Targeted therapy to β3 integrin reduces chemoresistance in breast cancer bone metastases

Abstract

Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Though β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and post-chemotherapy triple negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Further, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Introduction

Bone metastases remain a significant, unmet challenge in the treatment of breast cancer. The majority of patients with metastatic breast cancer will develop bone involvement (1), with predominantly osteolytic lesions accompanied by refractory pain, increased fracture risk, and decreased survival (2). Bone-targeted therapies such as bisphosphonates and the anti-RANKL monoclonal antibody denosumab have substantially improved quality of life, reducing fracture incidence and impeding bone metastatic progression. Unfortunately, these agents are associated with a survival benefit in only a subset of patients (3), and resistance to chemotherapy and radiation is common (4).

Interaction between the tumor microenvironment and cancer cells has been recognized as an important mechanism driving chemoresistance (5, 6), confounding studies that focus on in vitro treatment data. The bone represents a distinct metastatic niche, comprised of unique cell types, extracellular matrix (ECM) components, and soluble factors compared to visceral metastatic sites. Moreover, the progression from single, disseminated tumor cells on a quiescent bone surface to floridly outgrowing osteolytic lesions is a highly dynamic process, with the importance of individual microenvironmental factors likely varying over time (4, 7). Some critical factors have been identified at different stages of bone metastatic progression, but more targets are needed to enhance the efficacy of available therapies against clinically detectable lesions.

Integrins are heterodimeric transmembrane receptors that bind ligand moieties in the ECM, initiating signaling events with broad consequences for cell survival, proliferation, and migration (8). Integrin β3 (β3, as part of αvβ3 and αIIbβ3 heterodimers) can be a marker of tumor aggressiveness and is expressed on cells in the bone tumor microenvironment, including activated endothelium, osteoclasts, platelets, and immune cells (8–13). We recently showed that β3 is upregulated on breast cancer cells through TGF-β signaling in the bone microenvironment and can be exploited for bone-specific nanoparticle drug delivery (14). β3 has been identified as an important factor for bone colonization by breast cancer cells (9, 15), and has also been shown to promote resistance to EGFR inhibition across multiple cancer types (16). While studies have previously linked β3 signaling and chemotherapy resistance (17), its role in vivo, and particularly during therapy of established bone metastases, is poorly characterized.

In this study, we provide evidence for β3 as an important promoter of resistance to taxane chemotherapy in breast cancer bone metastases. We show that β3 expression is associated with an alternative metabolic response to taxanes in vitro and in vivo, and that β3-mediated resistance can be defused by combination therapy with mTORC1 inhibitors. Taken together, our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.

Materials and Methods

Animals

All animal studies were performed according to Washington University Institutional Animal Care and Use Committee (WU IACUC, Protocol# 20190104) guidelines. Female C57BL/6J (Jax, RRID:IMSR_JAX:000664) and BALB/c (Jax, RRID:IMSR_JAX:000651) mice were obtained from The Jackson Laboratory and injected at 6–7 weeks of age. All mice were housed under pathogen-free conditions according to the WU IACUC.

Cell lines and constructs

The C57BL/6 background PyMT-BO1-GFP-Luc murine breast tumor cell line was previously developed and validated as described (10). The BALB/c background 4T1-FL-GFP murine breast tumor cell line (derived from 4T1, RRID:CVCL_0125) was originally from Dr. David Piwnica-Worms (The University of Texas, Houston, TX) as previously described (18). All cell lines were cultured at low passage (used within 1–3 passages after thaw) and tested regularly for Mycoplasma-specific DNA by PCR amplification of cell or supernatant samples (last test January of 2020). For in vitro experiments involving coated culture dishes, non-tissue culture-treated plates were coated prior to cell seeding with either poly-L-lysine (Sigma: P4707) or Vitronectin XF (STEMCELL Technologies: 07180) according to manufacturer’s recommendations. In vitro drug treatments were performed using docetaxel (5mg/kg, LC Laboratories: RP 56976), everolimus (RAD001; Selleck Chemicals: Cat#: S1120), and doxorubicin hydrochloride (Sigma: PHR1789) at the stated concentrations.

CRISPR knockout of the Itgb3 gene in the PyMT-BO1 line was previously described (19). pMx, pMx-Δβ3, and pMx-hβ3 retroviral vectors used for rescue of β3 expression in the clone #1 β3KO PyMT-BO1 (β3KO1-BO1) line were a gracious gift from Steven Teitelbaum (Washington University School of Medicine, St. Louis, MO). Virus was packaged along with the pCMV-VSVG plasmid using the Plat-E cell line (RRID:CVCL_B488) (20). Tumor cell lines were transduced with viral supernatant for 12 hours at 37°C in 6-well tissue culture plates. Transduced cells were selected in 2ug/mL blasticidin (Sigma: 203350), and stable protein expression of the wild type (hβ3) and signaling mutant (Δβ3) integrin constructs was validated by western blot (below).

CRISPR knockout of Itgb3 in the 4T1 cell line was achieved by stable transduction of the Cas9 gene and the following gRNAs: 5’-CACCGCCGGGATAACCTCGTTGTTG-3’; 5’-AAACCAACAACGAGGTTATCCCGGC-3’, using the lentiCRISPR v2-Puro vector system (Addgene#: 98290). 293T cells (ATCC, RRID:CVCL_0063) were used for viral packaging with the pCMV-DR8.2 and pCMV-VSVG plasmids. Tumor cell lines were transduced with viral supernatant for 12 hours at 37°C in 6-well tissue culture plates. Transduced cells were selected in 10ug/mL puromycin (Sigma: P8833) and further purified by serial FACS sorting of TGF-β1 (2ng/mL, R&D Systems: 7666-MB-005) stimulated cells based on β3 expression. Itgb3 knockout cell lines were validated by sequencing and FACS.

Cell viability assays

Viability assays were performed as previously described (21, 22), with minor modifications (Supplementary Information).

For apoptosis assays, caspase-3/7 activity was determined using the Caspase-Glo 3/7 Assay System (Promega: G8090) and normalized to cell viability as reported by incubation with CellTiter-Blue (Promega: G8080). Colorimetric and luminescent readouts were measured with a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA).

