Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation

Marwan Moussa; David Mwin; Haixing Liao; M Fatih Atac; Aurelia Markezana; Eithan Galun; S Nahum Goldberg; Muneeb Ahmed

doi:10.1371/journal.pone.0266522

. 2022 Jul 20;17(7):e0266522. doi: 10.1371/journal.pone.0266522

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation

Marwan Moussa ¹, David Mwin ¹, Haixing Liao ^2,³, M Fatih Atac ¹, Aurelia Markezana ⁴, Eithan Galun ⁴, S Nahum Goldberg ^1,², Muneeb Ahmed ^1,^*

Editor: Yanlin Yu⁵

PMCID: PMC9299299 PMID: 35857766

Abstract

Radiofrequency ablation (RFA) of intrahepatic tumors induces distant tumor growth through activation of interleukin 6/signal transducer and activator of transcription 3 (STAT3)/hepatocyte growth factor (HGF)/tyrosine-protein kinase Met (c-MET) pathway. Yet, the predominant cellular source still needs to be identified as specific roles of the many types of periablational infiltrating immune cells requires further clarification. Here we report the key role of activated myofibroblasts in RFA-induced tumorigenesis and successful pharmacologic blockade. Murine models simulating RF tumorigenic effects on a macrometastatic tumor and intrahepatic micrometastatic deposits after liver ablation and a macrometastatic tumor after kidney ablation were used. Immune assays of ablated normal parenchyma demonstrated significantly increased numbers of activated myofibroblasts in the periablational rim, as well as increased HGF levels, recruitment other cellular infiltrates; macrophages, dendritic cells and natural killer cells, HGF dependent growth factors; fibroblast growth factor-19 (FGF-19) and receptor of Vascular Endothelial Growth Factor-1 (VEGFR-1), and proliferative indices; Ki-67 and CD34 for microvascular density. Furthermore, macrometastatic models demonstrated accelerated distant tumor growth at 7d post-RFA while micrometastatic models demonstrated increased intrahepatic deposit size and number at 14 and 21 days post-RFA. Multi-day atorvastatin, a selective fibroblast inhibitor, inhibited RFA-induced HGF and downstream growth factors, cellular markers and proliferative indices. Specifically, atorvastatin treatment reduced cellular and proliferative indices to baseline levels in the micrometastatic models, however only partially in macrometastatic models. Furthermore, adjuvant atorvastatin completely inhibited accelerated growth of macrometastasis and negated increased micrometastatic intrahepatic burden. Thus, activated myofibroblasts drive RF-induced tumorigenesis at a cellular level via induction of the HGF/c-MET/STAT3 axis, and can be successfully pharmacologically suppressed.

Introduction

Image-guided thermal ablation is an established clinical treatment that can achieve outcomes equivalent to surgical resection in well-selected patients [1–5], particularly for tumors less than 3 cm [6, 7]. Thermal ablation is now being used widely given it’s applicability to a growing cohort of non-surgical patients and a relatively lower economic cost [8–12]. While local treatment efficacy has continued to improve due to growing expertise and technological improvements, there is increasing evidence that effects from local ablation may result in unintentional downstream systemic side effects. Specifically, increased growth of distant untreated tumor following local ablation has been observed in both animal models [13–17] and with variable frequency in some clinical scenarios as unintended phenomena to both ablation [18, 19] and other interventional oncologic procedures [20–25].

Prior studies have linked post-ablation tumorigenesis to cytokinetic processes and accompanying inflammatory cellular infiltration that occur in the red peripheral zone surrounding an ablation. For liver ablation, Interlukin-6 (IL-6), hepatocyte growth factor and its high affinity receptor mesenchymal epithelial transition factor receptor (HGF/c-MET) axis [26–29] have been identified as major drivers of these effects. In HCC, IL-6 and the HGF/c-MET axis have been linked to increased angiogenesis through VEGF-1/A pathway, and proliferation through fibroblast growth factor-19 (FGF-19) [30–33]. Ablation-induced increase in IL-6 has been previously reported in clinical studies and in preclinical models, and successful blockade of this cytokine has curbed ablation induced distant tumor growth [14, 15, 17, 32, 34].

While specific cytokines have been identified as key promoters of post-ablation distant tumor growth and have been successfully blocked with pharmacologic therapy, the precise cellular sources of these cytokines have yet to be elucidated and potentially targeted. Accordingly, here we attempt to identify key cell populations driving post-ablation tumorigenesis. A growing body of literature implicates cancer-associated fibroblasts (CAFs) as a key cell population in promotion of tumorigenesis and proliferation pathways [31]. Given the prominence of fibroblasts in the periablational rim and their known potential to produce a host of growth factors including those found elevated post-RF ablation, we hypothesized that activated fibroblasts may play a key role in regulating unintentional distant tumor growth post-ablation. Moreover, we hypothesized that anti-fibroblast drugs such as atorvastatin may successfully attenuate tumorigenesis of distant metastasis. Accordingly, the purpose of this study was to characterize the role of myofibroblasts within the ablation zone on ablation-induced stimulation of distant tumors and micrometastatic disease in preclinical models, and to further study the use of an adjuvant anti-fibroblast agent, atorvastatin, on suppressing myofibroblast-driven ablation-induced distant tumor growth and metastatic implantation.

Material and methods

Overview

Animal studies were performed in accordance with protocols approved by the Institutional Animal Care Committees of Beth Israel Deaconess Medical Center, and Hadassah Hebrew University Medical Center, respectively. A total of 124 rats were implanted with a subcutaneous rat breast cancer cell line to characterize the role of myofibroblasts on both RFA-induced distant macrometastatic tumor growth and 64 mice were subject to intrahepatic implantation with two different murine colorectal cancer cell lines to characterize the role of micrometastatic tumor implantation and growth. The study was conducted over four phases.

Phase I

Determination of the role of myofibroblasts in hepatic RFA-induced HGF upregulation, periablational cell trafficking and downstream growth pathways modulation. First, 48 non-tumor bearing Fischer rats were randomized into the following treatment groups: 1) sham treatment, 2) sham with multiple doses of atorvastatin, 3) RFA of the liver, and 4) RFA of the liver with multiple doses of atorvastatin. Animals were sacrificed at 3 and 7 days post-treatment. Ablated and non-ablated liver tissues were collected to measure HGF. Liver tissue was also collected and analyzed for periablational infiltrating cell populations, tumor proliferation indices, and growth factors and growth factor receptors linked to RFA-induced tumorigenesis [R]. Analyzed infiltrating cell populations included alpha-smooth muscle actin positive activated myofibroblast (α-SMA), macrophages (CD68), natural killer cells (KIR3DL1), and mature dendritic cells (CD80). Tumorigenesis markers included Ki-67 (cell proliferation) and CD34 (microvascular density). Growth factors included the receptor for vascular endothelium growth factor-1 (VEGF-R1), a key marker for HCC angiogenesis pathways, and fibroblast growth factor-19 (FGF-19), an HGF/c-MET/STAT3 dependent tumorigenic promoter of HCC.

Next, 8 BALB/c and 8 C57BL/6 non-tumor bearing mice were randomized into two treatment groups: 1) hepatic RFA alone, and 2) hepatic RFA with a single dose of atorvastatin. Animals were sacrificed at 7d post-ablation. Liver tissues were harvested and analyzed for myofibroblast infiltration (α-SMA), cell proliferation (Ki-67), and microvascular density (CD34).

Phase II

Determination of the role of myofibroblasts in hepatic RFA tumorigenesis of macrometastasis. Here, 48 rats with established subcutaneous R3230 adenocarcinoma breast tumors were randomized into 4 treatment groups: 1) sham treatment, 2) sham with multiple doses of atorvastatin at 0, 24, and 48h, 3) hepatic RFA alone, and 4) hepatic RFA with multiple doses of atorvastatin. Animals were sacrificed at day7 post-treatment. Tumor growth curves were plotted and analyzed.

Phase III

Role of myofibroblasts in implantation and tumorigenesis of micrometastases following hepatic RFA. Two murine models of diffuse intrahepatic colorectal cancer micrometastases were used to study the role of myofibroblasts in RFA induced micrometastatic tumor cell implantation and growth. C-MET–positive murine colorectal cancer cell lines, CT26 and MC38, in syngeneic mouse strains, BALB/c and C57BL/6 mice, respectively, were used [17]. Animals (n = 24 per strain) were randomly assigned to 4 treatment groups: 1) sham procedure followed by intrasplenic tumor cell injection 1d later, 2) sham followed by intrasplenic tumor cell injection and daily atorvastatin for 3d; 1d prior to implantation, day of implantation and 1d after to implantation, 3) hepatic RFA followed by intrasplenic tumor cell injection, and 4) hepatic RFA followed by intrasplenic tumor cell injection and daily atorvastatin. Animals were sacrificed at 14d post-RFA for the CT26/BALB/c model and 21d post-RFA for the MC38/C57BL/6 model. Whole livers were harvested to assess for tumor load, myofibroblast infiltration (α-SMA), cell proliferation (Ki-67), and microvascular density (CD34).

