Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2–M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.

According to the World Health Organization, cancer is the second leading cause of death in the world, resulting in approximately 9.6 million deaths globally in 2018. Breast cancer accounts for around 2.09 million cases and 620,000 deaths, making it the second leading cause of cancer-related death in women. Breast cancer is classified into four major molecular subtypes by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) receptor expression: luminal A (ER/PR-positive/HER2-negative), luminal B (ER/PR-negative/HER2-positive), HER2-overexpressing, and triple-negative (TNBC, ER/PR/HER2-negative) (1). Each subtype has different treatment responses, diverse disease progression, sites of metastasis, and distinct clinical outcomes (1–3). The common treatment options for breast cancer include surgery, chemotherapy, radiotherapy, hormonal therapy, and immunotherapy.

TNBC accounts for nearly 15% to 20% of all diagnosed breast cancers (4) and can be subdivided into 6 different subtypes, which are basal-like (BL1 and BL2), immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor, and unstable subtype (5, 6). TNBCs are highly aggressive, associated with early recurrence and a tendency to spread to the lung, liver, and central nervous system, and patients have poor relapse-free survival rates (7, 8). The prognosis of TNBC patients is relatively poor because they cannot benefit from endocrine therapy or treatments targeting HER2. Therefore, TNBC breast cancer patients require unique treatments, and general cytotoxic chemotherapies are the basis of standard treatment regimens (2, 9).

Colchicine-binding site inhibitors (CBSI) are microtubule-destabilizing agents that target the interface of α,β-tubulin heterodimers (10). Extensive preclinical studies have shown that CBSIs are effective for the treatment of various tumor types (11–13). Compared with many microtubule-targeting agents (e.g., vincristine, or paclitaxel, interacting with tubulin through the vinca site or paclitaxel site, respectively) that are frequently used for treating cancer, CBSIs are less vulnerable to transporter-mediated drug resistance (e.g., overexpression of ABC transporters) (14–16), have better aqueous solubility (17, 18), possess potent anticancer effects against cancer cells overexpressing β3-tubulin (19, 20), and show vascular disrupting activities (21–23). These features highlight the clinical potential of new CBSIs for effective TNBC therapy while avoiding the limitations of current antitubulin drugs, including adverse side effects and the frequent onset of drug resistance due to drug efflux.

CH-2-77 is a CBSI with a nanomolar IC50 against a panel of cancer cell lines, including the metastatic TNBC cell line model MDA-MB-231. It has potent antitumor efficacy, inhibiting the growth of melanoma A375 xenografts in vivo (24). Our current study reports the efficacy of CH-2-77 in TNBC. In vitro, CH-2-77 showed nanomolar antiproliferative activity on a panel of TNBC cells, including paclitaxel-resistant TNBC cells, inhibited the migration and invasion, arrested cell cycle in the G2–M phase, and induced cancer cell death by disrupting microtubule dynamics. In vivo, CH-2-77 demonstrated acceptable pharmacokinetic properties and inhibited both primary tumor growth and lung metastasis in the MDA-MB-231 TNBC orthotopic xenograft model. In a direct head-to-head comparison with combretastatin A4 (CA4) phosphate (CA4P, also known as fosbretabulin), a CBSI that went through multiple clinical trials, CH-2-77 at an equivalent dose (20 mg/kg) had similar antitumor efficacy as CA4P (28 mg/kg, equivalent of 20 mg/kg for the active CA4). Together, these results strongly suggest that CH-2-77 is a promising drug candidate for the treatment of metastatic TNBC.

Chemical compounds and cell culture

Colchicine and paclitaxel were purchased from Sigma-Aldrich and LC Laboratories, respectively. CH-2-77 (purity >98%) was synthesized according to the previously reported method (24). Human MDA-MB-231, MDA-MB-468, and Mia PaCa-2 cells were purchased from ATCC in 2017 or 2019 and cultured in DMEM (Mediatech, Inc.) supplemented with FBS (10%, Atlanta Biologicals) and antibiotic–antimycotic solution (1%, AA, Sigma-Aldrich) in 5% CO2 at 37°C. Paclitaxel-resistant sublines (MDA-MB-231/TxR and MDA-MB-468/TxR) were generated by treating parental cells with paclitaxel, gradually increasing the concentration until the cells sustained growth in media containing 100 nmol/L of paclitaxel. Colchicine-resistant Mia PaCa-2 cells were generated by treating Mia PaCa-2 cells with colchicine gradually and continually until cells were stable in growth media containing 100 nmol/L of colchicine. Two TNBC cell lines were derived from patient-derived xenograft (PDX) tumors regenerated and excised from host mice, cHCI-2 (a treatment-naïve line), and cHCI-10 (a taxane-resistant line); the original PDX models were generously provided by Dr. Alana Welm through the Huntsman Cancer Institute (HCI) preclinical research resource. The cHCI-2 and cHCI-10 cells were described in (18) and cultured in the M87 growth medium supplemented with 10% FBS and 1% AA solution (25). All cells used in the experiments were in lower passages and were monthly screened for mycoplasma using the MycoAlert kit (Lonza) and were authenticated at the University of Arizona Genetics Core or HCI.

Cell proliferation assay

MDA-MB-231 (3,000 cells/well), MDA-MB-231/TxR (5,000 cells/well), MDA-MB-468 (7,500 cells/well), and MDA-MB-468/TxR (7,500 cells/well) were seeded overnight into 96-well plates and treated with each compound at increasing concentrations (0.1 nmol/L to 3 μmol/L) as described previously (18). cHCI-2 and cHCI-10 PDX cells were seeded overnight at 20,000 cells/well into 96-well plates and incubated with each compound for 5 days before the addition of the MTS reagent. During treatment, PDX cells were also imaged by an IncuCyte S3 live-cell imager (Sartorius), and representative images at the start time point and endpoint were captured. IC50 values were calculated as described before (18). All experiments were conducted using three biological replicates with at least three technical replicates per treatment per cell line.

