Trial Information

STELLA: a Feasibility Study on Stem Cells Sensitivity Assay

Patients Patients with LC, CRC and BC with good performance status and with tumor tissue
available, at failure of conventional therapies or without possibility to be treated with
therapy of proven efficacy, will be included in the study. Initial patient characterization
will include detailed information on the following items: gender, age, clinical history,
co-morbidity, physical examination, blood cell counts, complete blood analyses for liver and
renal function, coagulation, serological tumor markers, tumor location and stage, tumor
mass, metastases.

LC, CRC and BC CSCs identification Isolation and characterization of CSCs will be made
starting from samples of tumor tissue obtained from patients with LC, CRC and BC. Tumor
tissue will be collected before study entry, i.e tissue obtained during a diagnostic or
therapeutical procedure, like surgery or biopsies with other purposes than the protocol.The
surgical samples collected will be classified according to the specific histological
characteristics of the tumor. From each sample, by means of enzymatic and mechanical
procedures, the CSCs will be obtained and then cultivated in adequate culture mediums to be
subsequently used for biochemical and molecular studies. Each sample will be associated with
the patient history at the surgical time and an appropriate follow-up program consisting of
periodic clinical and instrumental controls that in order to assign a prognostic value to
the biological characteristics of the CSCs. Stem cells derived from the selected epithelial
tumors will then undergo analysis of surface and intracellular markers in order to provide a
definitive characterization of cellular phenotype. Stem cells derived from lung cancer, CRC
and BC are identified as a subset of tumor cells positive for the marker CD133.

Tumor specimens will be washed several times and left over night in DMEM:F12 medium
supplemented with high doses of Penicillin/Streptomycin and Fungizone in order to avoid
contamination. Tissue dissociation will be carried out by enzymatic digestion and recovered
cells cultured in serum-free medium containing 25 microg/ml insulin, 100 microg/ml
apo-transferrin, 10 microg/ml putrescine, 0.03 mM sodium selenite, 20nM progesterone, 0.6%
glucose, 5mM hepes, 0.1% sodium bicarbonate, 0.4% BSA, glutamine and antibiotics, dissolved
in DMEM-F12 medium and supplemented with 20 ng/ml EGF and 10 ng/ml bFGF. Flasks non-treated
for tissue culture will be used in order to reduce cell adherence and favourite growth of
undifferentiated tumour-spheres. These culture conditions select for immature tumour cells,
while non malignant or differentiated cells are negatively selected as assessed for CSCs of
different origin. Surviving immature tumor cells slowly proliferate giving rise to tumour
cell aggregates, "spheres", within 1-2 months in these culture conditions. Sphere-forming
cells can be expanded by mechanical dissociation of spheres, followed by re-plating of
single cells and residual small cell aggregates in complete fresh medium.

Differentiation of LC, CRC and BC sphere-forming cells will be obtained by cell culture in
specific medium (Cambrex). Phenotype of LC, CRC and BC spheres and their differentiated
progeny will be analyzed by flow cytometric analysis or immunofluorescence. In particular
stem cell markers such as CD133, CD34 and BCRP1 will be analyzed.

Successively, cancer spheres will be analyzed in order to define the status of pathways
involved in the process of proliferation, self-renewal and survival. In particular,
tumor-specific analysis will be carried out to investigate the activity and the possible
alteration of pathways responsible for stem cell homeostasis and global analysis
(phosphoproteomic and signal transduction analysis,innovative drug testing, analysis of
processes metastatization in vivo) aimed to provide an overall picture of the activation
state of the key cellular pathways.

Preclinical model By using cancer spheres the investigators will generate orthotopic
xenograft models that recapitulate the parental tumor behaviour, including the aggressive
features and the invasiveness potential. Orthotopic injection technique will be assessed in
5 weeks-old NOD/SCID mice. The injection procedure will be done with the support of a
dissecting microscope. After anesthetization, 200 to 500 cancer sphere cells, modified in
order to express a bioluminescent protein such as luciferase will be injected using a
Hamilton syringe and 32-gauge needle. Metastatic and local tumors will be compared for their
stem cell content through phenotypic analysis such as growth rate, or other stem cell
properties including clonogenic capacity in soft agar or through limiting dilution assays.
Infection of CSCs with lentiviral vector, coding for green fluorescent (GFP), as well as
luciferase reporter proteins, will allow CSCs tracking in vivo. Particularly, the
amphotropic packaging cell line 293T will be transfected by the
calcium-phosphate/chloroquine method. Culture supernatants containing viral particles will
be collected after 48h of transfection. Infection will be performed by culturing target
cells in 0.45 microm filtered viral supernatant for 3h in a CO2 incubator. Two infection
cycles will be performed to infect cells. Microscopic evaluation of GFP expression in viral
packaging and target cells will be performed by direct observation of cells using a reversed
fluorescence microscope equipped with a FITC filter. After infection, cells transduced with
luciferase will be sorted by flow cytometry to obtain a pure marked population. The local
tumor and the invasiveness development will be monitored through whole-body imaging
techniques, that will permit to detect, localize and quantify dynamically the optical signal
- bioluminescence - in a non invasive localization of the marked cell population. This
procedure will be performed using the Photon imager in vivo imaging system(Biospace Lab),
assisted by the most recent software for acquisition and image analysis. Thanks to this
system, it was recently acquired in the laboratory and characterized by a very high
sensitivity and 20 ms temporal resolution, the investigators will analyze non anesthetized
and freely moving animals. The bioluminescence signal will be acquired simultaneously as a
standard video of the animal. Once the investigators are sure of the success of tumour
growing, mice will be sacrificed. Tumor will be removed for morphological characterization
and phosphoproteomic analysis. This latter will be performed through reverse phase protein
microarray, which allows the achievement of a high degree of sensitivity, precision and
linearity, making possible to quantify the phosphorylated status of signal proteins in
immature and differentiated lung cancer cells. This system will provide information on
specific molecular pathways involved in LC, CRC and BC cell growth and spreading.

Tumor sensitivity to anti-tumoral agents To selectively discriminate the effective
therapeutic compounds against the putative tumor and invasion initiating cells, the
investigators will measure the viability of clonogenic LC, CRC and BC after the exposure to
several anti-tumor drugs differentially combined at singular time point up to 96 hours.