JOURNAL
OF SURGICAL
RESEARCH
53,
445-449
(19%)
Substance P Increases in Vitro Lymphokine-Activated-Killer (LAK) Cell Cytotoxicity against Fresh Colorectal Cancer Cells’ HELENE FLAGEOLE, M.D., Departments
of Pathology
and Surgery,
MARY SENTERMAN, M.D.,
AND JUDITH L. TRUDEL, M.D.’
The Montreal
McGill
General
Hospital,
University,
Montreal,
Quebec,
Canada
Submitted for publication November 27.1991 by lung cancer. In females, it is only preceeded by lung and breast cancers. Colon cancer is responsible for 20% of all deaths caused by cancer [ 11. Colon cancer has been resistant to the conventional modes of adjuvant therapy (radiotherapy and chemotherapy) until recently when the combination of 5-FU and Levamisole seemed to show some promise [21. Immunotherapy is a more innovative and experimental form of cancer treatment. Adoptive immunotherapy is a form of immunotherapy which consists of transferring to a tumor-bearing host active immunologic reagents, such as lymphokine-activated-killer (LAK) cells, with anti-tumor activity [3]. The LAK cell is an immune cell which becomes activated in the presence of interleukin-2 (IL-2). Rosenberg et al. [6] can be credited for performing most of the initial studies involving LAK cells and IL-2 both in animals and humans: experiments were done using LAK cells alone [5], others used IL-2 alone [8], and yet others used both agents [4, 6, 71 against a wide variety of tumors. The best responses are obtained when both LAK cells and IL-2 are infused together [4] and the response rate is very dependent on the type of tumor harbored by the host. In humans, the best responses occured in paEffl?CtO~ Target N 5:l 1O:l 25:l 50: 1 tients with renal cell carcinoma and melanoma [4-81. LAK HT-29 7 2.8 k 0.5 11.6 k 1.0 12.3 + 2.3 1’7.6 k 4.2 Colon cancer exhibits only minimal response to these LAK HT-29 11 1.2 * 0.5 4.1 t 0.9 9.3 i 1.0 16.9 k 3.1 highly competent immune cells [4, 6-91. Most clinical +sp LAK Fresh Ca 11 6.6 + 1.6 12.6 k 3.3 36.3 5 6.3. 69.4 * 9.58 studies utilized lymphocytes isolated from the periph+ SP eral blood to generate LAK cells. In a previous study LAK Fresh Ca 11 0.0 + 0.0. 0.1 k 0.18 0.1 f 0.1* 0.5 t 0.5’ [lo], we postulated that lymphocytes from the intestinal lamina propria could represent a more relevant source of We conclude that substance P significantly increases LAK cell cytotoxicity against fresh colon cancer cells, immune cells. Colon cancer cells turned out to be as rebut not against cultured C&S. 0 1992 Academic PMS, 1~. sistant to lysis by lymphocytes from an intestinal source as they had been to lymphocytes isolated from the peripheral blood [lo]. Such resistance could be attributable to insufficient LAK cell cytotoxicity against colon INTRODUCTION cancer cells. The purpose of our study was to enhance Colon carcinoma is a devastating disease. In males, it LAK cell cytotoxicity by hormonal manipulation. We ranks second as a cause of cancer death, preceeded only chose to use substance P to augment LAK cell cytotoxicity, because substance P stimulates human T lympho’ Presentedat the Annual Meetingof the Associationfor Academic cytes proliferation in vitro and because high-affinity Surgery,ColoradoSprings,CO, November20-23,1991. hormonal receptors for substance P are present on lym’ To whom reprint requestsshouldbe addressed at Montreal General Hospital,1650CedarAve., Room9802L.H., Montreal, Quebec, phocytes [ 11-141, suggesting a physiologic basis for horCanadaH3G lA4. monal interaction. Gut-associated lymphoid cells are modulated by several gut hormones. We postulated that lymphokine-associated-killer (LAK) cell cytotoxicity of lymphocytes isolated from the gut mucosa may be increased by substance P (SP). Intestinal lamina propria mononuclear cells (LPMC) and colonic cancer cells were isolated from operative specimens by successive mechanical and enzymatic dissociation methods. Effector LAK cells were induced by culturing LPMC with recombinant interleukin-2 at a concentration of 250 U/ml. Substance P (loM) was added to the culture medium. Targets consisted of fresh colon cancer cells, HT-29 (cultured human colon cancer cell line), and control cell lines. After 4 days of incubation, cytotoxicity was measured using a 4-h %r release assay. LAK cells alone showed moderate cytotoxicity against HT-29 and none against fresh colon cancer cells. LAK cells generated in the presence of substance P showed moderate cytotoxicity against HT-29 and strong cytotoxicity against fresh colorectal cancer cells. The percentage of cytotoxicity + SEM at various effector to target ratios was [(*) denotes P < 0.05 compared with above]:
445
Copyright0 1992 All
rights
of reproduction
oozz-4804/92 $4.00 by Academic Press, Inc. in any form reserved.
