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Surgical stress has been suggested to facilitate the growth of pre-existing micrometastases as well as small residual tumor postoperatively. The purpose of this study was to examine the effects of surgical stress following either mastectomy or laparotomy on ovarian cancer growth and to determine underlying mechanisms responsible for increased growth. In both HeyA8 and SKOV3ip1 models, the mice in the laparotomy and mastectomy groups had significantly greater tumor weight (P < 0.05) and nodules (P < 0.05) compared to anesthesia only controls. There was no increase in tumor weight following surgery in the β-adrenergic receptor (ADRB)-negative RMG-II model. To block the influence of sympathetic nervous system activation by surgical stress, we used propranolol infusion via Alzet pumps. Propranolol completely blocked the effects of surgical stress on tumor growth, indicating a critical role for ADRB signaling in mediating the effects of surgical stress on tumor growth. In the HeyA8 and SKOV3ip1 models, surgery significantly increased microvessel density (CD31) and VEGF expression, which were blocked by propranolol treatment. These results indicate that surgical stress could enhance tumor growth and angiogenesis, and β-blockade might be effective in preventing such effects.
Surgical cytoreduction is a critical component of cancer therapy for many tumor types including ovarian cancer. Some surgeons have observed rapid growth shortly after primary surgery. This observation has been further supported by several models that have demonstrated increased tumor growth and metastatic spread following surgery (1-6). There are several potential mechanisms for the effects on tumor growth by surgical stress including shedding of tumor cells due to physical manipulation (7, 8), a drop in the level of anti-angiogenic factors (9), local and systemic release of growth factors or cytokines (10), and suppression of cell mediated immunity (11). However, the mechanisms by which surgical stress promotes tumor growth are not fully understood.
To date, no reliable screening methods to detect ovarian cancer at an early stage have been developed. Thus, about 70% of women with newly discovered ovarian cancer have advanced disease at the time of diagnosis (12). Nevertheless, a complete clinical remission can be achieved in 80% of these patients with the use of maximal surgical cytoreduction and platinum-based combination chemotherapy (13). Unfortunately, upwards of 75% of those with complete clinical response will develop recurrent disease. Re-operation with secondary tumor resection has become an option for many ovarian cancer patients. Therefore, women with ovarian cancer will undergo several surgical procedures throughout the course of their disease. Given the paucity of information regarding the effects of surgery on ovarian cancer growth, here we examined the effects of laparotomy and extra-peritoneal surgery on ovarian cancer growth and angiogenesis using orthotopic animal models.
The ovarian cancer cell lines HeyA8, SKOV3ip1, and RMG-II were cultured in RPMI 1640 supplemented with 10% (RMG-II) to 15% (HeyA8 and SKOV3ip1) fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA) (14-16). For in vivo injections, the cells were washed twice with PBS, detached with 0.1% EDTA, centrifuged at 1,100 rpm for 7 min at 4°C, and reconstituted in serum-free HBSS (Life Technologies, Carlsbad, CA) at a concentration of 1.25 × 106 cells/mL (HeyA8), 5.0 × 106 cells/mL (SKOV3ip1), or 20 × 106 cells/mL (RMG-II) for 200 μL i.p. injection volume per one mouse. Only single-cell suspensions with >95% viability, as determined by trypan blue exclusion, were used for in vivo injections. All experiments were performed using cells grown to 60% - 80% confluence, and all cell lines were routinely tested to confirm absence of Mycoplasma.
Female athymic mice (NCr-nu) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center. The mice were housed and maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with the current regulations and standards of the U.S. Department of Agriculture, the U.S. Department of Health and Human Services, and the NIH. All studies were approved and supervised by the University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee. The mice used in these experiments were 8 to 12 weeks old. Mice (n = 10 per group) were monitored daily for tumor development and postoperative complications and were sacrificed on day 25-30 (HeyA8), day 35-40 (SKOV3ip1), or day 40-50 (RMG-II), or when any of the mice seemed moribund. Total body weight, tumor incidence and mass, and the number of tumor nodules were recorded. Tumors were fixed in formalin and embedded in paraffin or snap frozen in optimal cutting temperature compound (Sakura Finetek USA, Inc.) in liquid nitrogen.
For surgery, animals were exposed to an experimental mastectomy or laparotomy under isoflurane inhalation (Baxter, Deerfield, IL, USA) anesthesia. The surgical procedure for mastectomy consisted of a midline chest wall skin incision and removal of right mammary tissues from the chest wall. The skin of chest wall was closed with 3-4 surgical clips. The laparotomy consisted of a 4 cm midline abdominal incision, followed by the externalization of intestines for a period of 4 min as described previously by Ben-Eliyahu and colleagues (17, 18). During the laparotomy, the small intestine was rubbed with two saline-soaked cotton Q-tips in four locations to simulate a surgical procedure. The intestine was then returned to the abdominal cavity, irrigated with saline, and the abdominal wall was closed with surgical clips (19, 20).
