Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 12;10(4):1910-1922.
doi: 10.7150/thno.36936. eCollection 2020.

TGF-β inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity

Myrofora Panagi  1 , Chrysovalantis Voutouri  1 , Fotios Mpekris  1 , Panagiotis Papageorgis  1   2 , Margaret R Martin  3 , John D Martin  3 , Philippos Demetriou  4 , Chryso Pierides  4 , Christiana Polydorou  1 , Andreas Stylianou  1 , Maria Louca  1 , Laura Koumas  4   5 , Paul Costeas  4   5 , Kazunori Kataoka  6   7 , Horacio Cabral  3 , Triantafyllos Stylianopoulos  1
Affiliations

TGF-β inhibition combined with cytotoxic nanomedicine normalizes triple negative breast cancer microenvironment towards anti-tumor immunity

Myrofora Panagi et al. Theranostics. .

Abstract

Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

Keywords: immunostimulation; immunotherapy; nanomedicine; normalization; tumor microenvironment; vascular perfusion.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
TME normalization increases the efficacy of both chemo- and nanotherapy. Quantification of tumor growth rate, based on the time to reach double the initial volume, for orthotopic 4T1 (A) and E0771 (B) murine breast tumors implanted in female BALB/c and C57BL/6 mice, respectively. Mice were treated with Control (saline), tranilast (200mg/kg), doxorubicin (5mg/kg), Doxil (3mg/kg), tranilast-doxorubicin and tranilast-Doxil. Tumor volume was measured every 2 days until time of death or time to reach a tumor burden of 1200 mm3. Kaplan-Meier survival curves for 4T1 (C) and E0771 (D) tumor models treated as indicated (arrows). Statistical analyses were performed by comparing the treated groups with the control * and the tranilast-Doxil groups with all other treatment groups **, p≤0.05 (n=8-10).
Figure 2
Figure 2
Nanomedicine enhances tranilast-mediated normalization effects in the primary tumors. (A) Representative fluorescence images of Collagen I (green) and Hyaluronan (red) immunostaining of 4T1 breast tumors treated as indicated. (B) Representative fluorescence images of biotinylated tomato lectin (cyan), CD31 endothelial marker (red) and αSMA pericyte marker (green) immunostaining of 4T1 breast tumors treated as indicated. Quantification of Collagen I (C) and Hyaluronan (D) area fractions. (E) Pericyte coverage of perfused vessels was determined by the co-localization of CD31 and lectin positive staining with αSMA. Statistical analyses were performed by comparing the treated groups with the control * and the tranilast-Doxil group with all other treatment groups **, p≤0.05, (n=8-10). Scale bar: 200μm.
Figure 3
Figure 3
Enhanced tumor normalization by Doxil nanomedicine alleviates intratumoral fluid and solid pressure, increasing perfusion and enhancing tissue oxygenation. (A) Representative fluorescence images of 4T1 breast tumor slices immunostained for biotynilated tomato lectin (green) and CD31 (red) after various treatments as indicated. (B) Representative fluorescence images of biotynilated tomato lectin (green), pimonidazole (hypoxia, red) immunostaining and DAPI (blue) nuclear staining. (C) Quantification of CD31 and lectin co-expression indicating vascular perfusion. (D) Quantification of CD31 (red) positive staining indicating mean vessel diameter and (E) total blood vessel fraction. (F) Hypoxic fraction in E0771 tumors measured following pimonidazole (60 mg/kg) injection and staining. (G) Quantification of hydraulic conductivity and (H) interstitial fluid pressure measurements of 4T1 tumors treated as indicated. (I) Sample-blind measurements of tumor opening and (J) elastic modulus. Statistical analyses were performed by comparing the treated groups with the control * and the tranilast-Doxil group with all other treatment groups **, p≤0.05, (n=8-10). Scale bar: 200μm.
Figure 4
Figure 4
Tumor normalization in combination with nanomedicine reprograms macrophages by directing their polarization towards M1 phenotype. (A) Representative images of 4T1 tumor slices immunostained for the M1-like tumor associated macrophage (TAM) marker CD11c (green), the M2-like TAM marker CD206 (red) and the F4/80, which is a pan-macrophage marker (magenta). Quantification of anti-tumoral M1- to M2-like TAM ratio (B), and total TAMs area fraction (C) in the various treatment groups. (D) Ratio of M1-like CD45+ CD11b+ Gr1- F4/80+ CD11c+ CD206-) to M2-like (CD45+ CD11b+ Gr1- F4/80+ CD11c- CD206+) over total TAMs in orthotopic 4T1 breast tumors by flow cytometry analysis. (E) Histogram demonstrating distribution of distances between αSMA+ CAFs - CD3+ T cells. Statistical analyses were performed by comparing the treated groups with the control * and the tranilast-Doxil groups with all other treatment groups **, p≤0.05, (n=8-10). Scale bar: 200μm.
Figure 5
Figure 5
(A) Tumor volume curves of Balb/c mice bearing 4T1 tumors and (B) C57BL/6 mice bearing E0771 tumors treated with saline (Control), tranilast (200mg/kg), anti-CTLA-4/anti-PD-1 immunotherapy cocktail (5mg/kg and 10mg/kg, respectively), Doxil (3mg/kg), tranilast-anti-CTLA-4/anti-PD-1 immunotherapy cocktail, tranilast-Doxil, Doxil-anti-CTLA-4/anti-PD-1 immunotherapy cocktail and tranilast-Doxil-anti-CTLA-4/anti-PD-1 immunotherapy cocktail. Tranilast-Doxil induced TME normalization in combination with immunotherapy eradicates tumor growth. Statistical analyses were performed by comparing the treated groups with the control * and the tranilast-Doxil- anti-CTLA-4/anti-PD-1 immunotherapy cocktail groups with all other treatment groups **, p≤0.05 (n=8-10). (C) Flow cytometry analysis of CD3+CD4+CD127loCD25hiFoxp3+ Tregs amongst total CD4+ T cells and (D) cytotoxic CD8+ T cells/Tregs ratio of orthotopic 4T1 breast tumors. Tregs and CD8+ levels were assessed using established phenotypic criteria and total CD45+CD3+ cells were used as common denominator.
Figure 6
Figure 6
Schematic of the proposed tranilast-induced TME normalization in combination with Doxil nanomedicine mechanism of action. Tranilast-Doxil combinatorial treatment optimizes normalization of the TME by increasing tumor vessel functionality leading to improved perfusion. Improved perfusion results in increased tumor oxygenation and immune stimulation. Improved perfusion and oxygenation enhance the efficacy of ICBs inhibiting primary tumor growth.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Stylianopoulos T, Jain RK. Design considerations for nanotherapeutics in oncology. Nanomedicine. 2015;11:1893–907. - PMC - PubMed
    1. Jain RK, Stylianopoulos T. Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol. 2010;7:653–64. - PMC - PubMed
    1. Stylianopoulos T, Munn LL, Jain RK. Reengineering the Physical Microenvironment of Tumors to Improve Drug Delivery and Efficacy: From Mathematical Modeling to Bench to Bedside. Trends Cancer. 2018;4:292–319. - PMC - PubMed
    1. Golombek SK, May JN, Theek B, Appold L, Drude N, Kiessling F. et al. Tumor targeting via EPR: Strategies to enhance patient responses. Adv Drug Deliv Rev. 2018;130:17–38. - PMC - PubMed
    1. Jain RK. Taming vessels to treat cancer. Scientific American. 2008;298:56–63. - PubMed

Publication types

MeSH terms