Treatment with eFT-508 increases chemosensitivity in breast cancer cells by modulating the tumor microenvironment

The human BC cell lines, MDA-MB-231, MDA-MB-468, MCF7, T47D, and SUM149 (American Type Culture Collection, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, USA). The medium was supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% penicillin–streptomycin solution. All cell lines were incubated in 5% CO₂ at 37 °C where indicated cells were treated with indicated doses of adriamycin (Doxorubicin) and/or Tomivosertib (eFT-508) (Selleckchem) for 24 h.

Polysome profiling was done following a previously described protocol [18]. Briefly, cells were treated with 50 µg/mL with cycloheximide (Sigma-Aldrich, St. Louis, USA) for 30 min at 37 °C and then washed with cycloheximide-supplemented cold PBS. Cells were lysed, and post-nuclear extracts were layered on 10–50% sucrose gradients and centrifuged at 100,000g for 4 h. Gradient fractionation was performed using the UA-6 UV detector (Teledyne ISCO, US) based on 254 nm absorption over time.

After the indicated treatment regimen, puromycin (C58-58–2, Sigma Aldrich) was added at a final concentration of 1 μM (100-fold dilution) for 12 h, and the cells were cultured for an additional 30 min before harvesting and cell lysate preparation. Cell lysates were subjected to immunoblot analysis. Blots were probed with an anti-puromycin antibody (Kerafast) to assay relative puromycin incorporation. Finally, blots were stained with Coomassie to confirm equal loading across different samples.

Normal cells and BCs were plated in 6-well plates at a density of 3.0 × 10⁵ cells/well and cultured in CCSCs medium. After 24 h, cells were transferred to serum-free medium and incubated for 18 h. After the cells were treated according to the established groups, the cells were collected. Next, we added cell lysis buffer (50 mm tris–HCl, pH 7.5, 150 mm NaCl, 1 mm sodium-EDTA, 1 mm EDTA, 1% (v/v) Triton x-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM NaF, 1 μg/mL leupeptin, and 1 mM PMSF). The protein concentration was determined by the BIORAD reagent, and the loading volume of protein was calculated. Protein bands were separated based on molecular weights via 10% (w/v) SDS-PAGE (electrophoresis conditions: concentration gel 100 V, 25 min; separation gel 200 V, 40–50 min), and then transferred to PVDF membrane (transfer condition: 100 V, 2 h). The membrane was sealed with 5% (w/v) skimmed milk powder at room temperature for 1.5–2 h, followed by the addition of corresponding primary antibody and incubate at 4 °C overnight. Finally, the secondary antibody was incubated at room temperature for 1.5–2 h, and the protein image was obtained using a gel imager. The following primary antibodies were used: anti-P-eIF4E (ab1126), eIF4E (ab238519), and GAPDH (ab181603), all antibodies were purchased from Abcam China and used at 1:1000 dilution. GAPDH was used as the loading control.

We added 200 μL of assay buffer to the cells in the wells of the experimental titration plate and then placed on a shaking table for 10 min. The assay buffer was removed with a vacuum pump; 25 μL of assay buffer was added into the sample hole and appropriate matrix solution was added into the standard hole and quality control hole of back measuring hole. Finally, 25 μL of the sample was added into the appropriate hole, shook, and 25 μL of the microspheres were mixed into each hole. The board was closed with special plastic film and shook at 4 °C for 18–20 h. The residual liquid was gently removed with a vacuum pump. Next, 200 μL of the wave buffer was added to each hole for cleaning, followed by the addition of 25 μL of secondary antibody into each hole. The plate was incubated in a shaker for 2 h, then 25 µL of streptavidin phycoerythrin was added into the well containing the second antibody. The cells were incubated on shaking table for 30 min at room temperature, followed by the addition of 150 µL of sheath solution to all holes. The expressions of TNFα, IL-10, IL-8, CXCL-10, and IL-6 were measured by Luminex (Millipore) in plasma samples obtained from mice in different experimental groups.

Positive selection was used to isolate CD8 + T cells (10⁴ cells/hole) from tumors using magnetic-activated cell sorting using CD3 and CD8 microbeads (Miltenyi Biotec). The cells were cultured in 6-well plates coated with OKT3 (Ortho Biotech; 1 μg/mL) and anti-CD28 (clone CD28.2, Becton Dickinson; 1 μg/mL) antibodies. The cells were cultured in DMEM medium without double antibody for 4 days, and the supernatant was aspirated, and the PCR method and culture method were used to detect whether there was mycoplasma contamination. The cells in the culture flask with good growth condition and cell fusion degree of 75–80% were washed twice with PBS buffer and digested with trypsin. The degree of cell digestion was observed under a microscope. When the cells became round, and the intercellular space became larger, an appropriate amount of 10% (v/v) FBS 1640 medium was added to stop the digestion. The cells were gently blown down using a blow tube and transferred into a 15 mL centrifuge tube. After centrifugation at 800 rpm for 5 min, the supernatant was discarded and passaged at a ratio of 1:2.

