Nanoparticle modulation of the tumor microenvironment enhances therapeutic efficacy of cisplatin

Abstract

The tumor microenvironment (TME) serves as a multidrug resistant center for tumors under the assault of chemotherapy and a physiological barrier against the penetration of therapeutic nanoparticles (NPs). Previous studies have indicated the ability for therapeutic NP to distribute into, and deplete tumor-associated fibroblasts (TAFs) for improved therapeutic outcomes. However, a drug resistant phenotype gradually arises after repeated doses of chemotherapeutic NP. Herein, the acquisition of drug resistant phenotypes in the TME after repeated cisplatin NP treatment was examined. Particularly, this study was aimed at investigating the effects of NP damaged TAFs on neighboring cells and alteration of stromal structure after cisplatin treatment. Findings suggested that while off-targeted NP damaged TAFs and inhibited tumor growth after an initial dose, chronic exposure to cisplatin NP led to elevated secretion of Wnt16 in a paracrine manner in TAFs. Wnt16 upregulation was then attributed to heightened tumor cell resistance and stroma reconstruction. Results attest to the efficacy of Wnt16 knockdown in damaged TAFs as a promising combinatory strategy to improve efficacy of cisplatin NP in a stroma-rich bladder cancer model.

Keywords: Cisplatin; Nanoparticle; Tumor associated fibroblast; Tumor microenvironment; Wnt16.

Figure 1. Off-target delivery of cisplatin NP to TAF induces tumor cells resistance via secretion of Wnt16

a. Tumor growth inhibition of free cisplatin and cisplatin NP in a stroma rich bladder cancer (SRBC) model (n=5). b. Western blot analysis of Wnt16 protein levels in the resistant tumors. Intensity of the protein bands was quantified by using Image J (n=3, * p<0.05, Student’s t-test). c. Expression level of Wnt16 in different subtypes of bladder cancer. Data were collected and analyzed from The Cancer Genome Atlas (TCGA) database. (* p<0.05, one way ANOVA). d. Dose response curves of UMUC3 cells treated with different levels of recombinant Wnt16 protein (n=4, * p<0.05, ** p<0.01, one way ANOVA). IC₅₀ of cisplatin in UMUC3 treated with Wnt16 protein was shown in the inserted figure. eIn vitro western blot analysis of Wnt16 level in cisplatin treated UMUC3, cisplatin treated activated fibroblast NIH3T3 cells and the conditioned medium. f. DiI-labeled cisplatin NP distribution in the SRBC tumor. Tumors were collected 8h post DiI-NP injection. Presentative fluorescence images of TAF (α-SMA: green), blood vessels (CD31: magenta), and DiI-labeled NP (red) and nucleus (DAPI: blue) were shown. Scale Bar equates to 100 µm. g. Quantification of DiI-NP distribution in TAF by flow cytometry. SRBC model was established by co-inoculated UMUC3 with GFP labeled NIH3T3. h. Fluorescence and TUNEL staining of the SRBC tumor after multiple cisplatin NP treatments with α-SMA (red) and apoptotic cells (green). Yellow square boxes highlight the apoptotic fibroblasts (co-stained as yellow). The ratio of apoptotic fibroblasts over all apoptotic cells was calculated. Scale Bar is 100 µm.

Figure 2. Influences of specific Wnt16 knockdown on the neighboring tumor cells and fibroblasts

a. Cell viability (48h) of UMUC3 towards cisplatin (10 µM) when treated with different NIH3T3 conditioned medium (CM) (n=4, * p <0.05, ** p<0.01, one way ANOVA). b. A non-contact co-culture system was established with the upper chamber seeded with activated NIH3T3 of different treatments, and the lower chamber seeded with naïve UMUC3. This system was used in the scratch assay, β–catenin translocation assay and western blot. c. Scratch assay using the non-contact co-culture system with upper chamber NIH3T3 and lower UMUC3. * Moving distance of lower chamber UMUC3 compared to 0h, n=3. Scale bar is 100 µm. d. Confocal images of β-catenin nucleus translocation (Scale bars 20 µm) in low chamber UMUC3. e. Western blot analysis of Wnt pathway down-stream proteins (c-myc and cyclin D1), EMT markers (E-cadherin (E-cad) and N-cadherin (N-cad)) and apoptotic marker (cleaved PARP) in the lower UMUC3 chamber. f. A non-contact co-culture system was established with the upper chamber seeded with activated NIH3T3 and the lower chamber seeded with naïve activated NIH3T3. g. Assay of canonical Wnt pathway signaling through activation of luciferase labelled fibroblasts was performed in the lower chamber. Four h after co-culture, relative luciferase units (RLU) were quantified. Data are mean ± SD, n=3. h. Western blot analysis of a major ECM glycoprotein, fibronectin, level in the lower chamber 24h post co-culture. i. Confocal images of immunofluorescence staining of fibronectin in the lower chambers (fibronectin: green, cell nucleus: blue), scale bar represents 20 µm. j. Cell proliferation of activated NIH3T3 transfected with anti-Wnt16 or control siRNA at different concentrations (n = 4). k. Viability of activated NIH3T3 across a range of cisplatin concentrations with transfection of anti-Wnt16 or control siRNA (concentration of siRNA was 180 nM). (n=4, * p<0.05, Student’s t-test). Data points show mean ± SD.

