Vergleichende Etablierung und Charakterisierung eines orthotopen Kolonkarzinom-Xenograft-Modells

The human colon cancer cellline HT29 was investigated in various
models regarding different criteria. The in-vivo-experiments were
carried out with female SCID mice and consisted of subcutaneous
cell injection, orthotopic cell injection into the cecal wall and
orthotopic fixation of a tumor fragment onto the cecum. The
subcutaneous experiment took 41 days; the orthotopic animal
experiments were equally divided into three points of necropsy each
two weeks apart. On these days tumor, liver and lung were
withdrawn, fixed in 3,8% formaldehyde and analyzed histologically.
In addition blood samples of all animals of the orthotopic
experiments were taken on days of autopsy and the CEA
(carcinoembryonic antigen) content was determined using an ELISA.
The vascularization of the orthotopic primary tumors was examined
by staining of CD34, i.e. number and area of the tagged vessels
were ascertained. Additionally the green fluorescent protein (GFP)
was studied in view of its suitability as quantifiable reporter
gene in these models. Therefore not only HT29-wildtype but also
HT29 cells transfected with GFP were used in vitro and in the first
two in vivo assays. Advantages and disadvantages summarized: - The
subcutaneous model was realized easily, measurement of the primary
tumor was simple, the tumor take rate was 100 % and the laboratory
animals appeared to suffer only from a relative slight amount of
stress. Besides these advantages this setting cannot be used for
investigations regarding metastasis because of the low number of
metastases. - The orthotopic cell injection generated small, hardly
measurable primary tumors, but this approach is a suitable model of
metastasis because of the number of metastases detected. The
technical effort and the burden for the animals exceeded that found
in the subcutaneous setting but was below the effort of orthotopic
fixation of a tumor fragment. - Of all investigated models the
orthotopic fixation of a tumor fragment represents the model with
the greatest effort. The primary tumors were big enough to be
measured, but the number of metastases was too low to make
statistical valuable evaluations. - The CD34 staining successfully
marked the vessels of the primary tumor and facilitated a
computer-assisted quantitative analysis of the vascularization of
orthotopic colon tumors. It was assessed that a broad
neoangiogenesis occurred at the beginning of tumor growth prior to
the development of metastases. The number of metastases increased
with proceeding length of time, whereas the number of vessels
decreased. The continuous extension in tumor volume resulted in a
necrotic tumor center so that vessels were detectable in the border
area only. - The used HT29-GFP clone was not stable enough to
generate sufficient fluorescence. The cells both in vitro an in
vivo grew more slowly, the subcutaneous tumors showed necrotic
areas and there were less metastases after orthotopic injection of
GFP cells than after injection of wildtype cells. Because of the
insufficient fluorescence it was not possible to execute a
quantifiable analysis of metastasis. The application of GFP was not
advantageous within these models. - CEA suits to be a valuable
tumor marker for colon cancer in both investigated models. The CEA
content in the blood samples of tumor bearing animals increased
dependent on tumor burden and tumor invasiveness. A direct
correlation between tumor size and CEA might be established with an
improved measurement system or rather an advanced measuring of
tumor volume. Due to the comparative characterization of injection
and implantation techniques as well as other detailed examinations
carried out for this thesis, it is possible to select suitable
models for preclinical trials depending on the individual purpose.