Systemic inflammation enhances metastatic growth in a syngeneic neuroblastoma mouse model

doi:10.21203/rs.3.rs-4702299/v1

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Research Article

Systemic inflammation enhances metastatic growth in a syngeneic neuroblastoma mouse model

https://doi.org/10.21203/rs.3.rs-4702299/v1

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Journal Publication

published 17 Jul, 2024

Read the published version in Pediatric Surgery International →

You are reading this latest preprint version

Background

We previously showed that total tumor resection enhances metastatic growth in a syngeneic metastatic mouse model of neuroblastoma. In this study, we further investigated which surgical factors contributed most to metastatic growth.

Methods

Tumor cells derived from MYCN transgenic mice were subcutaneously injected into wild-type mice. Mice were randomly assigned to receive partial resection (PR group), subcutaneous implantation of a sponge (Sp group), or observation (Obs group). The lymph node metastasis volume and frequency of lung metastasis were compared 14 days after assignment by measuring C-reactive protein (CRP) and interleukin-6 (IL-6) levels.

Results

The lymph node metastasis volume in the Sp group was larger than in the Obs group (148.4 [standard deviation {SD}: 209.5] vs. 10.2 [SD 12.8] mm³). The frequency of lung metastasis was greater in the Sp group than in the PR group (11.9 [SD 12.2] vs. 6.6 [SD 4.0] counts/slide). The CRP level in the Sp group was higher than in the PR group (2.3 [SD 0.5] vs. 1.5 [SD 0.4] µg/mL), and the IL-6 level in the Sp group was higher than in the PR or Obs groups (28.4 [SD 34.5] vs. 12.4 [SD 19.0] vs. 5.4 [SD 8.1] pg/mL).

Conclusion

Metastatic growth may be enhanced by systemic inflammation.

Neuroblastoma

Metastasis

Surgical inflammation

Syngeneic mouse model

MYCN transgenic mouse

Neuroblastoma is the most common extracranial solid tumor in children, accounting for approximately 8% of all childhood cancers and 15% of childhood cancer mortality [1]. In a previous report, the incidence of metastatic sites at the diagnosis was as follows: bone marrow (70.5%), bone (55.7%), lymph nodes (30.9%), liver (29.6%), intracranial and orbital (18.2%), lung (3.3%), and central nervous system 0.6% [2]. Despite advances in multidrug therapy, the prognosis of patients with high-risk neuroblastoma (HR-NBL) with metastatic lesions at the diagnosis remains poor despite advances in multidrug therapy [3–5]. The recent results of the COG A3973 study of HR-NBL revealed that ≥ 90% resection resulted in a better event-free survival and a lower cumulative incidence of local progression. However, no differences were found in the overall survival or complication rates [6].

The impact of surgical interventions on the pathogenesis of distant metastasis of cancers has been the subject of various studies [7]. Such metastatic outgrowth rapidly becomes uncontrollable with chemoradiation and manages to seed additional metastatic colonies, resulting in disruption of vital organ function. Although the clinical importance of metastasis is apparent, its underlying mechanisms remain unclear [8]. Various perioperative factors may be responsible for the acceleration of metastasis during surgical manipulation. Possible factors include a decrease in concomitant tumor resistance (CR), which is a phenomenon in which a tumor-bearing host is resistant to the growth of secondary tumor implants [9–11], dissemination during tumor manipulation [12], and cytokines generated during the healing process in response to surgical invasiveness [13–15]. In neuroblastoma, factors affecting the growth of primary and metastatic lesions have not been fully identified [16].

As previously reported, we established a syngeneic neuroblastoma mouse model with a high frequency of metastatic spread using a method for primary culture of MYCN transgenic mouse (MYCN-TgM)-derived tumor cells [17]. Using this model, we investigated the effect of local tumor resection on metastatic growth and demonstrated that total tumor resection promotes metastatic growth [18]. However, this experiment did not allow us to determine which factors of surgical manipulation had the greatest impact on increases in the lymph node metastasis volume and frequency of lung metastasis.