446
JOURNAL
MATERIALS
AND
OF
SURGICAL
RESEARCH:
METHODS
Patient Population Freshly resected intestinal tissue was obtained from operative specimens of patients undergoing resection for cancer or benign colonic conditions. Lamina propria mononuclear cells (LPMC) were isolated from 19 patients. Ten of them had colorectal cancer: five had sigmoid lesions, three had rectal lesions, and two had cecal tumors. Three patients had surgery for excision of a villous tumor or multiple polyps, three had diverticular disease, one had Crohn’s disease, one had ulcerative colitis, and one had intractable lower gastrointestinal hemorrhage. The patients who were operated on for cancer also had part of the resected specimen utilized for isolation of fresh colon cancer cells.
VOL.
53, NO.
5, NOVEMBER
1992
Induction of LAK Cell Activity Purified LPMC were cultured in RPMI-1640 (GIBCO) with 10% human AB serum (GIBCO), 1.5% Hepes buffer (GIBCO), 1% gentamicin (GIBCO), and 2.5% penicillin-streptomycin-fungizone (PSF; GIBCO). Recombinant human IL-2 (Cetus Corp., Belmont, CA) was added at a concentration of 250 U/ml. The cells were cultured at a concentration of 0.5 X 106/ ml in upright tissue culture flasks at 37°C in 5% CO,95% humidified air atmosphere for a period of 4 days. Hormonal Enhancement of LPMC
Substance P (Peninsula Labs, San Carlos, CA) was added to the tissue flasks simultaneously with induction of LAK activity. A concentration of lop5 M was selected after going through the following determinations: in the gastrointestinal tract, concentrations as low as 10-l’ M exert a physiologic effect [27]. This concentration was therefore used as the starting point and was increased Isolation of LPMC and Fresh Cancer Cells until a response was obtained. Therefore, 10e5M repreLPMC were isolated using Fiocchi’s modification [ 151 sents the threshold dose in the dose-response curve; it of the original technique described by Bull and Bookwas selected over a higher dose because it is already pharman [ 161. A piece of normal mucosa was freed of mucus macological. Other in vitro studies using substance P as and epithelial cells in sequential steps using 75 mg of an immunomodulator used concentrations ranging bedithiothreitol (DTT; Sigma Chemical Co., St. Louis, tween 10-l’ and lop6 M [ll]. MO) in 50 ml of calcium and magnesium-free Hank’s balanced salt solution (CMF-HBSS; GIBCO) for 30 min Target Cell Lines and 37.5 mg of ethylenediaminetetraacetic acid (EDTA; Several target cell lines were used for cytotoxicity asSigma) in 90 ml of CMF-HBSS for two periods of 90 says. Daudi is a lymphoid cell line used as a LAK-sensimin. Overnight digestion in CMF-HBSS containing tive internal control. The cultured human colorectal cell 0.01% collagenase type III and 0.01% deoxyribonuclease line HT-29 was maintained in duplicate in a solution of (both from Worthington Diagnostic Systems, Inc., FreeRPMI-1640 supplemented with 10% fetal calf serum hold, NJ) was performed. The crude cell suspension was (FCS; GIBCO) and 1% PSF and split every third day. It purified over a Ficoll-Hypaque gradient (Pharmacia). was used as one of the experimental targets. HT-29 cells The isolated cells were almost exclusively LPMC (90are known to be slightly LAK sensitive [lo]. Fresh colon 95% purity, verified by microscopic examination), Viacancer cells isolated from surgical specimens were used bility of the isolated cells was verified using the trypan as the other experimental target. blue exclusion method (80-85% viability, by microsCOPY).
When the resection was done for malignant disease, fresh colon cancer cells were also isolated. The procedure is a combination of the methods of Brattain et al. [ 171 and Keder et al. [181. Nonnecrotic malignant tissue was rinsed in CMF-HBSS and minced in l- to 3-mm fragments. The fragments were placed in a solution of CMF-HBSS containing hyaluronidase (0.5 mg/ml; Sigma), collagenase type III, and DNAase (2 and 0.6 mg/ ml, respectively). This digestion process was repeated approximately five times. The cell suspension from the first digestion period was discarded to reduce contamination with red blood cells, dead cells, and debris. The cells were collected by centrifugation and further purified over a 20% Percoll gradient (Pharmacia). The isolated cells were usually 8590% viable (verified by microscopy using the trypan blue exclusion method) and were either used immediately or frozen in liquid nitrogen.
Microcytotoxicity
Assay
Target cells (3-5 million) were harvested and centrifuged (1700 rpm X 5 min.). All but 2 ml of supernatant was aspirated and 200 &i of sodium chromate (51Cr; ICN Chemicals) was added. The cells were incubated at 37°C for 90 min and agitated periodically. At the end of this period, the cells were washed three times and plated at lo4 cells/well in microtiter plates. The effector LAK cells, cultured with or without substance P, were aliquoted in triplicate to the wells containing target cells. Various effector to target (E:T) ratios were obtained by serial dilution of the effector cells added to a fixed number ( 104) of 51Cr-labeled target cells/well. Spontaneous and maximum release was determined by addition of culture medium and 1% sodium dodecyl sulfate (SDS; Sigma), respectively. The microtiter plates were spun at 500 rpm for 3 min, followed by incubation at 37°C in a
FLAGEOLE,
SENTERMAN,
AND
TRUDEL:
SUBSTANCE
447
P INCREASE
colon cancer cells were lysed by LAK cells cultured with substance P in a dose-responsive fashion (Fig. 2). These results are independent of the histological classification of the malignancy, the Duke’s staging of the disease, and the age and sex of the patient. The presence or absence of LAK cell activity was not influenced by the clinical indication for bowel resection, the location of the cancer, or the site which the LPMC were isolated from. The difference in cytotoxicity of LAK cells compared to LAK cells + SP was statistically significant (P < 0.01). Comparison of Activity of LAK Fresh Colon Cancer Cells 5:1
25:l
1O:l
5O:l
E:T ratio FIG.