Phase IV

Role of myofibroblasts in renal RFA-induced tumorigenesis of distant macrometastasis. To determine whether renal RFA-induced tumor growth is influenced by infiltrating myofibroblasts, RFA of the kidney was performed on 32 Fischer rats implanted with subcutaneous R3230 breast adenocarcinoma were randomized into 4 treatment groups: 1) sham, 2) sham combined with multiple doses of atorvastatin, 3) renal RFA alone, and 4) renal RFA combined with multiple doses of atorvastatin. Animals were sacrificed at 3d and 7d after sham or RFA procedures. As described in phase I, animals were analyzed utilizing similar end points, including tumor growth curves, ELISA to measure HGF in the ablated kidney, IHC of α-SMA and CD68 in periablational tissue, and Ki67, CD34, and VEGF-R1 in periablational kidney and breast adenocarcinoma tumor tissues.

Further detailed descriptions of animal models used, animal surgery, RFA, anesthesia and pain alleviation, sacrifice, sample collection, proteomic analysis and statistical analysis can be found in the supplemental section, and have been previously described in detail [17, 34–37].

Ethics statement

All of the methods and studies reported were approved by our Institutional Animal Care and Use Committee. Specifically, rat studies were performed at Beth Israel Deaconess Medical Center, while mouse experiments were performed at Hadassah Hebrew University Medical Center in accordance with protocols approved by the respective Institutional Animal Care Committees. For rat studies, the maximum permissible diameters for R3230 breast adenocarcninomas, as per approved IACUC protocol, was a mean diameter of 2 cm. Tumor sizes were assessed using manual caliper measurements of two largest perpendicular dimensions and calculating mean diameters. After surgical procedures, the animals were monitored on a daily basis to determine any specific clinical signs of postoperative pain and distress. Specific signs of weakening or distress included, but were not limited to: total combined tumor burden greater than 2 cm or 10% of body weight, decreased appetite, reduced ambulation (interfering with or hampering with the animal’s ability to obtain food and water, bear its own weight, or regain normal posture if placed on the back), weight loss (20% loss of body weight within 7 days, measured every 2-3d), tumor ulceration or necrosis for subcutaneous tumors. Post-surgically, if signs of distress were detected, a single dose of buprenorphine SR (72 hour duration of action) was administered at the site of surgery. In cases of tumor burden exceeding approved limits, animal were euthanized. For mouse experiments, general condition and animal well-being were used as metrics of tumor burden in lieu of a maximum permissible size due to microscopic nature of CRC liver deposits. Similar to rat experiments, post-operatively, animals were monitored on a daily basis to determine any specific clinical signs of postoperative pain and distress. For post-operative pain and distress, a single dose of buprenorphine SR was administered at the site of surgery. If signs of tumor burden overgrowth were detected, such as decreased appetite, reduced ambulation and or weight loss animals were euthanized.

Results

1. Atorvastatin reduces RFA-induced myofibroblast periablational infiltration

Immunofluorescence assays of activated myofibroblasts in rat liver demonstrated significantly higher intensity of infiltrating α-SMA positive cells in the RFA treatment group as compared to sham or sham with atorvastatin. Specifically, activated myofibroblasts accumulated in the periablational rim, increasing significantly from 3 to 7 days (p-value <0.001, RFA and RFA plus atorvastatin). Atorvastatin following RFA reduced myofibroblasts by 50% at 3d and 7d versus RFA treatment alone. (8,437,720.1± 694416.5 vs. 821117.8 ± 338805.1 vs. 1039511.7 ± 247990.3 vs. 4193656.4 ± 260689.6 Pixel Intensity (PI) at 3d, p-value < 0.001 for all comparisons with RFA and p-value <0.001 comparing control groups to RFA and atorvastatin and 12324537.7 ± 1478667.1 vs. 827895.6 ± 495894.0 vs. 1483688.6 ± 886000.3 vs. 5967565.4 ± 878778.3 PI at 7d, p-value < 0.001 for all comparisons with RFA and p-value <0.001 comparing control groups to RFA and atorvastatin). (Fig 1A).

Fig 1 — Animal livers were evaluated using immunofluorescent staining. Statistically significant increased immunofluorescent intensity of infiltrating α-SMA positive cells was detected in the RFA treatment group as compared to sham or sham plus atorvastatin. Atorvastatin treatment significantly reduced α-SMA cell positivity at 3 and 7 days post-treatment (p-value <0.001, both comparisons), while remaining elevated to control groups at both time points (p-value <0.001) (A). Immunohistochemical analysis of BALB/c and C57B/6 mice (B) livers 7 days after RF ablation with and without atorvastatin treatment likewise demonstrated significant reduction of α-SMA periablational rim thickness in the combined RFA and atorvastatin group in both models (p-value <0.001 and p-value <0.05, respectively).

In BALB/c and C57BL/6 mouse models, similar findings were noted. Specifically, a significant reduction of periablational rim thickness of α-SMA positive cells was demonstrated when comparing hepatic RFA alone to RFA with atorvastatin (78.98 μm ± 30.1 vs 38.61 μm ± 21.2, for BALB/c mice (p <0.001) and 74.13 μm ± 28.07 vs 43.11 μm ± 19.80, for C57BL/6 mice) (Fig 1B).

2. Myofibroblast inhibition suppresses RFA-induced upregulation of tumor promoting cytokines

Samples of both periablational and non-ablated liver 3d and 7d after RFA treatment demonstrated elevated levels of HGF when compared to control groups and RFA plus atorvastatin (p-value < 0.05, all comparisons; Table 1). There was no statistically significant difference between RFA and atorvastatin compared to control groups (p-value > 0.05 for all comparisons) (Fig 2A). Additionally, periablational rat liver at 7d post-treatment demonstrated elevated FGF19 and VEGRFR-1 levels in the RFA arm compared to sham or sham plus atorvastatin (p-value <0.001, for all comparisons), which were reduced by 50% in RFA plus atorvastatin (p-value <0.001, vs. RFA and p-value <0.001 vs control groups).

Table 1. Effect of myofibroblast blockade on Hepatocyte growth factor upregulation in the periablational rim and distant liver post-RFA.

		Sham	Sham + Ator	RFA	RFA + Ator
Periablational rim (OD ± SD)	3D	20 ± 3.2	15.5 ± 4.2	39.1 ± 6.7	19.7 ± 2
Periablational rim (OD ± SD)	7D	11.2 ± 3	7.2 ± 2	22.5 ± 2.8	7.3 ± 3.5
Distant liver (OD ± SD)	3D	15.8 ± 3.5	20.7 ± 2.7	22.6 ± 3.9	9.4 ± 3.3
Distant liver (OD ± SD)	7D	11.0 ± 2.4	10.6 ± 1.4	15.8 ± 3.0	9.7 ±1.2

Open in a new tab

Fig 2 — Ablated (and un-ablated, HGF only) livers were analyzed for known RF induced tumorigenic growth factors (HGF and VEGFR-1) as well as myofibroblast dependent growth tumorigenic growth factors (FGF-19). (A) Enzyme-Linked immunosorbent assays (ELISA) demonstrated statistically significant elevated levels of HGF in the RFA group when compared to control groups and RFA plus atorvastatin (p-value < 0.05 for all comparisons) in treated rat liver. No statistically significant difference was detected between RFA plus atorvastatin and control groups at 3 and 7 days (p-value > 0.1, all comparisons). ELISA also demonstrated statistically significantly elevated HGF levels in non-ablated rat liver with RFA treatment in comparison to control groups and combined atorvastatin with RFA at 3 and 7 days post-ablation (p-value < 0.05, all comparisons), with, no statistically significant difference detected between RFA plus atorvastatin treatment and control groups (p-value > 0.1, all comparisons). Significantly increased FGF-19 (B) and VEGRFR-1 (C) levels were demonstrated in RFA compared to sham or sham plus atorvastatin 7 day post-treatment (p-value <0.001, all comparisons) and were successfully reduced to half in RFA plus atorvastatin (p-value <0.001, all comparisons with RFA and p-value <0.001, for all comparisons to control groups).

3. Myofibroblast blockade eliminates hepatic RFA-induced tumorigenesis and proliferation of distant established tumors

RFA alone demonstrated significantly increased distant mean tumor diameter from 2d post-ablation to 7d post-ablation, when compared to sham, sham plus atorvastatin, or RFA plus atorvastatin (p-value <0.05, all comparisons). Additionally, RFA plus atorvastatin treatment did not show statistically significant different tumor growth when compared to sham or sham plus atorvastatin (p-value >0.05, both comparisons) (Fig 3A). At 7d post-ablation, hepatic RFA alone demonstrated significantly increased macrophages (CD68), mature dendritic cells (CD80), and natural killer cells (KIR3DL1) in the periablational rim when compared to sham, RFA plus atorvastatin, or sham plus atorvastatin (p-value <0.001, for all comparisons) (Table 2). Similarly, increased cellular proliferation (Ki-67) and microvascular density (CD34) were observed in the periablational rim at 3d and 7d following hepatic RFA alone, which were successfully reduced to 50% for Ki67 (p-value <0.001, vs RFA plus atorvastatin and p-value <0.001 vs control groups) and to baseline levels for CD34 in RFA plus atorvastatin (p-value <0.001, all comparisons between RFA vs RF plus atorvastatin or control groups; p-value >0.05 comparing RF plus atorvastatin and control groups). (Fig 3B and 3C and Table 2).