Colony formation assay

MDA-MB-231 or MDA-MB-468 cells were seeded in 12-well plates at a density of 500 or 800 cells per well, and once one single cell had proliferated to 4 cells, cells were treated with colchicine, paclitaxel, or CH-2-77 (1, 2, and 4 nmol/L) as in (18). Experiments were repeated by treating MDA-MB-231 or MDA-MB-468 cells with CH-2-77 (2.5, 5, and 10 nmol/L), and at the endpoint were fixed, stained, and quantified. Data are representative of two biological replicates with three technical replicates per treatment per cell line.

Immunofluorescence staining

MDA-MB-231 (105) cells or 2 × 105 MDA-MB-468 cells were seeded onto coverslips and allowed to attach overnight. A growth medium containing 2.5 nmol/L or 5 nmol/L of each compound was then added, and cells were treated for 24 hours following the steps described previously (18). Cells in interphase and mitosis were imaged using a Keyence BZ-X700 immunofluorescence microscope.

Western blot analysis

Cells grown to 70% confluence were treated with colchicine (10 nmol/L), paclitaxel (10 nmol/L), or CH-2-77 (2.5 nmol/L, 5 nmol/L and 10 nmol/L) for 24 hours. Protein samples were prepared as in (16). Primary antibodies included rabbit anticleaved-PARP (#5625, 1:1,000, Cell Signaling Technology, CST), rabbit anti-PARP (#9542, 1:1,000, CST), mouse anti–acetyl-α-tubulin (#12152, 1:1,000, CST), mouse anti–α-tubulin (#62204, 1:2,000, Invitrogen), rabbit anticleaved-caspase-9 (#7237, 1:1,000, CST), mouse anti–β-actin (#3700, 1:2,000, CST), rabbit anti-Bax (#2772, 1:1,000, CST), mouse anti-Bcl-2 (#sc-7382, 1:250, Santa Cruz Biotechnology, Inc.) and were detected with horse anti-mouse-IgG-HRP (#7076, 1:1,000, CST) or goat anti-rabbit-IgG-HRP (#7074, 1:1,000, CST) secondary antibodies. Membranes were washed 3 times with TBST, then incubated with ECL reagent (Bio-Rad) for 5 minutes and developed using X-ray film.

Migration and invasion

The potential of CH-2-77 (4 nmol/L) to inhibit migration or invasion of TNBC cells (5 × 104 for MDA-MB-231 and 105 for MDA-MB-468) was investigated by using Transwell noncoated inserts (#353097, Corning) or Transwell chambers coated with Matrigel (#354480) as in (18), respectively. Representative images of migrated or invaded cells were quantified using the Keyence microscope hybrid cell counting module. Data are representative of two biological replicates with three technical replicates per treatment per cell line.

Wound-healing assay

The antimigration activity of CH-2-77 was further evaluated in comparison with colchicine or paclitaxel treatment in a scratch assay (18). After creating a scratch wound, cells were treated with 4 nmol/L of each compound for 24 hours (MDA-MB-231) or 48 hours (MDA-MB-468). Representative wound images were acquired with an Evos microscope (Life Technologies) throughout treatment, and the percentage of the wound area was quantified by ImageJ. Data are representative of two biological replicates with three technical replicates per treatment per cell line.

Cell apoptosis and cell-cycle analysis

Cells were seeded overnight and incubated with colchicine (10 nmol/L), paclitaxel (10 nmol/L), and CH-2-77 (2.5 nmol/L, 5 nmol/L, and 10 nmol/L) for 24 hours. Then, cells were digested with trypsin-EDTA (#2520056, Gibco) and stained with Annexin-V–FITC (eBioscience, 5 μL) and propidium iodide (PI, 10 μL) in Annexin-V–FITC binding buffer (100 μL) for 10 minutes. Apoptotic cells were detected by flow cytometry. For cell-cycle distribution, cells with the same treatments as the apoptosis assay were harvested, washed, and fixed. After permeabilization, cells were stained with rabbit anti-phospho-histone H3 (Ser 10) antibody (#9701, 1:50, CST) for 1 hour at room temperature followed by a 30-minute incubation with the Alexa Fluor 488 goat anti-rabbit secondary antibody (#A-11008, 1:50, Molecular Probes). After washing 2 times with PBS, cells were resuspended in PI/RNase staining solution and detected by cytometry after a 5-minute incubation. Cells in the sub-G1, G1, S, and G2 phases were gated by a reported method (18). The experiment was conducted using three biological replicates with three technical replicates per treatment per cell line.

Pharmacokinetic assessment of CH-2-77

Male Sprague-Dawley rats (Envigo), weighing 200 to 250 g, were used in the pharmacokinetic studies. Groups of 5 animals were administered either 5 mg/kg CH-2-77 by intravenous injection via a femoral vein catheter or 20 mg/kg by oral gavage after an overnight fast. Both doses were formulated in PEG300/Tween80 (80%/20%), with a constant administration volume of the dosing solution of 1 mL/kg. Blood was collected in heparinized tubes via a jugular vein catheter at 10 predefined time points postdose after the intravenous administration (0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 hours), and at 9 times points after oral administration (0.25, 0.5, 1, 2, 4, 6, 8, 10, and 24 hours). Plasma was immediately separated by centrifugation (6,000 × g for 10 minutes at 4°C) and stored at −70°C until analysis. The study was conducted according to the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The study protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Tennessee Health Science Center (UTHSC).

Quantification of CH-2-77 concentrations in plasma was performed in duplicate by LC-MS/MS as in (26), except that the organic mobile phase used was methanol. Detection was performed using the mass transitions of m/z 308.3→184.1 for CH-2-77 and m/z 378.4→210.0 for the internal standard. The accuracy of the assay was within ± 10.3%, and the precision (coefficient of variation) was <14%, with a lower limit of quantitation of 0.98 ng/mL. The obtained CH-2-77 plasma concentration–time profiles were analyzed by standard noncompartmental pharmacokinetic procedures (27) using Phoenix WinNonlin v.8.1 (Certara).