1.
HT-29,
LAK
vs LAK
+ SP.
5% CO,-95% humidified atmosphere for 4 hr. Following incubation, plates were spun again at 1000 rpm for 5 min, 0.1 ml of supernatant was aspirated from each well, and radioactivity was measured as counts per min (cpm) in a gamma counter. Cytotoxicity was expressed as percentage-specific 51Cr release using the following formula: % specific release =
experimental CPM - spontaneous CPM maximum CPM - spontaneous CPM
An unpaired, two-tailed statistical analyses.
Student
x 100.
t test was used for all
RESULTS
LAK
Compared
DISCUSSION
The resulm of this study suggest that the gut mucosal immune system can be modulated by substance P. The net effect of this modulation results in an increase in gut-derived LAK cell cytotoxicity against fresh colon cancer cells, but not against the cultured human colon cancer cell line HT-29. The mechanism for modulation is uncertain at this time. Previous reports have demonstrated specific stimulation of human peripheral blood T-lympocytes by substance P [12-141. Other reports have demonstrated a stimulatory effect of SP on human thymus [al], and on human B lymphocytes [22]. Animal studies have also reported a modulatory effect of SP on the immune system: Moore et al. have worked on lymph nodes of sheep
60 -I l
p < 0.05 W
q
L/W LAK+SP
to LAK t SP against Fresh Colon Cancer
In this group, we conducted 11 assays with LAK cells alone and 11 with LAK cells + SP. Fresh colon cancer cells were found to be completely resistant to LAK cells alone, as previously demonstrated [lo]. However, fresh
and
We compared the activity of LAK cells + SP against HT-29 to that of LAK cells + SP against fresh colon cancer cells. We found that LAK cells cultured with SP exhibited a stronger degree of cytotoxicity against fresh colon cancer cells than they did against cultured colonic carcinoma. At higher E:T ratios (25:1, 50:1), the difference was statistically significant (P < 0.01) (Fig. 3).
to LAK + SE’ against HT-29
The effects of LAK cells from intestinal lamina propria against the cultured tumor cells HT-29 have been well characterized by Trudel et al. in previous work [lo]. Similar results were obtained in our laboratory. The cultured colon carcinoma cell line HT-29 showed the same susceptibility to lysis by LAK cells cultured with substance P as it had to LAK cells alone (Fig. 1) (in this arm of the experiment, there were seven controls and 11 assays with SP). There was no statistical difference in the degree of cytotoxicity toward HT-29 of LAK cells alone and LAK cells cultured with SP (P = 0.15). LAK Compared
+ SP against HT-29
5:l
1O:l
25:l
5O:l
E:T ratio FIG.
2.
Colon
cancer,
LAK
vs LAK
+ SP.
448
JOURNAL 60 l
50-
OF SURGICAL
RESEARCH:
colon ca
P HT-29
5:1
1O:l
25:l
5O:l
E:T ratio FIG. 3.
HT.29
vs colon
CA, LAK
+ SP.
[23, 241 and demonstrated an increase in CD4 cells mediated by SP. Croitoru et al. have done experiments with murine intestinal lymphocytes. They have shown an increase in the natural killer activity modulated by SP [25]. In addition to these several studies that demonstrated a stimulatory effect of SP on a number of lymphocyte subsets, the presence of high-affinity receptors for SP on lymphocytes has been suggested [ll, 14, 261. This body of information led to the hypothesis that SP exerts its effect on lymphocytes via a direct hormone-receptor interaction. This theory is supported by the fact that an antagonist of SP, the peptide [D-Pro', D-Phe7, D-Trp']SP, inhibits the proliferative effect of SP on human peripheral blood lymphocytes by a competitive mechanism [12]. This antagonist was first characterized as such when it was found to inhibit the SP-induced contraction of guinea pig ileal smooth muscle [19, 201. In summary, so far, the literature is capable of supporting the theory of a receptor-mediated stimulatory effect of substance P on human peripheral blood lymphocytes. However we have done a series of experiments using intestinal lymphocytes, not peripheral blood lymphocytes. To find out if the modulatory effect of SP on this particular subset of immune cells is also receptor-mediated, we will have to repeat the experiments in the presence of the SP antagonist and look for competitive inhibition. Our results showed increased LAK cell activity by hormonal manipulation with SP against a fresh but not against a cultured target. Why do fresh cancer cells and cultured cells respond differently to the same effector cells? To our knowledge there has been no work done on the effect of neuropeptides such as substance P on colorectal cancer. We do not know if there is a specific SP receptor on colon cancer cells. Should there be a specific SP receptor on human colon cancer, the presence of SP in the culture medium could improve the stereotaxic interaction between the effector and its target. Substance
VOL. 53, NO. 5, NOVEMBER
1992
P receptors might be down-regulated on cells kept in culture for extended periods, which would prevent the improved interaction to take place. We are presently culturing the HT-29 target cell line with substance P in order to perhaps up-regulate SP receptors in these cells. We will then repeat the microcytotoxicity assays with LAK cells + SP against HT-29 + SP. This should help to determine the mechanism of action of SP and its interactions with target cells. In summary we have demonstrated an enhancing effect of substance P on gut-derived cytotoxicity against fresh colon cancer cells. From these first experiments we conclude that the hypothesis of hormonally manipulating the gut mucosal immune system is viable. Previous work has shown a receptor-mediated effect of SP on peripheral blood lymphocytes but not on intestinal lymphocytes. We need to do additional work to determine if the effect of SP on the LPMC is also receptor-mediated by using SP’s antagonist. We need, in light of the results we obtained, to do a series of assays using peripheral blood lymphocytes of patients with colon cancer. Should we observe similar findings, blood lymphocytes represent a more accessible source of cells when clinical application is considered. We also need to characterize the way LPMC enhanced by SP interact with their target in order to explain the different results observed in fresh and cultured colon cancer cells. REFERENCES 1.