The cells of each group planted in the 96-well plate were taken, and after culturing for 48 h, 20 μL of MTT solution was added to the cells, and the incubation was continued for 4 h. Remove the supernatant solution with a pipette, add 150 μL of DMSO to each well, observe the dissolution of the crystals, and measure the absorbance (A) value of each well at 490 nm. A larger A value indicates higher cell viability.

All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University. MDA-MB-231 cells (0.5 × 10⁶) were trypsinized, washed with PBS, and resuspended in a 1:1 solution of PBS and Matrigel (phenol red-free and reduced growth factors, BD Biosciences) and injected into the 4th mammary fat pad of 8-week old female BALB/c mice (n = 20). Tumor volume was measured every day using a caliper. When the tumors reached 50 mm³ in size (around day 7), the mice were randomly divided into four experimental groups (n = 5/group)—vehicle (DMSO), adriamycin (3 mg/kg/day), eFT-508 (1 mg/kg/day), and eFT-508 (1 mg/kg/day) + adriamycin (3 mg/kg/day)—all injections were intratumoral. Bodyweight, tumor growth rate, and survival were analyzed for each group. The mice were sacrificed either when tumor volume reached 3000 mm³ or at the end of 8 weeks, whichever was earlier. The tumors and lungs were surgically removed, weighed, fixed in 4% polyformaldehyde, and stored at − 80 °C.

Next, 4 µm sections were either processed for H&E staining using routine methodologies or IHC. The sections were dewaxed using xylene I and II for 10 min, respectively. A cover glass was prepared in advance, covered with water, 100% (I, II), 90%, 80%, and 70% alcohol for 5 min, respectively, and rinsed with tap water for 15 min; dyed with hematoxylin for 5 min. The dyeing time could be appropriately increased or reduced. After differentiation with 5% acetic acid for 1 min, the sections were rinsed with running water, followed by the addition of acetic acid with a pipette, and the tissue was covered on the slide. After differentiation, the color became light and blue; Blue returning: Blue returning liquid, which is not available in the laboratory, can also be used; the sections were stained with Eosin for 1 min, dehydrated using 70%, 80%, 90%, and 100% alcohol for 10 s and xylene for 1 min. The sections were dried naturally in the fume hood and then sealed for about 5 min. Next, a drop of neutral gum was dropped to seal the film, and a straw was used to drop a very small drop; the tissue was covered after pressing the film to avoid bubbles in the middle. For IHC, sections were treated for antigen retrieval and incubated with primary antibodies (P-eIF4E, 1:200; Cell Signaling Technology) overnight at 4 °C. The images were obtained using an Olympus light microscope. We randomly selected three visual fields and analyzed three slices per sample at 20×magnification.

Cells were stained with cisplatin (DVS-Sciences) to identify live cells. Then chemoresistant cell lines were incubated with 19 metal-tagged anti-PD-L1 antibodies (Biolegend, 124,314), followed by an intercalator (DVS Sciences). Staining was done following guidelines of the Maxpar cell staining protocol (Fluidigm Corporation) and then incubated at 4 °C for 30 min [17]. CyTOF-II mass cytometer (Fluidigm) was used for sample runs. Post-run, the EQ Four Element Calibration Beads (EQ Beads, 201,078, Fluidigm) were used to normalize the signal. Events were initially gated for viability using Ir191 vs. Ir193, and double-positive cells were used in downstream analyses to identify different sub-populations post-dynamic downsampling.

Responders CD3 + CD8 + T cells, sorted from tumor-infiltrating immune cells, were stained with CFSE (Thermo Fisher Scientific) and seeded into 96-well plate format in RPMI. Also, 10% AB serum either alone or in combination with irradiated (40 Gy) allogeneic PBMCs (stimulators) at three different ratios—1:1, 2:1, and 5:1. CD14⁺ antigen was present in all wells. T cell proliferation was calculated by analyzing the CFSE dilution of the responders after 72 h.

Sorter tumor-infiltrating CD3 + CD8 + cells were cultured in medium ± PMA (20 ng/mL; Sigma-Aldrich, St. Louis, MO) and Ionomycin (1 µg/mL; Sigma-Aldrich). Luminex (Millipore) was used to quantify the levels of indicated cytokines in the supernatants collected before or after 3 days of PMA/Ionomycin stimulation.

Transcriptomic analyses of CD274 expression data in invasive breast carcinoma from The Cancer Genome Atlas (TCGA) were performed using the interactive web resource UALCAN (http://​ualcan.​path.​uab.​edu; Accession date: November 8, 2020) [19]. Survival probability based on low and high expression of CD274 was computed by Kaplan–Meier curve analysis and Log-rank text using USALCAN interactive portal.