In vivo efficacy of CH-2-77 in orthotopic MDA-MB-231 xenograft models

The maximum tolerable dose (MTD) study of CH-2-77 was conducted in NSG immunocompromised mice (Jax Labs, strain # 005557) using 4 doses (10, 20, 30, and 40 mg/kg, n = 4) daily with i.p. injection. Mouse body weight (b.w.) was recorded every day. The efficacy of CH-2-77 was assessed in an orthotopic TNBC xenograft model using methods previously reported (18). All protocols and treatments were approved by the UTHSC IACUC (protocol #: 17–080 or 20–0181). When the average tumor volume was ∼100 mm3, mice were randomized into 4 groups based on the tumor volume and b.w.: vehicle (PEG300:Tween 80:saline = 4:1:10; n = 6), CH-2-77 at 5 mg/kg (n = 6), 10 mg/kg (n = 6), or 20 mg/kg (n = 7). CH-2-77 treatment and vehicle were administered 5 times/week by i.p. injection (100 μL per 20 g/b.w.). Tumor volumes, measured by digital caliper and calculated by the formula tumor width2 × tumor length × 0.5, and mouse b.w. were recorded twice or three times a week, respectively. When the mean tumor volume reached ∼1,000 mm3 in the vehicle control group, mice were euthanized. Tumors and major organs were removed, photographed, and formalin-fixed for histopathology. Lungs were sent to the UTHSC Research Histology Core for paraffin embedding, sectioning, and staining with H&E. The in vivo efficacy of CH-2-77 was further evaluated in the same orthotopic TNBC xenograft model with a reference compound, CA4P. When the average tumor volume reached ∼100 mm3, mice were randomized into 3 groups: vehicle (n = 5), CH-2-77 at 20 mg/kg (n = 6), and CA4P at 28 mg/kg (n = 6). Vehicle, CH-2-77, and CA4P treatments were administered 5 times/week by i.p. injection. Tumor volumes and b.w. were recorded two or three times per week. When mean tumor volume reached ∼600 mm3 in the vehicle control group, mice were euthanized. Tumors and major organs were removed, measured, and photographed.

Statistical analysis

All experimental data were expressed as the mean ± SEM or mean ± SD and analyzed by GraphPad Prism. One-way ANOVA analysis followed by the Dunnett test was used for the cell apoptosis assays. Student t tests were used for in vitro migration and invasion assays. One-way ANOVA or two-way ANOVA, followed by the Dunnett multiple comparison test, was applied to in vivo experiments. Statistical significance is presented as *, P <0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Data availability statement

The data generated in this study are available within the article and its supplementary data files.

Antiproliferative effect of CH-2-77 on TNBC cells in vitro

CH-2-77 (Fig. 1A) possesses nanomolar potency against a panel of melanoma and breast cancer cell lines, as well as paclitaxel-resistant prostate or melanoma cell lines (24). To determine the effect on TNBC, the antiproliferative activity of CH-2-77 was first evaluated using two widely used TNBC cell lines, MDA-MB-231 and MDA-MB-468, and their paclitaxel-resistant sublines. MTS assays showed that CH-2-77 inhibited TNBC cell growth with an average IC50 of 2.5 nmol/L (Table 1). Moreover, CH-2-77 overcame paclitaxel resistance with similar cytotoxicity as for parental cells, whereas the potencies of colchicine and paclitaxel were decreased in paclitaxel-resistant cells (Table 1). The efficacy of CH-2-77 was also compared with that of colchicine or paclitaxel on two PDX cell lines from TNBC patients, cHCI-2 (treatment-naïve) and cHCI-10 (taxane refractory; Table 1). After 5 days of treatment, CH-2-77 (2.5 nmol/L) inhibited the proliferation of both cHCI-2 and cHCI-10 cells, whereas colchicine treatments showed limited effects in suppressing the growth of either PDX line at a concentration of 10 nmol/L (Supplementary Fig. S1A). Paclitaxel at 10 nmol/L reduced the proliferation of treatment-naïve cHCI-2 cells, but had no activity in suppressing the growth of taxane-resistant cHCI-10 cells. In addition, we tested the efficacy of CH-2-77 against colchicine-resistant cells using a colchicine-resistant pancreatic cancer cell line, Mia PaCa-2/ColxR. As shown in Table 1, whereas the IC50 of colchicine increased about 5 times and that of paclitaxel increased 2 times in Mia PaCa-2/ColxR compared with parental Mia PaCa-2 cells, CH-2-77 maintained its single-digit IC50 on Mia PaCa-2/ColxR cells with an IC50 of 3 nmol/L, suggesting the potential of CH-2-77 to overcome both paclitaxel and colchicine resistance in cancer cells.

Figure 1.

CH-2-77 impairs the colony formation and microtubule organization and stability of TNBC cells. A, Chemical structure of CH-2-77. B, Bar graphs show the anticolony formation effects of CH-2-77 on MDA-MB-231 or MDA-MB-468 cells compared with colchicine and paclitaxel in the low nanomolar range (1–4 nmol/L). The percentage (%) of colony area in each treatment group was quantified by comparing to the colony area observed in the control group. C, MDA-MB-231 cells treated with 2.5 nmol/L or 5 nmol/L of colchicine, paclitaxel, or CH-2-77 were imaged using a Keyence microscope (×20 magnification). Inserts in the bottom right corner show tubulin for mitotic cells (×40 magnification). Cells were stained for α-tubulin (red) and the nucleus (blue). D, The expression of α-tubulin and acylated-α-tubulin in CH-2-77–treated MDA-MB-231 cells (24 hours) was determined by western blot. GAPDH is the loading control. E–F, Immunocytochemistry and western blot experiments were repeated in MDA-MB-468 cells with similar conditions as in C and D.

Figure 1.

CH-2-77 impairs the colony formation and microtubule organization and stability of TNBC cells. A, Chemical structure of CH-2-77. B, Bar graphs show the anticolony formation effects of CH-2-77 on MDA-MB-231 or MDA-MB-468 cells compared with colchicine and paclitaxel in the low nanomolar range (1–4 nmol/L). The percentage (%) of colony area in each treatment group was quantified by comparing to the colony area observed in the control group. C, MDA-MB-231 cells treated with 2.5 nmol/L or 5 nmol/L of colchicine, paclitaxel, or CH-2-77 were imaged using a Keyence microscope (×20 magnification). Inserts in the bottom right corner show tubulin for mitotic cells (×40 magnification). Cells were stained for α-tubulin (red) and the nucleus (blue). D, The expression of α-tubulin and acylated-α-tubulin in CH-2-77–treated MDA-MB-231 cells (24 hours) was determined by western blot. GAPDH is the loading control. E–F, Immunocytochemistry and western blot experiments were repeated in MDA-MB-468 cells with similar conditions as in C and D.