2.
Silverman, A., Desai, T. K., and Luk, J. D. Colorectal cancer. Scope of the problem. GI Clin. North Am. 17: 655, 1988. Nelson, R. L. What is new in colorectal surgery? ACS Bull. 76: 12, 1991.
3. Rosenberg, cancer.
S. LAK cells: A new approach J. Clin. Znuest. 75: 4, 1985.
to immunotherapy
of
4. Van-Healst-Pisani,
5. 6. 7. 8. 9.
10.
11.
C. M., Pisani, R. J., and Kovach, J. S. Cancer immunotherapy: Current status of treatment with interleukin-2 and lympholime-activated-killer cells. Mayo Clin. Proc. 64: 451, 1989. Grimm, E. A. Human LAK cells as a potential immunotherapeutic modality. Biochem. Biophys. Actu 865: 267, 1986. Rosenberg, S. A., Lotze, M. T., et al. A Progress report on the treatment of 157 patients with advanced cancer using LAK cells and IL-2 or IL-2 alone. N. EngZ. J. Med. 316: 889,1987. Guillou, P. IL-2 and LAK cell therapy for gastrointestinal cancer. Actu Chir. Stand. 549: 26, 1989. Marshall, G. D. Adoptive immunotherapy in GI malignancies using IL-2. Current results and future prospects. Actu Chir. Stand. 549: 71, 1989. Lotze, M. T., Chang, A. E., Rosenberg, S. A., et al. High-dose recombinant interleukin-2 in the treatment of patients with disseminated cancer: responses, treatment-related morbidity, and histologic findings. JAMA 256: 3117, 1986.
Trudel, J. L., Fazio, V., Fiocchi, C., et al. LAK cells from human intestinal mucosa: Cytotoxicity against tumor cell lines and modified self but not autologous and allogenic colon cancer cells. J. Surg. Res. 44: 445, 1988. Payan, D. G., Brewster, D. R., and Goetzel, E. J. Specific stimulation of human T lympocytes by substance P. J. Zmmunol. 131: 1613,1983.
FLAGEOLE, 12.
13.
14.
15.
16.
SENTERMAN,
AND
Nordlind, K., and Mutt, V. Modulating effects of /3-endorphin, somatostatin, substance P and vasoactive peptide on the proliferative response of peripheral blood T lymphocytes of nickel allergic patients to nickel sulfate. Znt. Arch. Allergy Appl. Immun. 81: 368, 1986. Nordlind, K., and Mutt, V. Influence of P-endorphin, somatostatin, substance P and vasoactive intestinal peptide on the proliferative response of human peripheral blood lymphocytes to mercuric chloride. Znt. Arch. Allergy Appl. Immun. 80: 326,1986. Payan, D. G., Brewster, D. R., Missirian-Bastian, A., and Goetzel, E. J. Substance P recognition by a subset of human T lymphocytes. J. Clin. Znuest. 74: 1532, 1984. Fiocchi, C., Battisto, J. R., and Farmer, R. G. Gut mucosal lymphocytes in inflammatory bowel disease. Isolation and preliminary functional characterization. Dig. Dis. Sci. 24: 705, 1979. Bull, D. M., and Bookman, M. A. Isolation and functional characterization of human intestinal mucosa lymphoid cells. J. Clin. Invest. 59: 966, 1977.
17.
Brattain, M. G., Kimball, P. M., Pretlow, T. G., et al. Partial purification of human colonic carcinoma cells by sedimentation. Br. J. Cancer 35: 850, 1977.
18.
Keder, E., Ikejri, B. L., Bonnard, G. D., et al. A rapid technique for isolation of viable tumor cells from solid tumor cells for induction and measurement of cell-mediated cytotoxic responses. Eur. J. Clin. Oncol. 18: 991, 1982.
19.
Hanley, 1982.
M. R. Substance
P antagonists.
Trends
Neurosci.