In this study, we conducted two experiments to further investigate local tumor resection aggravating distant metastases to identify important perioperative factors associated with metastatic acceleration, which is critical for surgical treatment strategies for HR-NBL.

Ethics statement

All experimental procedures and protocols for animals conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. This study was approved by the Committee for Animal Research of our institution (Permit Number: M2019-5510).

Animals

MYCN-TgM was originally created by Prof. William A. Weiss (University of California, San Francisco, CA, USA) [19]. In this study, MYCN-TgM on a 129 + Ter/SvJcl background was kindly gifted by Prof. Kenji Kadomatsu (Nagoya University, Nagoya, Japan) [20] and maintained in our animal facility under specific-pathogen-free conditions.

Syngeneic mouse model

We cultured tumor spheres from primary tumors of MYCN-TgM [17]. MYCN-TgM-derived tumor cells were injected subcutaneously into two-month-old 129/SVJ wild-type mice following our previous experiment [18]. The size of the subcutaneous tumor was measured from the body surface with a vernier caliper, and the tumor volume was calculated using the following formula: (A × B2) / 2, where A is the largest dimension and B is the shortest dimension. When local tumors exceeded 1000 mm³ in size, mice were randomly assigned to each group.

The evaluation of metastases

The lymph node metastasis volume was measured from tumor diameters using the same formula as above (Fig. 1a). To quantify lung metastases, tissue sections of paraffin-embedded blocks containing all lobes of the lungs were stained for immunohistochemistry.

Immunohistochemistry

The lungs were fixed overnight in 4% paraformaldehyde at room temperature. They were embedded in paraffin, and 4-µm tissue sections were analyzed. The paraffin sections were deparaffinized and rehydrated. Antigens were retrieved by boiling the slides in 10 mM sodium citrate buffer (pH 6.0) for 10 min, and the endogenous peroxidase activity was quenched in 3% H₂O₂ for 10 min. The sections were blocked with a blocking solution (Blocking One; NACALAI TESQUE, Inc. Kyoto, Japan) and subsequently incubated overnight with rabbit anti-TH antibody (#2792, 1:300; CST, Danvers, MA, USA) in blocking solution at 4°C. The sections were then incubated with secondary biotin-labeled goat anti-rabbit IgG antibody (Boost IHC Detection Reagent#8114, 1:100; CST) at room temperature for 30 min. The DAB chromogen was developed in DAB solution (K3468; Dako, Santa Clara, USA) for 2 min. After counterstaining with hematoxylin (8656; Sakura Finetek, Nagoya, Japan), the slides were photographed in high-power fields under a microscope by randomly selecting three tissue areas, and the frequency of lung metastasis was obtained by counting numbers of TH-positive cells per slide (Fig. 1b).

The quantification of inflammatory cytokines (CRP and IL-6)

Blood samples were collected from all mice at the time of euthanization. To confirm the inflammatory response in mice, C-reactive protein (CRP) and interleukin-6 (IL-6) levels were measured using an enzyme-linked immunosorbent assay (ELISA).

Studying the effect of surgical manipulation on metastatic growth (pilot experiment)

Tumor cells derived from MYCN-TgM were subcutaneously injected into wild-type mice. When local tumors exceeded 1000 mm³ in size, mice were randomly assigned to the observation group (Observation group), complete resection group (Resection group), sham surgery with tumor manipulation group (Touched-sham group), or sham surgery through abdominal skin incision without tumor manipulation group (Untouched-sham group) (Fig. 2a). Before surgery, all mice were administered an intraperitoneal injection of 200 µL of anesthetic solution containing 15 µg medetomidine, 80 µg midazolam, and 100 µg butorphanol tartrate diluted in normal saline. In the Resection group, complete tumor resection, including that of the surrounding skin and subcutaneous tissue, was performed (Fig. 2b). In the Touched-sham group, a skin incision and tumor dissection with preservation of tumor feeder vessels were performed, and the skin was sutured without tumor resection (Fig. 2c). In the Untouched sham group, an abdominal skin incision with subcutaneous dissection, apart from the subcutaneous tumor, was made, and the skin was sutured (Fig. 2d). Fourteen days after the assignment, the mice were sacrificed, and axillary lymph node metastasis were harvested.