Fig 3 — (A)Tumor growth mean diameter measurements demonstrate RFA treatment significantly increased the distant tumor diameter when compared to sham, sham plus atorvastatin and RFA plus atorvastatin starting 3 days after ablation (p-value <0.05, all comparisons). Furthermore, no statistically significant different tumor growth was demonstrated in RFA plus atorvastatin when compared to sham or sham plus atorvastatin (p-value >0.05, both comparisons). Immunohistochemical analysis (B&C) and comparison of cellular proliferative (Ki-67) and microvascular density (CD34) indices was performed. Significantly elevated Ki-67 (B) and CD34 (C) levels were detected in RFA as compared to sham and sham plus atorvastatin groups at 7 days, which were successfully reduced to the baseline in RFA plus atorvastatin for CD34 (p-value <0.001, all comparisons between RFA alone vs. RFA plus atorvastatin or control groups; p-value >0.05 comparing RFA plus atorvastatin and control groups) and to half for Ki-67 (p-value <0.001, all comparisons with RFA and p-value <0.001, all comparisons to control groups).

Table 2. Effect of myofibroblast blockade on cellular trafficking, downstream tumorigenic growth factors and proliferation in rat livers’ periablational rim collected 7 days post hepatic ablation.

		Sham	Sham + Ator	RFA	RFA + Ator
Cellular infiltrates (PI/mm²±SD)	CD68	179.2 ± 40.5	136.2 ± 38.1	3521.2 ± 297.3	2266.1 ± 123.4
	CD80	11 ± 2	12 ± 6	325.2 ± 55.5	53 ± 18.2
	KIR3DL3	1049.1 ± 111.2	937.0 ± 92.2	27955.9 ± 2572.7	13822.5 ± 1447.7
Tumorigenic Growth Factors (PI/mm²±SD)	FGF-19	81.2 ± 8.6	85.7 ± 7.0	795.7 ± 56.9	251.8 ± 48.0
Tumorigenic Growth Factors (PI/mm²±SD)	VEGFR-1	94.4 ± 13.3	98.7 ± 10.9	2216 ± 158.7	918.7 ± 81.5
Prolif. indices (PI/mm²±SD)	Ki-67	76.5 ± 26.0	90.1 ± 27.4	1299.4± 140.5	571.9 ± 74.7
Prolif. indices (PI/mm²±SD)	CD34	77.2 ±11.8	69 ± 10.3	125.7 ± 8.7	85.8 ± 33.4

Open in a new tab

4. Myofibroblast blockade eliminates hepatic RFA-induced tumorigenesis of micrometastatic implants

In MC38 and CT26 colorectal metastatic models, hepatic RFA alone increased the total number of tumor nodules, tumor area ratio, and nodule size compared to sham, sham plus atorvastatin treatment and combined RFA with atorvastatin (p-value <0.05, all comparisons for MC38 and p-value <0.001, all comparisons for CT26). Combination RFA plus atorvastatin reduced tumor burden compared to RFA alone to baseline levels observed with sham or sham plus atorvastatin treatment (p-value >0.1 for all comparisons, both models) (Fig 4).

Fig 4 — RFA statistically significantly increased total number of tumor nodules, nodule size and tumor area ratio when compared to sham and sham plus multi-atorvastatin treatment and combination of RFA with atorvastatin in murine MC38 and CT26 colorectal metastatic models (p-value <0.05, all comparisons). Additionally, RFA plus atorvastatin treatment demonstrated statistically significant lower tumor burden compared to RFA, with no statistically significant increase in tumor burden noted when compared to sham or sham plus atorvastatin treatment (p-value >0.1 for all comparisons, both models).

In the hepatic RFA arm, increased activated myofibroblasts were observed in distant intrahepatic metastases at the time of sacrifice (MC38 and CT26 at 21 and 14 d) when compared to sham, sham plus atorvastatin, or RFA plus atorvastatin (p-value <0.001 for all comparisons). When combining RFA with atorvastatin, myofibroblast infiltration levels were reduced to baseline control groups’ levels (p-value >0.05 for all comparisons).

Similarly, in animals treated with hepatic RFA alone, metastatic tumors demonstrated increased cellular proliferation (Ki-67) and microvascular density (CD34) compared to sham, sham plus atorvastatin, and RFA plus atorvastatin (p-value <0.001, all comparisons). However, there was no difference in the assayed markers when comparing sham, sham plus atorvastatin and RFA plus atorvastatin indicating successful reduction of cellular markers to baseline levels with atorvastatin treatment (p-value >0.05 all comparisons). (Fig 5 and Table 3).

Fig 5 — Immunohistochemical analysis of MC38 and CT26 demonstrated increased infiltration of activated myofibroblasts, increased cell proliferative indices and microvascular density in RFA groups when compared to sham, sham plus atorvastatin and RFA plus atorvastatin (p-value <0.001 for all comparisons, for both models). Myofibroblast infiltration levels were reduced to baseline control groups’ levels in RFA with atorvastatin (p-value >0.05 for all comparisons, for both models). Likewise, RFA also demonstrated increased Ki-67 and CD34 signals in metastatic nodules compared to sham, sham plus atorvastatin and RFA with atorvastatin (p-value <0.001, all comparisons, both models), which were reduced to baseline levels in RFA plus atorvastatin treatment (p-value >0.05 all comparisons).

Table 3. Effect of atorvastatin on myofibroblast infiltration and proliferation in CRC metastasis after hepatic RF.

		Sham	Sham + Ator	RFA	RFA + Ator
Cellular infiltrates
α-SMA (%±SD)	MC38	7.1 ± 1.7	6.1 ± 1.8	19.6 ± 6.5	7.6 ± 1.6
α-SMA (%±SD)	CT26	5.3 ± 1.6	7.9 ± 2.4	15.7 ± 5.1	6.6 ± 2.2
Proliferative indices
Ki-67 (%±SD)	MC38	48.4 ± 10	43.2 ± 7.6	63.2 ± 11.7	40.7 ± 7.9
Ki-67 (%±SD)	CT26	30.9 ± 5.6	34.4 ± 7.3	51.5 ± 9.9	27.4 ± 4
CD34 (%±SD)	MC38	17.9 ± 3.4	18.5 ± 5.6	28.5 ± 4.1	15.4 ± 2.2
CD34 (%±SD)	CT26	13.6 ± 4	14.6 ± 2.9	23.7 ± 5.6	14.5 ± 3

Open in a new tab

6. Atorvastatin partially suppresses RFA-induced periablational myofibroblast infiltration in ablated kidney

Following renal RFA alone, increased periablational infiltration of activated myofibroblasts (α-SMA) (S1 Fig in S1 File) and macrophages (CD68) were observed compared to control groups and RFA with atorvastatin (p <0.05, all comparisons). However, adjuvant atorvastatin following renal RFA did not reduce infiltrating cells to baseline levels (p <0.05, all comparisons to control groups). (S1 Table in S1 File).

7. Myofibroblast blockade reduces renal RFA-induced tumor promoting cytokines, tumorigenesis of distant established tumors, and periablational and distant tumor proliferation

Renal RFA alone upregulated HGF at 3d and 7d compared to sham and sham plus atorvastatin (p <0.05 for all comparisons). Adjuvant atorvastatin reduced post-RFA HGF in periablational kidney compared to RFA alone (p <0.05, all comparisons). Compared to hepatic RFA, combination atorvastatin following renal RFA did not return completely to baseline, with HGF levels still elevated compared to sham treatment (p <0.05 for all comparisons) (S2A Fig and S1 Table in S1 File). VEGFR-1 level was also increased in both the renal periablational rim and distant tumor at 3d and 7d compared to sham, sham plus atorvastatin, or RFA plus atorvastatin (p <0.05, all comparisons). RFA plus atorvastatin demonstrating higher VEGFR-1 levels than control groups ((p <0.05, all comparisons) (S2B and S2C Fig and S1 Table in S1 File).

Renal RFA alone increased mean tumor diameter of the distant subcutaneous breast tumor from 3d to 7d post-ablation compared to sham, sham plus atorvastatin, or RFA plus atorvastatin (p <0.05, all comparisons). RFA plus atorvastatin treatment did not increase of mean tumor diameter when compared to control groups (p-value >0.05, all comparisons) (S3 Fig in S1 File).

Similarly, renal RFA alone increased periablational and distant tumor cellular proliferation and microvascular density compared to control groups. Atorvastatin partially attenuated increases in proliferation and microvascular density when combined with RFA (p <0.05, all comparisons). (S4 Fig and S1 Table in S1 File).

