Carcinoma-associated fibroblasts affect sensitivity to oxaliplatin and 5FU in colorectal cancer cells

The importance of tumor microenvironment (TME) as a relevant contributor to cancer progression and its role in the development of de novo resistance to targeted therapies has become increasingly apparent. However, the mechanisms of microenvironment-mediated drug resistance for nonspecific conventional chemotherapeutic agents, such as platinum compounds or antimetabolites, are still unclear. Here we describe a mechanism induced by soluble factors released by carcinoma-associated fibroblasts (CAFs) that induce the translocation of AKT, Survivin and P38 to the nucleus of tumor cells. These changes are guided to ensure DNA repair and the correct entrance and exit from mitosis in the presence of chemotherapy. We used conditioned media (CM) from normal-colonic fibroblasts and paired CAFs to assess dose response curves of oxaliplatin and 5-fluorouracil, separately or combined, compared with standard culture medium. We also evaluated a colony-forming assay and cell death to demonstrate the protective role of CAF-CM. Immunofluorescence confirmed the translocation of AKT, P38 and Survivin to the nucleus induced by CAF-soluble factors. We also have shown that STAT3 or P38 inhibition provides a promising strategy for overcoming microenvironment-mediated resistance. Conversely, pharmacologic AKT inhibition induces an antagonistic effect that relieves a cMET and STAT3-mediated compensatory feedback that might explain the failure of AKT inhibitors in the clinic so far.


Immunofluorescence staining
The cells were seeded in coverslip slides on 6-well plates, fixed with paraformaldehyde (PFA) 4% for 20 minutes and washed 3 times with PBS 1×, permeabilized with PBS 1× 0.1% Triton X-100 at room temperature for 5 minutes and blocked with BSA 2% for 45 minutes-2 hours. Cells were incubated with primary antibody in BSA 2% in a dilution 1:50 overnight and then incubated with 1:200 fluorescence secondary antibody in BSA 2% for 1 hour at room temperature. Coverslip slides were mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, USA).

Western blot analysis
Cells were plated onto different dishes at a density of 70% cells per dish and then treated according to the experimental design. After washing the cultures with PBS, cells were lysed with RIPA buffer.
Protein concentration was determined by the BCA Protein Assay (Pierce, Rockford, IL, USA). 30 μg of the protein extract was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes. After blocking for 1 hour with 5% dried non-fat milk in TBS 1× -Tween 0.1%, the membranes were incubated with primary antibody diluted 1:1000 in 1% bovine serum albumin (BSA) in TBS 1× -Tween 0.1%. Antibody binding was detected using a secondary antibody diluted 1:2000 in TBS 1× -Tween 0.1% and an enhanced chemiluminescence (ECL) detection kit (Amersham, Buckinghamshire, UK). α-tubulin expression was used as an endogenous control. A general protocol for nuclear and cytosolic fractions separation was used.

Cell death assessment
In order to assess necrosis or apoptosis by flow cytometry and immunofluorescence, cells were stained with the Promokine apoptotic/necrotic/healthy cell detection kit (cat #PK-CA707-30018, PromoCell, Heidelberg, Germany). First, the cells were detached from the cell culture plate using StemPro Accutase Cell Dissociation Reagent (cat #A1110501, Thermo Scientific), washed twice with 1X binding buffer and then incubated for 15 minutes at room temperature with staining solution (5 μL of FITC-Annexin V, 5 μL of Ethidium Homodimer III and 5 μL of Hoechst 33342 to 100 μL of 1× binding buffer). Cells were washed 1-2 times and mounted on a slide for microscope viewing, or resuspended in 400 μL in 1× binding buffer and then sent for flow cytometry analysis within 1 hour of staining in a Gallios Flow Cytometer (Beckman Coulter, Krefeld, Germany).
The Ac-DEVD-AMC Caspase-3 fluorogenic assay (BD Biosciences Pharmingen, San Agustin de Guadalix, Madrid, Spain) was performed to assess caspase-dependent apoptosis. Whole cellular extract (adherent cells and floating cells) was recovered with lysis buffer and, subsequently, 20 μM of Ac-DEVD-AMC and the cell lysate were replaced by 1 mL of protease assay buffer (described in the manufacturer's instructions). The reaction mixtures were incubated for 2 hours at 37°C in the dark and then measured at wavelengths of 380 nm excitation and 430-460 nm emission.
Immunofluorescence of cleaved caspase 3 was also carried out.

AKT inhibition by oral allosteric MK2206 inhibitor and P38 by VX-702 selective P38α inhibitor
MK-2206 2HCl and VX-702 were purchased from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO as a stock solution. CRC cells were seeded at a density of 2000/4000 cells per well in 96-well plates and incubated with DMEMF12 overnight. Inhibitors were then added to the cells in combination with 5FU or oxaliplatin. Cell proliferation was determined after 5 days by the WST-1 proliferation assay. The effect of the drug on each cell line in the presence or absence of inhibitors was calculated by normalizing the cell frequency after 5 days of continuous treatment with respect to the maximum frequency of cells in each treatment. Western blot was performed to confirm inhibition of pAKT Ser473 in colorectal cell lines. their respective IC50 for oxaliplatin (5 hours) and 5FU (24 hours). b. Ac-DEVD-AMC Capase-3 fluorogenic assay. HT29 and HCT116 cell lines were treated with drugs and their caspase-3 activity was measured at 8, 24, 48 and 72 hours in the presence of standard medium or CM-CAFs. Left and middle panel cells treated with 5FU; right panel cells treated with oxaliplatin. c. Cleaved-caspase 3 immunofluorescence. The HT29 cell line was treated in monoculture and coculture in the presence of 0.2 μM of 5FU. A decrease in cleaved Caspase-3 staining was noted in cells cocultured with CAFs. d. Picnotic nuclei and oncotic cells. Bars represent the magnitude of change between cells treated with oxaliplatin or 5FU when cultured with standard medium compared with CAF-conditioned medium at different times. DAPI staining shows the oncotic morphology of cells treated with 5FU for 24 and 72 hours in DMEMF12 and CAF-CM.