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Table 1.

Cytotoxicity of CH-2-77 in a panel of TNBC cell lines, including paclitaxel-resistant cell sublines and PDX models.

Mean IC50 ± SEM (nmol/L)
Cell linesCH-2-77ColchicinePaclitaxel
MDA-MB-231 2.1 ± 0.4 23.7 ± 3.1 0.9 ± 0.1 
MDA-MB-231/TxR 2.2 ± 0.5 36.8 ± 6.4 111.8 ± 37.8 
MDA-MB-468 3.2 ± 0.9 7.9 ± 1.4 2.0 ± 0.2 
MDA-MB-468/TxR 3.0 ± 0.6 41.9 ± 5.7 151.9 ± 23.8 
cHCI-2 5.6 ± 1.8 16.6 ± 6.9 12.7 ± 5.8 
cHCI-10 3.9 ± 1.7 23.4 ± 8.4 43.6 ± 8.2 
Mia PaCa-2 3.0 ± 0.7 20.3 ± 2.3 5.5 ± 0.6 
Mia PaCa-2/ColxR 3.2 ± 0.5 113.4 ± 18.4 10.6 ± 1.1 
Mean IC50 ± SEM (nmol/L)
Cell linesCH-2-77ColchicinePaclitaxel
MDA-MB-231 2.1 ± 0.4 23.7 ± 3.1 0.9 ± 0.1 
MDA-MB-231/TxR 2.2 ± 0.5 36.8 ± 6.4 111.8 ± 37.8 
MDA-MB-468 3.2 ± 0.9 7.9 ± 1.4 2.0 ± 0.2 
MDA-MB-468/TxR 3.0 ± 0.6 41.9 ± 5.7 151.9 ± 23.8 
cHCI-2 5.6 ± 1.8 16.6 ± 6.9 12.7 ± 5.8 
cHCI-10 3.9 ± 1.7 23.4 ± 8.4 43.6 ± 8.2 
Mia PaCa-2 3.0 ± 0.7 20.3 ± 2.3 5.5 ± 0.6 
Mia PaCa-2/ColxR 3.2 ± 0.5 113.4 ± 18.4 10.6 ± 1.1 

MDA-MB-231 and MDA-MB-468 cells were then treated with CH-2-77, colchicine, or paclitaxel in the low nanomolar range (1–4 nmol/L) to compare anticolony formation activity. CH-2-77 potently reduced the colony formation of both TNBC cell lines at a concentration of 4 nmol/L (Fig. 1B; Supplementary Fig. S1B). Consistent with the IC50 values, colchicine had no effect in inhibiting colony formation of TNBC cells at any concentration, whereas paclitaxel robustly inhibited colony formation in both cell lines, with activity in MDA-MB-231 cells even at 1 nmol/L. At higher concentrations, CH-2-77 repressed colony formation in a concentration-dependent manner in both cell lines (Supplementary Fig. S1C).

CH-2-77 disrupts microtubule stability in TNBC cells

Next, we determined the ability of CH-2-77 to disrupt microtubule assembly in TNBC cells. We treated MDA-MB-231 cells with 2.5 nmol/L or 5 nmol/L of CH-2-77, using colchicine and paclitaxel as controls. As a microtubule-stabilizing agent, paclitaxel condensed microtubules during interphase and the mitotic phase (Fig. 1C). Cells treated with 2.5 nmol/L of colchicine showed similarly organized and intact microtubule structures as untreated cells, suggesting the limited potency of colchicine. As expected, 5 nmol/L of colchicine caused disseminated and depolymerized microtubule arrangement in MDA-MB-231 cells during interphase, although it did not dissolve the microtubules in the mitotic cells. CH-2-77–treated cells showed similar disorganized and damaged microtubule networks to the 5 nmol/L of colchicine-treated cells, and 2.5 nmol/L of CH-2-77 induced unpolymerized microtubules in both interphase and mitotic MDA-MB-231 cells. Acetylation on Lys40 of α-tubulin was reported to stabilize microtubules, thus, we determined levels of acylated-α-tubulin versus α-tubulin on MDA-MB-231 cells treated with CH-2-77 (2.5, 5, and 10 nmol/L) by western blot (28, 29). A high concentration of CH-2-77 (10 nmol/L) decreased the levels of acylated-α-tubulin and α-tubulin, suggesting that CH-2-77 interferes with microtubule stability at high concentrations (Fig. 1D). Similar results were obtained in MDA-MB-468 cells (Fig. 1E and F).

CH-2-77 suppresses TNBC cell migration and invasion

Because microtubules are critical for cell migration (30–32), we next investigated the ability of CH-2-77 to impair random cell migration in both MDA-MB-231 and MDA-MB-468 cells. By the scratch wound assay, we compared the effect of CH-2-77 with colchicine and paclitaxel to delay filling the wounds. As displayed in Fig. 2A, after 24 hours of incubation, the wounds of untreated MDA-MB-231 cells were closed significantly, and 4 nmol/L of colchicine treatment closed similar to untreated control cells. However, CH-2-77 showed comparable anti-wound-healing effects as paclitaxel. Similar results were obtained in MDA-MB-468 cells treated with colchicine, paclitaxel, and CH-2-77 for 48 hours. We then performed Transwell assays to confirm the potency of CH-2-77 to inhibit TNBC cell migration (chemotaxis) and invasion through Matrigel (Fig. 2B and C). As anticipated, 4 nmol/L of CH-2-77 significantly inhibited the chemotactic migration and invasion of TNBC cells, with a 70% and a 63% inhibition of migration in MDA-MB-231 and MDA-MB-468 cells, respectively, and a 69% and a 53% suppression of invasion in MDA-MB-231 and MDA-MB-468 cells, respectively.

Figure 2.

