Faculty Bibliography

BACKGROUND:Our laboratory and others have previously demonstrated that tumors grow larger and are more easily established following laparotomy than after CO(2) pneumoperitoneum. The etiology of increased tumor growth after surgery is unknown. We hypothesized that, following laparotomy, a serum soluble factor(s) is generated that causes tumors to proliferate more rapidly. The purpose of the current study was to determine if in vitro tumor cells proliferate faster when incubated with serum from laparotomized mice than cells incubated with sera from mice who have undergone CO(2) pneumoperitoneum or anesthesia alone. METHODS:In the first experiment, female Balb/C mice (n = 84) were randomly divided into the following three groups: (a) control (AC), (b) CO(2) insufflation (INS), and (c) laparotomy (OPEN). The AC mice underwent no procedure. The INS group underwent CO(2) pneumoperitoneum at 4-6 mmHg for 20 min. The OPEN group had a midline incision from xiphoid to pubis. The serum of seven mice from each group were collected on postoperative days (POD) 1, 2, 4, and 7 via a cardiac puncture. The sera at each time point for each group were pooled. Twenty thousand C-26 colon cancer cells were incubated separately in growth media containing 10% mouse serum from each group (seven determinations/group) at each time point. In the second experiment, female Balb/C (n = 30) mice were divided into AC and OPEN groups. On POD4, sera were collected and pooled. Three separate studies were performed for the second experiment. In the first study, tumor cells were incubated with 10% AC sera or varying concentrations of OPEN mice sera (4-10%). In the second study, aliquots of sera from the OPEN group mice were then heated at 100 degrees C for 1 or 5 min. Tumors were then incubated separately in media with 10% AC, OPEN, or heated OPEN group sera. In the third study, aliquots of sera from the OPEN group mice were dialyzed against PBS through a 3.5-kD or an 8-kD dialysis membrane tubing for 24 h. Tumors were then incubated separately in media with 10% AC, OPEN, or dialyzed OPEN group sera. For both experiments, tumor proliferation was determined and compared between groups after 72 h of incubation. RESULTS:Tumor cells incubated with POD2 and POD4 sera from OPEN group mice proliferated twice as fast as those incubated with sera from either AC or INS group mice. The difference in proliferation was maximal on POD4 and started to decline by POD7. Proliferative activity from the OPEN group sera decreased significantly when heated for 1 min and was completely ablated after 5 min of heating. Proliferative activity from the OPEN group sera was completely ablated after dialysis. CONCLUSIONS:We conclude that there is a serum-soluble factor(s) present postoperatively that stimulates tumors to grow significantly faster after laparotomy. The mitogenic effect of laparotomized mice sera is dilutable. It is uncertain whether the factor is heat labile, since heating most likely destroys other necessary proteins in the sera. The size of the factor is undeterminable using the dialysis method. Further efforts to identify these factors are currently underway.

PURPOSE/OBJECTIVE:Our laboratory has previously shown that tumors are more easily established and grow larger after laparotomy vs. laparoscopy. The purpose of this study was to better characterize these differences in tumor growth by assessing tumor cell proliferation via the proliferating cell nuclear antigen assay, which has been shown to be a reliable marker of cellular proliferation. METHODS:Female C3H/He mice (N = 40) were inoculated intradermally in the dorsal skin with 10(6) cultured mouse mammary carcinoma cells <1 hour before interventions. Anesthesia control mice underwent no procedure. Laparotomy group mice had a midline incision from xiphoid to pubis that was closed after 20 minutes. Insufflation group mice underwent CO2 pneumoperitoneum (4-6 mmHg) for 20 minutes. On postoperative Day 6, tumors were excised from one-third of the mice in each group, and from the remaining mice on postoperative Day 12. Sections were made and stained immunohistochemically for proliferating cell nuclear antigen, and the proliferative index of each tumor was determined by taking the average of proliferating cell nuclear antigen-positive cells in five high-power fields (x450), counted in a blinded fashion with the aid of an optical grid. RESULTS:On postoperative Day 6, the mean proliferative index for the laparotomy group was significantly higher than those for both the insufflation (P < 0.04) and the control (P < 0.001) groups. Of note, the proliferative index of the insufflation group was significantly higher than that of the control (P < 0.01) group. Similarly, on postoperative Day 12, the mean proliferative index for the laparotomy group was significantly higher than for both the insufflation (P < 0.05) and the control (P < 0.005) groups. The proliferative index in the insufflation group was also significantly higher than that of the control (P < 0.04) group. CONCLUSIONS:We have demonstrated that there is a significantly higher rate of tumor cell proliferation with the mouse mammary carcinoma cell tumor line after laparotomy than after pneumoperitoneum or anesthesia alone at two postoperative times. Additionally, insufflation alone increases postoperative tumor cell proliferation but to a lesser extent than laparotomy. The mechanism underlying these findings is unclear.