In vivo modeling of metastasis and therapy

Distant metastases were established in mice by intracardiac (i.c.) inoculation of PyMT-BO1 or 4T1 cells into the left ventricle as previously described (14). Tumor burden was monitored by in vivo bioluminescence imaging (BLI). Mice were assigned randomly by cage to treatment groups; mouse weight and hindlimb tumor burden were compared to ensure no statistical differences between groups prior to treatment initiation. Docetaxel (5mg/kg, LC Laboratories: RP 56976) or equivalent vehicle was freshly prepared and administered by tail vein injection (Supplementary Information). Freshly prepared working solution of rapamycin (Sigma: R0395) or equivalent vehicle was administered 2mg/kg by i.p. injection (Supplementary Information). An equimolar equivalent of nanoparticle-encapsulated rapamycin or cargo-free nanoparticle control was administered by tail vein injection for nanoparticle experiments.

Bioluminescence imaging and radiography

BLI and X-ray assessment of tumor burden and bone lesion area were performed as previously described (14), with minor modifications (Supplementary Information).

Immunohistochemical staining

All slides were stained in parallel, using identical staining conditions, with anti-integrin β3 (clone: D7×3P, 1:200, Cell Signaling Technology, RRID:AB_2798136) using previously described protocols (14). Images were acquired on a NanoZoomer (Hamamatsu Photonics).

Post-chemotherapy biopsies from triple negative breast cancer patients

Primary breast cancer specimens were obtained from M0 patients with localized, triple negative disease at time of surgical resection and subsequently banked, curated, and assembled into a tissue microarray by the St. Louis Breast Tissue Registry. Clinical data were obtained in accordance with the Washington University Institutional Review Board (IRB #201102394) and WAIVER of Elements of Consent per 45 CFR 46.116 (d)., and deidentified prior to investigator access. IRB-directed human research activities were guided by principles set forth in the Belmont Report.

Areas of invasive tumor and tumor cell β3 expression by DAB staining were confirmed in consultation with a board-certified pathologist. β3 expression was scored by a group of investigators, all blinded to clinical annotation, using a bimodal classification system focused exclusively on positive staining in tumor cells. For each specimen, the percentage of identifiable tumor cells with β3 staining was jointly determined by the scoring group. Based on the range of tumoral β3 staining observed in the cohort as a whole, a cutoff of 10% was determined, with samples below this threshold assigned to the “Low” expressing group and those above assigned to the “High” expressing group. Samples for which the scoring group was not unanimous were referred to consulting pathologist for final resolution.

Survival analysis in human patients

Recurrence-free survival (RFS, defined as date of diagnosis to date of 1^st local or distant recurrence, otherwise censored at last known recurrence-free date) of β3 Low versus High triple negative breast cancer (TNBC) core samples after neoadjuvant chemotherapy was determined by Kaplan-Meier analysis using Cox proportional hazards and log rank test in consultation with a statistician in the Siteman Biostatistics Shared Resource. Relevant patient demographic data and tumor characteristics were compared between groups using Wilcoxon rank sum and Fisher’s exact tests. RFS in publicly available data was determined by Kaplan-Meier analysis through KM-Plotter (23), comparing TNBC patients receiving any chemotherapy in the lowest quartile of β3 expression to those in the three upper quartiles.

Transmission electron microscopy of murine bone metastases

Mice bearing 4T1 bone metastases were established, treated, and monitored as described above and in Fig. 2C. After tissue processing (Supplementary Information), X-ray microscopy (XRM Versa 520, Zeiss) was performed to identify tumor regions in hindlimb bone samples for thin sectioning. 70nm thin sections were prepared on grids, stained with 2% aqueous uranyl acetate followed by Reynold’s lead citrate, and imaged on a TEM (JEOL JEM-1400 Plus) at 120 KeV. Ultrastructural parameters were quantified using ImageJ (NIH, Bethesda, Maryland; RRID:SCR_003070).

Figure 2. Integrin β3 promotes docetaxel resistance in bone metastases.

A. Flow cytometry of overnight BrdU incorporation in β3lo and β3hi PyMT-BO1 cells in vitro. Cells were treated with DMSO or 10nM DTX for 24hrs, followed by a 48hr recovery period. Representative samples with integrin β3 gating (left), quantification of BrdU incorporation in β3lo and β3hi populations (right), n = 3 biological replicates per group, one of two independent experiments. Two-way ANOVA with Tukey post hoc test; ****, P<0.0001. B. MTT viability assay of 4T1 β3KO and β3WT cells treated with DTX for 48hrs. Assay on tissue culture-treated plate (left) and co-cultured with BMSCs (right, see Materials and Methods for details), n = 4 biological replicates per group, one independent experiment. Two-way ANOVA with Sidak post hoc test; *, P<0.05; ***, P<0.001. C. Ex vivo BLI of 4T1 β3KO and β3WT hindlimb tumor burden from mice receiving either vehicle or DTX (5mg/kg i.v.). Treatment schema (top), quantification of ex vivo BLI signal from hindlimbs (bottom left), representative BLI (bottom right), n = 8 mice per group. Data shown are log₂ transformed fold change in photons/s relative to the geometric mean of samples from vehicle treated mice. Each data point represents averaged signaling intensity from hindlimbs of one mouse. β3WT and β3KO experiments were performed independently. One-tailed unpaired t test with Welch’s correction; ***, P<0.001.

RNA sequencing and analysis

RNA-Seq was performed with the Genome Technology Access Center at Washington University School of Medicine (Supplementary Information). Data are accessible through NCBI GEO (GSE166315, GSE166536).

Flow cytometric analysis

After preparation as single cell suspensions, ex vivo and in vitro samples were stained and analyzed as previously described (14), with assay-specific modifications (Supplementary Information).

Western blot analysis

Western blot was performed as previously described (10) using anti-integrin β3 (D7×3P, RRID:AB_2798136), phospho-S6 ribosomal protein (Ser240/244) (D68F8, RRID:AB_10694233), phospho-S6 ribosomal protein (Ser235/236) (D57.2.2E, RRID:AB_916156), or β-actin loading control (13E5, RRID:AB_2223172), followed by horseradish peroxidase–conjugated anti-rabbit secondary antibody (all from Cell Signaling Technology) according to manufacturer’s protocols. Bands were developed via enhanced chemiluminescence and analyzed by densitometry in ImageJ (NIH, Bethesda, Maryland, RRID:SCR_003070). Blots in Supplementary Fig. S6A and Fig. 6C were run from samples harvested after 24 and 48 hours of treatment (one blot for each time point). After transfer, blots were cut in half. Top halves were blotted for integrin β3. Bottom halves were blotted sequentially for pS6 (S240/244), pS6 (S235/236), and β-actin, stripping between each with Restore Western Blot Stripping Buffer (ThermoFisher: 21059). Because of this, the β-actin loading control for 24hr panels in these two figures is the same.

Figure 6. mTORC1 inhibition reverses β3-mediated chemoresistance.