Discussion

There is a growing body of clinical reports of accelerated tumor growth after hepatic and renal RFA [18, 19, 34, 38] of primary and metastatic tumors [38, 39]. Studies investigating this phenomenon have revealed temporally-associated cytokine surges following liver RFA, specifically, interleukin-6 [28], a key pro-inflammatory marker associated with HGF/c-MET/STAT3 pathway upregulation, which is a known key pathway in tumorigenesis. Preclinical models have also correlated RFA-induced increases of inflammatory cytokines IL-6 [27], HGF [17], and VGEF [15], to upregulation of downstream key proliferative pathways [14], and resultant accelerated distant tumor growth peaking 6h to 3d following ablation [16]. At the same time, animal studies have demonstrated increased infiltrating immune cells in the periablational rim, namely polymorphic neutrophils, macrophages, and activated myofibroblasts [27]. Fibroblasts, specifically activated α-SMA–positive myofibroblasts, have been implicated as a crucial subset of the tumor microenvironment promoting gastrointestinal carcinomas [40, 41] promoting tumor growth [31], an immunosuppressive immunity [42], and are associated with chemo-resistance [43]. Myofibroblasts are also known as major producers of HGF [43] and interleukin-6 [44], key cytokines implicated in post-ablation tumorigenesis. Accordingly, in the current study we attempt to elucidate the role of myofibroblasts in the upregulation of the HGF/c-MET/STAT3 pathway and overall tumorigenesis following radiofrequency ablation.

Prior studies have shown that undesirable pro-tumorigenic effects can be successfully suppressed by selective inhibition of interleukin-6 [34], and c-MET, or STAT3 [15–17] in animal models. We postulated and demonstrate here that a similar approach can potentially be sought to modulate cell populations that orchestrate tumor growth and progression, such as myofibroblasts. Accordingly we tested the use of atorvastatin, an established lipid lowering medication, which has known inhibitory effects on myofibroblasts [45], in the post ablation setting. Through inhibition of small GTPases (RhoA and Ras) atorvastatin has demonstrated portal pressure lowering effects and attenuated fibrosis in preclinical models [46]. Additionally, atorvastatin has been shown in large retrospective studies to be associated with improved survival in non-small lung cancer [47] and prostate cancer [48], albeit the exact mechanism remains to be determined.

In the first phase of this study, increased myofibroblast infiltration in the periablational rim post-RFA was again demonstrated, but also now successfully attenuated by atorvastatin treatment in two different animal models. Furthermore, we demonstrate that atorvastatin markedly attenuates a host of post-RFA tumorigenesis-inducing phenomenon including release of HGF and key dependent growth factors such as FGF-19 and VEGFR-1, as well as tumor markers such as Ki-67 and CD34 implicating fibroblast proliferation in a cascade that leads to proliferation and angiogenesis. Interestingly, this is also associated with a decrease in other infiltrating cell populations observed which can be attributed to myofibroblast blockade, a known key regulator of the inflammatory cellular infiltrate [49]. Specifically, we report significant modulation of macrophages, mature dendritic cells, and natural killer cells. This underscores the complex interactions between infiltrating cells that ultimately govern tumorigenesis. We therefore hypothesize that despite selective myofibroblast inhibition a much broader cohort of cells are impacted due to the complex interdependence and regulatory roles between infiltrating periablational cells. Alternatively or simultaneously, atorvastatin may have a broad inhibitory effect on multiple inflammatory cells. Furthermore, this observation makes a strong case for cell specific targeting strategies, especially, of other abundant cell populations with known tumor promoting properties within the tumor microenvironment such as M2 macrophages.

Our subsequent tumor growth studies were in concert with immunohistochemical and proteomic findings. In a rat model, liver RF induced accelerated distant tumor growth which was successfully returned to baseline growth rates by a multi-day course of atorvastatin treatment. Similar effects were reproduced and confirmed in a second mouse colorectal model that simulates intrahepatic micrometastatic implantation from portal shower. Here too, atorvastatin treatment demonstrated successful attenuation of RF driven increased number of micrometastatic implants, nodule size, and overall hepatic tumor burden.

Finally, we confirmed similar findings for a second site in which RFA is commonly practiced. Specifically, we not only confirmed that renal ablation accelerates distant subcutaneous tumor growth [34], but also that this phenomenon can be suppressed, albeit only partially, by atorvastatin treatment. This suggests that RF induced distant tumorigenesis is not an isolated hepatic phenomenon and can most likely be observed with the ablation of any organ. It also reaffirms that organ specific interactions are at play after inflecting thermal injury upon it. For instance, atorvastatin treatment in liver RF models successfully attenuated any RF-driven HGF upregulation. While in kidney RF models, myofibroblast blockade significantly reduced HGF levels below RF groups but not to control groups. This highlights the potential role of organ specific cells and growth pathways in post ablation tumorigenesis.

Potential limitations of our study include those based upon experimental design. For example, we acknowledge that the subcutaneous implantation models used to ablate primary organs (liver or kidney) and study effects on a distant tumor of a separate origin (breast) represent an uncommon clinical scenario. Nevertheless, such a model can strengthen the argument that the observed phenomena transcend tumor origin and cellular homogeneity of the primary tumor and affected satellites. Likewise, our complementary intrasplenic injection model of tumor implantation via trans-splenic injection may create an artificial potentially greater portal cellular load than in a typical clinical scenario. Additionally, the timing of RF in relation to the portal tumor cell delivery does not necessarily reflect a longer tumor dwelling time in most clinical scenarios. Thus, although the tumor models used are well-characterized allowing comparison with other preclinical studies of RF ablation, careful interpretation and application of the results is warranted. Indeed, atorvastatin specific effects described in these models may vary in other models based on tissue susceptibility.

In conclusion, while radiofrequency ablation has demonstrated robust clinical efficacy in treatment of intrahepatic and renal tumors, the inadvertent ablation of normal parenchyma of the primary organ can stimulate distant tumor growth and proliferation. Our study offers three clinically pertinent insights. Activated myofibroblasts are a key orchestrating cell population in post RF ablation distant tumor growth, primarily through induction of the HGF/c-MET/STAT3 axis. Post RFA distant tumor growth is a phenomenon that transcends phenotype of ablated primary organ ablated (including liver or kidney) and phenotype of tumor (colorectal cancer or breast cancer) or size (micro or macrometastatic tumors). Finally, selective inhibition of RF induced distant tumor growth can be achieved with by targeted cellular inhibition of activated myofibroblasts. Mouse colorectal models demonstrated reduction of cellular, growth signal and tumor burden markers to baseline levels when combining atorvastatin with RF. However, in rat models, although tumor growth studies demonstrated complete negation of RF induced distant tumor growth with atorvastatin, cellular and growth signals were only partially curbed. Thus, further evaluation, especially, including long-term survival studies and combined targeted inhibition of key cellular and cytokine agents of established tumorigenesis pathways.

Supporting information

S1 File

(DOCX)