BACKGROUND:Previous work has demonstrated that cell-mediated immune function in rats is better preserved after laparoscopic than open surgery. We have also shown that tumors are more easily established in mice and grow larger after sham laparotomy than after pneumoperitoneum. The purpose of this study is to determine if the functional status of the cell-mediated immune system influences postoperative tumor growth. METHODS:Immunocompetent (study 1) and T-cell deficient athymic (study 2) mice were injected with mouse mammary carcinoma cells in the dorsal skin. Mice then underwent either no procedure, midline laparotomy, or carbon dioxide pneumoperitoneum. Tumor masses on postoperative day 12 were compared. RESULTS:In immunocompetent mice, laparotomy group tumors were nearly twice as large as laparoscopy group tumors (p < 0.02), which were 1.5 times as large as control group tumors (NS). In the athymic model, however, differences between the sham laparotomy and pneumoperitoneum groups were lost (p > 0.5). Tumors grew much larger in the athymic control mice than in the immunocompetent control mice (p < 0.01). CONCLUSION/CONCLUSIONS:We conclude that T-cell function plays a significant role in host containment of mouse mammary carcinoma and in the mechanism of differences in tumor growth observed after laparotomy and pneumoperitoneum.

BACKGROUND:Many cellular elements responsible for wound healing are affected by laparotomy. The aim of this study was to evaluate the effects of laparotomy and CO2 pneumoperitoneum on wound healing. METHODS:Male Sprague Dawley rats were randomly assigned to one of three experimental groups. Anesthesia control rats underwent no procedure. Pneumoperitoneum group rats were insufflated with CO2 gas. Laparotomy group rats underwent a 7-cm midline laparotomy incision. The interventions were 30 min long. For the incisional study (n = 30), a 4-cm dorsal full-thickness skin incision was made on each rat and then closed with staples. On postoperative days 7 and 14, an equal number of rats were sacrificed from each group, and wound tensile strength measurements were performed. For the excisional study (n = 45), each group of 15 rats underwent a 2-cm diameter circular dorsal full-thickness skin excision. Blinded measurements of wound area were performed every other day until wounds closed. RESULTS:Wound tensile strength values were not significantly different among experimental groups at either time point. The study had a power of 80% to find a 30% difference at POD 7 and a power of 80% to find a 23% difference at POD 14 to a confidence level of p < 0.05. Wound contraction data from the excisional model were analyzed with the Generalized Estimation Equations statistical approach. When we modeled the treatment group as a covariate, no statistical difference was found between groups, demonstrating equal slopes across time. CONCLUSIONS:From the results of these studies, we conclude that wound healing in this model is not significantly diminished following laparotomy or peritoneal insufflation, as compared to anesthesia control.

The results from the majority of the reviewed studies support the hypothesis that abdominal surgery, performed via either a large incision or CO2 pneumoperitoneum, systemically encourages tumor growth in the postoperative period. A full laparotomy incision appears to have a significantly greater effect than CO2 pneumoperitoneum on postoperative tumor growth. Whether the large tumor observed in the surgical groups are the result of increased tumor cell proliferation or diminished tumor cell death remains unclear. There is some evidence pointing to both mechanisms. The loss of the postoperative tumor growth differences between the open and pneumo animals in the athymic mouse experiment suggests that cell-mediated immune function plays a role in tumor containment. The proliferation study results, however, suggest that other stimulatory influence(s) are also at work. Clearly, much research needs to be done regarding the etiology of these tumor growth differences. Other tumor cell lines need to be studied, and investigations regarding tumor growth in an intra-abdominal location need be performed as well. This body of research suggests that the manner in which the surgeon gains access to the abdominal cavity may have an impact on the propensity of tumor cells to implant, survive, and grow in the period immediately after surgery. If true, this may be the most compelling justification for the use of minimally invasive techniques for the curative resection of malignancies. However, it remains to be proven that human tumors will demonstrate differences in tumor growth similar to those noted in some of these animals models. Furthermore, it is not all clear that slight differences in tumor growth postoperatively will translate into significant differences in long-term survival or recurrence rates. At first glance, the existence of port-site tumors would appear to contradict totally the conclusions of many studies discussed in this synopsis. If laparoscopic methods are associated with decreased rates of tumor growth and establishment, then why do port-site tumors form? This is a complex issue calling for discussion that goes far beyond the scope of this article. However, several brief comment on this topic follow. The etiology of port tumors is unknown, although traumatization of the tumor during mobilization, resection, or removal is likely to play a significant role in the liberation of tumor cells from the primary. A relatively small protective benefit, in terms of slower tumor growth rates in laparoscopic patients, will likely not be sufficient to prevent a large inoculum of viable tumor cells in an abdominal wound from establishing a metastasis. Furthermore, as suggested earlier, the systemic effects on tumor growth may be different from the local (i.e., intra-abdominal or abdominal wound) effects. Finally, the true incidence of port tumors remains unknown. It has not been definitively established that the laparoscopic wound tumor incidence is significantly higher than the open rate, although this is the assumption of most surgeons. Several relatively large recently published laparoscopic series have reported port tumor incidences of 0 to 1.2%, which is in the same "ballpark" as the 0.6 1.0% abdominal wound tumor incidences mentioned in several open colectomy series. Clearly, much more research in this area is needed to understand port tumors better and to reconcile the port tumor results with the systemic tumor growth benefits that may be associated with minimally invasive methods.