Quantitative data were presented as mean ± standard deviation (SD). Statistical analysis between groups was performed using Student’s t-test (two-group comparison) or ANOVA (multiple group comparisons). Kaplan–Meier curves were computed to analyze survival rates, and Log-rank (Mantel-Cox) test was used to evaluate statistical significance. A P-value < 0.05 was considered to be statistically significant.

We initially determined CD274 (encodes PD-L1) mRNA expression within the TCGA dataset in invasive breast carcinoma (BRCA). The expression of CD274 mRNA was slightly significantly lower in primary tumor tissues compared to the normal tissues (Fig. 1A; P = 0.049305). However, there was no correlation between CD274 expression and the overall survival in patients with BC (Fig. 1B; P = 0.8). There was no correlation between CD274 expression and disease stage (Additional file 1: S1A) and race (Additional file 1: Figure S1B).

Next, we evaluated CD274 expression in BRCA based on BC subclasses, luminal, HER2 + , and TNBC. The expression of CD274 was higher compared to both normal (P = 0.0101171) and luminal (P = 0.023656), but not HER2 + (P = 0.103021) (Fig. 2A). Then we determined CD274 expression within the different subtypes of TNBC. The expression of CD274 was statistically significant different in TNBC-IM compared to normal (P = 0.0138987), luminal (P = 0.016004), HER2 + (P = 0.020079), TNBC-BL1 (P = 0.025676), TNBC-LAR (P = 0.010937), TNBC-MSL (P = 0.0119235), and TNBC-UNS (P = 0.030065) (Fig. 2B). Thus, PD-L1 mRNA expression had a significant correlation with the receptor-negative status [13, 14], especially to the TNBC-IM subtype.

Since PD-L1 mRNA expression was significantly higher in TNBC patient samples, we next evaluated if cancer cell lines representing different subtypes had different chemosensitivity to adriamycin and eFT-508 alone and in combination. Luminal cell lines MCF7 and T47D, basal-like, EGFR + SUM149, and TNBC cell lines MDA-MB-231 and MDA-MB-468 cells were used. The exponentially growing cultures of these cell lines were treated with increasing concentrations of adriamycin for 24 h. All cell lines were chemoresistant with a high IC₅₀ ranging from 48.3 to 49.2 nM (Fig. 3A). However, when these cells were treated with increasing concentration of eFT-508, MDA-MB-231 (IC₅₀ = 0.31 ± 0.02 nM) and MDA-MB-468 (IC₅₀ = 1.21 ± 0.01 nM) cell lines exhibited significantly higher chemosensitivity compared to MCF7 (IC₅₀ = 11.38 ± 1.02 nM), T47D (IC₅₀ = 10.19 ± 0.01 nM), or SUM149 (IC₅₀ = 9.14 ± 0.14 nM) cell lines (Fig. 3B; P < 0.05 in each case). MDA-MB-231 cells were significantly more chemosensitive compared to MDA-MB-468 cells (Fig. 3B). Combination treatment with adriamycin and eFT-508 revealed higher in vitro chemosensitivity with significantly lower IC₅₀ in all cell lines compared to either monotherapy (Fig. 3C). Again, the IC₅₀ was significantly lower in MDA-MB-231 cells compared to each of the other four cell lines (Fig. 3C). Western blot results showed that PD-L1 (Proteintech, Mouse Monoclonal| Catalog number: 66248–1-Ig, Fig. 3D) and PD-L2 (Proteintech, Rabbit Polyclonal, Catalog number: 18251–1-AP, Fig. 3E) were highly expressed in BC cells. In addition, five cell lines were treated with eFT-508; the results of WB showed a decreased expression of PD-L1 and PD-L2 (Fig. 3F). Immunoblot analysis confirmed a robust decrease in P-eIF4E (S209) in each of the cell lines treated with eFT-508 without any change in total eIF4E expression (Fig. 3G). Polysome profiling indicated the inhibition of global translation following eFT-508 treatment in these cells, as evident by a decrease in both 80S and polysome peaks (Fig. 3H and data not shown). Polysome profiling results were further corroborated by puromycin labeling. Treatment with eFT-508 (Fig. 3I) but not Adriamycin (Fig. 3J) resulted in a robust decrease in puromycin labeling in both MDA-MB-231 and MCF7 cells. To determine if the observed results were due to clonality of the cell lines tested, we next determined the response of the parental MDA-MB-231 and multidrug resistant MDA-MB-231/ADR cells to adriamycin and eFT-508 treatment, alone or in combination. Indeed, the synergistic effect of adriamycin and eFT-508 was conserved in this model too (Fig. 4A–C). Again, the difference in chemosensitivity was not due to the differential effect of eFT-508 on translational inhibition as evidenced by puromycin labeling (Fig. 4D). Hence, the observed difference in chemosensitivity of the TNBC cell line MDA-MB-231 compared to the other cell lines tested was not due to the differential effect of eFT-508 on eIF4E phosphorylation and translational inhibition, indicating that the difference was instead dictated by the receptor negativity of the BC cell lines.

