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. 2017 Apr 11;8(15):24506-24517.
doi: 10.18632/oncotarget.15534.

Pirfenidone normalizes the tumor microenvironment to improve chemotherapy

Christiana Polydorou  1 , Fotios Mpekris  1 , Panagiotis Papageorgis  1   2 , Chrysovalantis Voutouri  1 , Triantafyllos Stylianopoulos  1
Affiliations

Pirfenidone normalizes the tumor microenvironment to improve chemotherapy

Christiana Polydorou et al. Oncotarget. .

Abstract

Normalization of the tumor microenvironment by selectively targeting components of the tumor extracellular matrix has been recently proposed to have the potential to decompress tumor blood vessels, increase vessel perfusion and thus, improve drug delivery and the efficacy of cancer therapy. Therefore, we now need to identify safe and well tolerated pharmaceutical agents that are able to remodel the microenvironment of solid tumors and enhance chemotherapy. In this study, we repurposed Pirfenidone, a clinically approved anti-fibrotic drug for the treatment of idiopathic pulmonary fibrosis, to investigate its possible role on tumor microenvironment normalization. Using two orthotopic mammary tumor models we demonstrate that Pirfenidone reduces collagen and hyaluronan levels and, as a result, significantly increases blood vessel functionality and perfusion and improves the anti-tumor efficacy of doxorubicin. Reduction of extracellular matrix components were mediated via TGFβ signaling pathway inhibition due to downregulation of TGFβ1, COL1A1, COL3A1, HAS2, HAS3 expression levels. Our findings provide evidence that repurposing Pirfenidone could be used as a promising strategy to enhance drug delivery to solid tumors by normalizing the tumor microenvironment.

Keywords: biomechanics; breast cancer; drug delivery; tumor perfusion; vessel compression.

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Conflict of interest statement

CONFLICTS OF INTEREST

None

Figures

Figure 1
Figure 1. Pirfenidone remodels the tumor microenvironment by reducing collagen and hyaluronan levels in a dose-depended manner
(A–D) Representative immunofluorescent images from whole tumor samples of control and Pirfenidone-treated tumors for collagen, hyaluronan (HA), CD31 and lectin. (E, F) Area fraction analysis shows that 500 and 650 mg/kg Pirfenidone reduces collagen and hyaluronan levels significantly compared to the control group. (G–I) The 500 and 650 mg/kg dose of Pirfenidone increased significantly mean vessel diameter, improving fraction of perfused vessels but had no effect on number of tumor blood vessels (n = 6–8). Asterisks indicate a statistically significant difference between compared groups (p < 0.05). Scale bar: 100 μm.
Figure 2
Figure 2. Pirfenidone decreases the levels of ECM components and normalizes blood vessel perfusion in 4T1 breast tumors
(A–D) Representative immunofluorescent images from histological analysis show the effect of Pirfenidone on collagen, hyaluronan (HA), CD31 and lectin. (E, F) Quantification of area fractions of collagen and hyaluronan indicates that hyaluronan but not collagen levels are significantly reduced compared to control tumors. (G–I) Quantification of blood vessel diameter, perfused vessels fraction and number of vessels in control and Pirfenidone-treated tumors indicate that Pirfenidone increases significantly blood vessel functionality by decompressing tumor vessels without affecting the number of vessels (n = 6–8). Asterisks indicate statistically significant difference between groups (p < 0.05). Scale bar: 100 μm.
Figure 3
Figure 3. Pirfenidone normalizes solid and fluid stress in tumors
(A) Representative images from tumor opening experiment indicating that a cut made at ∼80% of tumor thickness along the main axis results in opening of the tumor due to stress release by tissue relaxation. (B) Schematic of the unconfined compression set up. Stiffer tumors have a higher elastic modulus and resist stronger to compression. Tumors with larger values of hydraulic conductivity allow easier fluid flow through their mass and thus, more fluid exits the tissue during compression. (C) Measurements of tumor opening for the two different tumor models indicate that treatment with 500 mg/kg Pirfenidone leads to lower values of relaxation compared to control and hence to lower levels of solid stress. (D) The values of the elastic modulus for the Pirfenidone-treated tumors are shown to be significantly lower in both models. (E) Pirfenidone treatment increases the hydraulic conductivity of both tumor types, which in turn causes a decrease in the (F) interstitial fluid pressure.
Figure 4
Figure 4. Pirfenidone increases intratumoral delivery of doxorubicin
Quantification of doxorubicin concentration in control and Pirfenidone-treated 4T1 breast tumors as well as in kidney, liver and heart tissues. 9 mg/kg doxorubicin was injected intravenously to the animals 4 hours prior to sacrifice. Pirfenidone improves the delivery of doxorubicin in tumors by ∼ 50% without affecting delivery to normal tissues. Asterisk indicates statistically significant differences between compared groups (p < 0.05, n = 6).
Figure 5
Figure 5. Pirfenidone increases anti-tumor efficacy of chemotherapy
Tumor volume growth rates of (A) orthotopic MCF10CA1a human breast tumors and (B) 4T1 murine breast tumors implanted in female CD1 nude or BALB/c mice, respectively. Control, 500 mg/kg Pirfenidone or doxorubicin alone treated-animals had no effect on tumor growth in both tumor models. Combination treatment of 500 mg/kg Pirfenidone and 4 mg/kg doxorubicin for the MCF10CA1a or 5 mg/kg for the 4T1 tumors showed a significant reduction in tumor growth (n = 8–10). The error bars denote the standard error of the mean. Asterisks indicate a statistically significant difference between compared groups (p < 0.05).
Figure 6
Figure 6. Pirfenidone suppresses human COL1A1, HAS2, HAS3, COL3, TGFB1 and FN1 gene expression levels
Real-time PCR gene expression analysis for the quantification of human collagen I, (hCOL1A1), hyaluronan synthase 1 (hHAS1), hyaluronan synthase 2 (hHAS2), hyaluronan synthase 3 (hHAS3), periostin (hPOSTN), collagen 3 (hCOL3A1), collagen 4 (hCOL4A1), transforming growth factor-beta 1 (hTGFβ1), vimentin (hVIM), fibronectin (hFN1), and lysyl oxidase (hLOX) mRNA levels in control-treated compared to Pirfenidone-treated MCF10CA1a tumors. Data indicate that Pirfenidone downregulates expression of COL1A1, HAS2, HAS3, COL3A1, TGFB1 and FN1 genes. Data represent the average expression of each gene normalized to beta-actin from at least 3 independent experiments from 5 control and 5 Pirfenidone-treated tumors. Asterisks indicate statistically significant differences between compared groups (p < 0.05).
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