CH-2-77 inhibits the cell migration and invasion of TNBC cells. A, The wound-healing assay showed the effects of colchicine, paclitaxel, and CH-2-77 to prevent wound closure in MDA-MB-231 cells or MDA-MB-468 cells at a concentration of 4 nmol/L. Representative images were captured at indicated time points after drug treatment for both cell lines. The percentage of the wound area of each treatment group was compared with their counterparts at the start point (0 hours). P values were determined relative to the control group. B and C, A Transwell chemotaxis migration using Transwell noncoated inserts (B) or invasion assay using Transwell chambers coated with Matrigel (C) was performed using TNBC cells treated with 4 nmol/L of CH-2-77. Representative cell images were acquired from the lower part of the membranes. The migration or invasion potential of CH-2-77–treated TNBC cells was calculated as the percentage of migrated or invaded cells relative to the cells in the control group (set to 100%).

Figure 2.

CH-2-77 inhibits the cell migration and invasion of TNBC cells. A, The wound-healing assay showed the effects of colchicine, paclitaxel, and CH-2-77 to prevent wound closure in MDA-MB-231 cells or MDA-MB-468 cells at a concentration of 4 nmol/L. Representative images were captured at indicated time points after drug treatment for both cell lines. The percentage of the wound area of each treatment group was compared with their counterparts at the start point (0 hours). P values were determined relative to the control group. B and C, A Transwell chemotaxis migration using Transwell noncoated inserts (B) or invasion assay using Transwell chambers coated with Matrigel (C) was performed using TNBC cells treated with 4 nmol/L of CH-2-77. Representative cell images were acquired from the lower part of the membranes. The migration or invasion potential of CH-2-77–treated TNBC cells was calculated as the percentage of migrated or invaded cells relative to the cells in the control group (set to 100%).

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CH-2-77 treatment causes G2–M phase arrest and apoptosis of TNBC cells

We previously reported the CBSI VERU-111 as a potent tubulin-destabilizing agent in TNBC cells by inducing G2–M cell-cycle arrest and apoptosis (16, 18). As a structurally modified analogue of VERU-111, CH-2-77 was then evaluated for similar cell-cycle disruption and apoptosis induction in TNBC cells. As displayed in Fig. 3A and Supplementary Fig. S2A, CH-2-77 treatment arrested both TNBC cell lines at the G2–M phase in a concentration-dependent manner. MDA-MB-231 cells were primarily arrested in the M phase, whereas MDA-MB-468 cells accumulated in the G2 phase. Colchicine treatment had little effect on cell-cycle progression in TNBC cells, whereas paclitaxel arrested the cells into the M phase and the sub-G1 phase, which was consistent with its widely reported potent cytotoxicity in vitro. We also determined the effect of CH-2-77 in inducing apoptosis using Annexin-V/PI costaining (Fig. 3B; Supplementary Fig. S2B). Apoptosis was significantly induced by CH-2-77 at a concentration of 10 nmol/L, with greater efficacy compared with colchicine or paclitaxel. Next, to determine if CH-2-77 treatment led to the TNBC cell apoptosis, western blotting for a panel of apoptosis markers was performed, including PARP, a known cell death protein (33, 34). We first compared the capacity of CH-2-77 to induce the cleavage of PARP after treatment with colchicine or paclitaxel (Fig. 3C). Consistent with the Annexin-V/PI results shown in Fig. 3B, CH-2-77 increased the levels of cleaved-PARP, with greater induction than observed for either colchicine or paclitaxel (at 10 nmol/L). A clear increase in cleaved-PARP levels was observed in CH-2-77–treated TNBC cells along with a concomitant decrease in full-length PARP levels, indicating CH-2-77 causes apoptosis through the cleavage of PARP (Fig. 3D). Bax is a proapoptotic marker, and Bcl-2 is an antiapoptotic marker (35–37). In CH-2-77–treated MDA-MB-231 cells, the levels of Bax, cleaved-caspase-3, and cleaved-caspase-9 were upregulated in a concentration-dependent manner (Fig. 3E), and CH-2-77 was simultaneously able to decrease levels of Bcl-2, demonstrating that CH-2-77 induces apoptosis in MDA-MB-231 cells through the intrinsic apoptosis pathway.

Figure 3.

CH-2-77 induces cell-cycle arrest and cell apoptosis in MDA-MB-231 and MDA-MB-468 cells. A, Effects of increasing concentrations of CH-2-77 (2.5, 5, and 10 nmol/L), or 10 nmol/L of colchicine (COL) or 10 nmol/L of paclitaxel (PTX) on TNBC cell-cycle distribution after 24-hour treatment as detected by phospho-histone H3 (Ser10)/PI costaining. B, Annexin-V/PI costaining evaluated the extent of apoptosis in TNBC cells with the same drug treatments as in A. C, Expression of cleaved-PARP was compared in both TNBC cell lines by western blot after treatment as in A. GAPDH was used as a loading control. D, Western blot analysis was repeated after 24 hours of exposure to CH-2-77 treatment to compare levels of full-length PARP and the cleaved active PARP counterpart (cleaved-PARP). β-Actin was used as a loading control. E, Expression of a panel of apoptosis-related proteins, cleaved-caspase-9, cleaved-caspase-3, Bax, and Bcl-2 was compared in MDA-MB-231 cells after CH-2-77 treatments for 24 hours. Either GAPDH or β-actin was used as a loading control.

Figure 3.

CH-2-77 induces cell-cycle arrest and cell apoptosis in MDA-MB-231 and MDA-MB-468 cells. A, Effects of increasing concentrations of CH-2-77 (2.5, 5, and 10 nmol/L), or 10 nmol/L of colchicine (COL) or 10 nmol/L of paclitaxel (PTX) on TNBC cell-cycle distribution after 24-hour treatment as detected by phospho-histone H3 (Ser10)/PI costaining. B, Annexin-V/PI costaining evaluated the extent of apoptosis in TNBC cells with the same drug treatments as in A. C, Expression of cleaved-PARP was compared in both TNBC cell lines by western blot after treatment as in A. GAPDH was used as a loading control. D, Western blot analysis was repeated after 24 hours of exposure to CH-2-77 treatment to compare levels of full-length PARP and the cleaved active PARP counterpart (cleaved-PARP). β-Actin was used as a loading control. E, Expression of a panel of apoptosis-related proteins, cleaved-caspase-9, cleaved-caspase-3, Bax, and Bcl-2 was compared in MDA-MB-231 cells after CH-2-77 treatments for 24 hours. Either GAPDH or β-actin was used as a loading control.