5: 138,
TRUDEL:
SUBSTANCE
449
P INCREASE
20.
Folkers, K., Horig, design of antagonists 505,198l.
J., Rosell, S., and Bjorkroth, of Substance P. Acta Physiol.
U. Chemical &and. 111:
21.
Piantelli, M., Maggiano, N., Larocca, L. M., Ricci, R., Ranalletti, F. O., Lauriola, L., and Capelli, A. Neuropeptide-immunoreactive cells in human thymus. Brain Behuu. Immun. 4: 189, 1990.
22.
Laurenzi, M. A., Persson, M. A., Dalsgaard, C. J., and Ringd, 0. Stimulation of human B lymphocyte proliferation by the neuropeptides substance P and neurokinin A. &and. J. Immunol. 30: 695, 1989.
23.
Moore, T. C., Whitley, G. A., Lami, J. L., and Said, S. I. Substance P increases and prolongs increased output of T4 (CD4) lymphocytes from lymph nodes of sheep in vivo: Is it a mediator of immunological memory? Zmmunopharmacology 20: 207,199O.
24.
Moore, T. C., Lami, J. L., and Spruck, C. H. Substance P increases lymphocyte traffic and lymph flow through peripheral lymph nodes of sheep. Immunology 67: 109, 1989.
25.
Croitoru, K., Ernst, P. B., Bienenstock, J., Padol, I., and Stanisz, A. M. Selective modulation of the natural killer activity of murine intestinal leukocytes by the neuropeptide substance P. Zmmunology 71: 196, 1990.
26.
Pascual, D. W., Blalock, J. E., and Bost, K. L. Antipeptide bodies that recognize a lymphocyte substance P receptor. munol. 143:3697,1989.
27.
Payan, D. G. Substance P: A modulator of neuroendocrine-immune function. Hosp. Pruct. 15: 24, 67, 1989.
antiJ. Zm-
OF SURGICAL
RESEARCH
53,
445-449
(19%)
Substance P Increases in Vitro Lymphokine-Activated-Killer (LAK) Cell Cytotoxicity against Fresh Colorectal Cancer Cells’ HELENE FLAGEOLE, M.D., Departments
of Pathology
and Surgery,
MARY SENTERMAN, M.D.,
AND JUDITH L. TRUDEL, M.D.’
The Montreal
McGill
General
Hospital,
University,
Montreal,
Quebec,
Canada
Submitted for publication November 27.1991 by lung cancer. In females, it is only preceeded by lung and breast cancers. Colon cancer is responsible for 20% of all deaths caused by cancer [ 11. Colon cancer has been resistant to the conventional modes of adjuvant therapy (radiotherapy and chemotherapy) until recently when the combination of 5-FU and Levamisole seemed to show some promise [21. Immunotherapy is a more innovative and experimental form of cancer treatment. Adoptive immunotherapy is a form of immunotherapy which consists of transferring to a tumor-bearing host active immunologic reagents, such as lymphokine-activated-killer (LAK) cells, with anti-tumor activity [3]. The LAK cell is an immune cell which becomes activated in the presence of interleukin-2 (IL-2). Rosenberg et al. [6] can be credited for performing most of the initial studies involving LAK cells and IL-2 both in animals and humans: experiments were done using LAK cells alone [5], others used IL-2 alone [8], and yet others used both agents [4, 6, 71 against a wide variety of tumors. The best responses are obtained when both LAK cells and IL-2 are infused together [4] and the response rate is very dependent on the type of tumor harbored by the host. In humans, the best responses occured in paEffl?CtO~ Target N 5:l 1O:l 25:l 50: 1 tients with renal cell carcinoma and melanoma [4-81. LAK HT-29 7 2.8 k 0.5 11.6 k 1.0 12.3 + 2.3 1’7.6 k 4.2 Colon cancer exhibits only minimal response to these LAK HT-29 11 1.2 * 0.5 4.1 t 0.9 9.3 i 1.0 16.9 k 3.1 highly competent immune cells [4, 6-91. Most clinical +sp LAK Fresh Ca 11 6.6 + 1.6 12.6 k 3.3 36.3 5 6.3. 69.4 * 9.58 studies utilized lymphocytes isolated from the periph+ SP eral blood to generate LAK cells. In a previous study LAK Fresh Ca 11 0.0 + 0.0. 0.1 k 0.18 0.1 f 0.1* 0.5 t 0.5’ [lo], we postulated that lymphocytes from the intestinal lamina propria could represent a more relevant source of We conclude that substance P significantly increases LAK cell cytotoxicity against fresh colon cancer cells, immune cells. Colon cancer cells turned out to be as rebut not against cultured C&S. 0 1992 Academic PMS, 1~. sistant to lysis by lymphocytes from an intestinal source as they had been to lymphocytes isolated from the peripheral blood [lo]. Such resistance could be attributable to insufficient LAK cell cytotoxicity against colon INTRODUCTION cancer cells. The purpose of our study was to enhance Colon carcinoma is a devastating disease. In males, it LAK cell cytotoxicity by hormonal manipulation. We ranks second as a cause of cancer death, preceeded only chose to use substance P to augment LAK cell cytotoxicity, because substance P stimulates human T lympho’ Presentedat the Annual Meetingof the Associationfor Academic cytes proliferation in vitro and because high-affinity Surgery,ColoradoSprings,CO, November20-23,1991. hormonal receptors for substance P are present on lym’ To whom reprint requestsshouldbe addressed at Montreal General Hospital,1650CedarAve., Room9802L.H., Montreal, Quebec, phocytes [ 11-141, suggesting a physiologic basis for horCanadaH3G lA4. monal interaction. Gut-associated lymphoid cells are modulated by several gut hormones. We postulated that lymphokine-associated-killer (LAK) cell cytotoxicity of lymphocytes isolated from the gut mucosa may be increased by substance P (SP). Intestinal lamina propria mononuclear cells (LPMC) and colonic cancer cells were isolated from operative specimens by successive mechanical and enzymatic dissociation methods. Effector LAK cells were induced by culturing LPMC with recombinant interleukin-2 at a concentration of 250 U/ml. Substance P (loM) was added to the culture medium. Targets consisted of fresh colon cancer cells, HT-29 (cultured human colon cancer cell line), and control cell lines. After 4 days of incubation, cytotoxicity was measured using a 4-h %r release assay. LAK cells alone showed moderate cytotoxicity against HT-29 and none against fresh colon cancer cells. LAK cells generated in the presence of substance P showed moderate cytotoxicity against HT-29 and strong cytotoxicity against fresh colorectal cancer cells. The percentage of cytotoxicity + SEM at various effector to target ratios was [(*) denotes P < 0.05 compared with above]:
445
Copyright0 1992 All
rights
of reproduction
oozz-4804/92 $4.00 by Academic Press, Inc. in any form reserved.
446
JOURNAL
MATERIALS
AND
OF
SURGICAL
RESEARCH:
METHODS
Patient Population Freshly resected intestinal tissue was obtained from operative specimens of patients undergoing resection for cancer or benign colonic conditions. Lamina propria mononuclear cells (LPMC) were isolated from 19 patients. Ten of them had colorectal cancer: five had sigmoid lesions, three had rectal lesions, and two had cecal tumors. Three patients had surgery for excision of a villous tumor or multiple polyps, three had diverticular disease, one had Crohn’s disease, one had ulcerative colitis, and one had intractable lower gastrointestinal hemorrhage. The patients who were operated on for cancer also had part of the resected specimen utilized for isolation of fresh colon cancer cells.
VOL.
53, NO.
5, NOVEMBER
1992
Induction of LAK Cell Activity Purified LPMC were cultured in RPMI-1640 (GIBCO) with 10% human AB serum (GIBCO), 1.5% Hepes buffer (GIBCO), 1% gentamicin (GIBCO), and 2.5% penicillin-streptomycin-fungizone (PSF; GIBCO). Recombinant human IL-2 (Cetus Corp., Belmont, CA) was added at a concentration of 250 U/ml. The cells were cultured at a concentration of 0.5 X 106/ ml in upright tissue culture flasks at 37°C in 5% CO,95% humidified air atmosphere for a period of 4 days. Hormonal Enhancement of LPMC
Substance P (Peninsula Labs, San Carlos, CA) was added to the tissue flasks simultaneously with induction of LAK activity. A concentration of lop5 M was selected after going through the following determinations: in the gastrointestinal tract, concentrations as low as 10-l’ M exert a physiologic effect [27]. This concentration was therefore used as the starting point and was increased Isolation of LPMC and Fresh Cancer Cells until a response was obtained. Therefore, 10e5M repreLPMC were isolated using Fiocchi’s modification [ 151 sents the threshold dose in the dose-response curve; it of the original technique described by Bull and Bookwas selected over a higher dose because it is already pharman [ 161. A piece of normal mucosa was freed of mucus macological. Other in vitro studies using substance P as and epithelial cells in sequential steps using 75 mg of an immunomodulator used concentrations ranging bedithiothreitol (DTT; Sigma Chemical Co., St. Louis, tween 10-l’ and lop6 M [ll]. MO) in 50 ml of calcium and magnesium-free Hank’s balanced salt solution (CMF-HBSS; GIBCO) for 30 min Target Cell Lines and 37.5 mg of ethylenediaminetetraacetic acid (EDTA; Several target cell lines were used for cytotoxicity asSigma) in 90 ml of CMF-HBSS for two periods of 90 says. Daudi is a lymphoid cell line used as a LAK-sensimin. Overnight digestion in CMF-HBSS containing tive internal control. The cultured human colorectal cell 0.01% collagenase type III and 0.01% deoxyribonuclease line HT-29 was maintained in duplicate in a solution of (both from Worthington Diagnostic Systems, Inc., FreeRPMI-1640 supplemented with 10% fetal calf serum hold, NJ) was performed. The crude cell suspension was (FCS; GIBCO) and 1% PSF and split every third day. It purified over a Ficoll-Hypaque gradient (Pharmacia). was used as one of the experimental targets. HT-29 cells The isolated cells were almost exclusively LPMC (90are known to be slightly LAK sensitive [lo]. Fresh colon 95% purity, verified by microscopic examination), Viacancer cells isolated from surgical specimens were used bility of the isolated cells was verified using the trypan as the other experimental target. blue exclusion method (80-85% viability, by microsCOPY).
