• Avdic Erma B.Sc., master student (
  • Maeding Nicole B.Sc., master student (
  • Verwanger Thomas Dr., assistant (


Expression of immune stimulating factors of CT26 mouse colon carcinoma cells under hypoxic conditions

Previous investigations showed complete regression of CT26 induced tumors in BALB/c mice after low-dose hypericin-PDT, very likely by vascular damage. In addition, mice showed an anti-tumor response against re-challenge with CT26 tumor cells. This response, likely based on a cytotoxic T-cell reaction, bears the potential to eliminate metastases or tumor cells surviving PDT, a shortcoming of the local PDT. It is therefore of major interest to investigate this potential in detail.
From these previous results it can be concluded that CT26 tumor cells were damaged and killed not directly by, but as a consequence of hypericin-PDT: by photo-destruction of the tumor-supporting vessels the tumor cells are cut from oxygen supply, by this inducing hypoxic conditions.
Hypoxia in turn is known to induce a wide range of cellular reactions, ranging from altered gene expression for e.g. adaptive or repair mechanisms up to lethal damage.
The “message” from damaged tumor cells to the immune system, which causes it to attack the same cell type arises inter alia 1) from tumor-associated macrophages (TAMs), which clear dead tumor cells by phagocytosis - mediated by pro-inflammatory cytokines in the case of necrosis. For the kind of reaction of the TAMs to dead tumor cells the cell death mode (apoptosis or necrosis) is decisive. 2) The “message” may be induced by immunogenic factors such as damage-associated molecular pattern (DAMPs) of the tumor cells, which have to be presented on the cell surface or released from the cells in order to induce an immune response.

DAMPs may indeed be responsible for the observed immune response, but their presence may only be proved by site-correlated measurements as present in tissue sections. Since this requires new time-consuming as well as costly animal experimentation, it cannot be performed in the present master thesis.
The first approach is therefore to determine in-vitro the expression of immunogenic genes, which have the potential to act as immune stimulating factors, possibly as DAMPs, under conditions given in the previous mouse study.
As analysed in a recent master thesis, low-dose hypericin-PDT on  mouse tumor tissue from the in vivo study, or direct low-dose hypericin-PDT on CT26 cells in vitro induces a number of changes in the expression of genes such as thrombospondins, VEGF, HIF-1alpha and cytokines.
Based on the two studies, the question emerged, how CT26 gene expression will be influenced by hypoxic conditions, which probably governed the in vivo tumor elimination. In addition, the cellular response to hypoxia, apparent on changes in the cell cycle and cell death should be investigated. The cell death mode probably could indicate the kind and amount of TAM involvement.
Genes of interest will include: TSP-1 and -2, VEGF, HIF-1alpha, IL-6, TNF-alpha, TGF-beta, IL-8, HSPa13, HSP90b1, the expression of which will be measured by real-time RT-PCR.
Hypoxic conditions will be established using an AtmosBag (Sigma-Aldrich) filled with a gas mixture of 1% O2 and 99% N2. The cells will be exposed for at least 10 hours before analysis of gene expression, cell cycle, cell death and apoptosis rate.


Sanovic R, Verwanger T, Hartl A, Krammer B. Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma. Photodiagnosis Photodyn Ther. 2011 Dez; 8(4): 291-6.

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