A. Significantly enriched pathways (FDR q < 0.250) from hallmark GSEA comparing transcriptomic profiles from hβ3 DTX and pMx DTX groups. mTOR and mTOR-associated pathways (orange). B. MTT viability assay of 4T1 β3KO and β3WT cells treated with 100nM everolimus (mTORCi) for 48hrs. n = 8 biological replicates per group, one of two independent experiments. Two-way ANOVA with Tukey post hoc test; ****, P<0.0001. C. Western blot analysis of β3 expression in 4T1 cells after 24hr of everolimus (mTORCi) treatment. Representative blot (left), quantitation (right). n = 2 biological replicates per group, one of two independent experiments. One-tailed unpaired t test with Welch’s correction; *, P<0.05. D. Flow cytometric analysis of de novo protein synthesis in 4T1 β3KO and β3WT cells. Cells were treated with DMSO or 10nM DTX for 24hrs, followed by 48hrs of either DMSO or 100nM everolimus (mTORCi). Quantification of AF549 ClickIt-HPG fluorescence positivity (left), quantification of percentage decrease in positivity between vehicle and combination-treated samples for each genotype (right). n = 3 biological replicates per sample, one of two independent experiments. Three-way ANOVA with Tukey post hoc test; **, P<0.01; ****, P<0.0001. E. Ex vivo BLI of β3WT PyMT-BO1 hindlimb tumor burden from mice receiving either vehicle, DTX alone (5mg/kg i.v.), rapamycin alone (RAPA, 2mg/kg i.p.), or both combined (COMBO). Treatment schema (top), representative BLI (middle), quantification of ex vivo BLI signal from hindlimbs (bottom), n = 5–6 mice per group. Data shown are log2 transformed fold change in photons/s relative to the geometric mean of samples from vehicle-treated mice. Each data point represents averaged signaling intensity from hindlimbs of one mouse. Two-way ANOVA with Tukey post hoc test; **, P<0.01; ***, P<0.001. F. Schematic of αvβ3-targeted, rapamycin-loaded nanoparticles. G. Ex vivo BLI of β3WT PyMT-BO1 hindlimb tumor burden and X-ray radiography of tibiofemoral joint osteolytic area from mice receiving either αvβ3-CF-NP, αvβ3-CF-NP and free DTX (5mg/kg i.v.), or αvβ3-RAPA-NP particles (2mg/kg equivalent rapamycin dose) and free DTX. Combination treatment strategy (top), quantification of ex vivo BLI signal from hindlimbs (middle left), quantification of osteolytic lesion area (middle right), representative radiographs from each group (bottom), n = 14–15 mice per group. Scale bar = 1.25mm. Red arrows indicate areas of significant bone erosion. Data shown are log2 transformed fold change in photons/s or total lesion area relative to the geometric mean of samples from αvβ3-CF-NP mice. One-way ANOVA with Tukey post hoc test; *, P<0.05; **, P<0.01; ****, P<0.0001.

Oxygen consumption analyses

Cells were seeded and treated in 6-well plates as indicated, then lifted with trypsin and re-seeded onto Seahorse XF96 V3 PS Cell Culture Microplates (Agilent: 101085–004) overnight at experimentally optimized density. Extracellular flux analysis of oxygen consumption rate (OCR) was performed on the Seahorse Biosciences XF96 Flux Analyzer (Agilent) at baseline and after serial injection of oligomycin (1.5uM), FCCP (0.5 or 1uM), and antimycin A/rotenone (0.5uM) (Seahorse XF Cell Mito Stress Test Kit, Agilent: 103015–100) according to manufacturer’s recommendations. After analysis, tumor cell sample luciferase activity was determined for normalization using a SpectraMax i3 plate reader (Molecular Devices, Sunnyvale, CA). Data normalization, analysis, and calculation of maximum OCR were performed using Wave Desktop v2.6 (Agilent, RRID:SCR_014526).

Galuminox imaging of radical oxygen species

Live cell fluorescence imaging was performed as previously described (24), with minor modifications (Supplementary Information).

Synthesis of αvβ3-RAPA nanoparticles

Phospholipid/polysorbate 80 micelle nanoparticles (NP) were prepared as a microfluidized suspension of 20% (v/v) combining polysorbate 80 (NOF America) with a 2.0% (w/v) commixture and 1.7% (w/v) glycerin in pH 6.5 carbonate buffer. The commixture included 2 mole% rapamycin, 0.15 mole% αvβ3-PEG2000-PE (Supplementary Information), and high-purity phosphatidylcholine (Lipoid). Rapamycin was excluded from commixture for targeted, drug-free nanoparticles. The lipid commixtures were combined with the polysorbate, buffer, and glycerin and homogenized at 20,000 psi for 4 minutes at 4°C with a microfluidics homogenizer (M110s or LV1, Microfluidics, Inc). Nanoparticles were sterile filtered and preserved under inert gas in sterile sealed vials until use. Dynamic light scattering (Zeta Plus, BrookHaven) showed nominal particle size of 23.9 nm, with polydispersity of 0.258 and an average electrophoretic zeta potential of −−1.61mv for αvβ3-RAPA-NPs, which were closely similar to αvβ3-CF-NP control.

Serum chemistry analysis

Blood was obtained by submandibular venous puncture and collected in Microtainer serum separator tubes (BD Biosciences: BD365967) for serum chemistry analysis using the Liasys 330 AMS Diagnostic liquid chemistry analyzer. Investigators were blinded to treatment groups during analysis.

Statistical analysis

All sample sizes reported in the study are the minimum number of samples. For animal studies, sample sizes were decided based on our previous work in these models. Statistical differences were analyzed using either one- or two-tailed unpaired t test with Welch’s correction, ANOVA with Tukey or Sidak test for post hoc multiple comparisons, or ANOVA with test for linear trend using Prism 8 (GraphPad Software Inc., RRID:SCR_002798). Results were considered to reach significance at P ≤ 0.05, indicated with asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001). Data are presented as mean values; error bars represent ± SD.

Results

Integrin β3 expression is increased in breast cancer cells after chemotherapy

Dysregulated expression of integrin β3 (β3) is associated with increased aggressiveness and drug resistance in cancer (16). We first asked if exposure to the chemotherapeutic agent docetaxel (DTX) alters the proportion of β3-expressing cells in tumor populations. To test this, the 4T1 and PyMT-BO1 murine breast cancer cell lines (modeling triple negative and luminal B disease, respectively) were administered DTX in vitro and cell surface β3 expression was assessed by flow cytometry. We observed an increase in the percentage of β3+ cells in both cell lines after DTX treatment (Fig. 1A, Supplementary Fig. S1A), with a stronger dose-dependent response in the PyMT-BO1 line. In vitro exposure to doxorubicin, another chemotherapeutic agent frequently administered in breast cancer, yielded similar increases in the percentage of β3+ cells (Supplementary Fig. S1B).