When the resection was done for malignant disease, fresh colon cancer cells were also isolated. The procedure is a combination of the methods of Brattain et al. [ 171 and Keder et al. [181. Nonnecrotic malignant tissue was rinsed in CMF-HBSS and minced in l- to 3-mm fragments. The fragments were placed in a solution of CMF-HBSS containing hyaluronidase (0.5 mg/ml; Sigma), collagenase type III, and DNAase (2 and 0.6 mg/ ml, respectively). This digestion process was repeated approximately five times. The cell suspension from the first digestion period was discarded to reduce contamination with red blood cells, dead cells, and debris. The cells were collected by centrifugation and further purified over a 20% Percoll gradient (Pharmacia). The isolated cells were usually 8590% viable (verified by microscopy using the trypan blue exclusion method) and were either used immediately or frozen in liquid nitrogen.
Microcytotoxicity
Assay
Target cells (3-5 million) were harvested and centrifuged (1700 rpm X 5 min.). All but 2 ml of supernatant was aspirated and 200 &i of sodium chromate (51Cr; ICN Chemicals) was added. The cells were incubated at 37°C for 90 min and agitated periodically. At the end of this period, the cells were washed three times and plated at lo4 cells/well in microtiter plates. The effector LAK cells, cultured with or without substance P, were aliquoted in triplicate to the wells containing target cells. Various effector to target (E:T) ratios were obtained by serial dilution of the effector cells added to a fixed number ( 104) of 51Cr-labeled target cells/well. Spontaneous and maximum release was determined by addition of culture medium and 1% sodium dodecyl sulfate (SDS; Sigma), respectively. The microtiter plates were spun at 500 rpm for 3 min, followed by incubation at 37°C in a
FLAGEOLE,
SENTERMAN,
AND
TRUDEL:
SUBSTANCE
447
P INCREASE
colon cancer cells were lysed by LAK cells cultured with substance P in a dose-responsive fashion (Fig. 2). These results are independent of the histological classification of the malignancy, the Duke’s staging of the disease, and the age and sex of the patient. The presence or absence of LAK cell activity was not influenced by the clinical indication for bowel resection, the location of the cancer, or the site which the LPMC were isolated from. The difference in cytotoxicity of LAK cells compared to LAK cells + SP was statistically significant (P < 0.01). Comparison of Activity of LAK Fresh Colon Cancer Cells 5:1
25:l
1O:l
5O:l
E:T ratio FIG.
1.
HT-29,
LAK
vs LAK
+ SP.
5% CO,-95% humidified atmosphere for 4 hr. Following incubation, plates were spun again at 1000 rpm for 5 min, 0.1 ml of supernatant was aspirated from each well, and radioactivity was measured as counts per min (cpm) in a gamma counter. Cytotoxicity was expressed as percentage-specific 51Cr release using the following formula: % specific release =
experimental CPM - spontaneous CPM maximum CPM - spontaneous CPM
An unpaired, two-tailed statistical analyses.
Student
x 100.
t test was used for all
RESULTS
LAK
Compared
DISCUSSION
The resulm of this study suggest that the gut mucosal immune system can be modulated by substance P. The net effect of this modulation results in an increase in gut-derived LAK cell cytotoxicity against fresh colon cancer cells, but not against the cultured human colon cancer cell line HT-29. The mechanism for modulation is uncertain at this time. Previous reports have demonstrated specific stimulation of human peripheral blood T-lympocytes by substance P [12-141. Other reports have demonstrated a stimulatory effect of SP on human thymus [al], and on human B lymphocytes [22]. Animal studies have also reported a modulatory effect of SP on the immune system: Moore et al. have worked on lymph nodes of sheep
60 -I l
p < 0.05 W
q
L/W LAK+SP
to LAK t SP against Fresh Colon Cancer
In this group, we conducted 11 assays with LAK cells alone and 11 with LAK cells + SP. Fresh colon cancer cells were found to be completely resistant to LAK cells alone, as previously demonstrated [lo]. However, fresh
and
We compared the activity of LAK cells + SP against HT-29 to that of LAK cells + SP against fresh colon cancer cells. We found that LAK cells cultured with SP exhibited a stronger degree of cytotoxicity against fresh colon cancer cells than they did against cultured colonic carcinoma. At higher E:T ratios (25:1, 50:1), the difference was statistically significant (P < 0.01) (Fig. 3).
to LAK + SE’ against HT-29
The effects of LAK cells from intestinal lamina propria against the cultured tumor cells HT-29 have been well characterized by Trudel et al. in previous work [lo]. Similar results were obtained in our laboratory. The cultured colon carcinoma cell line HT-29 showed the same susceptibility to lysis by LAK cells cultured with substance P as it had to LAK cells alone (Fig. 1) (in this arm of the experiment, there were seven controls and 11 assays with SP). There was no statistical difference in the degree of cytotoxicity toward HT-29 of LAK cells alone and LAK cells cultured with SP (P = 0.15). LAK Compared
+ SP against HT-29
5:l
1O:l
25:l
5O:l
E:T ratio FIG.
2.
Colon
cancer,
LAK
vs LAK
+ SP.
