Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX characterization. (A) Scanning transmission electron microscopy coupled with electronic energy-dispersive x-ray spectroscopy (STEM-EDX) atomic mapping of Si (green), O (red), N (blue), and S (purple) atoms in NP-FAP. Weight (%) composition was calculated in 52.8% of O 2 , 46.7% of Si, and 0.6% of S. (B) Doxorubicin release study from NP-FAP-DOX in the absence or presence of glutathione (GSH) at different concentrations, determined by measuring doxorubicin fluorescence vs. time in phosphate-buffered saline (PBS) (mean ± SD). (C) Hemolytic activity of the NP-FAP-DOX. Red blood cells were incubated with PBS (negative control), 1% Triton X-100 (positive control), or NP-FAP-DOX at different concentrations (mean ± SD).

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: Transmission Assay, Electron Microscopy, Spectroscopy, Fluorescence, Saline, Activity Assay, Incubation, Negative Control, Positive Control

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX targeting efficacy. (A and B) MCF-10A, MDA-MB-468, MDA-MB-231, cancer-associated fibroblasts (CAFs), and MDA-MB-436 cells were treated with NP-FAP-DOX (25 μg/ml, 3 h). Intracellular doxorubicin was visualized by fluorescence microscopy. (A) Representative images. (B) Quantification of doxorubicin fluorescence intensity (mean ± SEM). Statistical comparisons were made against MCF-10A. (C to J) Comparative internalization of NP-CTR-DOX vs. NP-FAP-DOX (25 μg/ml, 3 h). Representative images and corresponding quantification of intracellular doxorubicin fluorescence in (C and D) MDA-MB-436 cells, (E and F) CAFs, and (G to J) patient-derived organoids (PDOs). Fluorescence intensity normalized to NP-CTR-DOX (mean ± SEM). **** P < 0.0001. n.s., nonsignificant.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: Fluorescence, Microscopy, Derivative Assay

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX cytotoxic effect. (A to D) MDA-MB-436 cells, (E and F) CAFs, and (G) PDO65 were treated with NP-CTR, NP-FAP, or NP-FAP-DOX for 72 h. Cell viability was measured using the WST-1 assay for MDA-MB-436 (A) and CAFs (E), and CellTiter-Glo for PDOs (G). Apoptosis was assessed by flow cytometry using Annexin V-FITC staining (B and F). (C and D) Clonogenic assay in MDA-MB-436 cells treated with NP-CTR, NP-FAP, or NP-FAP-DOX for 24 h. (C) Quantification of colony number normalized to the negative control. (D) Representative images of colony formation after treatment with 5 μg/ml. Mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n.s., nonsignificant.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: WST-1 Assay, Flow Cytometry, Staining, Clonogenic Assay, Negative Control

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX in vivo antitumoral efficacy. (A) Tumor growth curve of mice following treatment with PBS (vehicle), NP-CTR-DOX, and NP-FAP-DOX ( n = 12 per group). The arrows indicate the days of treatment administration. (B) Tumor volume at the treatment endpoint (mean ± SEM). (C) Mki67 expression in tumors at the treatment endpoint determined by real-time quantitative polymerase chain reaction (RT-qPCR) normalized to PBS (mean ± SEM). (D) Representative images of internalized doxorubicin in paraffin-embedded tumor sections at the treatment endpoint. Blue: nucleus, red: doxorubicin. Scale bar: 50 μm. (E) Representative images of immunofluorescence staining of cleaved-caspase 3 (cCASP3) in paraffin-embedded tumor sections at the treatment endpoint. Blue: nucleus, green: cCASP3. Scale bar: 50 μm. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX in vivo TME modulation. (A) Fap-α expression in tumors at the treatment endpoint was determined by RT-qPCR normalized to PBS ( n = 12 per group). Mean ± SEM. (B) Representative images of immunofluorescence staining of alpha-smooth muscle actin (α-SMA) in paraffin-embedded tumor sections at the treatment endpoint. Blue: nucleus, green: α-SMA. Scale bar: 50 μm. (C) Representative images of Masson’s trichrome staining in paraffin-embedded tumor sections at the treatment endpoint. Blue: collagen, pink: fibrin, red: muscle fibers, black: nucleus. Scale bar: 50 μm. (D) Representative second-harmonic generation (SHG) microscopy images of tumor sections showing fibrillar collagen structure. (E to H) Percentage of (E) T lymphocytes (CD45 + /CD3ε + ), (F) cytotoxic T cells (CD45 + /CD3ε + /CD8a + ), (G) M1-like macrophages (CD45 + /F4/80 + /CD86 + ), and (H) M2-like macrophages (CD45 + /F4/80 + /CD206 + ) in xenograft tumors analyzed by flow cytometry at the treatment endpoint ( n = 12 per group). (I) Ratios of M1 to M2 (M1/M2) were calculated as the percentage of M1-like macrophages divided by the percentage of M2-like macrophages on xenograft tumors analyzed by flow cytometry at the treatment endpoint ( n = 12 per group). Mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Flow Cytometry