Click here for additional data file.^{(1.8MB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work is supported by National Cancer Institute (1R01CA197081-01A1), Israel Ministry of Science and Technology (3-12063), and Israel Science Foundation (1277/15). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, et al. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg. 2004. Jun;239(6):818,25; discussion 825–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Alexander ES, Machan JT, Ng T, Breen LD, DiPetrillo TA, Dupuy DE. Cost and effectiveness of radiofrequency ablation versus limited surgical resection for stage I non-small-cell lung cancer in elderly patients: is less more? J Vasc Interv Radiol. 2013. Apr;24(4):476–82. doi: 10.1016/j.jvir.2012.12.016 [DOI] [PubMed] [Google Scholar]
3.Loveman E, Jones J, Clegg AJ, Picot J, Colquitt JL, Mendes D, et al. The clinical effectiveness and cost-effectiveness of ablative therapies in the management of liver metastases: systematic review and economic evaluation. Health Technol Assess. 2014. Jan;18(7):vii,viii, 1–283. doi: 10.3310/hta18070 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Schlijper RC, Grutters JP, Houben R, Dingemans AM, Wildberger JE, Van Raemdonck D, et al. What to choose as radical local treatment for lung metastases from colo-rectal cancer: surgery or radiofrequency ablation? Cancer Treat Rev. 2014. Feb;40(1):60–7. doi: 10.1016/j.ctrv.2013.05.004 [DOI] [PubMed] [Google Scholar]
5.Stintzing S, Grothe A, Hendrich S, Hoffmann RT, Heinemann V, Rentsch M, et al. Percutaneous radiofrequency ablation (RFA) or robotic radiosurgery (RRS) for salvage treatment of colorectal liver metastases. Acta Oncol. 2013. Jun;52(5):971–7. doi: 10.3109/0284186X.2013.766362 [DOI] [PubMed] [Google Scholar]
6.Groeschl RT, Gamblin TC, Turaga KK. Ablation for hepatocellular carcinoma: validating the 3-cm breakpoint. Ann Surg Oncol. 2013. Oct;20(11):3591–5. doi: 10.1245/s10434-013-3031-5 [DOI] [PubMed] [Google Scholar]
7.Tabrizian P, Jibara G, Shrager B, Schwartz M, Roayaie S. Recurrence of hepatocellular cancer after resection: patterns, treatments, and prognosis. Ann Surg. 2015. May;261(5):947–55 doi: 10.1097/SLA.0000000000000710 [DOI] [PubMed] [Google Scholar]
8.de Baere T, Tselikas L, Yevich S, Boige V, Deschamps F, Ducreux M, et al. The role of image-guided therapy in the management of colorectal cancer metastatic disease. Eur J Cancer. 2017. Apr;75:231–42. doi: 10.1016/j.ejca.2017.01.010 [DOI] [PubMed] [Google Scholar]
9.Kurup AN, Callstrom MR. Increasing Role of Image-Guided Ablation in the Treatment of Musculoskeletal Tumors. Cancer J. 2016. Nov/Dec;22(6):401–10. doi: 10.1097/PPO.0000000000000233 [DOI] [PubMed] [Google Scholar]
10.Lencioni R, Crocetti L. Image-guided ablation for hepatocellular carcinoma. Recent Results Cancer Res. 2013;190:181–94. doi: 10.1007/978-3-642-16037-0_12 [DOI] [PubMed] [Google Scholar]
11.Petre EN, Solomon SB, Sofocleous CT. The role of percutaneous image-guided ablation for lung tumors. Radiol Med. 2014. Jul;119(7):541–8. doi: 10.1007/s11547-014-0427-7 [DOI] [PubMed] [Google Scholar]
12.Wah TM. Image-guided ablation of renal cell carcinoma. Clin Radiol. 2017. Aug;72(8):636–44. doi: 10.1016/j.crad.2017.03.007 [DOI] [PubMed] [Google Scholar]
13.Nikfarjam M, Muralidharan V, Christophi C. Altered growth patterns of colorectal liver metastases after thermal ablation. Surgery. 2006. Jan;139(1):73–81. doi: 10.1016/j.surg.2005.07.030 [DOI] [PubMed] [Google Scholar]
14.Kumar G, Goldberg SN, Gourevitch S, Levchenko T, Torchilin V, Galun E, et al. Targeting STAT3 to Suppress Systemic Pro-Oncogenic Effects from Hepatic Radiofrequency Ablation. Radiology. 2018. Feb;286(2):524–36. doi: 10.1148/radiol.2017162943 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Kumar G, Goldberg SN, Wang Y, Velez E, Gourevitch S, Galun E, et al. Hepatic radiofrequency ablation: markedly reduced systemic effects by modulating periablational inflammation via cyclooxygenase-2 inhibition. Eur Radiol. 2017. Mar;27(3):1238–47. doi: 10.1007/s00330-016-4405-4 [DOI] [PubMed] [Google Scholar]
16.Ahmed M, Kumar G, Moussa M, Wang Y, Rozenblum N, Galun E, et al. Hepatic Radiofrequency Ablation-induced Stimulation of Distant Tumor Growth Is Suppressed by c-Met Inhibition. Radiology. 2016. Apr;279(1):103–17. doi: 10.1148/radiol.2015150080 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Liao H, Ahmed M, Markezana A, Zeng G, Stechele M, Galun E, et al. Thermal Ablation Induces Transitory Metastatic Growth by Means of the STAT3/c-Met Molecular Pathway in an Intrahepatic Colorectal Cancer Mouse Model. Radiology. 2020. Feb;294(2):464–72. doi: 10.1148/radiol.2019191023 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Kotoh K, Enjoji M, Arimura E, Morizono S, Kohjima M, Sakai H, et al. Scattered and rapid intrahepatic recurrences after radio frequency ablation for hepatocellular carcinoma. World J Gastroenterol. 2005. Nov 21;11(43):6828–32. doi: 10.3748/wjg.v11.i43.6828 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Mori Y, Tamai H, Shingaki N, Moribata K, Shiraki T, Deguchi H, et al. Diffuse intrahepatic recurrence after percutaneous radiofrequency ablation for solitary and small hepatocellular carcinoma. Hepatol Int. 2009. Sep;3(3):509–15. doi: 10.1007/s12072-009-9131-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Anderson P, Dische S. Local tumor control and the subsequent incidence of distant metastatic disease. Int J Radiat Oncol Biol Phys. 1981. Dec;7(12):1645–8. doi: 10.1016/0360-3016(81)90186-3 [DOI] [PubMed] [Google Scholar]
21.Fagundes H, Perez CA, Grigsby PW, Lockett MA. Distant metastases after irradiation alone in carcinoma of the uterine cervix. Int J Radiat Oncol Biol Phys. 1992;24(2):197–204. doi: 10.1016/0360-3016(92)90671-4 [DOI] [PubMed] [Google Scholar]
22.Strong MS, Vaughan CW, Kayne HL, Aral IM, Ucmakli A, Feldman M, et al. A randomized trial of preoperative radiotherapy in cancer of the oropharynx and hypopharynx. Am J Surg. 1978. Oct;136(4):494–500. doi: 10.1016/0002-9610(78)90268-4 [DOI] [PubMed] [Google Scholar]
23.Vilalta M, Rafat M, Graves EE. Effects of radiation on metastasis and tumor cell migration. Cell Mol Life Sci. 2016. Aug;73(16):2999–3007. doi: 10.1007/s00018-016-2210-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Alieva M, van Rheenen J, Broekman MLD. Potential impact of invasive surgical procedures on primary tumor growth and metastasis. Clin Exp Metastasis. 2018. Apr;35(4):319–31. doi: 10.1007/s10585-018-9896-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Tohme S, Simmons RL, Tsung A. Surgery for Cancer: A Trigger for Metastases. Cancer Res. 2017. Apr 1;77(7):1548–52. doi: 10.1158/0008-5472.CAN-16-1536 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol. 2016. Jun;64(6):1403–15 doi: 10.1016/j.jhep.2016.02.004 [DOI] [PubMed] [Google Scholar]
27.Rozenblum N, Zeira E, Bulvik B, Gourevitch S, Yotvat H, Galun E, et al. Radiofrequency Ablation: Inflammatory Changes in the Periablative Zone Can Induce Global Organ Effects, including Liver Regeneration. Radiology. 2015. Aug;276(2):416–25. doi: 10.1148/radiol.15141918 [DOI] [PubMed] [Google Scholar]
28.Erinjeri JP, Thomas CT, Samoilia A, et al. Image-guided thermal ablation of tumors increases the plasma level of interleukin-6 and interleukin-10. J Vasc Interv Radiol. 2013;24(8):1105‐1112. doi: 10.1016/j.jvir.2013.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Ahmad F, Gravante G, Bhardwaj N, et al. Changes in interleukin-1β and 6 after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections. Am J Surg. 2010;200(4):500‐506. doi: 10.1016/j.amjsurg.2009.12.025 [DOI] [PubMed] [Google Scholar]
30.Zhou Z, Xu MJ, Gao B. Hepatocytes: a key cell type for innate immunity. Cell Mol Immunol. 2016. May;13(3):301–15. doi: 10.1038/cmi.2015.97 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Tao L, Huang G, Song H, Chen Y, Chen L. Cancer associated fibroblasts: An essential role in the tumor microenvironment. Oncol Lett. 2017. Sep;14(3):2611–20. doi: 10.3892/ol.2017.6497 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Rozenblum N, Zeira E, Scaiewicz V, Bulvik B, Gourevitch S, Yotvat H, et al. Oncogenesis: An "Off-Target" Effect of Radiofrequency Ablation. Radiology. 2015. Aug;276(2):426–32. doi: 10.1148/radiol.2015141695 [DOI] [PubMed] [Google Scholar]
33.Zhou M, Yang H, Learned RM, Tian H, Ling L. Non-cell-autonomous activation of IL-6/STAT3 signaling mediates FGF19-driven hepatocarcinogenesis. Nat Commun. 2017. May 16;8:15433. doi: 10.1038/ncomms15433 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Ahmed M, Kumar G, Navarro G, Wang Y, Gourevitch S, Moussa MH, et al. Systemic siRNA Nanoparticle-Based Drugs Combined with Radiofrequency Ablation for Cancer Therapy. PLoS One. 2015. Jul 8;10(7):e0128910. doi: 10.1371/journal.pone.0128910 ; PMCID: PMC4495977. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.D’Ippolito G, Ahmed M, Girnun GD, Stuart KE, Kruskal JB, Halpern EF, et al. Percutaneous tumor ablation: reduced tumor growth with combined radio-frequency ablation and liposomal doxorubicin in a rat breast tumor model. Radiology. 2003. Jul;228(1):112–8. doi: 10.1148/radiol.2281020358 [DOI] [PubMed] [Google Scholar]
36.Moussa M, Goldberg SN, Kumar G, Sawant RR, Levchenko T, Torchilin VP, et al. Nanodrug-enhanced radiofrequency tumor ablation: effect of micellar or liposomal carrier on drug delivery and treatment efficacy. PLoS One. 2014. Aug 18;9(8):e102727. doi: 10.1371/journal.pone.0102727 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Moussa M, Goldberg SN, Tasawwar B, Sawant RR, Levchenko T, Kumar G, et al. Adjuvant liposomal doxorubicin markedly affects radiofrequency ablation-induced effects on periablational microvasculature. J Vasc Interv Radiol. 2013. Jul;24(7):1021–33 doi: 10.1016/j.jvir.2013.03.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Kang TW, Lim HK, Cha DI. Aggressive tumor recurrence after radiofrequency ablation for hepatocellular carcinoma. Clin Mol Hepatol. 2017. Mar;23(1):95–101. doi: 10.3350/cmh.2017.0006 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Schuld J, Richter S, Oberkircher LW, Seeland U, Debnar-Daumler KI, Rauch J, et al. Evidence for tumor cell spread during local hepatic ablation of colorectal liver metastases. J Surg Res. 2012. Nov;178(1):268–79. doi: 10.1016/j.jss.2012.03.019 Epub 2012 Mar 31. . [DOI] [PubMed] [Google Scholar]
40.Kobayashi H, Enomoto A, Woods SL, Burt AD, Takahashi M, Worthley DL. Cancer-associated fibroblasts in gastrointestinal cancer. Nat Rev Gastroenterol Hepatol. 2019. May;16(5):282–95. doi: 10.1038/s41575-019-0115-0 [DOI] [PubMed] [Google Scholar]
41.Yin Z, Dong C, Jiang K, Xu Z, Li R, Guo K, et al. Heterogeneity of cancer-associated fibroblasts and roles in the progression, prognosis, and therapy of hepatocellular carcinoma. J Hematol Oncol. 2019. Sep 23;12(1):101,019-0782-x [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Liu T, Han C, Wang S, Fang P, Ma Z, Xu L, et al. Cancer-associated fibroblasts: an emerging target of anti-cancer immunotherapy. J Hematol Oncol. 2019. Aug 28;12(1):86. doi: 10.1186/s13045-019-0770-1 ; PMCID: PMC6714445. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Ireland LV, Mielgo A. Macrophages and Fibroblasts, Key Players in Cancer Chemoresistance. Front Cell Dev Biol. 2018. Oct 9;6:131. doi: 10.3389/fcell.2018.00131 ; PMCID: PMC6189297. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Wu X, Tao P, Zhou Q, Li J, Yu Z, Wang X, et al. IL-6 secreted by cancer-associated fibroblasts promotes epithelial-mesenchymal transition and metastasis of gastric cancer via JAK2/STAT3 signaling pathway. Oncotarget. 2017. Mar 28;8(13):20741–50. doi: 10.18632/oncotarget.15119 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Klein S, Klösel J, Schierwagen R, Körner C, Granzow M, Huss S, et al. Atorvastatin inhibits proliferation and apoptosis, but induces senescence in hepatic myofibroblasts and thereby attenuates hepatic fibrosis in rats. Lab Invest. 2012. Oct;92(10):1440–50. doi: 10.1038/labinvest.2012.106 [DOI] [PubMed] [Google Scholar]
46.Trebicka J, Hennenberg M, Schulze Pröbsting A, Laleman W, Klein S, Granzow M, et al. Role of beta3-adrenoceptors for intrahepatic resistance and portal hypertension in liver cirrhosis. Hepatology. 2009. Dec;50(6):1924–35. doi: 10.1002/hep.23222 [DOI] [PubMed] [Google Scholar]
47.Ung MH, MacKenzie TA, Onega TL, Amos CI, Cheng C. Statins associate with improved mortality among patients with certain histological subtypes of lung cancer. Lung Cancer. 2018. Dec;126:89–96. doi: 10.1016/j.lungcan.2018.10.022 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Huang BZ, Chang JI, Li E, Xiang AH, Wu BU. Influence of Statins and Cholesterol on Mortality Among Patients With Pancreatic Cancer. J Natl Cancer Inst. 2016. Dec 31;109(5):doi: 10.1093/jnci/djw275 Print 2017 May. [DOI] [PubMed] [Google Scholar]
49.Van Linthout S, Miteva K, Tschöpe C. Crosstalk between fibroblasts and inflammatory cells. Cardiovasc Res. 2014. May 1;102(2):258–69. doi: 10.1093/cvr/cvu062 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0266522.r001

