Therapy targeting tumor blood vessels ought to inhibit tumor growth. However, tumors become refractory to antiangiogenic drugs. Therefore, therapeutic solutions should be sought to address cellular resistance to antiangiogenic therapy. In this regard, reversal of the proangiogenic and immunosuppressive phenotype of cancer cells, and the shift of the tumor microenvironment towards more antiangiogenic and immune-stimulating phenotype may hold some promise. In our study, we sought to validate the effects of a combination therapy aimed at reducing tumor blood vessels, coupled with the abrogation of the immunosuppressive state. To achieve this, we developed an oral DNA vaccine against endoglin. This antigen was carried by an attenuated Salmonella Typhimurium and applied before or after tumor cell inoculation into immunocompetent mice. Our results show that this DNA vaccine effectively inhibited tumor growth, in both the prophylactic and therapeutic settings. It also activated both specific and nonspecific immune responses in immunized mice. Activated cytotoxic T-lymphocytes were directed specifically against endothelial and tumor cells overexpressing endoglin. The DNA vaccine inhibited angiogenesis but did not affect wound healing. In combination with interleukin-12-mediated gene therapy, or with cyclophosphamide administration, the DNA vaccine resulted in reduced microvessel density and lowered the level of T reg lymphocytes in the experimental tumors. This effectively inhibited tumor growth and prolonged survival of the treated animals. Polarization of tumor milieu, from proangiogenic and immunosuppressive, towards an immunostimulatory and antiangiogenic profile represents a promising avenue in anticancer therapy.

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Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.

Hypoxia is an important feature of tumor microenvironment, exerting far-reaching effects on cells and contributing to cancer progression. Previous studies have established substantial differences in hypoxia response between various cell lines. Investigating this phenomenon in melanoma cells contributes to a better understanding of cell lineage-specific hypoxia response and could point out novel hypoxia-regulated genes. We investigated transcriptional activity of B 16(F10) murine melanoma cells cultured for 24 h under hypoxic (nominal 1% O2, 15 samples including controls) and hypoxia-mimicking conditions (cobalt chloride, 100 or 200 microM, 6 samples including controls). Gene expression profiles were analyzed using MG-U74Av2 oligonucleotide microarrays. Data analysis revealed 2541 probesets (FDR <5%) for 1% oxygen experiment and 364 probesets (FDR <5%) for cobalt chloride, which showed differences in expression levels. Analysis of hypoxia-regulated genes (true hypoxia, 1% O2) by stringent Family-Wise Error Rate estimation indicated 454 significantly changed transcripts (p < 0.05). The most upregulated genes were Lgals3, Selenbpl, Nppb (more than ten-fold increase). We observed significant differences in expression levels of genes regulating glycolysis (Pfkp, Hk2, Aldo3, Eno2), apoptosis (Bnip3, Bnip31, Cdknla), transcription (Bhlhb2, Sap30, Atf3, Mxil), angiogenesis (Vegfa, Adm, Anxa2, Ctgf), adhesion (Pkp2, Itga4, Mcam), migration (Cnn2, Tmsb4x), and other processes. Both true hypoxia and hypoxia mimicry induced HIF-1-regulated genes. However, unsupervised analysis (Singular Value Decomposition) revealed distinct differences in gene expression between these two experimental conditions. Contrary to hypoxia, cobalt chloride caused suppression of gene expression rather than stimulation, especially concerning transcripts related to proliferation, immune response, DNA repair, and melanin biosynthesis.

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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.

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Vasculature targeting agents have been tested as cancer therapeutics for the past few years. Such therapy could be accomplished using, for example, bifunctional (two-domain) peptides. RGD-4C-GG-D (KLAKLAK) 2 , a peptide designed by Ellerby and coworkers (1999) (full sequence: ACD-CRGDCFCGGKLAKLAKKLAKLAK), binds selectively to α V β 3 integrin receptors expressed in tumor neovasculature and, after internalization, effectively induces apoptosis of endothelial cells. The aim of this study was to examine if RGD-4C-GG-D (KLAKLAK) 2 would efficiently target cells, among them B16(F10), that overexpress α V β 3 receptors, and whether it would be suitable for therapeutic treatment of primary B16(F10) murine melanoma tumors. Thus, the peptide would target two distinct tumor compartments: that formed by endothelium of blood vessels and that made up of neoplastic cells. The therapeutic peptide was recognized and did induce apoptosis in B16(F10) cell line. Tumor growth inhibition was observed following direct intratumoral administration. However, cessation of peptide administration led to rapid tumor growth and death of the animals.