Figure 1. Integrin β3 expression is increased in breast cancer cells after chemotherapy.

A. Flow cytometry of integrin β3 expression in PyMT-BO1 and 4T1 cells harvested 48 hours after overnight treatment with DTX. One of three independent experiments, each with n = 2 biological replicates per treatment group. One-way ANOVA with test for linear trend; ****, P<0.0001. B. Representative X-ray radiographs of tibiofemoral joints from vehicle and DTX-treated mice bearing either PyMT-BO1 or 4T1 metastases established by i.c. injection. Scale bar = 1.25mm. Red arrows indicate areas of significant bone erosion. n = 8–9 mice per group. C. Ex vivo flow cytometry of live, GFP+ PyMT-BO1 cells harvested from established bone metastases treated with either vehicle or DTX (5mg/kg i.v.). n = 7–8 mice per group. Two-tailed unpaired Welch’s t test; ****, P<0.0001. D. Design of tissue microarray with 38 primary TNBC biopsies obtained after neoadjuvant chemotherapy. Summary of β3-stratified patient demographics (right). E. Integrin β3 IHC in human TNBC patients after chemotherapy. Representative images of low and high tumor β3 staining (left), summary of scoring (right, see Materials and Methods). Scale bars = 100 μm (20x) or 50 μm (40x). F. Kaplan-Meier analysis of β3-stratified recurrence-free survival in patients from tissue microarray. Swimmer’s plot of individual time to recurrence (left), Kaplan-Meier curves and statistics (right). Hazard ratio (HR) and confidence intervals determined by Cox proportional hazards model; significance determined by log rank test. G. Kaplan-Meier analysis of β3-stratified recurrence-free survival in 315 high-risk TNBC patients receiving any chemotherapy obtained from publicly available microarray data. Hazard ratio (HR) and confidence intervals determined by Cox proportional hazards model; significance determined by log rank test.

We next evaluated integrin β3 expression after DTX in the bone metastatic environment. We had previously demonstrated increased tumoral β3 in bone metastases compared to primary breast tumors, both in human patients and in 4T1 and PyMT-BO1 preclinical models (14). We found that DTX failed to attenuate osteolytic lesions generated by 4T1 and PyMT-BO1 (Fig. 1B), indicating that both cell lines were fairly chemoresistant. This prompted us to measure β3 expression in the resistant tumor cells that remained. Given their greater β3 response to DTX in vitro, we focused on PyMT-BO1 bone metastases, harvesting live, GFP+ tumor cells for assessment of β3 expression by ex vivo flow cytometry. We found a significant increase in the proportion of GFP+β3+ tumor cells in bone metastasis samples from mice receiving DTX compared to those from mice receiving vehicle (vehicle: 33% β3+; DTX: 54% β3+, p<0.0001) (Fig. 1C, Supplementary Fig. S1C).

To gauge translational relevance, we assessed tumoral β3 expression in a tissue microarray (TMA) of high-risk, post-chemotherapy clinical specimens taken from 38 patients with localized TNBC who did not achieve pathological complete response (pCR) after neoadjuvant chemotherapy (Fig. 1D). We evaluated tumor cell-specific β3 expression by immunohistochemistry (IHC), designating samples as either Low or High based on staining intensity and β3+ cell frequency (see Materials and Methods). 97% of patient specimens had positive tumoral β3 staining after chemotherapy. 27 (71%) were characterized as Low tumoral β3 expression, while 11 (29%) were High (Fig. 1D and E). As expected, we observed a consistent vascular pattern of strong β3 expression on tumor neoangiogenic endothelium, serving as a positive staining control (Supplementary Fig. S1D) (25). Kaplan-Meier analysis of differences in recurrence-free survival (RFS) between patients with β3 Low and High post-chemotherapy residual tumors revealed a trend toward increased risk of recurrence in the High group, particularly after the first 1.5 years after diagnosis (note curve cross in Fig. 1F), though this was not statistically significant in our relatively small sample (HR 1.75, 0.66–4.74; p=0.254) (Fig. 1F). To validate this finding in a larger cohort, we used publicly available microarray data to perform a separate RFS analysis in 315 TNBC patients who had received chemotherapy (23). In this data set, patients with increased tumoral β3 expression (High, upper three quartiles) were twice as likely to experience recurrence compared to patients in the lowest quartile of expression (Low) (HR = 2.01, logrank p<0.0095) (Fig. 1G). Together, these data suggest that tumoral β3 expression is increased and associated with worse outcomes after chemotherapeutic challenge.

Integrin β3 promotes docetaxel resistance in bone metastases

We next considered functional differences in β3+ tumor cells that might drive poor outcomes after treatment, measuring proliferation changes in cells with high and low β3 expression after chemotherapy in vitro. PyMT-BO1 cells were exposed to DTX in vitro and BrdU incorporation was assessed by flow cytometry in β3hi (High) and β3lo (Low) populations. The β3lo population of DTX-treated PyMT-BO1 cells exhibited significantly reduced BrdU incorporation compared to β3lo cells receiving vehicle (48% reduction, p<0.0001). Interestingly, BrdU incorporation by β3hi cells present in the same cultures was unchanged between DTX and vehicle-treated samples (Fig. 2A), suggesting that β3-expressing cells respond differently to DTX. To address this, we employed CRISPR/Cas9 technology to generate Itgb3 knockout (β3KO) derivatives of the 4T1 (Supplementary Fig. S2A) and PyMT-BO1 (Supplementary Fig. S2C) (19) murine breast cancer cell lines.

Cell viability of β3KO derivatives was measured by MTT assay. Both β3WT and β3KO 4T1 cells were sensitive to DTX administration in vitro, with β3WT cells showing only modestly higher viability (Fig. 2B). Given that integrins enhance cell adhesion (8), and that tumor cell co-culture with bone marrow stromal cells (BMSCs) increases chemoresistance (26), we next assessed the DTX viability of β3WT and β3KO 4T1 derivatives in BMSC co-culture. In these conditions, β3WT 4T1 cells exhibited enhanced resistance to DTX compared to single culture, while BMSC co-cultured β3KO 4T1 derivates remained sensitive (Fig. 2B). We found similar in vitro DTX viability trends in β3WT and β3KO PyMT-BO1 derivatives (Supplementary Fig. S2D).