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Pharmacokinetics of CH-2-77

To ensure sufficient in vivo metabolic stability and systemic exposure for antitumor activity, the pharmacokinetics of CH-2-77 were investigated in rats (Fig. 4). CH-2-77 concentrations followed reproducible pharmacokinetics with multiphase behavior. After intravenous administration, concentrations fell rapidly over the first 2 hours with a half-life (t½) of approximate 22 minutes, followed by an intermediate phase between 2 and 8 hours where concentrations declined with a t½ of approximately 111 minutes, and a long terminal phase after 8 hours with a terminal t½ of 799 ± 245 minutes. The area under the concentration–time curve (AUC) as a measure of systemic exposure after a 5 mg/kg dose was 51.1 ± 19.8 minutes × μg/mL with peak concentrations (Cmax) of 1,440 ± 361 ng/mL. The clearance of CH-2-77 was 110 ± 41.3 mL/min/kg, or 12-fold higher compared with VERU-111 (9.05 mL/min/kg) (38), and the tissue distribution was more pronounced with a volume of distribution at a steady state (Vss) of 11.3 ± 5.46 L/kg (compared with 1.8 ± 0.2 L/kg for VERU-111). Oral bioavailability was minimal at 0.3%. Overall, these pharmacokinetic properties leave room for future improvements but were deemed sufficient to embark on in vivo efficacy studies for CH-2-77, acknowledging that frequent dosing to achieve systemic exposure near the MTD would likely be required for antitumor activity.

Figure 4.

Plasma concentration–time profile (geomean ± 95% CI) of CH-2-77 in rats (n = 5) after intravenous administration of 5 mg/kg.

Figure 4.

Plasma concentration–time profile (geomean ± 95% CI) of CH-2-77 in rats (n = 5) after intravenous administration of 5 mg/kg.

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Efficacy of CH-2-77 in an orthotopic metastatic TNBC xenograft model

Before conducting the in vivo study, we completed an MTD study to identify a safe maximum dose of CH-2-77. As shown in Supplementary Fig. S3, at the dose of 30 mg/kg, CH-2-77 caused around 10% of body weight loss in mice after only 3 consecutive days of treatment, and 100% of mortality in all NSG mice (n = 4) after only 5 doses. Thus, based on promising in vitro potencies, the acceptable pharmacokinetic parameters, and the MTD results, and because the duration of treatment was expected to last approximately three weeks, the antitumor effects of CH-2-77 on TNBC were next determined in vivo using a maximum dose of 20 mg/kg. An orthotopic TNBC xenograft model, MDA-MB-231, with high penetrance of lung metastasis from the mammary fat pad was used. The average tumor volume in CH-2-77–treated mice was significantly smaller than the vehicle group, in a dose-dependent manner (Fig. 5A). There was no significant body weight loss observed in any treatment group, showing the absence of cumulative toxicity of CH-2-77 in this model (Fig. 5B). When the tumors in the vehicle group grew to a mean of ∼1,000 mm3, all mice were euthanized and tissues were harvested. Compared with the vehicle control, the reduction in mean final tumor volume and mean final tumor wet weight of tumors from the CH-2-77 treatment groups confirmed its in vivo potency. The maximum inhibitory effect of CH-2-77 on primary tumor growth was observed at the 20 mg/kg dose (Fig. 5C and D). As reported in our previous study using VERU-111, orthotopic MDA-MB-231 xenografts efficiently metastasize to the lungs (18). Because CH-2-77 inhibits the migration and invasion of TNBC cells in vitro, all lungs were sectioned and stained with H&E to evaluate the antimetastatic activity of CH-2-77. Multiple lung metastases with large nodule sizes were observed in the vehicle group (Fig. 5E and F). The 5 mg/kg CH-2-77 treatment regimen reduced the average number of lung nodules from 48 to 36, without statistical significance. However, the 10 mg/kg and 20 mg/kg doses suppressed lung metastasis, with the mean number of lung metastases as 16 and 4, respectively. In the 20 mg/kg CH-2-77 cohort, only sparsely located small lesions were observed.

Figure 5.

CH-2-77 inhibits primary tumor growth and lung metastasis in an orthotopic MDA-MB-231 TNBC xenograft model in a dose-dependent manner. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. CH-2-77 was given to mice 5 times a week intraperitoneally. At the study endpoint, ex vivo tumor volume ± SD (C) and tumor wet weights (in grams) ± SD (D) were measured. The mean values are shown above each scatter bar graph. E, Lung metastases ± SD of each group after manually counting nodules in H&E-stained whole lung sections. The mean number of metastases is indicated above each bar. F, Representative images of H&E-stained lung images; metastases are indicated by black arrows.

Figure 5.

CH-2-77 inhibits primary tumor growth and lung metastasis in an orthotopic MDA-MB-231 TNBC xenograft model in a dose-dependent manner. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. CH-2-77 was given to mice 5 times a week intraperitoneally. At the study endpoint, ex vivo tumor volume ± SD (C) and tumor wet weights (in grams) ± SD (D) were measured. The mean values are shown above each scatter bar graph. E, Lung metastases ± SD of each group after manually counting nodules in H&E-stained whole lung sections. The mean number of metastases is indicated above each bar. F, Representative images of H&E-stained lung images; metastases are indicated by black arrows.

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Efficacy of CH-2-77 in an orthotopic metastatic TNBC xenograft model in comparison with CA4P as a reference compound

To compare the efficacy of CH-2-77 with a CBSI that entered clinical trials, the in vivo efficacy of CH-2-77 was determined in a MDA-MB-231 xenograft model using a potent CBSI, CA4P (also known as fosbretabulin, a prodrug of CA4). The percent change in tumor growth over time in response to CH-2-77 (20 mg/kg) was similar between the two MDA-MB-231 xenograft experiments shown in Figs. 5 and 6, demonstrating the reproducibility of the in vivo CH-2-77 results (Supplementary Fig. S4A and S4B). The average tumor volume of both CH-2-77 (20 mg/kg) and CA4P (28 mg/kg, equivalent to 20 mg/kg of the active parent compound CA4) treatment groups was significantly reduced in comparison with the vehicle group, but with no significant loss in body weight (Fig. 6A and B; Supplementary Fig. S4C–S4H). When compared with the vehicle control group, the ex vivo tumor volume and final wet weights of both treatment groups also confirmed that both treatments were similarly potent in vivo (Fig. 6C and D). A representative picture of tumors from each cohort is shown in Fig. 6E. These results demonstrate that CH-2-77 at a dose of 20 mg/kg has equivalent in vivo efficacy relative to the clinical trial reference compound CA4P (28 mg/kg).