448
JOURNAL 60 l
50-
OF SURGICAL
RESEARCH:
colon ca
P HT-29
5:1
1O:l
25:l
5O:l
E:T ratio FIG. 3.
HT.29
vs colon
CA, LAK
+ SP.
[23, 241 and demonstrated an increase in CD4 cells mediated by SP. Croitoru et al. have done experiments with murine intestinal lymphocytes. They have shown an increase in the natural killer activity modulated by SP [25]. In addition to these several studies that demonstrated a stimulatory effect of SP on a number of lymphocyte subsets, the presence of high-affinity receptors for SP on lymphocytes has been suggested [ll, 14, 261. This body of information led to the hypothesis that SP exerts its effect on lymphocytes via a direct hormone-receptor interaction. This theory is supported by the fact that an antagonist of SP, the peptide [D-Pro', D-Phe7, D-Trp']SP, inhibits the proliferative effect of SP on human peripheral blood lymphocytes by a competitive mechanism [12]. This antagonist was first characterized as such when it was found to inhibit the SP-induced contraction of guinea pig ileal smooth muscle [19, 201. In summary, so far, the literature is capable of supporting the theory of a receptor-mediated stimulatory effect of substance P on human peripheral blood lymphocytes. However we have done a series of experiments using intestinal lymphocytes, not peripheral blood lymphocytes. To find out if the modulatory effect of SP on this particular subset of immune cells is also receptor-mediated, we will have to repeat the experiments in the presence of the SP antagonist and look for competitive inhibition. Our results showed increased LAK cell activity by hormonal manipulation with SP against a fresh but not against a cultured target. Why do fresh cancer cells and cultured cells respond differently to the same effector cells? To our knowledge there has been no work done on the effect of neuropeptides such as substance P on colorectal cancer. We do not know if there is a specific SP receptor on colon cancer cells. Should there be a specific SP receptor on human colon cancer, the presence of SP in the culture medium could improve the stereotaxic interaction between the effector and its target. Substance
VOL. 53, NO. 5, NOVEMBER
1992
P receptors might be down-regulated on cells kept in culture for extended periods, which would prevent the improved interaction to take place. We are presently culturing the HT-29 target cell line with substance P in order to perhaps up-regulate SP receptors in these cells. We will then repeat the microcytotoxicity assays with LAK cells + SP against HT-29 + SP. This should help to determine the mechanism of action of SP and its interactions with target cells. In summary we have demonstrated an enhancing effect of substance P on gut-derived cytotoxicity against fresh colon cancer cells. From these first experiments we conclude that the hypothesis of hormonally manipulating the gut mucosal immune system is viable. Previous work has shown a receptor-mediated effect of SP on peripheral blood lymphocytes but not on intestinal lymphocytes. We need to do additional work to determine if the effect of SP on the LPMC is also receptor-mediated by using SP’s antagonist. We need, in light of the results we obtained, to do a series of assays using peripheral blood lymphocytes of patients with colon cancer. Should we observe similar findings, blood lymphocytes represent a more accessible source of cells when clinical application is considered. We also need to characterize the way LPMC enhanced by SP interact with their target in order to explain the different results observed in fresh and cultured colon cancer cells. REFERENCES 1.
2.
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C. M., Pisani, R. J., and Kovach, J. S. Cancer immunotherapy: Current status of treatment with interleukin-2 and lympholime-activated-killer cells. Mayo Clin. Proc. 64: 451, 1989. Grimm, E. A. Human LAK cells as a potential immunotherapeutic modality. Biochem. Biophys. Actu 865: 267, 1986. Rosenberg, S. A., Lotze, M. T., et al. A Progress report on the treatment of 157 patients with advanced cancer using LAK cells and IL-2 or IL-2 alone. N. EngZ. J. Med. 316: 889,1987. Guillou, P. IL-2 and LAK cell therapy for gastrointestinal cancer. Actu Chir. Stand. 549: 26, 1989. Marshall, G. D. Adoptive immunotherapy in GI malignancies using IL-2. Current results and future prospects. Actu Chir. Stand. 549: 71, 1989. Lotze, M. T., Chang, A. E., Rosenberg, S. A., et al. High-dose recombinant interleukin-2 in the treatment of patients with disseminated cancer: responses, treatment-related morbidity, and histologic findings. JAMA 256: 3117, 1986.
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Piantelli, M., Maggiano, N., Larocca, L. M., Ricci, R., Ranalletti, F. O., Lauriola, L., and Capelli, A. Neuropeptide-immunoreactive cells in human thymus. Brain Behuu. Immun. 4: 189, 1990.
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Moore, T. C., Lami, J. L., and Spruck, C. H. Substance P increases lymphocyte traffic and lymph flow through peripheral lymph nodes of sheep. Immunology 67: 109, 1989.
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Croitoru, K., Ernst, P. B., Bienenstock, J., Padol, I., and Stanisz, A. M. Selective modulation of the natural killer activity of murine intestinal leukocytes by the neuropeptide substance P. Zmmunology 71: 196, 1990.
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Pascual, D. W., Blalock, J. E., and Bost, K. L. Antipeptide bodies that recognize a lymphocyte substance P receptor. munol. 143:3697,1989.
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antiJ. Zm-