BACKGROUND:Surgery can suppress immune function and facilitate tumor growth. Several studies have demonstrated better preservation of immune function following laparoscopic procedures. Our laboratory has also shown that tumors are more easily established and grow larger after sham laparotomy than after pneumoperitoneum in mice. The purpose of this study was to determine if the previously reported differences in tumor establishment and growth would persist in the setting of an intraabdominal manipulation. METHODS:Syngeneic mice received intradermal injections of tumor cells and underwent either an open or laparoscopic cecal resection. In study 1, the incidence of tumor development was observed after a low dose inoculum; whereas in study 2, tumor mass was compared on postoperative day 12 after a high-dose inoculum. RESULTS:In study 1, tumors were established in 5% of control mice, 30% of laparoscopy mice, and 83% of open surgery mice (p < 0.01 for all comparisons). In study 2, open surgery group tumors were 1.5 times as large as laparoscopy group tumors (p < 0.01), which were 1.5 times as large as control group tumors (p < 0.02). CONCLUSION/CONCLUSIONS:We conclude that tumors are more easily established and grow larger after open laparoscopic bowel resection in mice.

BACKGROUND:Reports of port site tumor recurrences after laparoscopic-assisted resection of colon tumors have raised concerns about the safety of laparoscopic cancer surgery. Tumor cell suspension studies in animals have implicated the CO2 pneumoperitoneum (pneumo) in the etiology of port tumors. Unfortunately, in several ways, the cell suspension model is unrealistic and does not permit assessment of how tumor cells become liberated from the primary tumor. The purpose of this study was to establish a more realistic splenic tumor model and to determine the relative importance of the CO2 pneumo and excessive surgical manipulation in the development of port site and incisional tumor recurrences. METHODS:Splenic tumors were established in female Balb/C mice (n = 134) via a subcapsular injection of 10(5) C-26 colon adenocarcinoma cells (0.1 ml volume) via a left-flank incision at the initial procedure. Ten days later, the animals were reexplored via a 1-cm left subcostal incision. Those with isolated splenic tumors (95%) were randomized into one of four groups: (a) control, (b) CO2 pneumo, (c) crushed tumor, or (d) crushed tumor with pneumo. Ports were placed in the left lower, right lower, and right upper quadrants of each mouse. In groups 1 and 2, the mice underwent a meticulously performed splenectomy; in groups 3 and 4, the tumor capsule was crushed intraabdominally prior to splenectomy. In groups 1 and 3, the subcostal incision was closed and the ports were removed after 15 min of anesthesia. Following splenectomy, group 2 and group 4 mice underwent closure of the subcostal incision and a 15-min CO2 pneumo (4-6 mm Hg) after which the ports were removed. Twelve days later, the mice were killed and examined for abdominal wall tumor implants. RESULTS:Significantly more animals in group 3 (crushed tumor) developed port site and incisional tumors than those in group 1 (control) (p < 0.002 for both comparisons). The same results were found when group 4 (crush plus pneumo) was compared to group 2 (pneumo) (p < 0.002 for both comparisons). Regarding the port wounds, when the ports are considered individually (number of ports with tumors/total number of ports for each group), there were significantly more port tumors in the two crush groups than in the noncrush groups. No significant differences were noted when the port site and incisional tumor rates for group 1 (control) and group 2 (pneumo) were compared or when the results for group 2 (crush) and group 4 (crush pneumo) were compared. CONCLUSIONS:A splenic tumor model was successfully established. When compared to meticulous technique, purposefully traumatic handling of the splenic tumor before resection resulted in significantly more port wound and incisional tumors. In contrast, the addition of a pneumo after splenectomy did not significantly influence the incidence of port tumors in either the "good" or the "poor" technique groups. These results suggest that surgical technique plays a larger role in the development of port site tumors than the CO2 pneumoperitoneum.

