Combination of polymeric micelle formulation of TGFβ receptor inhibitors and paclitaxel produces consistent response across different mouse models of Triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of targetable receptors and sometimes poor response to chemotherapy. The transforming growth factor beta (TGFβ) family of proteins and their receptors (TGFRs) are highly expressed in TNBC and implicated in chemotherapy-induced cancer stemness. Here, we evaluated combination treatments using experimental TGFR inhibitors (TGFβi), SB525334 (SB), and LY2109761 (LY) with paclitaxel (PTX) chemotherapy. These TGFβi target TGFR-I (SB) or both TGFR-I and TGFR-II (LY). Due to the poor water solubility of these drugs, we incorporated each of them in poly(2-oxazoline) (POx) high-capacity polymeric micelles (SB-POx and LY-POx). We assessed their anticancer effect as single agents and in combination with micellar PTX (PTX-POx) using multiple immunocompetent TNBC mouse models that mimic human subtypes (4T1, T11-Apobec and T11-UV). While either TGFβi or PTX showed a differential effect in each model as single agents, the combinations were consistently effective against all three models. Genetic profiling of the tumors revealed differences in the expression levels of genes associated with TGFβ, epithelial to mesenchymal transition (EMT), TLR-4, and Bcl2 signaling, alluding to the susceptibility to specific gene signatures to the treatment. Taken together, our study suggests that TGFβi and PTX combination therapy using high-capacity POx micelle delivery provides a robust antitumor response in multiple TNBC subtype mouse models.

FIGURE 1

Characterization of single TGFβi and co‐loaded TGFβi/PTX in POx micelles by DLS and TEM. (a) DLS intensity size distributions and the corresponding TEM images of the nanoassemblies formed in drug‐loaded PMs of various compositions (a1) SB‐POx (8/20); (a2) LY‐POx (8/20); (a3) PTX‐POx (8/20); (a4) SB/PTX‐POx (5.2/8/20); (a5) LY/PTX‐POx (4/8/20); (a6) LY/PTX‐POx (5.2/8/20). The numbers in the brackets represent the mass ratios TGFβi/POx, PTX/POx, or TGFβi/PTX/POx. (b) Drug concentration, loading capacity (LC [%] = M _drug/[M _drug + M _excipient] × 100 [%]) and DLS characteristics (z‐average hydrodynamic diameter, PDI, diameters at intensity size distribution maxima) of the formulations presented in (a). Drug concentration in the solution was measured by HPLC. Data represent mean ± SE. For DLS measurements, the samples prepared at POx 20 mg/mL were diluted 10 times. DLS, dynamic light scattering; PDI, polydispersity index; PM, polymeric micelles; POx, poly(2‐oxazoline); PTX, paclitaxel; TEM, transmission electron microscopy; TGFβi, transforming growth factor beta inhibitors.

FIGURE 2

TGFβi treatment suppresses TGFβ signaling in NIH‐3T3 fibroblasts. (a) Schematic of canonical TGFβ signaling. (b) Capillary‐based western blot analysis of expression of p‐SMAD2/3 (~62 kDa) and housekeeping protein, β‐actin (~48 kDa) in NIH‐3T3 cells treated with TGFβi in POx PMs (SB‐POx [8/20] and LY‐POx [8/20]) or dissolved in DMSO. Control groups were treated with the same amounts of POx or DMSO. The images were generated using Compass for Simple Western (version 6.0). (c) The quantification of p‐SMAD2/3 bands in (b). *p < 0.05 computed by one‐way ANOVA with Tukey's multiple comparisons test. (d, e) The p‐SMAD2/3 expression analyzed by flow cytometry in NIH‐3T3 cells treated with TGFβi (d) formulated in POx PMs or (e) dissolved in DMSO. The (d) and (e) panels were run in the same experiment and split for the clarity of presentation to avoid overlap between the free and micelle‐formulated drug histograms. The panels have common histograms for USC, unstimulated, TGFβ‐stimulated controls. The mean florescence intensities for (d) and (e) are included in the parentheses. ANOVA, analysis of variance; PM, polymeric micelles; POx, poly(2‐oxazoline); TGFβ, transforming growth factor beta; TGFβi, TGFβ inhibitors; USC, unstained control.