To examine the influence of β-adrenergic signaling for surgical stress, we used the nonspecific β-adrenergic receptor (ADRB) antagonist s-propranolol hydrochloride with 2 mg/kg/d (Sigma, St. Louis, MO) and ALZET mini-osmotic pumps (Model 2004, DURECT Corporation, Cupertino, CA, USA) (21, 22). We inserted the mini-pumps containing PBS or propranolol on the nape of neck 7 days before surgical stress (23).
To quantify angiogenesis, microvessel density (MVD) was ascertained by counting CD31-positive vessels as described previously (14). In brief, 8-μm sections of fresh frozen samples were fixed and incubated with anti-mouse CD31 (1:800, PharMingen) at 4°C overnight. Immunohistochemical procedures of VEGF and PCNA were done as described previously (24). In brief, for detecting VEGF and PCNA immunoreactivity, formalin-fixed, paraffin-embedded serial sections were deparaffinized by sequential washing of xylene followed by descending grades of ethanol. Depending on the antibody used, antigen retrieval was achieved by either citrate buffer (pH 6.0) in a steamer (PCNA) or pepsin in a 37°C humidified incubator (VEGF). Endogenous peroxidases were blocked with 3% H2O2 in methanol. Nonspecific proteins and exposed epitopes were blocked with 5% normal horse serum/1% normal goat serum, and slides were incubated at 4(C overnight with the respective primary antibody at the following dilutions: 1:100 VEGF (Santa Cruz Biotechnology) or 1:50 PCNA (Dako). After PBS washing, the appropriate secondary antibody was applied for 1 h at room temperature. Visualization was achieved with 3,3’-diaminobenzidine chromagen. All counterstaining was done with Gill’s hematoxylin (Sigma-Aldrich).
To quantify MVD, five random fields at ×100 magnification per slide were examined for each tumor (one slide per mouse, five slides per each treatment group) and the number of microvessel per field was counted by two investigators (JWL, HSK) in a blinded fashion. A single microvessel was defined as a discrete cluster or single cell stained positive for CD31 with the presence of a lumen (16). To quantify PCNA expression, the number of PCNA-positive cells and the total number of tumor cells were counted in five random fields at ×100 magnification followed by calculation of the percentage of positive cells (16).
To check the cytokines and chemokines in serum of mice, we used multiplexing xMAP technology to analyze mouse Cytokine/Chemokine 17 Plex Panel (Bio-Rad Laboratories, Hercules, CA) that included G-CSF, GM-CSF, IFN-g, IL-1a, IL-1B, IL-6, IL-10, IL-12 (p70), IL-15, MCP-1, M-CSF, MIG, MIP-1a, MIP-1B, MIP-2, RANTES, and TNFa. Blood samples were taken from hearts during inhalation anesthesia to minimize further stress. Serum samples were obtained from mice with control (anesthesia with isoflurane inhalation alone), 6 hr, 1 day, 3 days, and 5 days later after laparotomy (n=2 of each group). Multiplex xMAP assays were performed according to manufacturer’ protocols as previously described. Samples were analyzed using the Bio-Plex suspension array system (Bio-Rad Laboratories). For each analyte, 100 beads were analyzed and means were calculated. Analysis of experimental data was performed using four-parametric-curve fitting to the standard analyte curves
Continuous variables were compared with the Student’s t test or ANOVA if normally distributed and the Mann-Whitney rank sum test if distributions were non-parametric using Statistical Package for the Social Sciences (SPSS, Inc.). A P value of < 0.05 was considered statistically significant.
To mimic the effects of surgery, we performed a laparotomy on mice four days after tumor cell injection. In addition, to account for possibility of local effects, we also performed a mastectomy on a separate group of animals. In the ADRB-positive HeyA8 and SKOV3ip1 models, laparotomy resulted in a showed 370% (HeyA8) and 263% (SKOV3ip1) increase in tumor weight (P < 0.05, both) (Fig. 1). Mastectomy also increased tumor weight by 203% (HeyA8) and 226% (SKOV3ip1, P < 0.05) compared with anesthesia only controls (Fig. 1). Similarly, the number of tumor nodules was also significantly greater following a laparotomy in both models (P < 0.05, data not shown). Since the sympathetic nervous system (SNS) is known to be activated during surgery (18, 25), we next asked whether tumor cells with ADRB expression might be responsible for the increased tumor growth. To address this question, we first utilized an ADRB-null model using the RMG-II cells (23). In this model, there was no difference in tumor weight between the control and laparotomy groups (0.32 vs. 0.28g, P = 0.42, Fig. 1). There were no significant differences in the body weight of animals between the groups, suggesting that surgery did not adversely affect the overall well-being the animals. Next, we examined whether a β-blocker would abrogate the increased tumor growth. For these experiments, we used the SKOV3ip1 model and inserted a mini-osmotic pump containing propranolol into the mice to deliver the drug continuously. There were no significant complications associated with pump insertion into the mice. Fig. 2 shows that either mastectomy or laparotomy resulted in significantly increased tumor weight compared with controls in the group with PBS containing pumps. Treatment with propranolol had no significant effect on tumor growth in the anesthesia only controls. However, treatment with continuous propranolol infusion was able to block the increased tumor growth observed in response to either mastectomy or laparotomy (Fig. 2).