Studying the effect of surgical invasiveness on metastatic growth (main experiment)

As previously mentioned, syngeneic mouse models were created. When local tumors exceeded 1000 mm³ in size, the mice were randomly assigned to receive partial resection (PR group) or inflammation was induced by subcutaneously implanting sterile polyvinyl acetate sponges without tumor manipulation (Sp group) or observation (Obs group) (Fig. 3a). Before surgery, all mice were anesthetized as described in the pilot study. In the PR group, 80%-90% of the local tumors were excised (Fig. 3b). Sterile polyvinyl acetate sponges were subcutaneously implanted into the Sp group (Fig. 3c). Sponge implantation is a well-established model of wounding and the subsequent wound-healing response with enhanced systemic inflammation [21]. Fourteen days after assignment, the mice were sacrificed, and axillary lymph node metastasis, lungs, and blood were harvested.

Statistical analyses

Statistical analyses were performed using the JMP® software program, ver. 14.2.0 (SAS Institute Inc., North Carolina, USA.). The Kruskal-Wallis test and Wilcoxon tests were used for the statistical analyses. p < 0.05 was considered significant.

Tumor manipulation and surgical invasiveness as well as the decreased CR accelerating metastatic growth (pilot experiment)

MYCN-TgM tumor cells were subcutaneously injected into 55 mice. Two mice were excluded because they presented no axillary lymph node metastasis when the local tumors exceeded 1000 mm³ in size. As a result, 53 tumor-bearing mice were assigned to 4 groups: Observation (n = 14), Resection (n = 14), Touched-sham (n = 16), and Untouced-sham (n = 9). Four mice from the Observation group and two each from the Touched-sham and the Untouched-sham groups died on postoperative day (POD) 14 due to tumor burden. Ultimately, 14 mice in the Observation and the Touched-sham groups, 10 mice in the Resection group, and 7 mice in the Untouched-sham group survived and were included in the analysis.

In the measurement of axillary lymph node metastasis, the lymph node metastasis volumes were 28.3 mm³ (standard deviation [SD] 18.4) in the Observation group, 311.2 mm³ (SD 330.5) in the Resection group, 339.1 mm³ (SD 241.6) in the Touched-sham group, and 22.3 mm³ (SD 11.0) in the Untouched-sham group. The lymph node metastasis volume in the Resection group was significantly higher than that in the Observation group (p = 0.0002) and Untouched-sham group (p = 0.0014). The lymph node metastasis volume in the Touched-sham group was significantly higher than that in the Observation group (p = 0.0388) (Fig. 4).

Surgical invasiveness with systemic inflammation enhancing metastatic growth (main experiment)

MYCN-TgM tumor cells were subcutaneously injected into 40 mice. Six mice were excluded because they presented no axillary lymph node metastasis when the local tumors exceeded 1000 mm³ in size. As a result, 34 tumor-bearing mice were assigned to 3 groups: the Obs group (n = 12), PR group (n = 11), and Sp group (n = 11). Three mice each from the PR and Obs groups and two mice from the Sp group died on POD 14 because of surgical recovery failure. Nine mice in the Sp and Obs groups and eight mice in the PR group ultimately survived and were included in the analysis.

In the measurement of axillary lymph node metastasis, lymph node metastasis volume was 92.8 mm³ (SD 146.7) in the PR group, 148.4 mm³ (SD 209.5) in the Sp group, and 10.2 mm³ (SD 12.8) in the Obs group (Fig. 5a). The lymph node metastasis volume in the Sp group was significantly larger than that in the Obs group (p = 0.0297), while there was no significant difference in lymph node metastasis volume between the Sp and PR groups (p = 0.8473) or between the PR and Obs groups (p = 0.0658).