FIGURE 3

TGFβ inhibition synergizes with chemotherapy to inhibit primary tumor growth and lung metastases in 4T1 TNBC tumor‐bearing mice (n = 3 ~ 4). (a) Tumor growth curves following treatments with single‐drug SB‐POx (8/20) i.p., single‐drug PTX‐POx (8/20) i.v., or separately administered combinations SB‐POx (8/20) i.p. and PTX‐POx (8/20) i.v. using different SB‐POx schedules (daily or eod) at 32 mg/kg. See Table S3 for the complete statistical comparison between all groups. (Statistical comparisons were computed by two‐way ANOVA with Tukey's multiple comparisons test.) (b) Percent tumor growth inhibition corresponding to tumor growth curves. See Table S4 for the complete statistical comparison between all groups (Statistical comparisons were computed by one‐way ANOVA with Tukey's multiple comparisons test.) (c) Images of lungs with metastatic nodules (left) and corresponding H&E stain images (right) with metastatic nodules indicated with black arrows from representative mice in each group. (d) Histopathological scoring of lung metastatic burden. Statistical comparisons were computed by one‐way ANOVA with Tukey's multiple comparisons test. (e) Body weight changes (percent of initial) in mice treated with TGFβi‐POx and/or PTX‐POx. ANOVA, analysis of variance; eod, every other day; i.p., intraperitoneal; i.v., intravenous; PDI, polydispersity index; POx, poly(2‐oxazoline); PTX, paclitaxel; TGFβ, transforming growth factor beta; TNBC, triple‐negative breast cancer.

FIGURE 4

Tumor growth inhibition with i.p. versus i.v. routes of TGFβi delivery (n = 3–4). (a) Tumor growth curves in 4T1 tumor‐bearing mice treated with either single drug or separately administered TGFβi and PTX combinations, or (b) co‐loaded micelles of TGFβi and PTX (common saline and PTX‐POx groups were used for experiments (a) and (b) since the experiments were conducted in parallel); see Table S5 for the complete statistical comparison between all groups. (Statistical comparisons were computed by two‐way ANOVA with Tukey's multiple comparisons test.). (c) Percent tumor growth inhibition by treatments corresponding to (a) and (b). *p < 0.05 and **p < 0.01 computed by one‐way ANOVA with Tukey's multiple comparisons test. (d) Histopathological scoring of lung metastatic burden. Data represent mean ± SD. N = 2 with duplicate sections. *p < 0.05 computed by one‐way ANOVA with Tukey's post hoc test. ANOVA, analysis of variance; eod, every other day; i.p., intraperitoneal; i.v., intravenous; POx, poly(2‐oxazoline); PTX, paclitaxel; TGFβi, transforming growth factor beta inhibitor.

FIGURE 5

Tumor growth inhibition with oral route of TGFβi delivery (n = 5) (a) Tumor growth curves in 4T1 tumor‐bearing mice treated with orally delivered (o.g.) TGFβi formulated either in POx or 0.5% NaCMC and 0.25% Tween 80. Data represent mean ± SD. N = 4. **p < 0.003, ****p < 0.0001 computed by two‐way ANOVA with Tukey's post hoc test. See Table S6 for the complete statistical comparison between all groups. (Statistical comparisons were computed by two‐way ANOVA with Tukey's multiple comparisons test.). (b) Percent tumor growth inhibition by treatments corresponding to (a). See Table S7 for the complete statistical comparison between all groups. (Statistical comparisons were computed by one‐way ANOVA with Tukey's multiple comparisons test.) (c) Histopathological scoring of lung metastatic burden. Data represent mean ± SD. N = 2 with duplicate sections. ****p < 0.0001 computed by one‐way ANOVA with Tukey's post hoc test. (d) Body weight changes (percent of initial) in mice treated with TGFβi‐POx and/or PTX‐POx. ANOVA, analysis of variance; i.g., oral gavage; POx, poly(2‐oxazoline); PTX, paclitaxel; NaCMC, sodium carboxymethylcellulose; TGFβi, transforming growth factor beta inhibitor.

FIGURE 6

Differential sensitivity of Claudin‐low TNBC subtypes to TGFβi and PTX. Tumor growth curves following treatments with single‐drug SB‐POx (8/20) i.p., single‐drug PTX‐POx (8/20) i.v., or separately administered combinations SB‐POx (8/20) i.p. and PTX‐POx (8/20) i.v. in mice bearing (a) T11‐Apobec (n = 5) and (b) T‐11 UV tumors (n = 5). Data represent mean ± SD. N = 4. **p < 0.003, ****p < 0.0001 computed by two‐way ANOVA with Tukey's post hoc test. See Tables S8 and S9 for the complete statistical comparison between all groups. (c, d) Percent tumor growth inhibition by treatments corresponding to (a) and (b) as determined at the endpoint of tumor growth experiments. Statistical comparisons were computed by one‐way ANOVA with Tukey's multiple comparisons test. (e, f) Body weight changes (percent of initial) in mice treated with TGFβi‐POx and/or PTX‐POx. ANOVA, analysis of variance; i.p., intraperitoneal; i.v., intravenous; POx, poly(2‐oxazoline); PTX, paclitaxel; TGFβi, transforming growth factor beta inhibitor; TNBC, triple‐negative breast cancer.