BACKGROUND:We investigated the ability of pressurized CO2 gas to aerosolize B16 melanoma (B16) tumor cells in an in vitro model. METHODS:The experimental apparatus consisted of an 18.9-L plastic cylindrical vessel and a compliant latex pouch was attached to the top. Two 5-mm ports penetrated the vessel; insufflation and desufflation were carried out through them. A culture dish containing 20 million B16 cells in liquid culture media was placed at the base within the container. In the first experiment, the vessel was insufflated with CO2 gas to a static pressure of 15 or 30 mm Hg with the outflow port closed. After 10 min, the outflow port was opened and the gas was desufflated through a collecting device containing sterile culture medium. In a second experiment, a continuous flow of CO2 through the vessel was maintained after a pressure of 15 or 30 mm Hg was established. A total of 10 L CO2 was cycled through the vessel. In both experiments, 24 determinations were carried out at each pressure. Each experimental culture dish was microscopically scanned for 2 weeks for the presence of tumor cells. The third and fourth experiments tested for the presence of aerosolized nonviable tumor cells in the expelled gas. Using the model described above, after 10 mins of 30 mm Hg static pressure, the CO2 gas was expelled directly onto a glass slide and cytofixed. Alternately, after 10 mins at 30 mm Hg static pressure, the gas was expelled through a saline-filled Soluset (Abbott Laboratories), centrifuged, and the residue cytofixed onto a glass slide. Each of the five slides per experiment were examined microscopically for the presence of cells. RESULTS:In the first and second experiments, no cells or growth were observed in any of the 96 experimental dishes. In experiments three and four, no cells were detected on any of the slides. CONCLUSIONS:It was not possible with this model to aerosolize tumor cells in a pressurized CO2 environment. Our results suggest that aerosolization of tumor cells is not the mechanism of port site recurrences after laparoscopic surgery for malignant disease.

PURPOSE/OBJECTIVE:Mouse mammary carcinoma tumors are established more easily and grow larger after sham laparotomy and open bowel resection than after CO2 pneumoperitoneum and laparoscopic-assisted bowel resection. The purpose of this study was to determine whether similar differences in tumor growth would be found when sham laparotomy and pneumoperitoneum were compared for the colon-26 mouse adenocarcinoma and B-16 mouse melanoma tumor lines. METHODS:In all three studies, a high-dose injection of tumor cells was used, which resulted in tumors in almost all control mice. In Study 1, female BALB/C mice (n = 127) were injected intradermally in the dorsal skin with 10(6) colon-26 cells in a 0.1-ml volume before interventions. In Study 2, female C57 BL/6 mice (n = 140) were inoculated similarly with 10(6) B-16 melanoma cells. Study 2 consisted of three separate trials conducted on different days. Study 3 was performed because considerable differences in mean tumor size were observed in each of these trials. In Study 3, the B16 experiment was repeated with a larger n (n = 82) on a single day. In each study, after tumor cell injections, mice were randomly assigned to one of three groups: 1) anesthesia control (no procedure); 2) full laparotomy (4-cm midline incision x 20 minutes, staple closure); or 3) CO2 pneumoperitoneum (4-6 mmHg X 20 minutes). Tumors were excised and weighed on postoperative day 12. RESULTS:In Studies 1 and 3, mean tumor sizes of the laparotomy groups were significantly larger than both the control group and pneumoperitoneum group lesions (P values by Student's t-test). In Study 2, laparotomy group tumors, although significantly larger than control group lesions, were not significantly larger than pneumoperitoneum group tumors. For all three studies, there was no significant difference between mean tumor sizes of the pneumoperitoneum and control groups. CONCLUSION/CONCLUSIONS:Both colon-26 adenocarcinoma and B-16 melanoma tumors grow larger after laparotomy than after pneumoperitoneum in a murine model. The mechanism of these postoperative tumor growth differences remains to be elucidated.