Decision Letter 0

Yanlin Yu

7 Jan 2022

PONE-D-21-21027

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation.

PLOS ONE

Dear Dr. Ahmed,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Hanna Landenmark

Senior Editor, PLOS ONE

on behalf of

Yanlin Yu

Academic Editor, PLOS ONE

Journal Requirements:

1) Please include a complete ethics statement in the Methods section. Please ensure that you state for each experiment conducted the name of the ethics committee that approved the study, the method of euthanasia, the method of anesthesia and any efforts to alleviate suffering. Please also provide for each experiment the following information: 1. maximum sizes tumors grew, 2. how tumor size was calculated, 3. the post-operative and post tumor implantation care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Please also state whether the IACUC provided approval for any large tumor sizes.

2) Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

3) Please note that concerns were raised about image quality, however, in the event of publication, all images will be uploaded in the original resolution rather than that seen in the PDF, so there is no need to enhance your figures if the original resolution is sufficiently clear.

Additional Editor Comments (if provided):

The authors report the role of activated myofibroblasts in RFA-induced tumorigenesis using murine models. The study is well designed, the results are interesting. The manuscript is well written, but the images of the figures are not clear. So, I suggest publishing the vision of the manuscript with clear figures images.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: RF ablation was reported to increase growth of distant untreated tumor foci following local tumor ablation. Thus the researchers were looking to demonstrate that undesirable pro-tumorigenic effects induced by RF ablation, is carried out by myofibroblasts and that their suppression might affect tumor growth and progression.

They report on the key role of activated myofibroblasts in RFA-induced distant metastases growth and successful pharmacologic blockade in mouse and rat models of colon, renal and breast experimental models.

Activated myofibroblasts are a key cell population in post RF ablation distant tumor growth, primarily through induction of the HGF/c-MET/STAT3 axis. Post RFA distant tumor growth was observed in different phenotypes of ablated primary organ such as liver or kidney or phenotype of tumors such as colorectal cancer or breast cancer. They also observed that RF induced distant tumor growth can be achieved by inhibition of activated myofibroblasts using atorvastatin, an established lipid lowering medication, and anti-fibroblast agent.

This is a well executed research with interesting and important results.

I suggest that the authors will discuss the possibility that macrophage inhibition will achieve similar effects?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Yona Keisari

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2022 Jul 20;17(7):e0266522. doi: 10.1371/journal.pone.0266522.r002

Author response to Decision Letter 0

20 Jan 2022

Dear Drs. Landemark and Yu:

Thank you very much for your favorable review of our submitted original manuscript, PONE-D-21-21027, entitled “Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation”. Enclosed you will find our revised manuscript in which we have made the necessary requested changes. We will now detail the changes made in response to all comments on a point-by-point basis.

Journal Requirements:

1. Please include a complete ethics statement in the Methods section. Please ensure that you state for each experiment conducted the name of the ethics committee that approved the study, the method of euthanasia, the method of anesthesia and any efforts to alleviate suffering. Please also provide for each experiment the following information: 1. maximum sizes tumors grew, 2. how tumor size was calculated, 3. the post-operative and post tumor implantation care received by the animals, including the frequency of monitoring and the criteria used to assess animal health and well-being. Please also state whether the IACUC provided approval for any large tumor sizes.

Response: We now include a complete ethics statement in the Methods section. All of the methods and studies reported were approved by our Institutional Animal Care and Use Committee. We now include the additional information requested, including maximum tumor sizes (all of which were within the approved tumor size limit in our study protocol), how these were calculated, and the post-operative and post-tumor implantation care received.

“All of the methods and studies reported were approved by our Institutional Animal Care and Use Committee. Specifically, rat studies were performed at Beth Israel Deaconess Medical Center, while mouse experiments were performed at Hadassah Hebrew University Medical Center in accordance with protocols approved by the respective Institutional Animal Care Committees. For rat studies, the maximum permissible diameters for R3230 breast adenocarcninomas, as per approved IACUC protocol, was a mean diameter of 2 cm. Tumor sizes were assessed using manual caliper measurements of two largest perpendicular dimensions and calculating mean diameters. After surgical procedures, the animals were monitored on a daily basis to determine any specific clinical signs of postoperative pain and distress. Specific signs of weakening or distress included, but were not limited to: total combined tumor burden greater than 2 cm or 10% of body weight, decreased appetite, reduced ambulation (interfering with or hampering with the animal's ability to obtain food and water, bear its own weight, or regain normal posture if placed on the back), weight loss (20% loss of body weight within 7 days, measured every 2-3d), tumor ulceration or necrosis for subcutaneous tumors. Post-surgically, if signs of distress were detected, a single dose of buprenorphine SR (72 hour duration of action) was administered at the site of surgery. In cases of tumor burden exceeding approved limits, animal were euthanized. For mouse experiments, general condition and animal well-being were used as metrics of tumor burden in lieu of a maximum permissible size due to microscopic nature of CRC liver deposits. Similar to rat experiments, post-operatively, animals were monitored on a daily basis to determine any specific clinical signs of postoperative pain and distress. For post-operative pain and distress, a single dose of buprenorphine SR was administered at the site of surgery. If signs of tumor burden overgrowth were detected, such as decreased appetite, reduced ambulation and or weight loss animals were euthanized.”

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: There are no changes in the reference list.

3. Please note that concerns were raised about image quality, however, in the event of publication, all images will be uploaded in the original resolution rather than that seen in the PDF, so there is no need to enhance your figures if the original resolution is sufficiently clear.

Response: We have now uploaded images of higher resolution. We are happy to work with the editorial staff on these as required.

Additional Editor Comments:

Response: See response above.

Reviewer #1 - Specific comments:

“RF ablation was reported to increase growth of distant untreated tumor foci following local tumor ablation. Thus the researchers were looking to demonstrate that undesirable pro-tumorigenic effects induced by RF ablation, is carried out by myofibroblasts and that their suppression might affect tumor growth and progression.

This is a well-executed research with interesting and important results.

I suggest that the authors will discuss the possibility that macrophage inhibition will achieve similar effects?”

Response: We appreciate the Reviewer’s comments and enthusiasm for our work. We now address the potential role of macrophage suppression as well in the Discussion.

