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. 2017 Sep 10:261:105-112.
doi: 10.1016/j.jconrel.2017.06.022. Epub 2017 Jun 27.

Sonic-hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

Fotios Mpekris  1 , Panagiotis Papageorgis  2 , Christiana Polydorou  1 , Chrysovalantis Voutouri  1 , Maria Kalli  1 , Athanassios P Pirentis  1 , Triantafyllos Stylianopoulos  3
Affiliations

Sonic-hedgehog pathway inhibition normalizes desmoplastic tumor microenvironment to improve chemo- and nanotherapy

Fotios Mpekris et al. J Control Release. .

Abstract

Targeting the rich extracellular matrix of desmoplastic tumors has been successfully shown to normalize collagen and hyaluronan levels and re-engineer intratumoral mechanical forces, improving tumor perfusion and chemotherapy. As far as targeting the abundant cancer-associated fibroblasts (CAFs) in desmoplastic tumors is concerned, while both pharmacologic inhibition of the sonic-hedgehog pathway and genetic depletion of fibroblasts have been employed in pancreatic cancers, the results between the two methods have been contradictory. In this study, we employed vismodegib to inhibit the sonic-hedgehog pathway with the aim to i) elucidate the mechanism of how CAFs depletion improves drug delivery, ii) extent and evaluate the potential use of sonic-hedgehog inhibitors to breast cancers, and iii) investigate whether sonic-hedgehog inhibition improves not only chemotherapy, but also the efficacy of the most commonly used breast cancer nanomedicines, namely Abraxane® and Doxil®. We found that treatment with vismodegib normalizes the tumor microenvironment by reducing the proliferative CAFs and in cases the levels of collagen and hyaluronan. These modulations re-engineered the solid and fluid stresses in the tumors, improving blood vessel functionality. As a result, the delivery and efficacy of chemotherapy was improved in two models of pancreatic cancer. Additionally, vismodegib treatment significantly improved the efficacy of both Abraxane and Doxil in xenograft breast tumors. Our results suggest the use of vismodegib, and sonic hedgehog inhibitors in general, to enhance cancer chemo- and nanotherapy.

Keywords: Breast cancer; Drug delivery; Nanomedicine; Pancreatic cancer; Re-engineering cancer; Tumor microenvironment; Tumor perfusion.