Figure 6.

CH-2-77 inhibits primary tumor growth in an orthotopic MDA-MB-231 TNBC xenograft model in comparison with CA4P. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. At the study endpoint, ex vivo tumor volume ± SEM (C) and tumor wet weights (in grams) ± SEM (D) were measured. The mean values are shown above each scatter bar graph. E, Tumor images representative of each group's mean ex vivo tumor volume and tumor wet weight.

Figure 6.

CH-2-77 inhibits primary tumor growth in an orthotopic MDA-MB-231 TNBC xenograft model in comparison with CA4P. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. At the study endpoint, ex vivo tumor volume ± SEM (C) and tumor wet weights (in grams) ± SEM (D) were measured. The mean values are shown above each scatter bar graph. E, Tumor images representative of each group's mean ex vivo tumor volume and tumor wet weight.

Close modal

Systemic chemotherapy is often the only viable option for early-stage and advanced TNBC patients to reduce and to prevent tumor progression and metastasis, including anthracyclines, platinum-based regimens, taxanes, and other cytotoxic agents (39–41). Although taxanes are highly successful in breast cancer treatment, their clinical use is often limited by narrow therapeutic windows, poor aqueous solubility, and drug resistance mediated by ABC transporters (42). To address these limitations, numerous studies have focused on developing CBSIs, which are reported to have significantly less susceptibility to multidrug resistance and to show additional therapeutic activities as compared with taxanes and vinca alkaloids, such as tumor vasculature disruption effects (43, 44).

We have previously reported the efficacy of a well-tolerated CBSI, VERU-111, in suppressing tumor growth and metastasis in vitro and in vivo (18). Recently, a novel series of VERU-111 analogues were generated and evaluated by our team (24). Among all the analogues tested, CH-2-77 [compound 4v, in the original report (24)] was the most potent one and significantly inhibited melanoma growth in vivo. Because CH-2-77 showed an IC50 value of 1.7 nmol/L against MDA-MB-231 cells in the abovementioned report, we determined the efficacy of CH-2-77 in TNBC models. The antiproliferative activity of CH-2-77 was first determined using a panel of diverse TNBC cell lines, including paclitaxel-resistant cells and patient-derived cell lines (cHCI-2 and cHCI-10). CH-2-77 maintained cytotoxicity in all cell lines tested in the nanomolar range and was more potent than colchicine. Although CH-2-77 was less cytotoxic than paclitaxel in paclitaxel-sensitive parental TNBC cells, it was more effective in both PDX cell lines, and more importantly, retained efficacy in the paclitaxel-resistant MDA-MB-231 and MDA-MB-468 TNBC sublines. Additionally, we showed that CH-2-77 could overcome colchicine resistance, whereas the efficacy of paclitaxel was reduced in colchicine-resistant cells, demonstrating a distinct advantage of CH-2-77 to overcome colchicine resistance. Interestingly, although CH-2-77 consistently shows high potency against cancer cells in vitro with nanomolar potency, its affinity to microtubules is in the micromolar range as determined in our previous study (24). It is not clear at this point why this inconsistency exists. CH-2-77 as a new molecule has not been thoroughly characterized at this stage. Although our current experimental data clearly showed that it targets the colchicine site in tubulin, we cannot rule out the possibility that it has additional high-affinity drug target(s), as many small-molecule drugs do. Alternatively, it may also be possible that CH-2-77 may undergo certain intracellular transformation, leading to a more active molecule to exert its potent nanomolar cytotoxicity. We will investigate this inconsistency in future studies to further elucidate its mechanisms of action.

Previous crystal structure and tubulin polymerization studies have shown that CH-2-77 targets the colchicine-binding site of tubulin and inhibits tubulin polymerization (24). To confirm this effect in TNBC, we visualized the microtubule structure of TNBC cells treated with CH-2-77 by immunofluorescence. As expected, CH-2-77 treatment induced microtubule depolymerization on both MDA-MB-231 and MDA-MB-468 cells. At the concentration of 2.5 nmol/L, CH-2-77 induced the formation of multipolar spindles and disorganized the microtubule assembly in mitotic MDA-MB-231 and MDA-MB-468 cells. However, we did not observe any dividing TNBC cells in the CH-2-77 treatment groups, presumably due to the cytotoxic activity of CH-2-77 as 5 nmol/L of CH-2-77 killed all mitotic cells. Using CH-2-77–treated TNBC cells, we also determined the levels of acylated-α-tubulin and α-tubulin. CH-2-77 induced the degradation of acylated-α-tubulin and α-tubulin at the concentration of 10 nmol/L, whereas 2.5 nmol/L or 5 nmol/L of CH-2-77 seemed to have a limited effect. This observation was in contrast to the obvious microtubule disorganization observed by immunofluorescence caused by CH-2-77 treatment, suggesting that α-tubulin acetylation might be indirectly affected by microtubule-destabilizing agents with an unclear mechanism and may require a high concentration of antitubulin agents to be observed (45, 46).