“Furthermore, this observation makes a strong case for cell specific targeting strategies, especially, of other abundant cell populations with known tumor promoting properties within the tumor microenvironment such as M2 macrophages.”

We have uploaded clean and annotated versions of the manuscript with tracked changes and figures, as requested.

Once again we thank you for a favorable reply.

Sincerely,

Muneeb Ahmed, M.D. on behalf of all authors

Attachment

Submitted filename: PONE-D-21-21027.Response to Reviewers.doc

Click here for additional data file.^{(54.5KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0266522.r003

Decision Letter 1

Yanlin Yu

23 Mar 2022

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation.

PONE-D-21-21027R1

Dear Dr. Ahmed,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Yanlin Yu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Dr. Ahmed,

Thank you for submitting your revised manuscript to PLOS ONE. I am delighted to inform you that your manuscript, " Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation", has now been accepted for publication in PLOS ONE.

Kind regards,

Yanlin

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0266522.r004

Acceptance letter

Yanlin Yu

24 May 2022

PONE-D-21-21027R1

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation.

Dear Dr. Ahmed:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yanlin Yu

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File

(DOCX)

Click here for additional data file.^{(1.8MB, docx)}

Attachment

Submitted filename: PONE-D-21-21027.Response to Reviewers.doc

Click here for additional data file.^{(54.5KB, doc)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0266522.ref001] 1.Abdalla EK, Vauthey JN, Ellis LM, Ellis V, Pollock R, Broglio KR, et al. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg. 2004. Jun;239(6):818,25; discussion 825–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref002] 2.Alexander ES, Machan JT, Ng T, Breen LD, DiPetrillo TA, Dupuy DE. Cost and effectiveness of radiofrequency ablation versus limited surgical resection for stage I non-small-cell lung cancer in elderly patients: is less more? J Vasc Interv Radiol. 2013. Apr;24(4):476–82. doi: 10.1016/j.jvir.2012.12.016 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref003] 3.Loveman E, Jones J, Clegg AJ, Picot J, Colquitt JL, Mendes D, et al. The clinical effectiveness and cost-effectiveness of ablative therapies in the management of liver metastases: systematic review and economic evaluation. Health Technol Assess. 2014. Jan;18(7):vii,viii, 1–283. doi: 10.3310/hta18070 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref004] 4.Schlijper RC, Grutters JP, Houben R, Dingemans AM, Wildberger JE, Van Raemdonck D, et al. What to choose as radical local treatment for lung metastases from colo-rectal cancer: surgery or radiofrequency ablation? Cancer Treat Rev. 2014. Feb;40(1):60–7. doi: 10.1016/j.ctrv.2013.05.004 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref005] 5.Stintzing S, Grothe A, Hendrich S, Hoffmann RT, Heinemann V, Rentsch M, et al. Percutaneous radiofrequency ablation (RFA) or robotic radiosurgery (RRS) for salvage treatment of colorectal liver metastases. Acta Oncol. 2013. Jun;52(5):971–7. doi: 10.3109/0284186X.2013.766362 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref006] 6.Groeschl RT, Gamblin TC, Turaga KK. Ablation for hepatocellular carcinoma: validating the 3-cm breakpoint. Ann Surg Oncol. 2013. Oct;20(11):3591–5. doi: 10.1245/s10434-013-3031-5 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref007] 7.Tabrizian P, Jibara G, Shrager B, Schwartz M, Roayaie S. Recurrence of hepatocellular cancer after resection: patterns, treatments, and prognosis. Ann Surg. 2015. May;261(5):947–55 doi: 10.1097/SLA.0000000000000710 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref008] 8.de Baere T, Tselikas L, Yevich S, Boige V, Deschamps F, Ducreux M, et al. The role of image-guided therapy in the management of colorectal cancer metastatic disease. Eur J Cancer. 2017. Apr;75:231–42. doi: 10.1016/j.ejca.2017.01.010 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref009] 9.Kurup AN, Callstrom MR. Increasing Role of Image-Guided Ablation in the Treatment of Musculoskeletal Tumors. Cancer J. 2016. Nov/Dec;22(6):401–10. doi: 10.1097/PPO.0000000000000233 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref010] 10.Lencioni R, Crocetti L. Image-guided ablation for hepatocellular carcinoma. Recent Results Cancer Res. 2013;190:181–94. doi: 10.1007/978-3-642-16037-0_12 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref011] 11.Petre EN, Solomon SB, Sofocleous CT. The role of percutaneous image-guided ablation for lung tumors. Radiol Med. 2014. Jul;119(7):541–8. doi: 10.1007/s11547-014-0427-7 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref012] 12.Wah TM. Image-guided ablation of renal cell carcinoma. Clin Radiol. 2017. Aug;72(8):636–44. doi: 10.1016/j.crad.2017.03.007 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref013] 13.Nikfarjam M, Muralidharan V, Christophi C. Altered growth patterns of colorectal liver metastases after thermal ablation. Surgery. 2006. Jan;139(1):73–81. doi: 10.1016/j.surg.2005.07.030 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref014] 14.Kumar G, Goldberg SN, Gourevitch S, Levchenko T, Torchilin V, Galun E, et al. Targeting STAT3 to Suppress Systemic Pro-Oncogenic Effects from Hepatic Radiofrequency Ablation. Radiology. 2018. Feb;286(2):524–36. doi: 10.1148/radiol.2017162943 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref015] 15.Kumar G, Goldberg SN, Wang Y, Velez E, Gourevitch S, Galun E, et al. Hepatic radiofrequency ablation: markedly reduced systemic effects by modulating periablational inflammation via cyclooxygenase-2 inhibition. Eur Radiol. 2017. Mar;27(3):1238–47. doi: 10.1007/s00330-016-4405-4 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref016] 16.Ahmed M, Kumar G, Moussa M, Wang Y, Rozenblum N, Galun E, et al. Hepatic Radiofrequency Ablation-induced Stimulation of Distant Tumor Growth Is Suppressed by c-Met Inhibition. Radiology. 2016. Apr;279(1):103–17. doi: 10.1148/radiol.2015150080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref017] 17.Liao H, Ahmed M, Markezana A, Zeng G, Stechele M, Galun E, et al. Thermal Ablation Induces Transitory Metastatic Growth by Means of the STAT3/c-Met Molecular Pathway in an Intrahepatic Colorectal Cancer Mouse Model. Radiology. 2020. Feb;294(2):464–72. doi: 10.1148/radiol.2019191023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref018] 18.Kotoh K, Enjoji M, Arimura E, Morizono S, Kohjima M, Sakai H, et al. Scattered and rapid intrahepatic recurrences after radio frequency ablation for hepatocellular carcinoma. World J Gastroenterol. 2005. Nov 21;11(43):6828–32. doi: 10.3748/wjg.v11.i43.6828 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref019] 19.Mori Y, Tamai H, Shingaki N, Moribata K, Shiraki T, Deguchi H, et al. Diffuse intrahepatic recurrence after percutaneous radiofrequency ablation for solitary and small hepatocellular carcinoma. Hepatol Int. 2009. Sep;3(3):509–15. doi: 10.1007/s12072-009-9131-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref020] 20.Anderson P, Dische S. Local tumor control and the subsequent incidence of distant metastatic disease. Int J Radiat Oncol Biol Phys. 1981. Dec;7(12):1645–8. doi: 10.1016/0360-3016(81)90186-3 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref021] 21.Fagundes H, Perez CA, Grigsby PW, Lockett MA. Distant metastases after irradiation alone in carcinoma of the uterine cervix. Int J Radiat Oncol Biol Phys. 1992;24(2):197–204. doi: 10.1016/0360-3016(92)90671-4 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref022] 22.Strong MS, Vaughan CW, Kayne HL, Aral IM, Ucmakli A, Feldman M, et al. A randomized trial of preoperative radiotherapy in cancer of the oropharynx and hypopharynx. Am J Surg. 1978. Oct;136(4):494–500. doi: 10.1016/0002-9610(78)90268-4 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref023] 23.Vilalta M, Rafat M, Graves EE. Effects of radiation on metastasis and tumor cell migration. Cell Mol Life Sci. 2016. Aug;73(16):2999–3007. doi: 10.1007/s00018-016-2210-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref024] 24.Alieva M, van Rheenen J, Broekman MLD. Potential impact of invasive surgical procedures on primary tumor growth and metastasis. Clin Exp Metastasis. 2018. Apr;35(4):319–31. doi: 10.1007/s10585-018-9896-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref025] 25.Tohme S, Simmons RL, Tsung A. Surgery for Cancer: A Trigger for Metastases. Cancer Res. 2017. Apr 1;77(7):1548–52. doi: 10.1158/0008-5472.CAN-16-1536 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref026] 26.Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol. 2016. Jun;64(6):1403–15 doi: 10.1016/j.jhep.2016.02.004 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref027] 27.Rozenblum N, Zeira E, Bulvik B, Gourevitch S, Yotvat H, Galun E, et al. Radiofrequency Ablation: Inflammatory Changes in the Periablative Zone Can Induce Global Organ Effects, including Liver Regeneration. Radiology. 2015. Aug;276(2):416–25. doi: 10.1148/radiol.15141918 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref028] 28.Erinjeri JP, Thomas CT, Samoilia A, et al. Image-guided thermal ablation of tumors increases the plasma level of interleukin-6 and interleukin-10. J Vasc Interv Radiol. 2013;24(8):1105‐1112. doi: 10.1016/j.jvir.2013.02.015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref029] 29.Ahmad F, Gravante G, Bhardwaj N, et al. Changes in interleukin-1β and 6 after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections. Am J Surg. 2010;200(4):500‐506. doi: 10.1016/j.amjsurg.2009.12.025 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref030] 30.Zhou Z, Xu MJ, Gao B. Hepatocytes: a key cell type for innate immunity. Cell Mol Immunol. 2016. May;13(3):301–15. doi: 10.1038/cmi.2015.97 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref031] 31.Tao L, Huang G, Song H, Chen Y, Chen L. Cancer associated fibroblasts: An essential role in the tumor microenvironment. Oncol Lett. 2017. Sep;14(3):2611–20. doi: 10.3892/ol.2017.6497 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref032] 32.Rozenblum N, Zeira E, Scaiewicz V, Bulvik B, Gourevitch S, Yotvat H, et al. Oncogenesis: An "Off-Target" Effect of Radiofrequency Ablation. Radiology. 2015. Aug;276(2):426–32. doi: 10.1148/radiol.2015141695 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref033] 33.Zhou M, Yang H, Learned RM, Tian H, Ling L. Non-cell-autonomous activation of IL-6/STAT3 signaling mediates FGF19-driven hepatocarcinogenesis. Nat Commun. 2017. May 16;8:15433. doi: 10.1038/ncomms15433 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref034] 34.Ahmed M, Kumar G, Navarro G, Wang Y, Gourevitch S, Moussa MH, et al. Systemic siRNA Nanoparticle-Based Drugs Combined with Radiofrequency Ablation for Cancer Therapy. PLoS One. 2015. Jul 8;10(7):e0128910. doi: 10.1371/journal.pone.0128910 ; PMCID: PMC4495977. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref035] 35.D’Ippolito G, Ahmed M, Girnun GD, Stuart KE, Kruskal JB, Halpern EF, et al. Percutaneous tumor ablation: reduced tumor growth with combined radio-frequency ablation and liposomal doxorubicin in a rat breast tumor model. Radiology. 2003. Jul;228(1):112–8. doi: 10.1148/radiol.2281020358 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref036] 36.Moussa M, Goldberg SN, Kumar G, Sawant RR, Levchenko T, Torchilin VP, et al. Nanodrug-enhanced radiofrequency tumor ablation: effect of micellar or liposomal carrier on drug delivery and treatment efficacy. PLoS One. 2014. Aug 18;9(8):e102727. doi: 10.1371/journal.pone.0102727 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref037] 37.Moussa M, Goldberg SN, Tasawwar B, Sawant RR, Levchenko T, Kumar G, et al. Adjuvant liposomal doxorubicin markedly affects radiofrequency ablation-induced effects on periablational microvasculature. J Vasc Interv Radiol. 2013. Jul;24(7):1021–33 doi: 10.1016/j.jvir.2013.03.006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref038] 38.Kang TW, Lim HK, Cha DI. Aggressive tumor recurrence after radiofrequency ablation for hepatocellular carcinoma. Clin Mol Hepatol. 2017. Mar;23(1):95–101. doi: 10.3350/cmh.2017.0006 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref039] 39.Schuld J, Richter S, Oberkircher LW, Seeland U, Debnar-Daumler KI, Rauch J, et al. Evidence for tumor cell spread during local hepatic ablation of colorectal liver metastases. J Surg Res. 2012. Nov;178(1):268–79. doi: 10.1016/j.jss.2012.03.019 Epub 2012 Mar 31. . [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref040] 40.Kobayashi H, Enomoto A, Woods SL, Burt AD, Takahashi M, Worthley DL. Cancer-associated fibroblasts in gastrointestinal cancer. Nat Rev Gastroenterol Hepatol. 2019. May;16(5):282–95. doi: 10.1038/s41575-019-0115-0 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref041] 41.Yin Z, Dong C, Jiang K, Xu Z, Li R, Guo K, et al. Heterogeneity of cancer-associated fibroblasts and roles in the progression, prognosis, and therapy of hepatocellular carcinoma. J Hematol Oncol. 2019. Sep 23;12(1):101,019-0782-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref042] 42.Liu T, Han C, Wang S, Fang P, Ma Z, Xu L, et al. Cancer-associated fibroblasts: an emerging target of anti-cancer immunotherapy. J Hematol Oncol. 2019. Aug 28;12(1):86. doi: 10.1186/s13045-019-0770-1 ; PMCID: PMC6714445. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref043] 43.Ireland LV, Mielgo A. Macrophages and Fibroblasts, Key Players in Cancer Chemoresistance. Front Cell Dev Biol. 2018. Oct 9;6:131. doi: 10.3389/fcell.2018.00131 ; PMCID: PMC6189297. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref044] 44.Wu X, Tao P, Zhou Q, Li J, Yu Z, Wang X, et al. IL-6 secreted by cancer-associated fibroblasts promotes epithelial-mesenchymal transition and metastasis of gastric cancer via JAK2/STAT3 signaling pathway. Oncotarget. 2017. Mar 28;8(13):20741–50. doi: 10.18632/oncotarget.15119 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref045] 45.Klein S, Klösel J, Schierwagen R, Körner C, Granzow M, Huss S, et al. Atorvastatin inhibits proliferation and apoptosis, but induces senescence in hepatic myofibroblasts and thereby attenuates hepatic fibrosis in rats. Lab Invest. 2012. Oct;92(10):1440–50. doi: 10.1038/labinvest.2012.106 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref046] 46.Trebicka J, Hennenberg M, Schulze Pröbsting A, Laleman W, Klein S, Granzow M, et al. Role of beta3-adrenoceptors for intrahepatic resistance and portal hypertension in liver cirrhosis. Hepatology. 2009. Dec;50(6):1924–35. doi: 10.1002/hep.23222 [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref047] 47.Ung MH, MacKenzie TA, Onega TL, Amos CI, Cheng C. Statins associate with improved mortality among patients with certain histological subtypes of lung cancer. Lung Cancer. 2018. Dec;126:89–96. doi: 10.1016/j.lungcan.2018.10.022 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0266522.ref048] 48.Huang BZ, Chang JI, Li E, Xiang AH, Wu BU. Influence of Statins and Cholesterol on Mortality Among Patients With Pancreatic Cancer. J Natl Cancer Inst. 2016. Dec 31;109(5):doi: 10.1093/jnci/djw275 Print 2017 May. [DOI] [PubMed] [Google Scholar]