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Figures

Figure 1
Figure 1. Vismodegib inhibits the activity of cancer-associated fibroblasts in pancreatic tumors.
Representative images from immunofluorescence analysis of MiaPaCa2 or BxPC3 tumors that were mock or vismodegib-treated (40 mg/kg). (A) Tumor sections were stained for the CAF's marker a-SMA (green) and the proliferation marker Ki67 (red). Magnified images from selected tumor areas indicate the presence of Ki67+/a-SMA (yellow) cells. (B) Quantification of Ki67+/a-SMA (yellow) tumor area fraction in MiaPaCa2 or BxPC3 control or vismodegib-treated tumors (n=8-10). Asterisks indicate statistically significant difference between compared groups (p<0.05). Scale bar: 25 μm.
Figure 2
Figure 2. Remodeling of tumor microenvironment using vismodegib.
Representative images from immunofluorescence staining showing the effect of vismodegib (40 mg/kg) on (A) collagen (green) and (B) hyaluronan (HA, red), (C) CD31/Lectin and (D) CD31 levels compared to control-treated MiaPaCa2 or BxPC3 tumors. Quantification of collagen (E) area fractions were found to be significantly lower in vismodegib-treated compared to control-treated MiaPaCa2 tumors (p = 0.0001), whereas in BxPC3 tumors no significant changes were observed. Quantification of hyaluronan (F) area fractions were found to be significantly lower in both vismodegib-treated MiaPaCa2 and BxPC3 tumors (p = 0.02 and p = 3E-5, respectively), compared to control-treated tumors. For both vismodegib-treated pancreatic tumors the fraction of perfused vessels (G) increased compared to control tumors (p = 0.03, MiaPaCa2; p = 0.03, BxPC3). In contrast, quantification of CD31+ area (H) showed no significant change between the groups. Asterisks indicate statistically significant difference between compared groups (n = 8-10). Scale bar: 100 μm.
Figure 3
Figure 3. Vismodegib acts as a stress-alleviating agent.
(A) Schematic of the unconfined compression experiment. Desmoplastic tumors become stiff as they grow, exhibiting a higher elastic modulus and resisting stronger to compression. Desmoplasia reduces tumors hydraulic conductivity, resisting to fluid flow through their mass and thus, less fluid exits the tissue during compression. (B) Vismodegib-treated tumors in BxPC3 decreased the elastic modulus compared to control treatment (p = 0.002) whereas in MiaPaCa2 had no effect. Vismodegib-induced reduction in ECM content resulted in lower values of hydraulic conductivity of the tumor interstitial space (p = 0.005 for MiaPaCa2 and p = 0.04 for BxPC3) (C), which in turn caused alleviation of the interstitial fluid pressure (p = 4E-9 for MiaPaCa2 and p = 5E-5 for BxPC3) (D). Asterisks indicate statistically significant difference between compared groups (n = 8-10).
Figure 4
Figure 4. Vismodegib increases anti-tumor efficacy of chemotherapy.
Tumor volume growth rates of (A) MiaPaCa2 and (B) BxPC3 pancreatic human tumors implanted in male NOD/SCID mice. Control treatment (MCT -0.5% methylcellulose, 0.2% Tween 80), vismodegib (40 mg/kg) or gemcitabine (50 mg/kg) alone had no effect on tumor growth in both pancreatic cancer cell lines. (A) Combination of vismodegib and gemcitabine significantly decreased tumor growth of MiaPaCa2 pancreatic tumors compared to gemcitabine monotherapy (p = 0.005 on day 60, n = 8-10). (B) In BxPC3 model combination of vismodegib and gemcitabine significantly delayed tumor growth compared to gemcitabine monotherapy (p = 7E-7 on day 26, n = 8-10). Asterisks indicate a statistically significant difference between compared groups (p < 0.05).
Figure 5
Figure 5. Vismodegib increases intratumoral delivery of doxorubicin.
Quantification of doxorubicin (Dox) concentration in control and vismodegib-treated MiaPaCa2 pancreatic tumors as well as in kidney, liver and heart tissues. Doxorubicin (20 mg/kg) was injected intravenously to the animals 4 hours prior to sacrifice. Vismodegib increased the delivery of doxorubicin by 2-fold in vismodegib-treated compared to control-treated tumors (p < 0.05, n = 9). No significant differences were observed in drug delivery to normal tissues. Asterisk indicates statistically significant differences between compared groups (p < 0.05).
Figure 6
Figure 6. Vismodegib suppresses Sonic hedgehog signaling in stromal cells in vivo.
Real-time PCR gene expression analysis and quantification of mouse (A) or human-specific (B) Gli1, Gli2, Gli3, Ptch1, Ptch2, Smo and Shh mRNA levels extracted from control-treated compared to vismodegib-treated MiaPaCa2 tumors, indicated that vismodegib suppressed Gli1 and Gli2 expression in mouse stromal cells but not in MiaPaCa2 human cancer cells. Relative expression for all genes in both groups was normalized based on the expression of beta-actin. Data represent the average of at least 3 independent experiments from 4 control and 4 vismodegib-treated tumors ± S.E. values and asterisks indicate statistically significant differences between compared groups (p < 0.05).
Figure 7
Figure 7. Vismodegib improves anti-tumor efficacy of nanotherapeutics.
Tumor volume growth rates of orthotopic MCF10CA1a breast tumors implanted in CD1 nude mice that were either mock-treated (0.9% NaCl for Abraxane, saline for Doxil and MCT-0.5% methylcellulose, 0.2% Tween 80- for vismodegib), vismodegib-treated alone (40 mg/kg daily via gavage), Doxil alone (3 mg/kg on days 14 and 21 post-implantation, i.v.), Abraxane alone (20 mg/kg on days 14 and 21 post-implantation, i.v.), or vismodegib in combination with either Doxil or Abraxane, as described above. Abraxane alone had no effect on tumor volume, compared to combined administration of vismodegib and Abraxane, which significantly delayed tumor growth (p = 0.02 on day 28, n = 6-8). Additionally, whereas Doxil (3mg/kg) monotherapy had no effect, combination of vismodegib and Doxil significantly decreased tumor volume (p = 2E-6 on day 28, n = 6-8) compared to Doxil alone. Asterisks indicate a statistically significant difference between compared groups (p < 0.05).
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References

    1. Chauhan VP, Stylianopoulos T, Boucher Y, Jain RK. Delivery of molecular and nanomedicine to tumors: Transport barriers and strategies. Annual Reviews Chemical and Biomolecular Engineering. 2011;2:281–298. - PubMed
    1. Jain RK, Martin JD, Stylianopoulos T. The role of mechanical forces in tumor growth and therapy. Annu Rev Biomed Eng. 2014;16:321–346. - PMC - PubMed
    1. Stylianopoulos T, Martin JD, Chauhan VP, Jain SR, Diop-Frimpong B, Bardeesy N, Smith BL, Ferrone CR, Hornicek FJ, Boucher Y, Munn LL, et al. Causes, consequences, and remedies for growth-induced solid stress in murine and human tumors. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:15101–15108. - PMC - PubMed
    1. Stylianopoulos T, Martin JD, Snuderl M, Mpekris F, Jain SR, Jain RK. Coevolution of solid stress and interstitial fluid pressure in tumors during progression: Implications for vascular collapse. Cancer research. 2013;73:3833–3841. - PMC - PubMed
    1. Voutouri C, Polydorou C, Papageorgis P, Gkretsi V, Stylianopoulos T. Hyaluronan-Derived Swelling of Solid Tumors, the Contribution of Collagen and Cancer Cells, and Implications for Cancer Therapy. Neoplasia. 2016;18:732–741. - PMC - PubMed

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