor microenvironment-forming cells substantially contribute to the progression of a neoplasm. Inhibited angiogenesis thus makes an immune response, both nonspecific and specific, possible. The remarks presented here may prove helpful in devising novel anticancer strategies involving antiangiogenic in combination with immunomodulatory drugs.

[Peptides: a new class of anticancer drugs]. / Peptydy--nowa klasa leków przeciwnowotworowych.

Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

Human ADSC xenograft through IL-6 secretion activates M2 macrophages responsible for the repair of damaged muscle tissue.

BACKGROUND: Adipose-derived mesenchymal stromal cells (ADSCs) are multipotent stromal cells. The cells secrete a number of cytokines and growth factors and show immunoregulatory and proangiogenic properties. Their properties may be used to repair damaged tissues. The aim of our work is to explain the muscle damage repair mechanism with the utilization of the human adipose-derived mesenchymal stromal cells (hADSCs). METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hind limb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6NCrl and NOD SCID mice. The mice received PBS- (controls) or 1 × 106 hADSCs. One, 3, 7, 14 and 21 days after the surgery, we collected the gastrocnemius muscles for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: The retention time of hADSCs in the limb lasted about 14 days. In the mice receiving hADSCs, the improvement in the functionality of the damaged limb occurred faster than in the control mice. More new blood vessels were formed in the limbs of the mice receiving hADSCs than in limbs of the control mice. hADSCs also increased the infiltration of the macrophages with the M2 phenotype (7-AAD-/CD45+/F4/80+/CD206+) into the ischemic limbs. hADSCs introduced into the limb of mice secreted interleukin-6. This cytokine stimulates the emergence of the proangiogenic M2 macrophages, involved, among others, in the repair of a damaged tissue. Both macrophage depletion and IL-6 blockage suppressed the therapeutic effect of hADSCs. In the mice treated with hADSCs and liposomes with clodronate (macrophages depletion), the number of capillaries formed was lower than in the mice treated with hADSCs alone. Administration of hADSCs to the mice that received siltuximab (human IL-6 blocker) did not cause an influx of the M2 macrophages, and the number of capillaries formed was at the level of the control group, as in contrast to the mice that received only the hADSCs. CONCLUSIONS: The proposed mechanism for the repair of the damaged muscle using hADSCs is based on the activity of IL-6. In our opinion, the cytokine, secreted by the hADSCs, stimulates the M2 macrophages responsible for repairing damaged muscle and forming new blood vessels.

The effect of culture media on large-scale expansion and characteristic of adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare. METHODS: ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated. RESULTS: All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest. CONCLUSIONS: Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.

Monitoring of diffusion properties and transverse relaxation time of mouse ischaemic muscle after administration of human mesenchymal stromal cells derived from adipose tissue.

OBJECTIVES: Application of non-invasive imaging methods plays an important role in the assessment of cellular therapy effects in peripheral artery disease. The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing ischaemic hindlimb muscle after administration of human mesenchymal stromal cells derived from adipose tissue (hADSC) in mice. MATERIALS AND METHODS: MRI experiments were performed on a 9.4T Bruker system. The measurement protocol included transverse relaxation time mapping and diffusion tensor imaging. The monitoring period encompassed 14 days after femoral artery ligation and subsequent cell administration. The effect of hADSC transplantation was compared with the effect of normal human dermal fibroblasts (NHDFs) and phosphate-buffered saline injection. RESULTS: The most significant differences between the hADSC group and the remaining ones were observed around day 3 after ischaemia induction (increased transverse relaxation time in the hADSC group in comparison with the control group) and around day 7 (increased transverse relaxation time and decreased third eigenvalue of the diffusion tensor in the hADSC group in comparison with the control and NHDF groups) at the site of hADSC injection. Histologically, it was associated with increased macrophage infiltration at days 3-7 and with the presence of small regenerating fibres in the ischaemic tissue at day 7. CONCLUSIONS: Our results underscore the important role of macrophages in mediating the therapeutic effects of hADSCs and confirm the huge potential of magnetic resonance imaging in monitoring of cellular therapy effects.

Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.