[pone.0266522.ref049] 49.Van Linthout S, Miteva K, Tschöpe C. Crosstalk between fibroblasts and inflammatory cells. Cardiovasc Res. 2014. May 1;102(2):258–69. doi: 10.1093/cvr/cvu062 [DOI] [PubMed] [Google Scholar]

PERMALINK

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation

Marwan Moussa

David Mwin

Haixing Liao

M Fatih Atac

Aurelia Markezana

Eithan Galun

S Nahum Goldberg

Muneeb Ahmed

Roles

Abstract

Introduction

Material and methods

Overview

Phase I

Phase II

Phase III

Phase IV

Ethics statement

Results

1. Atorvastatin reduces RFA-induced myofibroblast periablational infiltration

Fig 1. Atorvastatin reduces RFA-induced myofibroblast periablational infiltration in treated liver in three rodent models.

2. Myofibroblast inhibition suppresses RFA-induced upregulation of tumor promoting cytokines

Table 1. Effect of myofibroblast blockade on Hepatocyte growth factor upregulation in the periablational rim and distant liver post-RFA.

Fig 2. Myofibroblast blockade inhibits RFA-induced upregulation of tumor promoting cytokines.

3. Myofibroblast blockade eliminates hepatic RFA-induced tumorigenesis and proliferation of distant established tumors

Fig 3. Myofibroblast blockade eliminates hepatic RF driven tumorigenesis and of proliferation distant established tumors.

Table 2. Effect of myofibroblast blockade on cellular trafficking, downstream tumorigenic growth factors and proliferation in rat livers’ periablational rim collected 7 days post hepatic ablation.

4. Myofibroblast blockade eliminates hepatic RFA-induced tumorigenesis of micrometastatic implants

Fig 4. Myofibroblast blockade eliminates hepatic RF driven tumorigenesis of micrometastatic implants.

Fig 5. Myofibroblast blockade suppresses RF induced tumor proliferation of micrometastatic implants in murine colorectal cancer models.

Table 3. Effect of atorvastatin on myofibroblast infiltration and proliferation in CRC metastasis after hepatic RF.

6. Atorvastatin partially suppresses RFA-induced periablational myofibroblast infiltration in ablated kidney

7. Myofibroblast blockade reduces renal RFA-induced tumor promoting cytokines, tumorigenesis of distant established tumors, and periablational and distant tumor proliferation

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Yanlin Yu

Roles

Author response to Decision Letter 0

Decision Letter 1

Yanlin Yu

Roles

Acceptance letter

Yanlin Yu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases