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Isoflurane Impacts Murine Melanoma Growth in a Sex-Specific, Immune-Dependent Manner: A Brief Report

Meier, Angela, MD, PhD*; Gross, Emilie T. E., PhD; Schilling, Jan M., MD*,‡; Seelige, Ruth, PhD; Jung, Yujin, BS; Santosa, Endi, MS; Searles, Stephen, BA; Lin, Tuo, BS§; Tu, Xin M., PhD§; Patel, Hemal H., PhD*,‡; Bui, Jack D., MD, PhD

doi: 10.1213/ANE.0000000000002902
Anesthetic Clinical Pharmacology: Brief Report
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The impact of volatile anesthetics on cancer progression has been observed for decades, but sex differences have not been described. Male and female immune systems vary considerably, and the immune system plays an important role in limiting cancer growth. Currently, mouse models describing the impact of volatile anesthetics on cancer growth are limited to same-sex models. In this brief report, we describe a sex-specific impact of isoflurane on melanoma growth observed in wild-type but not in immune-deficient mice. Future experimental designs related to anesthesia and cancer should evaluate the biological variable of sex in a systematic manner.

From the Departments of *Anesthesiology

Pathology, University of California San Diego, San Diego, California

Department of Veterinary Affairs, San Diego Health Care System, San Diego, California

§Department of Family Medicine and Public Health, University of California San Diego, San Diego, California.

Published ahead of print March 21, 2018.

Accepted for publication February 1, 2018.

Funding: A.M. is supported by a grant from the International Anesthesia Research Society and was supported by a grant from the University of California, San Diego Faculty Senate. J.D.B. is supported by grants from the NCI (CA157885) and The Hartwell Foundation. X.M.T. and T.L. are supported by the National Institutes of Health (NIH), grant UL1TR001442 of CTSA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conflicts of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Angela Meier, MD, PhD, Department of Anesthesiology, University of California San Diego, 200 W Arbor Dr, San Diego, CA 92103. Address e-mail to anmeier@ucsd.edu.

Emerging clinical data highlight the importance of anesthetic choice during tumor surgery on subsequent cancer survival.1 Specifically, the use of inhalational anesthesia has been suggested to hasten death in cancer patients.1 The contribution of the immune system in controlling tumor growth is considered a “hallmark of cancer,”2 and inhalational anesthetics can significantly modulate the immune response.3 Biological differences between men and women are plentiful and complex, and immune-related sex differences directly translate into differences in human disease incidence and survival.4 However, studies describing the interactions of anesthesia and the immune system have not taken sex into consideration, and rodent models in this field of study use same-sex animals only5 or do not specify sex.6 Here, we communicate our observation that isoflurane has a sex-specific and immune-dependent effect on murine melanoma growth, impacting male but not female tumor growth. Sex differences should be taken into consideration when studying the impact of inhalational anesthesia on cancer progression.

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METHODS

The University of California San Diego Animal Care and Use Committee approved all of the described animal studies.

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Animals

Animals used in our studies were either bred at our facility or ordered from Charles River. They were provided with food and water ad libitum.

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Experiments

In Vivo Anesthetic Exposure.

For tumor growth experiments, male and female wild-type (WT) C57BL/6 mice, male C57BL/6 RAG1/− mice, or male C57BL/6 RAG2−/− × γc−/− mice between 8 and 12 weeks of age were injected subcutaneously into the right flank with 1 × 106 cells of the melanoma cell line B16F1ova harvested at approximately 80% confluence. After injection, mice were anesthetized with 1%–1.5% of vaporized isoflurane (Fluriso [VetOne, Boise, ID], M1000 [Supera Anesthesia Innovations, Clackamas, OR] or SurgiVet Vaporizer [Smiths Medical, Minneapolis, MN]) in oxygen in a Plexiglas (Evonik Performance Materials GmbH, Essen, Germany) chamber while continuously monitored. Their body temperature was maintained by using a temperature-controlled pad. Mice were subsequently emerged and kept at our facility for the remainder of the experimental time as described above. Tumors were measured blinded whenever possible on day 7, 10, and 13/14 unless the tumor size exceeded the permitted parameters by our animal protection protocol in which case the animals were euthanized before the end of the experiment to alleviate suffering. Tumor size was recorded on the day of assessment and/or on the day of euthanization.

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In Vitro Anesthetic Exposure.

For examining the impact of isoflurane on the tumor directly, B16 melanoma cells were exposed either for 2 hours to 1%–1.5% of isoflurane in an incubator chamber with continuous 5% carbon dioxide–air mix gas flow (Billups-Rothenberg, Inc, San Diego, CA) or to gas flow without isoflurane, and subsequently injected into male and female C57BL/6 WT mice.

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Statistics

Changes of tumor size over time and difference of such changes between experimental and control as well as between male and female mice were modeled using the generalized estimating equations (GEEs), in which treatment (isoflurane versus control, with control serving as the reference group), time (day 7, 10, and 14, with day 7 serving as reference level), sex (male versus female, with female serving as reference level), and their interactions formed the predictors. If there were significant interactions between 2 (or 3) factors, we assessed factor effects within levels of the interacting factor(s). If no significant interaction was present between any factors, we reported main effects for each factor collapsing over the other factors. The semiparametric GEEs require no distribution assumption, providing valid inference for a broad class of data distribution.7 All analyses were set at type I error α = .05.

Before fitting the GEEs, missing data were imputed for those that were euthanized due to large tumors by using the largest observed tumor size at assessments on the day of their euthanization (n = 1 in the control group and n= 4 in the isoflurane group for the WT in vivo experiments and n = 1 in the female control group in the in vitro experiments). Missing data due to any other reason unrelated to tumor growth were not imputed (n = 1 for 1 mouse in the male in vivo control group that was euthanized due to fighting injuries). We also imputed data for the mice that were euthanized using the last observation carried forward method. Because results from the 2 methods are quite similar, we only report the ones from the first approach.

The sample size of the male mice was able to detect a large between-group effect size (Cohen d = 0.88) with 80% power and a 2-sided α = .05. The actual effect size observed in our data was d = 0.96 for the difference between the isoflurane-exposed group and control group at day 14 within the male mice, slightly exceeding the detectable effect size. Power was actually larger than indicated by the power analysis because of modeling the repeated assessments using the GEEs.

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RESULTS

Overall, we observed that melanoma grew faster in male mice treated with isoflurane compared to control male mice. This effect was not seen in female mice. Given the potential impact of time, sex, and treatment group, we proceeded to perform a GEE analysis to formally test the impact of these parameters on tumor growth rate. The GEEs showed significant main effects (P < .001 for time, P = .003 for treatment groups, and P < .001 for sex), 2-way interactions (P < .05 for time-by-treatment, P < .001 for time-by-sex, and P = .016 for treatment-by-sex), and 3-way interaction (P = .038). As expected, tumor size increased significantly over time (significant increase from day 7 to day 10 [P < .001; confidence interval {CI}, 38.6–78.0] and to day 14 [P < .001; CI, 42.7–121.5]), but no significant difference in tumor growth was seen between the male and female mice in the control group (P = .37; CI, −40.4 to 25.1 at day 10, and P = .89; CI, −15.3 to 108.5 at day 14). Within the females, there was no significant difference between the isoflurane-exposed group and control group (P = .37; CI, −34.4 to 12.8 at day 10, and P = .89; CI, −46.3 to 53.1 at day 14). Within the male group, there was a significantly higher increase in the isoflurane group (P = .01; CI, 13.7–107.7 at day 10, and P = .042; CI, 2.9–159.1 at day 14), compared to the control group. The specific effect of isoflurane in male but not in female mice is indicated in the Figure, panel A, which shows observed tumor size (without any imputed data) for the control versus isoflurane-exposed mice. This effect of isoflurane on melanoma growth was absent in male mice lacking functional B and T cells (RAG1−/−; Figure, panel B) or in male mice lacking functional B, T, and natural killer cells (RAG2−/− × γc−/−; Figure, panel C), suggesting that the intact male immune system participated in translating the isoflurane exposure to a clinical phenotype.