Given the contribution of the tumor microenvironment to therapeutic responses (6), we next interrogated the role of β3 for chemoresistance in murine bone metastases. Mice bearing disseminated β3WT or β3KO 4T1 cells were administered either DTX or vehicle and assessed for organ tumor burden by ex vivo BLI. Across all organs analyzed (kidneys, lung, liver, and hindlimb bones) in β3WT tumors, there was no significant difference in bioluminescence between vehicle and DTX-treated groups (Fig. 2C, Supplementary Fig. S2B). By contrast, hindlimb bones from DTX-treated mice bearing β3KO cells exhibited significantly (50.6-fold) reduced tumor burden compared to vehicle, with visceral organs also exhibiting trends toward decrease (kidneys: 5.3-fold; lung: 1.2-fold; liver: 5.7-fold) (Fig. 2C, Supplementary Fig. S2B). Parallel experiments using β3WT and β3KO PyMT-BO1 cells revealed similar findings, with β3KO bone metastases showing the greatest decrease after DTX (Supplementary Fig. S2E and F). Taken together, these results suggest that tumoral β3 plays a functional role in the chemoresistant phenotype.

Rescue of integrin β3 expression restores chemoresistance in a signaling-dependent manner

Having established that β3 deletion sensitizes bone metastases to DTX, we next asked if β3 rescue in β3KO breast cancer cells was sufficient to restore DTX resistance. To do this, clone #1 β3KO PyMT-BO1 cells (β3KO1-BO1) were retrovirally engineered to express either an empty vector (pMx), a functional human integrin β3 construct (hβ3), or the DiYF integrin β3 mutant (Δβ3), which can bind ligand but is incapable of downstream signaling (Fig. 3A) (27). In vitro, hβ3-expressing cells were significantly more viable than pMx-expressing cells by MTT after DTX exposure (~3.6nM vs. ~1.6nM IC50, p<.0001). Rescue with signaling-deficient Δβ3 mutant, by contrast, had no effect on viability (~1.2nM vs. ~1.6nM IC50, p=0.3678) (Fig. 3B). We also observed diminished apoptosis and enhanced proliferation in hβ3-expressing cells compared to both empty vector and Δβ3-rescue after DTX exposure (Fig. 3C and D). In BMSC co-culture, hβ3 rescue significantly increased DTX resistance compared to wild type PyMT-BO1 cells (Supplementary Fig. S3A), which have lower β3 expression at baseline.

Figure 3. Rescue of integrin β3 expression restores chemoresistance in a signaling-dependent manner.

A. Retroviral recue of β3KO1-BO1 cells with empty vector (pMx), functional human β3 (hβ3), or signaling-deficient Δβ3. Construct schematic (left), western blot confirmation of integrin β3 rescue (right). B. Docetaxel IC₅₀ from 72hr MTT viability assay in vitro using pMx, hβ3, and Δβ3 β3KO1-BO1 cells, n =24 per genotype spread across 8 drug concentrations, one of two independent experiments. Data represent mean ± SEM. One-way ANOVA with Tukey post hoc test; ****, P<0.0001. C. Luminometric assessment of caspase-3/7 activity in vitro. Cells were treated with DMSO or 30nM DTX for 40hr. Data represent fold increase in luminescent caspase-3/7 activity compared to untreated controls of the same genotype and normalized to cell viability by CellTiter Blue, n = 4 biological replicates per group, one of three independent experiments. One-way ANOVA with Tukey post hoc test; **, P<0.001. D. Flow cytometric analysis of BrdU incorporation in vitro. Cells were treated with DMSO or 10nM DTX for 40hrs, followed by 2hrs of BrdU incorporation. n = 2 biological replicates per group, one of two independent experiments. One-way ANOVA with Tukey post hoc test; *, P<0.05, **, P<0.001. E. Ex vivo bioluminescent tumor burden in hindlimb bone, lung, and kidney of mice bearing pMx, hβ3, or Δβ3 β3KO1-BO1 tumors receiving either vehicle or DTX (5mg/kg i.v.). Tumor establishment by intracardiac injection and treatment schema (top), quantification of ex vivo BLI signal from organs (bottom), n = 6–8 mice per group. Data shown are log₂ transformed fold change in photons/s relative to the geometric mean of samples from vehicle-treated mice. pMx, hβ3, and Δβ3 experiments were performed independently. One-way ANOVA with Tukey post hoc test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

To confirm this in vivo, we established β3KO1-BO1 derivative metastases in mice and administered DTX or vehicle. Empty vector β3KO metastases of the kidneys, lungs, and hindlimbs were sensitive to DTX (4.8-fold, 2.5-fold, and 6.3-fold decrease from vehicle, respectively). Organs harboring hβ3-expressing tumors, meanwhile, exhibited no significant difference in BLI between DTX and vehicle-receiving mice. Importantly, signaling-deficient Δβ3-rescued tumors were notably sensitive to DTX, with statistically similar fold decreases to empty vector groups (Fig. 3E). Taken together, these results suggest that β3 expression is sufficient to promote increased resistance to DTX in vitro and in vivo, and that this phenotype requires intact integrin signaling.

Integrin β3 mediates an alternative metabolic response to docetaxel

Our results suggested that β3-mediated chemoresistance was most evident in the bone metastatic microenvironment. To evaluate the role of β3 expression in the DTX response of individual tumor cells in this context, we analyzed DTX-treated β3WT and β3KO 4T1 murine bone metastases by transmission electron microscopy (TEM) (Fig. 4A). Vehicle-treated bone metastases were grossly similar, with no evident β3-dependent differences in organelle morphology or ECM composition. In DTX-treated β3KO tumors, many breast cancer cells exhibited membrane blebbing and fragmentation, consistent with a higher level of cell death. Individual mitochondrial area was increased compared to vehicle in β3KO (Supplementary Fig. S4A), but the ratios of neither total mitochondrial area nor rough endoplasmic reticulum (ER) area to cytosolic area was altered (Fig. 4B and C). In DTX-treated β3WT bone metastases, breast cancer cells remained largely intact, and were notably embedded in abundant fibrillar ECM not observed in vehicle-treated tumors. In contrast to β3KO, rough ER area was markedly increased from vehicle, with pronounced cisternae clearly visible (WT vehicle: 4.5% rough-ER-to-cytosol; WT DTX: 14.9% rough-ER-to-cytosol, p<0.0001) (Fig. 4B). Similar to DTX-treated β3KO tumors, individual mitochondrial area was increased in DTX-treated β3WT (Supplementary Fig. S4A), while total mitochondrial area was unchanged. Together, these results suggest that DTX administration elicits tumoral β3-dependent changes in the cellular and microenvironmental ultrastructure of bone metastases.