On the basis of our prior findings related to the effects of stress hormones on tumor growth and angiogenesis (23), we considered whether tumor angiogenesis might be stimulated following surgery. When we examined the MVD in mice tissues, surgical stress resulted in significantly greater MVD counts (P < 0.05) in the HeyA8 (Fig. 3A) and SKOV3ip1 (Supplementary Fig. S1) models. But, these findings were not present in the ADRB-null RMG-II model (Fig. 3B). Moreover, surgical stress showed increased VEGF staining compared with control in HeyA8 (Fig. 3A) and SKOV3ip1 (Supplementary Fig. S1) models. There was no difference in VEGF expression in the RMG-II model (Fig. 3B). We also examined the effects of surgery on tumor cell proliferation using PCNA staining. In the HeyA8 (Fig. 3A) and SKOV3ip1 (Supplementary Fig. S1) models, positive staining of PCNA expression was significantly increased in mastectomy and laparotomy compared with control (P < 0.05, both). But in RMG-II model, there was no significant difference between two groups (Fig. 3B).
To evaluate the effect of propranolol for angiogenesis in surgical stress on in vivo tumor growth, we performed the immunohistochemistry for angiogenesis markers in tumor samples. Increased MVD and VEGF by surgery were blocked by propranolol (Fig. 4).
It is likely that other cytokines may be activated during the physiological response to surgery. Therefore, we examined a panel of 17 cytokines and chemokines including G-CSF, GM-CSF, IFN-γ, IL-1a, IL-1B, IL-6, IL-10, IL-12 (p70), IL-15, MCP-1, M-CSF, MIG, MIP-1a, MIP-1B, MIP-2, RANTES, and TNFα in mouse serum after surgical stress. Serum samples were obtained from mice with control (anesthesia with isoflurane inhalation alone), 6 hours, 1 day, 3 days, and 5 days later after laparotomy (n=2 of each group). Concentrations of G-CSF, IL-1a, IL-6, and IL-15 were significantly higher during the perioperative period (Fig. 5).
The key findings from our study are that increased angiogenic processes mediated by surgical stress could promote ovarian cancer growth in vivo. This increase in tumor growth was abrogated by blocking ADRB-mediated angiogenesis and peri-operative use of propranolol could have preventive effects for the surgical stress-induced tumor growth.
Surgery, as a stressor, could increase the risk of postoperative metastases and their mechanisms can be divided as local or systemic effects. Some reports have suggested that the surgical wound has important roles in surgical stress as the source of the tumor-stimulating factors (10, 26, 27). These reports suggested that growth factors or cytokines released by wounding could contribute to creating a suitable environment for tumor growth (10). Wound healing and tumor progression both involve processes of cell proliferation, inflammation, and angiogenesis. In contrast to local effects, Ben-Eliyahu et al. suggested that surgical stress results in activation of the hypothlamic-pituitary-adrenal axis and the SNS and influences the immune system, especially surgery-induced suppression of natural killer (NK) cell activity (28, 29). They reported that marginating pulmonary NK activity was suppressed by surgery and a combined use of a β-blocker and a prostagladin-synthesis inhibitor restored NK function (18).
We recently reported that chronic (immobilization) stress promotes tumor development and progression via β-adrenergic activation of the cAMP-PKA signaling pathway in an orthotopic murine model of ovarian carcinoma (23). In addition to chronic stress, understanding the mechanisms responsible for mediating the effects of surgical stress on human tumor tissues is crucial for determining the full impact of this stressor on tumorigenesis and for devising effective interventions.
Our results indicate that surgical stress likely results in systemic sympathetic responses that promote tumor growth, in part, by activating ADRB on tumor cells. Biologically, the acceleration in tumor growth is mediated by increased angiogenesis resulting from increased VEGF production. This premise is supported by our finding that even surgery at an extraperitoneal site (i.e., mastectomy) promoted intraperitoneal tumor growth (Fig. 1). Our findings related to the efficacy of a beta-blocker in abrogating the surgically-induced tumor growth suggest that such an approach may hold utility for cancer patients undergoing surgery (Fig. 2). Our findings regarding activation of angiogenesis by surgical stress are consistent with work from our and other groups demonstrating the effects of stress on angiogenic factors (Fig. 3 and and4)4) (23, 30).
Although we focused on sympathetic influence and angiogenesis in the current study, it is possible that other mechanisms might be operative as well in mediating the effects of stress. Therefore, we analyzed several cytokines and chemokines in serum following surgery (Fig. 5). Indeed, several cytokines were elevated, which might also be involved in the effects of surgical stress on tumor growth (31, 32).
In summary, increased angiogenic processes mediated by surgical stress promote tumor growth in vivo. These effects can be effectively blocked by a β-blocker such as propranolol. These findings could have significant implications for the perioperative management of cancer patients.
Immunohistochemistry for CD31, VEGF, and PCNA in SKOV3ip1 model. All photographs were taken at original magnification ×100. The bars in the graphs correspond sequentially to the labeled columns of images at left. Error bars represent s.e.m. *P < 0.05
The authors would like to thank Dr. Robert Langley for helpful discussion. We also thank Donna Reynolds for assistance with immunohistochemistry.