Figure

Figure

To further corroborate this concept, the tumor cells were exposed to 2 hours of 1%–1.5% isoflurane in vitro before tumor injection, thereby limiting the anesthetic exposure to cancer cells and not immune cells or any other host cells. When applied to these in vitro data, the GEEs showed significant main effect of time (P < .001), but no significant difference in main effect of isoflurane treatment (P = .93), main effect of sex (P = .25), time-by-treatment interaction (P = .58), time-by-sex interaction (P = .26), treatment-by-sex interaction (.29), and time-by-treatment-by-sex interaction (.47). The lack of direct isoflurane effect on tumor growth shown in the Figure D for the observed tumor sizes for male and female mice across all time points again indicates an immune system–dependent mechanism rather than a direct anesthetic effect on melanoma cells.

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DISCUSSION

Our observational studies demonstrate that isoflurane impacts melanoma growth in male mice only when an intact immune system is present, while no such effects on tumor growth occurred in WT, immune-competent female mice. A direct effect of isoflurane on tumor cells was proposed previously,8 but this was not apparent on our melanoma model: tumor growth was not affected if the tumor was exposed to isoflurane before transplantation into WT males or females. The effect of inhalational anesthetics on tumor progression via its impact on the immune system has been studied and discussed in mice and humans.9 Mouse models have previously demonstrated a detrimental effect of inhalational anesthetics on tumor spread.6,10 Interestingly, published literature on the effect of anesthesia exclusively used male rodent models10 or does not specify sex of mice,6 and the effect of sex in this process has not been reported. Differences in male and female immune functions are well established, and our understanding of their clinical implications is expanding rapidly.4 Little attention, however, has been directed to examine sex-specific effects of anesthetics and how these relate to cancer progression. Our results indicate that male and female immune function may be affected differently by anesthetics, and further studies taking not only sex, but also phases of the female estrous cycle into account are warranted. We do not suggest that human women are not affected by the detrimental effects of anesthesia on the immune system, and an effect of inhalational anesthetics on cancer growth in human women should not be excluded. In fact, human clinical studies examining the benefits of intravenous anesthesia versus inhalational anesthesia found benefits in avoiding volatiles in both men and women, and additional studies describing the benefits of regional anesthesia demonstrate such effects in males as well as in females.11 When studying the effects of volatile anesthesia on the immune system and their subsequent effect on cancer growth, especially in murine models, careful consideration should be given to the sex of the species being studied and comparative experiments are warranted when working with both rodent and human samples.

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DISCLOSURES

Name: Angela Meier, MD, PhD.

Contribution: This author helped design and conduct the experiments; collect, assemble, and analyze the data; and write the manuscript.

Conflicts of Interest: A. Meier received consulting fees from Millennium Health unrelated to this work.

Name: Emilie T. E. Gross, PhD.

Contribution: This author helped with murine experiments and helped edit the manuscript.

Conflicts of Interest: None.

Name: Jan M. Schilling, MD.

Contribution: This author helped with experimental setup (isoflurane chamber), statistical analysis, and helped edit the manuscript.

Conflicts of Interest: None.

Name: Ruth Seelige, PhD.

Contribution: This author helped with the experiments and helped edit the manuscript.

Conflicts of Interest: None.

Name: Yujin Jung, BS.

Contribution: This author helped with the experiments.

Conflicts of Interest: None.

Name: Endi Santosa, MS.

Contribution: This author helped with the experiments.

Conflicts of Interest: None.

Name: Stephen Searles, BA.

Contribution: This author helped with the experiments.

Conflicts of Interest: None.

Name: Tuo Lin, BS.

Contribution: This author helped perform the statistical analysis of all the data.

Conflicts of Interest: None.

Name: Xin M. Tu, PhD.

Contribution: This author helped perform the statistical analysis of all the data, provide critical feedback, and edit the manuscript.

Conflicts of Interest: None.

Name: Hemal H. Patel, PhD.

Contribution: This author helped edit the manuscript and provide critical feedback.

Conflicts of Interest: None.

Name: Jack D. Bui, MD, PhD.

Contribution: This author helped design and interpret the experiments, provide mentorship and laboratory space, edit the manuscript, and provide critical feedback.

Conflicts of Interest: None.

This manuscript was handled by: Markus W. Hollmann, MD, PhD.

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