Figure 4. Docetaxel treatment elicits rough ER expansion and extracellular matrix production in β3WT bone metastases.

A. Representative transmission electron micrographs of 4T1 β3KO and β3WT bone metastases treated with either vehicle or DTX (5mg/kg). Tumor establishment and i.v. drug administration were performed as indicated in Fig. 2C. Scale bars = 500nm. n = 1–3 tumors per group. Nuclei (orange), mitochondria (blue), rough ER (red). B. Quantification of mitochondrial area and rough ER area from individual tumor cells in bone metastases. Data shown as percentage of total cytosolic area per cell, n = 7–43 cells evaluated per group. Two-way ANOVA with Tukey post hoc test; ****, P<0.0001. C. Top 10 normalized enrichment scores from GSEA analysis of GO cellular compartment terms comparing 4T1 β3WT DTX and 4T1 β3KO DTX RNA-Seq samples. Terms related to rough ER and ECM displayed in red.

To identify mechanistic links between β3 expression and chemoresistance, we generated RNA-Seq transcriptomic profiles of β3WT versus β3KO 4T1 cells and hβ3-rescued versus empty vector β3KO1-BO1 cells after DTX or DMSO exposure in vitro. Gene set enrichment analysis (GSEA) of biological process and cellular compartment gene ontology (GO) terms in 4T1 profiles revealed β3-dependent enrichment of genes associated with ER, the unfolded protein response, and collagen-containing ECM after DTX administration (Fig. 4C, Supplementary Fig. S4B). Using hallmark GSEA (28), we next isolated functional pathways of interest, focusing on those where β3-expressing and β3KO DTX responses were most different. A group of metabolism-related pathways was the most enriched during the β3-mediated DTX response in hβ3-rescued β3KO1-BO1 cells, many of which were also positive in the 4T1 analysis (Fig. 5A, see dashed line box). Interestingly, the hallmark pathway with the greatest positive difference in both 4T1 and PyMT-BO1 was OXPHOS (4T1: +3.2 net NES; BO1: +7.3 net NES) (Fig 5A).

Figure 5. Integrin β3 mediates an alternative metabolic response to docetaxel.

A. Comparison of hallmark GSEA results between β3KO1-BO1 and 4T1. For each cell line, normalized enrichment scores (NES) were separately determined for the DTX response (DTX v. DMSO) in β3-expressing and β3KO cells. The difference between these scores (β3WT response – β3KO response) was calculated, and all values were internally normalized to a 0–100 scale for that line, with 0 corresponding to the smallest NES difference and 100 corresponding to the largest. B. Extracellular flux analysis of maximum OCR after serial addition of the indicated drugs (see Materials and Methods). Cells were treated with DMSO or 10nM DTX for 24hrs, followed by a 48hr recovery period. β3KO1-BO1 (left), 4T1 (right), OCR readings over time (top), maximum OCR calculation (bottom, see Materials and Methods). Data shown are pmol O2 consumed per minute, normalized to constitutive luciferase activity measured after assay completion, n = 3–10 biological replicates per group, one of two independent experiments for each cell type. Two-way ANOVA with Tukey post hoc test comparing DTX versus DMSO within each genotype; for β3-expressing cells: *, P<0.05, **, P<0.01; ****, P<0.0001; for β3KO cells: #, P<0.05. C. Fluorescence staining of ROS by Galuminox (see Materials and Methods). β3WT and β3KO 4T1 cells treated with DMSO or 10nM DTX overnight, followed by a 48hr recovery period. Representative DIC and fluorescence images (left), quantitation of Corrected Total Cell Fluorescence (CTCF, see Supplementary Information) (right). Scale bar = 20μm, data are mean ± SEM and represent 3 independent experiments. Two-way ANOVA with Tukey post hoc test; P<0.0001, ****.

To functionally validate OXPHOS enrichment during the β3WT DTX response, we performed in vitro extracellular flux analysis of oxygen consumption rate (OCR) in both 4T1 and PyMT-BO1 lines. We found significantly increased maximum OCR after DTX in hβ3-rescued β3KO1-BO1 cells, while empty vector (pMx) exhibited no or minimal increase compared to vehicle treatment (Fig. 5B). Likewise, in 4T1, we found significant increases in maximum OCR after DTX in β3WT not seen in β3KO cells (Figure 5B). Interestingly, the differences in OCR between 4T1 β3WT and β3KO were observed on plates coated with vitronectin (a ligand recognized by activated αvβ3 integrin) but not on regular tissue culture-treated plates (Supplementary Fig. S5A and B).

These differences in bulk oxygen handling after chemotherapy prompted investigation of reactive oxygen species (ROS), another pathway identified in our hallmark analysis (Fig. 5A). Galuminox, a novel fluorescent metalloprobe (24), allowed us to directly image hydrogen peroxide and superoxide in live 4T1 cells by confocal microscopy. These studies revealed a nearly 5-fold increase in β3WT ROS after DTX, while ROS after DTX in β3KO cells was not significantly different (Fig. 5C). Taken together, our results suggest that β3 mediates an alternative metabolic response to DTX treatment in breast cancer cells.

mTORC1 inhibition reverses β3-mediated chemoresistance

We searched our hallmark GSEA for metabolically relevant signaling pathways that could be targeted in combination with DTX to overcome β3-mediated resistance. We found mTORC1 activity and its target E2F, both established regulators of mitochondrial metabolism and protein synthesis (29), to be among the most significantly enriched signaling pathways in hβ3-expressing cells exposed to DTX (mTORC1 NES 3.7, q<0.0001; E2F NES 4.1, q<0.0001) (Fig. 6A). To functionally validate the importance of mTORC1 activity in β3WT 4T1 cells without chemotherapy, we assessed viability after exposure to the mTORC1 inhibitor everolimus (mTORCi). Though everolimus abrogated phosphorylation of ribosomal protein S6 in both β3WT and β3KO 4T1 cells (Supplementary Fig. 6A), β3WT 4T1 cells exhibited significant viability reduction compared to DMSO control, while β3KO viability was unaffected (Fig. 6B). We next asked if, similar to our docetaxel experiments, β3 expression is increased after everolimus treatment. Indeed, western blot analysis of 4T1 cells after in vitro exposure to everolimus demonstrated significantly higher total β3 protein expression compared to cells receiving vehicle (Fig. 6C). Flow cytometry analysis showed that the proportion of β3+ PyMT-BO1 cells was likewise increased after in vitro everolimus treatment (Supplementary Fig. 6B).

