Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT02300883 |
| Other study ID # |
ONC/OSS-02/2011 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
April 2011 |
| Est. completion date |
January 2018 |
Study information
| Verified date |
September 2022 |
| Source |
Istituto Clinico Humanitas |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
This is an observational prospective analysis of biological characteristics of malignant
mesothelioma (MM) patients. Frozen and paraffin-embedded tumor specimens and pleural effusion
of patients will be collected and than will be analyzed with the following analyses:
1Purification of Tumour Associated Macrophages (TAM) and tumour cells from pleural effusions.
2.Mass spectrometry analysis of TAMs and tumor cells 3.Co focal microscopy analysis of
macrophages, tumour cells and specimens derived from patients.
Description:
This is an observational prospective analysis of biological characteristics of malignant
pleural mesothelioma (MPM) patients. Frozen and paraffin-embedded tumor specimens and pleural
effusion of patients will be collected and than will be analyzed with the following analyses:
1. Purification of TAMs and tumour cells from pleural effusions. Pleural effusions will be
subjected to centrifugation in order to separate cellular and non-cellular components.
Cell-free supernatant will be collected and subjected to further analysis for the
identification and characterization of secreted proteins (ELISA, Western Blot). Pleural
effusion cell components will be characterized by cytofluorimetry in order to evaluate
leukocytes composition (e.g. macrophages, neutrophils, lymphocytes). Next, to isolate
TAMs from the cellular components, the investigators will adopt a standard protocol
currently performed in the investigators group to purify TAMs from ascites derived from
ovarian carcinoma patients . Briefly, TAMs will be separated by their selective capacity
to adhere in culture plates in serum-free conditions. After 1 hour of incubation,
non-adherent cells (mainly tumour cells and other inflammatory cells) will be thoroughly
washed off with jets of medium. After adherence, cells were rested for 1 hour in
standard culture conditions and subsequently lysed to extract proteins. To isolate
tumour cells from leukocytes, a cell sorting-based approach will be used. Briefly,
inflammatory cells will be stained with lymphocyte common antigen (CD45) antibody and
negative cells (tumour cells) will be separated by cell sorting. Next tumour cells will
be grown and amplified in standard culture conditions.
2. Mass spectrometry analysis of TAMs and tumor cells Generation of proteome and the
phospho-proteomic maps of TAMs and tumour cells will be accomplished by quantitation of
both the entire proteome and the phospho-proteome. Results obtained with TAMs will be
compared to untreated, M1 and M2 polarized Monocytes-derived macrophages (M-DM), while
tumor cells purified from patients' pleural effusion will be analysed with respect to
human mesothelial cells. Protein samples obtained from the purified TAMs and tumour
cells, derived from the patient's pleural effusions, will be extracted using
lysis/acetone precipitation. Cell extracts will be subsequently subjected to trypsin
digestion and the resulting peptides will be labelled with the "iTRAQ" reagents.
Labelled peptides will be separated with the first dimension of the separation being
strong cation exchange (SCX) chromatography. Fractions will be collected and
phospho-enriched, when required. The pooled mixture of peptides will be submitted to a
second dimension chromatographic separation, organized as a two-step process: a
desalting/concentrating pre-column, and an analytical column for the separation. System
is a nano-flow configuration, with direct interfacing to a mass spectrometer. Tandem
mass spectrometry will be performed on a hybrid ion-trap (IT) - Fourier transform
ion-cyclotron-resonance mass spectrometer. Quantitative and identification data analysis
will be carried out with Proteome Discoverer software (Thermo), using SEQUEST as the
search engine. Statistical analysis and data classification will be carried out with aid
of in-house written script for the freeware statistical analysis package.
3. Co focal microscopy analysis of macrophages, tumour cells and specimens derived from
patients. The analysis will be conducted on the cellular components derived from pleural
effusion (as previously described) and from frozen and paraffin-embedded tissues.
Immunofluorescence staining will be performed with markers of DNA damage and polarized
inflammation. Furthermore, immunofluorescence analysis will be held to validate putative
new MM molecular markers obtained from the mass spectrometry studies.