In the analysis of lung metastasis, the frequency of lung metastasis was 4.0 counts/slide (SD 6.6) in the PR group, 11.9 counts/slide (SD 12.2) in the Sp group, and 3.6 counts/slide (SD 2.4) in the Obs group (Fig. 5b). The frequency of lung metastasis was higher in the Sp group than in the PR group (p = 0.0283).

In the ELISA, the CRP level was 1.5 µg/mL (SD 0.4) in the PR group, 2.3 µg/mL (SD 0.5) in the Sp group, and 1.96 µg /dL in the Obs group (SD 0.6] (Fig. 6a). The CRP level in the Sp group was significantly higher than that in the PR group (p = 0.0042), while there was no significant difference in CRP levels between the PR and Obs groups (p = 0.1611) or between the PR and Sp groups (p = 0.2135).

The IL-6 level was 12.4 pg/mL (SD 19.0) in the PR group, 28.4 pg/mL (SD 34.5) in the Sp group, and 5.4 pg/mL in the Obs group (SD 8.1) (Fig. 6b). The IL-6 level in the Sp group was significantly higher than that in the PR group (p = 0.047) and the Obs group (p = 0.0151), whereas there was no significant difference in the IL-6 level between the PR and Obs groups (p = 0.5535).

The effect of primary tumor resection on metastatic lesions in neuroblastoma remains unknown. In this study, we attempted to elucidate the mechanisms of metastasis and how surgical manipulation accelerates neuroblastoma metastasis. One possible mechanism is the decrease in the CR hypothesis, in which the immune response to tumor cells inhibits metastatic growth accelerated by tumor resection [9–11]. Second, metastases are activated by surgical dissemination, inflammatory responses, and wound healing processes [21]. As shown in our previous study, the group with local recurrence had a longer postoperative duration from local tumor resection to the appearance of metastases than the group without local recurrence. The enhanced metastatic growth after local tumor resection was thus assumed to have been caused by decreased CR rather than surgical invasiveness [18].

A pilot experiment was conducted to examine how surgical manipulation could accelerate metastatic growth. In this experiment, the Resection group was representative of decreased CR, the Touched-sham group was representative of tumor manipulation, and the Untouched-sham group represented surgical manipulation apart from the tumor. Considering the results from the pilot experiment, the Resection and Touched-sham groups showed significant lymph node enlargement, and we speculated that tumor manipulation, surgical invasiveness, and decreased CR were significant trigger factors for the outgrowth of metastasis. However, the involvement of surgical invasion alone could not be determined from this experimental system, as the Untouched-sham manipulation seemed to be far less invasive than the Touched-sham and Resection manipulations. Therefore, we planned the main experiment to study enhanced surgical invasiveness and tumor manipulation.

Possible factors influencing surgical manipulation include intraoperative dissemination and systemic inflammation induced by the tumor manipulation. We considered the partial resection model (PR group) in the main experiment to reflect these factors more strongly than the total resection model (resection group) in the pilot experiment. In addition, sponge-implantation surgery has been reported to reflect wound healing and the subsequent wound-healing response with enhanced systemic inflammation [21]. This technique may be an excellent surgical invasiveness model without tumor manipulation, and tumor acceleration with this model surgery has been reported in other carcinomas [22]. We applied sponge-implanting surgery to a mouse model of neuroblastoma (Sp group) as a representative of more highly systemic inflammation conditions during the wound healing process compared with the Untouched-sham group. The Sp group showed a significantly greater frequency of lung metastasis than the PR group, and the same trend was observed for the lymph node metastasis volume. This result implied that surgical invasiveness was more strongly involved than decreased CR in accelerating metastasis.