Our in vivo TEM images revealed that β3WT cells undergo rough ER expansion after exposure to DTX, possibly as part of an unfolded protein stress response (Fig. 4). To determine the effect of combination DTX and mTORCi on this phenotype in vitro, we assessed de novo protein production in β3WT and β3KO 4T1 cells exposed to either DTX, mTORCi, or both. At baseline, β3WT cells incorporated almost 65% more HPG-methionine than β3KO. DTX reduced HPG-methionine incorporation by 25% in β3WT cells, but did not affect de novo protein production in β3KO. Importantly, while mTORCi alone had no effect on HPG-methionine incorporation by β3WT cells, combination with DTX resulted in a 60% reduction compared to vehicle, almost twice the reduction observed in β3KO (Fig. 6D). Given previous demonstration of a link between β3 signaling and mTORC1 activity (30, 31), in addition to the clinically approved use of mTORC1 inhibitors in breast cancer patients, we decided to pursue it as a candidate for combination therapy with DTX in breast cancer bone metastases.

To evaluate this combination strategy, we established β3WT PyMT-BO1 bone metastases by i.c. injection. Mice were randomized to receive either vehicle, DTX alone, the mTORC1 inhibitor rapamycin alone (RAPA), or combined treatment (COMBO). While ex vivo BLI bone tumor burden in groups receiving DTX or RAPA alone was not significantly different from vehicle, combination therapy synergistically attenuated bone metastases (5.5-fold reduction compared to vehicle, p<0.01) (Fig. 6E). This effect was not observed in visceral metastases (Supplementary Fig. S6C).

αvβ3-targeted nanoparticles loaded with mTOR inhibitor enhance docetaxel efficacy in bone metastases

To confirm β3-dependent synergy and provide proof of principle for this strategy in a precision medicine setting, we modified our αvβ3-targeted micelle nanoparticle (previously described in (14)) with a rapamycin cargo (αvβ3-RAPA-NP) to specifically deliver rapamycin to cells expressing activated αvβ3 integrin heterodimers (Fig. 6F, Supplementary Fig. S6D). Mice bearing β3WT PyMT-BO1 bone metastases were randomized to receive either cargo-free control nanoparticles (αvβ3-CF-NP), combination αvβ3-CF-NP and free DTX, or combination αvβ3-RAPA-NP and free DTX. By ex vivo BLI and X-ray analysis, we found that combination αvβ3-RAPA-NP and free DTX was significantly more effective to decrease bone tumor burden and tumor-induced bone loss (osteolysis) than cargo-free nanoparticles and free DTX (Fig 6G). As before, the effect on tumor burden was not significant in visceral metastases (Supplementary Fig. S6E). Additionally, rapamycin loading did not increase serum markers of therapy-induced toxicity compared to cargo-free particles in combination with DTX (Supplementary Fig. S6F). Taken together, these data suggest mTORC1 inhibition as a strategy for enhancing response to taxane therapy in αvβ3-expressing bone metastases.

Discussion

Bone metastases are common in breast cancer (32), manifesting as osteolytic lesions with a fundamentally different biology than the primary tumor or visceral metastatic sites (4). Bone-targeted agents such as bisphosphonates and denosumab have improved patient quality of life, but these therapies are not curative and largely spare the tumor itself (3).

Exposure to the bone microenvironment modulates tumor cell phenotype (5, 33). In previous studies, we found that bone-induced TGF-β signaling upregulates β3 expression in breast cancer cells (14). The current study expands on this finding, demonstrating that tumoral β3 expression itself promotes chemoresistance characterized by an alternative metabolic response to DTX. We further showed that combination rapamycin and DTX overcomes β3-mediated resistance. Finally, administration of rapamycin-loaded, αvβ3-targeted nanoparticles specifically improved DTX response in murine bone metastases, providing proof of principle for an effective strategy that might circumvent possible toxicities associated with combination therapy.

β3+ murine breast cancer cells were increased after in vitro chemotherapy, corroborating results in human cells reported by Vellon and colleagues (34). DTX in vitro failed to reduce proliferation in the β3hi population, suggesting that DTX selects for resistant cells with higher β3 expression. Our in vivo findings supported this, with β3+ tumor cells enriched in bone metastases remaining after systemic DTX treatment. In human patients, incomplete response to neoadjuvant chemotherapy is associated with significantly worse outcomes (35), likely driven by selection for and reprogramming toward resistance in the cells that survive (36). Using publicly available data, we found that β3 expression at diagnosis was associated with a higher recurrence risk in TNBC patients receiving any chemotherapy. To our knowledge, this is the first report of a correlation between αvβ3 expression and recurrence-free survival in TNBC patients receiving chemotherapy. We would expect this effect to be most significant in patients with bone metastases, where β3 expression is known to be increased. Unfortunately, large databases of bone metastases are not readily available, possibly contributing to this association in primary breast tumors, where absolute β3 expression is low, going unreported until now. We found populations of β3+ residual tumor cells in 97% of post-chemotherapy primary tumor specimens we analyzed from high-risk patients with localized TNBC who failed to achieve a pCR after neoadjuvant chemotherapy. While survival significance in this TMA cohort was limited by sample size, we found a trend toward increased risk of recurrence in patients with High β3 expression. This difference was especially pronounced after a curve crossing event ~1.5 years after diagnosis, raising the possibility that β3 expression might be more relevant to recurrence later in the course of TNBC. Studies are planned to analyze a larger cohort of patient samples with longer follow-up times.

β3 is an important promoter of bone metastasis (15), is upregulated in bone metastases compared to the primary and visceral sites (14), is important to protumor microenvironment cells such as osteoclasts (9) and tumor blood vessels (11, 12), and has previously been implicated in resistance to therapies across several cancer types (17, 37, 38). Despite this, direct pharmacological blockade of αvβ3 has not shown significant activity in clinical trials of aggressive and advanced cancers (39). Further, the addition of the RGD-mimetic integrin αvβ3/αvβ5-inhibitor cilengitide to temozolomide chemotherapy was not associated with clinical benefit in a phase III trial of glioblastoma patients (40). Trial design and advanced patient stage may have contributed to this lack of efficacy; however, off-target effects of pharmacological αvβ3 blockade on host cells, such as enhancing pro-tumor neoangiogenesis (41) and promoting immunosuppression (10), could also be at play. Interestingly, a preclinical study in lung and pancreatic cancer models demonstrated that low doses of cilengitide could enhance the activity of gemcitabine chemotherapy in vivo by simultaneously increasing tumor cell-extrinsic delivery and tumor cell-intrinsic drug uptake (42), raising the possibility that the effects of these agents on the tumor microenvironment might still be exploited for synergistic anti-tumor effects when used in the right context.