An orthotopic model of spontaneous metastasis was established by injecting MDA-MB-231 cells in the mammary fat pad of BALB/c mice. Once tumors reached a volume of 50 mm³, mice were randomly divided into four groups (n = 5/group)—vehicle (DMSO), adriamycin (3 mg/kg/day), eFT-508 (1 mg/kg/day), and eFT-508 (1 mg/kg/day) + adriamycin (3 mg/kg/day)—all injections were intratumoral. Tumors reached the size of 50 mm³ at approximately day 7 in all mice. Tumor growth, as well as metastatic dissemination, was followed for up to 8 weeks after the initial injection. Tissues were harvested either when mice were dead or when tumor volume reached 3000 mm³. All mice in the vehicle group died by the end of week 6. Hence body weights were measured for 5 weeks. No significant difference in body weight was observed among the animals from the different experimental groups (Fig. 4E). The tumor growth rate was significantly higher in the vehicle and adriamycin groups compared to eFT-508 or combination therapy groups (P < 0.0001 in each case). Combination therapy exhibited synergism in attenuating tumor growth rate compared to eFT-508 monotherapy (Fig. 4F; P < 0.05).

After the animals were sacrificed, we counted the micrometastatized lung nodules and plotted them. Both eFT-508 monotherapy and combination therapy significantly reduced the incidence of macrometastatic lung nodules (Fig. 4G; P < 0.0001 compared to either vehicle or adriamycin group). No statistical difference was observed between eFT-508 monotherapy and combination groups (Fig. 4G; P > 0.05). Similarly, eFT-508 monotherapy or combination therapy significantly improved survival compared to vehicle or adriamycin groups (Fig. 4H). Thus, these results showed potent anti-tumorigenic and anti-metastatic effects of eFT-508 and revealed synergism with adriamycin only in the context of tumor growth rate. H&E staining of lung tissues (Fig. 5A–D). H&E staining showed that the combined drug group had fewer lung metastases than the single adriamycin group, which was consistent with the results in Fig. 4G. IHC staining confirmed the suppression of phosphorylation of eIF4E in the eFT-508 monotherapy (Fig. 5G) and combination (Fig. 5H) groups compared to vehicle (Fig. 5E) and adriamycin (Fig. 5F) groups.

Next, we evaluated the tumor microenvironment for PD-L1 and PD-L2 expression using mass cytometry. Compared to tumors in the vehicle (Fig. 6A, E) or adriamycin treatment group (Fig. 6B, E), both eFT-508 monotherapy (Fig. 6C, E) and combination therapy (Fig. 6D, E ) significantly downregulated PD-L1 + and PD-L2 + breast tumor cells. Even though there was no significant difference in PD-L1 + and PD-L2 + breast tumor cells between vehicle and adriamycin groups, combination therapy with adriamycin and eFT-508 showed a synergistic effect compared to both adriamycin and eFT-508 monotherapy groups (Fig. 6C–E). These results indicated that treatment with eFT-508 was perhaps inducing immune modulation potentiating tumor cell clearance.

Since the overexpression of PD-L1 was associated with tumor exhaustion and functional impairment [20, 21], we hypothesized that eFT-508-mediated inhibition of PD-L1 expression could restore the effector function of tumor-infiltrating CD8 + T cells (TILs). Hence, the functional capacity of tumor-infiltrating CD8 + T cells in mice treated with vehicle and eFT-508 monotherapy was assessed. CD8 + T cells isolated from mice treated with vehicle demonstrated attenuated expression of Th1/Th2 cytokines, interleukin (IL)-17, interleukin (IL)-10 interferon-gamma (IFN- γ), and tumor necrosis factor-alpha (TNFα), both in the native state and after polyclonal stimulation with PMA and ionomycin (Fig. 7A). In comparison, the expression of all four cytokines was higher in tumor-infiltrating T cells isolated from mice treated with eFT-508 in the native state and significantly induced after polyclonal stimulation (Fig. 7A). Furthermore, the effector function of tumor-infiltrating CD8 + T was determined using the mixed lymphocyte reaction (MLR) assay. The tumor-infiltrating CD8 + T cells in mice treated with vehicle control had impaired alloreactivity compared to those in mice treated with eFT-508 (Fig. 7B), consistent with the altered effector function. Thus, these results established that treatment with eFT-508 alone or in combination with adriamycin had a synergistic effect on tumor growth, metastatic disease progression, potentially due to its ability to restore effector function of tumor-infiltrating cytotoxic CD8 + T cells.