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX cardio and systemic toxicity evaluation. (A) Schematic representation of the in vivo study. 4T1 cells were injected into the mammary fat pad of BALB/C mice. The tumors, with an initial size of approximately 50 mm 3 , were intravenously treated with PBS (vehicle) or 50 mg/kg of nanoparticles (NPs: NP-CTR-DOX or NP-FAP-DOX) 4 times per week for 15 d ( n = 12 per group). Free doxorubicin (DOX) (3.75 mg/kg) was intravenously administered for 8 d. The mice were sacrificed 3 d after the last treatment, and the tumor and organs were collected. (B) Tumor growth curve mice treated with PBS (vehicle), NP-FAP-DOX, and free DOX ( n = 12 per group; mean ± SEM) (C) Body weight changes in tumor-bearing mice during treatment with PBS (vehicle), NP-FAP-DOX, or free DOX (mean ± SEM). (D) Myh7 expression in heart tissue at the treatment endpoint was determined by RT-qPCR and normalized to PBS (mean ± SEM). (E) Representative images of hematoxylin and eosin staining in paraffin-embedded heart, liver, spleen, and kidney tissue sections at the treatment endpoint. Thin arrows indicate cytoplasmic lysosomes. Thick arrows indicate degeneration of hepatocytes. Dashed areas in the spleen represent the white pulp. Dotted areas in the kidney represent the glomerular capsule, the asterisk represents tubule dilations, and the hash sign represents cellular infiltrations. Scale bar: 50 μm (heart, liver, and kidney) or 200 μm (spleen). DOX: Free doxorubicin. ****P < 0.0001.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, Expressing, Quantitative RT-PCR, Staining

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: Schematic representation of the NP-FAP-DOX design and the proposed mechanism of action. MSNs were loaded with doxorubicin and functionalized with a FAP-α ligand peptide to enable selective recognition of FAP-α-expressing CAFs and tumor cells. Following cellular internalization, intracellular glutathione (GSH)-triggered cleavage of disulfide bonds induces controlled doxorubicin release. This results in CAF depletion, extracellular matrix degradation, induction of tumor cell apoptosis, and remodeling of tumor microenvironment, ultimately leading to potent antitumor activity in TNBC.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay

Journal: Biomaterials Research

Article Title: Fibroblast Activation Protein Alpha-Targeted Nanoparticles for Tumor Microenvironment Remodeling and Antitumor Therapy in Triple-Negative Breast Cancer

doi: 10.34133/bmr.0347

Figure Lengend Snippet: NP-FAP-DOX in vivo TME modulation. (A) Fap-α expression in tumors at the treatment endpoint was determined by RT-qPCR normalized to PBS ( n = 12 per group). Mean ± SEM. (B) Representative images of immunofluorescence staining of alpha-smooth muscle actin (α-SMA) in paraffin-embedded tumor sections at the treatment endpoint. Blue: nucleus, green: α-SMA. Scale bar: 50 μm. (C) Representative images of Masson’s trichrome staining in paraffin-embedded tumor sections at the treatment endpoint. Blue: collagen, pink: fibrin, red: muscle fibers, black: nucleus. Scale bar: 50 μm. (D) Representative second-harmonic generation (SHG) microscopy images of tumor sections showing fibrillar collagen structure. (E to H) Percentage of (E) T lymphocytes (CD45 + /CD3ε + ), (F) cytotoxic T cells (CD45 + /CD3ε + /CD8a + ), (G) M1-like macrophages (CD45 + /F4/80 + /CD86 + ), and (H) M2-like macrophages (CD45 + /F4/80 + /CD206 + ) in xenograft tumors analyzed by flow cytometry at the treatment endpoint ( n = 12 per group). (I) Ratios of M1 to M2 (M1/M2) were calculated as the percentage of M1-like macrophages divided by the percentage of M2-like macrophages on xenograft tumors analyzed by flow cytometry at the treatment endpoint ( n = 12 per group). Mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific, MA, USA) using 2 μl of resulting complementary DNA (cDNA), 5 μl of TaqMan Universal Master Mix (Applied Biosystems), 0.5 μl of TaqMan 20× assays (human FAP-α : Hs00990791_m1, human GAPDH : Hs03929097_g1, mouse Fap-α : Mm01329177_m1, mouse Gapdh : Mm99999915_g1, mouse Mki67 : Mm01278617_m1, mouse Myh7 : Mm00600555_m1; Applied Biosystems), and 2.5 μl of RNase-free water, according to the manufacturer’s instructions.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Flow Cytometry