Similar to VERU-111, CH-2-77 significantly inhibited the migration and invasion of both TNBC cell lines but exhibited twice the potency of VERU-111 (18). CH-2-77 also induced the G2–M phase arrest and apoptosis of MDA-MB-231 and MDA-MB-468 cells in the low nanomolar range. We determined the protein levels of cleaved-PARP, an indicator of cell apoptosis, on TNBC cells treated with CH-2-77. CH-2-77 caused a marked increase of cleaved-PARP in TNBC cells, and its effect was stronger than observed for paclitaxel at the same concentration (10 nmol/L). We also compared the protein levels of cleaved-caspase-3, which is induced by intrinsic and extrinsic apoptosis pathways, but we only observed the upregulation of cleaved-caspase-3 in response to CH-2-77 in MDA-MB-231 cells. Therefore, MDA-MB-231 cells were profiled for additional apoptosis marker evaluations (47). Both cleaved-caspase-8 and cleaved-caspase-9 are upstream regulators of caspase-3 cleavage, whereas the caspase-8 pathway is involved mainly in the extrinsic pathway and the caspase-9 pathway is involved mainly in the intrinsic pathway (48, 49). Because only cleavage of caspase-9 was observed on MDA-MB-231 cells after CH-2-77 treatment, but cleavage of caspase-8 was not, we speculate that CH-2-77 treatment induces apoptosis of MDA-MB-231 cells through the intrinsic mitochondrial pathway. This conclusion is consistent with observations of changes in the protein levels of the antiapoptotic protein, Bcl-2 (repressed), and the proapoptotic protein, Bax (induced) in CH-2-77–treated MDA-MB-231 cells.

Before evaluating the antitumor efficacy of CH-2-77 in vivo, we evaluated the pharmacokinetics of CH-2-77 in rats. Although the oral bioavailability of CH-2-77 was poor and the clearance was relatively high as compared with VERU-111, plasma exposure after intraperitoneal administration was deemed to be potentially sufficient to result in in vivo efficacy in an orthotopic metastatic TNBC xenograft model based on previously reported hepatic microsomal stability in mice (50). Therefore, we designed our in vivo efficacy study to dose the mice 5 times per week at three dose levels. We chose the highly metastatic MDA-MB-231 orthotopic xenograft model to demonstrate the anticancer effects of CH-2-77 in TNBC. In comparison with the strong primary tumor growth suppression observed in an A375 melanoma xenograft model, CH-2-77 was not able to fully inhibit the growth of the MDA-MB-231 xenografts, suggesting the relatively enhanced malignancy of TNBC (24). However, CH-2-77 significantly inhibited the growth of MDA-MB-231 xenografts in a dose-dependent manner, without gross symptoms of cumulative toxicities, even at the highest dose of 20 mg/kg. Moreover, CH-2-77 repressed spontaneous lung metastasis originating from the mammary fat pad tumors.

Overall, CH-2-77 as a novel CBSI shows great promise in overcoming both paclitaxel resistance and colchicine resistance. Moreover, its in vivo efficacy (20 mg/kg) in an aggressive xenograft TNBC model is similar to a CBSI that went through multiple clinical trials, CA4P (28 mg/kg). From our previous studies using a melanoma model (24), CH-2-77 induced 88.5% of tumor inhibition at a dose of 20 mg/kg, suggesting that CH-2-77 is efficacious for other solid tumors in addition to TNBC. Because of potent cytotoxicity, the efficacy of CH-2-77 could likely be further enhanced by encapsulation in nanoparticles or used as a warhead in antibody–drug conjugates targeting TNBC in the future. In addition to CH-2-77, we recently developed a new series of 6-aryl-2-benzoyl-pyridines, some of which have improved metabolic stability and pharmacokinetic parameters as compared with CH-2-77 in vitro and in vivo (50). Therefore, we are currently devoting efforts to remedy any metabolic shortcomings of CH-2-77, while maintaining its low nanomolar potency, and we expect that more efficacious chemotherapeutic drug candidates for TNBC will be developed soon.

In summary, we report herein that CH-2-77 potently inhibits the proliferation, migration, and invasion of TNBC cells. In particular, in vitro studies showed that CH-2-77 induces G2–M phase cell-cycle arrest and expedites TNBC cell apoptosis primarily through the intrinsic mitochondrial apoptosis pathway. Moreover, CH-2-77 demonstrated remarkable antitumor and antimetastatic efficacy in an orthotopic TNBC xenograft model. CH-2-77 was able to suppress lung metastasis derived from the primary tumor in a dose-dependent manner without overt toxicities. Overall, further development of the CH-2-77 scaffold holds potential as a first-line or second-line chemotherapy for patients who progress on taxanes to more effectively treat TNBC patients and to improve survival from metastatic disease.

B. Meibohm reports grants from NIH during the conduct of the study. D.D. Miller reports other support from VERU during the conduct of the study; in addition, D.D. Miller has a patent for CH-2-77 issued, licensed, and with royalties paid from VERU. W. Li reports personal fees and other support from Veru, Inc. during the conduct of the study; other support from SEAK Therapeutics, LLC outside the submitted work; in addition, W. Li has a patent for the tubulin inhibitor portfolio owned by the University of Tennessee Research Foundation and licensed to Veru Inc. for commercial development issued, licensed, and with royalties paid from Veru, Inc. No disclosures were reported by the other authors.

S. Deng: Conceptualization, data curation, formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing. R.I. Krutilina: Data curation, investigation, writing–review and editing. K.L. Hartman: Data curation, writing–review and editing. H. Chen: Methodology, writing–review and editing. D.N. Parke: Resources, data curation, methodology. R. Wang: Data curation. F. Mahmud: Data curation. D. Ma: Data curation. P.B. Lukka: Data curation, formal analysis, methodology. B. Meibohm: Resources, methodology, writing–review and editing. T.N. Seagroves: Resources, supervision, methodology, writing–review and editing. D.D. Miller: Resources, investigation, writing–review and editing. W. Li: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, methodology, writing–original draft, project administration, writing–review and editing.

All flow cytometry data were generated in the Flow Cytometry and Cell Sorting core at UTHSC with the assistance of Dr. Tony Marion and Dr. Deidre Daria. We thank Dr. Alana Welm at the Huntsman Cancer Institute (HCI) for providing the original PDX models for generating cHCI-2 and cHCI-10 cells. This work is supported by DoD grants W81XWH2010011 (W. Li) and W81XWH2010019 (T.N. Seagroves). Additional support is provided by NIH grants R01CA148706 (W. Li and D.D. Miller), R01CA240447, 1S10OD010678, and 1S10RR026377 to W. Li, and NIH grant 1S10OD016226 to B. Meibohm. The contents of the article are solely the responsibility of the authors and do not necessarily represent the official views of the DoD or the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data