Figure 3. Single dose cisplatin NP attenuate TME function and improve NP penetration

Single dose siCont NP (1), cisplatin NP/siCont NP (2), siWnt NP (3) and cisplatin NP/siWnt NP (4) were IV administered to mice separately with cisplatin 1.0 mg/kg and siRNA 0.6 mg/kg. Tissues were collected 2 days after injections. a. Immunofluorescence staining of fibronectin, α-SMA. The nuclei were stained with DAPI (blue) and fibronectin and α-SMA were stained red. Another ECM component, collagen, was stained by using Masson trichrome. The blue color represents collagen content, while the cytoplasm is stained red. Scale bar is 100 µm. b. Quantitative analysis of fibronectin, α-SMA and collagen content using Image J from 5 randomly selected microscopic fields. (n=5, * p<0.05; ** p<0.01; *** p<0.001, Student’s t-test) c. Western blot analysis of FAP α, α-SMA, fibronectin and Wnt16 protein levels in the tumors two days after single dose NP treatment. d. NIH3T3 in the SRBC model was further labeled with GFP. The ratio of GFP positive TAF was quantified by flow cytometry two days after NP treatment. (n=8, *** p<0.001, Student’s t-test) e. DiI-labeled liposomes (~70nm) were IV injected one day before sacrificing the mice. Fluorescent liposome accumulations and penetrations in the SRBC tumors were quantified by flow cytometry (n=3, ** p<0.01, Student’s t-test). f. The amount of accumulated platinum in cisplatin NP/siCont NP (2) and cisplatin NP/siWnt NP (4) were measured by ICP-MS and shown as ng cisplatin/mg tumor tissue. (n=3, * p<0.05, Student’s t-test). Data in all charts show mean ± SD.

Figure 4. siWnt NP overcome the cisplatin NP induced TME stiffening after multiple doses and improve the overall NP accumulation

siCont NP (1), cisplatin NP/siCont NP (2), siWnt NP (3) and cisplatin NP/siWnt NP (4) were IV administered to mice separately with cisplatin 1.0 mg/kg and siRNA 0.6 mg/kg, every other day for a total of 4 injections. a. Immunofluorescence staining of fibronectin (red), α-SMA (red) and cell nucleus (DAPI: blue). Collagen was stained via Masson trichrome staining (blue). Scale bar represents 100 µm. b. Quantitative analysis of fibronectin, α-SMA and collagen content using Image J from 5 randomly selected microscopic fields. (* p<0.05; ** p<0.01; *** p<0.001, Student’s t-test) c. Western-blot analysis of FAPα, α-SMA, fibronectin and Wnt16 levels in the tumors 2 days after multiple NP treatment. d. NIH3T3 in the SRBC model was further labeled with GFP. The ratio of GFP positive TAF was quantified by flow cytometry 2 days after NP treatment. (*** indicated p<0.001, Student’s t test, n=8) e. DiI-labeled liposomes (~70nm) were IV injected 1 day before sacrificing the mice. Fluorescent liposome accumulations and penetrations in the SRBC tumors were quantified by flow cytometry (** p<0.01, Student’s t-test, n=3). f. The amount of accumulated platinum in cisplatin NP/siCont NP (2) and cisplatin NP/siWnt NP (4) were measured by ICP-MS and shown as ng cisplatin/mg tumor tissue (*p<0.05, Student’s t-test, n=3). Data in all charts show mean ± SD.

Figure 5. Wnt16 downregulation attenuates cisplatin NP induced angiogenesis for bladder cancer treatment

A non-contact co-culture study was performed to evaluate tube formation in vitro. The upper chamber NIH3T3 cells were given different treatments and co-cultured with HUVEC cells seeded in the lower chambers. Tube formation was monitored in HUVEC cells as indicators for angiogenesis 4h after co-culture. HUVEC cells were stained by calcium AM (green) (a) and the number of formed tubes was calculated (b). Five randomly selected microscopic fields were quantitatively analyzed by Image J (* p<0.05, ** p<0.01, *** p<0.001, Student’s t-test). Scale bar represents 100 µm. Influence on angiogenesis was further evaluated in vivo by immunofluorescence staining endothelial cells with CD31 (red) after multiple treatments (c). Quantitative analysis was calculated based on 5 randomly selected microscopic fields (* p<0.05, ** p<0.01, Student’s t-tests, scale bar indicates 100 µm) (d). Distribution of DiI labeled cisplatin NP 8h after IV injection in different treatments of the SRBC model (e). Five images were quantified and the result is shown on right (* p<0.05, ** P<0.01, *** P<0.001, Student’s t-tests) (f).

Figure 6. IV injection of siWnt NP with cisplatin NP inhibited SRBC tumor growth

a. IV injection of siWnt NP with cisplatin NP inhibited SRBC tumor growth when the first injection started on day 9 post inoculation (small tumor volume ~150 mm³, n=5–7). Data show mean ± SD (** p<0.01; *** p<0.001, ns, no significant difference, one way ANOVA). b. IV injection of siWnt NP with cisplatin NP led to tumor regression when the first injection started on day 14 post inoculation (large tumor volume ~700 mm³, n=4). c. Scheme of the proposed hypothesis. d. Immunohistochemistry staining of Wnt16 on tumor tissues at the end point of small tumor inhibition study. Wnt16 was stained brown and the cell nuclei were stained blue. The scale bar represents 100 µm. The Wnt16 content of 5 randomly selected microscopic fields was quantified using Image J. The quantification bar chart is shown on right (** p<0.01, *** p<0.001, Student’s t-test). e. Western blot analysis of Wnt16 levels in tumors after treatment. Three samples were taken randomly from three mice in each treatment group. The intensity of the Wnt16 western band was analyzed by Image J and calculated based on content of GAPDH. Quantification is shown on right. * p<0.05, Student’s t-test. f. Effect of NP on the SRBC model apoptosis using TUNEL assay. Five images were quantified and the data is shown on right (* p<0.01, *** p<0.001, Student’s t-test). Same scale bar used as d.