β3 studies in breast cancer have hinged primarily on in vitro characterization with pharmacological blockade or on its role in promoting metastasis (15, 17). Using CRISPR/Cas9 technology, we abrogated tumoral β3 expression in the setting of an intact immune system. Because manipulation of tumoral integrin β3 could also affect tumor growth (15, 43), we used each genetic line as its own control, only comparing response to chemotherapy across genotypes. We show here that bone metastases lacking integrin β3 were significantly more sensitive to docetaxel than wild type metastases.

In vitro, β3WT and β3KO breast cancer cells were both highly sensitive to docetaxel, but in vivo β3WT bone metastases were relatively resistant. In vitro survival increased when β3WT cells were plated on BMSCs, suggesting ligand availability as a potential factor in αvβ3 integrin-mediated resistance. Integrin activation, which induces a conformational change that exposes the ligand binding domain, is required for ligand binding and signaling (44), and it is possible that β3 activation in the bone microenvironment results in easier access to ECM ligands. The bone metastatic microenvironment also has a lower pH and oxygen concentration, higher stress modulus, and distinct nutrient and chemical milieu (4), all of which can influence cancer cell reliance on αvβ3 signaling (31, 45, 46).

While BLI, histology, and X-ray indicated that docetaxel had minimal effect on β3WT bone metastases, TEM analysis uncovered a dramatic metabolic response to chemotherapy in these resistant cells characterized by profound increases in protein production and ECM deposition. Experiments are underway to profile these ECM proteins, determine the mechanism through which β3 ligand binding and signaling are involved, and more specifically exploit this metabolic vulnerability to enhance therapeutic efficacy.

We identified protein synthesis, ECM enrichment, and OXPHOS as potential downstream targets of β3 signaling in response to chemotherapy. Although no β3-mediated changes in mitochondrial ultrastructure were evident by TEM, our in vitro studies showed that DTX consistently increased OCR in β3WT compared to β3KO cells. Live cell imaging of 4T1 cells further demonstrated robust β3-mediated increases in DTX-induced ROS generation, suggesting an alternative metabolic response that drives therapeutic resistance (47, 48).

Consistent with increased ER observed in vivo, we found that in vitro protein production was higher at baseline and more responsive to docetaxel in β3WT cells. Recent evidence indicates that tumoral ER stress is a common feature of breast cancer bone metastases (49), and others have shown that integrin signaling bolsters in vitro protein production during hypoxia (31). These data suggest that diminished tolerance for ER stress (50) could drive the large chemosensitizing effect we observe with β3KO in the bone compared to other metastatic sites, where tumoral β3 expression is not as high. Future studies, including single-cell RNA and ribosomal sequencing of tumor cells collected directly from bone metastases, are planned to more specifically evaluate ER stress and unfolded protein response during β3-mediated chemoresistance in vivo.

mTORC1 pathway genes were enriched after DTX treatment of β3WT cells and are demonstrated targets of αvβ3 signaling in breast cancer (29–31). Administration of either the mTORC1 inhibitor rapamycin or DTX alone had little effect on PyMT-BO1 bone tumor burden, while rapamycin and DTX together significantly attenuated bone metastases. Notably, this synergistic effect was exclusive to bone, where tumor expression of β3 is much higher than in visceral metastases (14). mTORC1 inhibition alone has been shown to restrict tumor growth in bone micrometastases (51), suggesting that combination with DTX may provide additional benefit for adjuvant metastasis prevention (52). While single-agent mTOR inhibition does not attenuate tumor growth in bone macrometastases (51, 53), it has been shown to protect against osteolytic bone loss, which might contribute indirectly to our findings (53).

To test the specific effect of rapamycin inhibition in αvβ3-expressing cells, we co-administered free DTX with rapamycin-loaded, αvβ3-targeted nanoparticles (14). Combination of mTOR inhibitors and taxane chemotherapy is clinically challenging due to toxicity (54), but αvβ3-NPs can reduce drug availability in the circulation by influencing release kinetics (14, 55). In the current study, combination therapy with docetaxel and αvβ3-RAPA-NP was more effective than docetaxel alone against PyMT-BO1 bone metastases, demonstrating that the efficacy of mTORC1 inhibition is in part mediated by its specific activity in cells expressing activated αvβ3 integrin. The combination of αvβ3-RAPA-NP with DTX did not significantly affect serum toxicity markers in mice, which holds potential for clinical translation.

The bone-specificity of rapamycin/DTX combination therapy was a striking finding from our study. We have previously shown that tumoral integrin β3 expression is increased in bone metastases compared to the primary or visceral metastatic sites, facilitating increased drug delivery to the bone by αvβ3-targeted nanoparticles (14). Beyond enhancement of drug delivery, our transcriptomic and functional studies suggest that mTOR activity is also more important for β3-expressing cells, particularly in the setting of docetaxel treatment. Consistent with this, increased tumoral β3 expression could render bone metastases more dependent on mTOR activity for proliferation after exposure to docetaxel. The effect of mTOR inhibition on visceral metastases is likely less pronounced because β3-expressing tumor cells represent a significantly lower proportion of the overall tumor burden than what we observe in the bone. In our aggressive preclinical metastatic models, experimental duration is likely not long enough for mTOR-dependent β3+ cells to sufficiently accumulate. Overexpression of β3 in β3KO1-BO1 cells resulted in increased chemoresistance in visceral metastases. Experiments are underway to determine the role of mTOR for chemoresistance in β3-expressing cells at these visceral sites. In addition to what we observe in bone metastases, we propose that patients with visceral metastases who have been previously treated with chemotherapy and would be predicted to have increased β3+ cells could benefit from combination mTORC1 inhibition and DTX.

Finally, although it is of high experimental utility, intracardiac injection exhibits limitations as a model of breast cancer bone metastasis. It overlooks selection processes that occur at the primary site and during intravasation into the circulatory system, and has lower latency rates between dissemination and frank macrometastatic outgrowth. These differences all have the potential to impact therapeutic response, but currently, no genetic mouse models of spontaneous breast cancer bone metastasis exist. Studies are in progress to evaluate the efficacy of our combination strategy on bone metastasis following resection of mammary fat pad tumors and monitoring for spontaneous metastases. Nevertheless, our findings here highlight the need for therapeutic strategies that consider the microenvironmental context of the tumor when targeting metastatic cells.