To evaluate the systemic inflammatory response, IL-6 and CRP levels were measured in the main experiment. Infection or external invasion causes macrophages to release IL-6, which acts on hepatocytes to produce CRP [23]. In various inflammatory diseases, IL-6 in the blood shows an early rise preceding CRP and a rapid disappearance, reflecting inflammatory conditions more acutely than CRP. Blood test results showed that both CRP and IL-6 levels were higher in the Sp group than in the PR group. Given that the blood tests were performed 14 days after each manipulation, this could be due to a high inflammatory response in the Sp surgery itself or long-term inflammation due to a foreign body reaction. Although continuous postoperative blood tests are necessary to determine whether the factor more strongly influencing the acceleration of metastases is the duration or intensity of inflammation, blood samples were taken at the time of sacrificial death; thus, this was impossible to determine in our experimental settings. In contrast, the CRP level was high, but the IL-6 level was not as high in the Obs group. We considered CRP level elevation in the Obs group to be indicative of chronic inflammation caused by the tumor.

There have been reports that IL-6 affects the microenvironment in which cancer cells reside in other carcinomas; IL-6 increases cancer cell migration and invasive potential [24–26]. In tumor progression, activated STAT3 upregulates the expression of MMP-2, MMP-7, and MMP-9, which enhances the invasive potential of the tumor. Epithelial-mesenchymal transition (EMT) is a developmental switch of tumor cells from an epithelial phenotype to a mesenchymal phenotype, seen in the early stage of tumorigenesis, and is involved in the progression of primary tumors toward metastasis promotion. IL-6 has been shown to promote EMT by activating the STAT3 signaling pathway [27–29]. The lack of progression of metastases in the Obs group and the acceleration of metastases in the other groups suggested that IL-6 strongly enhances metastasis. In the future, we will study the utility of postoperative continuous blood tests for IL-6 levels to further our understanding of the relationship between metastasis and IL-6 levels.

Several limitations associated with the present study warrant mention. Metastases were evaluated only for early postoperative metastasis within a short period (14 days after treatment), and late postoperative metastases were not evaluated. Although sponge-implanting surgery represents a well-established model of wound healing, it can also induce foreign body reaction and is not suitable in real-world clinical settings.

The results of this study suggest that local tumor resection before complete control of metastases should be avoided, and delayed local treatment of HR-NBL with metastases may be justified. The metastatic growth of unresectable HR-NBL may be enhanced by systematic surgical invasiveness due to surgical complications. Therefore, we plan to use anti-inflammatory drugs in the postoperative period to evaluate possible metastasis suppression, which may contribute to the perioperative management of neuroblastoma [30]. Our analyses will help further our understanding of the relationship between surgical manipulation and metastasis in advanced HR-NBL with metastatic lesions.

Conflict of Interest

The authors declare no conflicts of interest in association with the present study.

Funding

This work was supported by JSPS KAKENHI Grant Numbers JP23K19638, JP23K08054.

Author Contribution

KM and KY designed the study, analyzed the data, and wrote the manuscript. MI and SF designed the study, interpreted the data, revised it, and finally approved the manuscript. All authors have commented on the manuscript. All authors have read and approved the final manuscript.

Acknowledgement

The English language used in this manuscript was reviewed by Brian Quinn (Editor-in-Chief, Japan Medical Communication).

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No competing interests reported.

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Journal Publication

published 17 Jul, 2024

Read the published version in Pediatric Surgery International →

Editorial decision: Accepted
12 Jul, 2024
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You are reading this latest preprint version

Systemic inflammation enhances metastatic growth in a syngeneic neuroblastoma mouse model

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials & Methods

Ethics statement

Animals

Syngeneic mouse model

The evaluation of metastases

Immunohistochemistry

The quantification of inflammatory cytokines (CRP and IL-6)

Studying the effect of surgical manipulation on metastatic growth (pilot experiment)

Studying the effect of surgical invasiveness on metastatic growth (main experiment)

Statistical analyses

Results

Surgical invasiveness with systemic inflammation enhancing metastatic growth (main experiment)

Discussion

Conclusions

Declarations

Conflict of Interest

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Journal Publication

Version 1