Progesterone receptor expression contributes to gemcitabine resistance at higher ECM stiffness in breast cancer cell lines

Abstract

Chemoresistance poses a great barrier to breast cancer treatment and is thought to correlate with increased matrix stiffness. We developed two-dimensional (2D) polyacrylamide (PAA) and three-dimensional (3D) alginate in vitro models of tissue stiffness that mimic the stiffness of normal breast and breast cancer. We then used these to compare cell viability in response to chemotherapeutic treatment. In both 2D and 3D we observed that breast cancer cell growth and size was increased at a higher stiffness corresponding to tumours compared to normal tissue. When chemotherapeutic response was measured, a specific differential response in cell viability was observed for gemcitabine in 2 of the 7 breast cancer cell lines investigated. MCF7 and T-47D cell lines showed gemcitabine resistance at 4 kPa compared to 500 Pa. These cell lines share a common phenotype of progesterone receptor (PGR) expression and, indeed, pre-treatment with the selective progesterone receptor modulator (SPRM) mifepristone abolished resistance to gemcitabine at high stiffness. Our data reveals that combined treatment with SPRMs may therefore help in reducing resistance to gemcitabine in stiffer breast tumours which are PGR positive.

Fig 1. Validating the polyacrylamide (PAA) hydrogel model.

(A) Schematic of the PAA model. (B) Brightfield microscopy of MCF-7 cells cultured for 48 hrs on the PAA model. (C) Immunofluorescent imaging of vinculin and actin in MCF-7 cells cultured on 500 Pa and 4 kPa stiffness hydrogels for 16 hrs. (D) Median and individual values for nuclear and cytoplasmic areas counting for 17–25 cells per condition. Significance was calculated using a Mann-Whitney U-test. (E) MCF-7 cell viability as measured by alamarBlue assay over 144 hrs culture on 500 Pa and 4 kPa stiffness hydrogels. The mean and SEM of 2 independent repeats are shown. (F) MCF-7 cell growth after 120 hrs culture on 500 Pa and 4 kPa stiffness hydrogels as measured by DAPI stained nuclei. The mean and SEM of 3 independent repeats are shown. Significance was calculated using a Student’s paired t-test.

Fig 2. Matrix stiffness modulates chemotherapeutic resistance to gemcitabine but this is not attributed to classical EMT.

(A) MCF-7 cells were cultured on 500 Pa and 4 kPa hydrogels for 24 hrs prior to the addition of chemotherapeutic agents as indicated; 5-fluroruracil (5-FU), hydroxyurea (HU), and gemcitabine. After 72 hrs cell viability was measured by alamarBlue assay. The means and SEMs are plotted for >3 independent repeats. Significance was calculated using a Student’s paired t-test. (B) E-cadherin and β-catenin visualised by immunofluorescence in MCF-7 cells cultured on 500 Pa and 4 kPa stiffness hydrogels for 24 hrs. (C) Western blot for E-cadherin and a GAPDH control of whole cell extracts from MCF-7 cells cultured on 500 Pa and 4 kPa stiffness hydrogels for 72 hrs. (D) Immunofluorescent visualisation of YAP in MCF-7 cells cultured on 500 Pa and 4 kPa stiffness hydrogels for 24 hrs. (E) Nuclear YAP intensity (integrated density) measured in approximately 100 nuclei for each condition for each of 3 independent repeats. The median and individual cells from pooled data are plotted. Significance was calculated using a Mann-Whitney U-test.

Fig 3. Stiffness specific gemcitabine resistance is not associated with altered levels of replication stress or DNA damage levels.

The MCF-7 cell line was cultured on 500 Pa and 4 kPa stiffness hydrogels for 24 hrs prior to treatment with 5 nM gemcitabine (Gem) or a vehicle control for a further 24 hrs. Cells were then fixed and stained using immunofluorescence for (A) RPA34 or (B) γH2AX. Representative images are shown for each stain. Nuclear intensity (integrated density) was measured in 100 nuclei for each of 3 independent repeats. The median and pooled data from the repeats are shown. Statistical significance was calculated using a Kruskal-Wallis test.

Fig 4. Resistance to gemcitabine at higher stiffness occurred only in progesterone receptor positive cell lines.

(A-E) The T-47D, MDA-MB-468, CAL-51, SKBR-3, and ZR-75-1 cell lines were cultured on 500 Pa and 4 kPa stiffness hydrogels for 24 hrs prior to the addition of gemcitabine (gem). After 72 hrs treatment cell viability was measured by alamarBlue assay. Means and standard errors of the mean are plotted for three independent repeats. Significance was calculated using a Student’s paired T-test. (F) Summary of hormone receptor and p53 expression status in the breast cancer cell lines. (G) Hormone expression investigated relative to β-actin as determined by RT-q-PCR. The delta CT values for cells grown on glass, are plotted in grey for 3 independent cell pellets performed in triplicate. The mean and standard error of the mean are plotted in black.

Fig 5. Resistance to gemcitabine can be reversed by addition of the SPRM–mifepristone.

The MCF-7 cell line was cultured on 500 Pa and 4 kPa hydrogels for 24 hrs prior to the addition of mifepristone as indicated for 24 hrs. Gemcitabine was then added in combination with mifepristone or its vehicle control for a further 72 hrs. Cell viability was measured by alamarBlue assay. The means and SEMs are plotted for 3 independent repeats. Significance was calculated using a Student’s paired t-test.

Fig 6. Resistance to gemcitabine at higher stiffnesses also occurs in a 3D model and is reversed by mifepristone.

(A) Schematic of the alginate model where different alginate percentages are used to give different stiffnesses. Beads were formed by dropping alginate cell suspension into calcium chloride solution. Alginate beads were then washed in medium and cells allowed to grow by incubating at 37°C. (B) Representative brightfield images of MCF-7 cells 96 hrs post alginate bead gelation. (C) Rounded average measured elastic modulus measured for 6 gels pooled from 2 independent repeats measured 1 hr after gelation at a frequency of 1 Hz by rheometry. (D) MCF-7 cells were cultured in alginate beads for 24 hrs prior to the addition of gemcitabine, after 72 hrs treatment cell viability was measured by alamarBlue assay. The means and SEMs are plotted for 4 independent repeats. (E) MCF-7 cells were cultured in alginate hydrogels for 24 hrs prior to the addition of mifepristone. After 24 hrs 5 nM gemcitabine (Gem) was added. Cells were incubated for 72 hrs before cell viability was measured by alamarBlue assay. The means and SEMs are plotted for 3 independent repeats. Significance was calculated using a Student’s paired t-test.