Cancer Immunol Immunother (1987) 25:242-244 ancer mmunolggy mmunotherapy

© Springer-Verlag 1987

Abrogation of the tumor promoting effect of allogeneic blood transfusion by polyadenylic-polyuridylic acid (poly A-poly u)

S. K. Singh, R. L. Marquet, D. L. Westbroek, and J. Jeekel

Department of Surgery and Laboratory of Experimental Surgery, Erasmus Universiteit Rotterdam, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands

Summary. Studies from several centers have shown an im- munosuppressive effect of surgical procedures, whilst others have shown blood transfusion in association with cancer surgery to have an adverse effect on ultimate prog- nosis. We have previously demonstrated enhanced growth of tumor metastases, in rats following allogeneic blood transfusion and surgery. Polyadenylic-polyuridylic acid (poly A-poly U) has been reported to stimulate immune re- sponses. In this report, we have investigated the effective- ness of poly A-poly U as an adjuvant to blood transfusion and surgical procedures in BN rats bearing artificial lung metastases. Significantly reduced tumor growth was ob- served, following poly A-poly U adjuvant treatment. These results lead to serious contemplation of the use of this drug as adjuvant therapy in blood transfused and surgically treated patients.

Introduction

Current therapeutic approaches to cancer include surgery, radiotherapy, chemotherapy and immunotherapy. Many studies have indicated an immunosuppressive effect asso- ciated with several treatment modalities. Stratton et al [22] showed the profound effect of irradiation on the lympho- cyte subpopulation in cancer patients. Various chemother- apeutic regimes are known to produce an immunosuppres- sive effect [14]. Numerous reports have indicated the most commonly employed therapy for cancer patients, i.e., sur- gical resection, to have immunosuppressive effects [6, 17]. Anesthesia, the inevitable accompaniment to a surgical procedure, has also been shown to produce similar effects on the immune apparatus [23]. Furthermore, surgery in cancer patients is often accompanied with substantial blood loss, necessitating administration of blood transfu- sions. Blood loss on its own has been shown to produce decreased immunocompetence [7, 21]. Several recent retro- spective studies have indicated a relationship between blood transfusion and adverse prognosis of disease-free survival of surgically treated cancer patients [5, 18]. This has subsequently been confirmed in animal studies [9, 16]. We have earlier demonstrated a detrimental effect of sur- gery in combination with allogeneic blood transfusion on the growth of established artificial tumor metastases, in a rat model [19, 20].

Offprint requests to: S. K. Singh

A double-stranded synthetic polynucleotide, polyaden- ylic-polyuridylic acid (poly A-poly U) has been reported to stimulate immune responses. It has been shown to posses an antitumor effect, and to be a potent immunom- odulator of both the humoral and cellular immune re- sponse and to result in enhancement of natural killer (NK) cell activity [1, 3, 11, 12, 24). Furthermore, even at high dosage, no toxic or pyrogenic effects have been reported on use of poly A-poly U as an adjunct in cancer therapy [4, 8].

Previously we have shown enhanced growth of LS 175 tumor metastases in BN rats undergoing abdominal sur- gery in combination with allogeneic blood transfusion. Enhanced metastatic growth has also been observed fol- lowing allogeneic blood transfusion alone [16, 19, 20]. This enhanced growth correlated with decreased T-cell activity. This manuscript reports on the effect of adjuvant treat- ment with poly A-poly U in tumor-bearing rats under- going abdominal surgery and treatment with blood trans- fusions.

Materials and methods

Animals. Male rats of the inbred BN and WAG strains were used. The animals were bred under specific patho- gen-free conditions and were 16-20 weeks old.

Tumor. Tumor LS 175 is an spontaneous, nonimmunogen- ic sarcoma in BN rats. The tumor is maintained as a sta- tionary culture in Dulbecco's minimum essential medium, supplemented with 10% fetal calf serum. To obtain cells for the vivo experiments free floating LS 175 cell clumps were harvested from the tissue culture flasks and, after washing were resuspended in Hanks balanced salt solu- tion. Single cells were prepared by rinsing the suspension through a pipette. Viability was assessed by trypan blue ex- clusion and was between 90% and 95%.

Lung colony assay. Tumor cells (2.5 x 105), suspended in a volume of 1 ml, were injected i.v. into experimental and control rats. The number of colonies developing in the lungs was counted after 24 days. The lungs were excised, rinsed in tap water, and subsequently fixed in Bouin's so- lution. Tumor nodules on the lung surface, visible to the naked eye, were counted.

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Blood transfusions. BN rats received a single i.v. injection of 1 ml heparinized whole allogeneic blood or 1 ml of syn- geneic blood. Control animals received no transfusions.

Abdominal surgery. Under ether anesthesia a resection of a 5 -6 cm segment of ileum and subsequent end-to-end anas- tomoses using continuous suturing with 7.0 silk was per- formed. The procedure lasted 15-20 min, with an estimat- ed blood loss of + 2 ml.

Poly A-poly U. Poly A-poly U (Sigma P3259) St. Louis, USA powder was dissolved in phosphate-buffered saline and adjusted to concentration of 3000 txg/ml. Then 0.5 ml (1500 gg) of this solution was administered i.v. to experi- mental animals on days +7, + 14, and +21

Experimental design. The effectiveness of poly A-poly U in abrogating the adverse effect of allogeneic blood transfu- sion alone or in combination with surgery, on the growth of established lung metastases was investigated. BN rats were inoculated with 2.5 x 105 LS 175 tumor cells on day 0. The control animals did not receive any blood transfusion or poly A-poly U and did not undergo abdominal surgery. On day + 7, experimental groups of animals received syn- geneic or allogeneic blood transfusion alone or in combi- nation with abdominal surgery. A group of rats underwent abdominal surgery but received no blood transfusion and another group was given poly A-poly U alone without un- dergoing surgery or blood transfusion. All experimental groups were given poly A-poly U at a dosage of 1500 gg/ week, on days +7, + 14, and +21. Animals were sacri- ficed on day + 24 and lung colonies counted. Each group comprised of 9-15 animals. The results were statistically analyzed using Student's t-test.

Results

On day + 24 the mean number of colonies in the control group, i.e., animals inoculated with tumor cells on day 0 not undergoing any further experimental procedure, was 60 +_ 14 (Table 1). All other experimental groups were treat-

Table I. Effect of polyadenylic A-polyuridylic U (Poly A-poly U) as adjuvant therapy on tumor metastatic growth

Treatment on day + 7 No. of Mean no. of * animals lung colonies

(+SD)

No treatment 15 60+_ 14

Poly A-poly U only 10 39+_ 7 S

Syngeneic transfusion + poly A-poly U 10 20+_ 4 S

Allogeneic transfusion + poly A-poly U 10 33+ 10 S

Surgery + poly A-poly U 9 26 +_ 6 S

Surgery + syngeneic transfusion + poly A-poly U 9 40+_ 9 S

Surgery + allogeneic transfusion + poly A-poly U 9 28 +_ 4 S

Poly A-poly U administered in doses of 1500 pg/week on days +7, + 14, and +21 after tumor inoculation * Significantly different as compared to control group, with P < 0.005 in Student's t-test

ed with poly A-poly U on days +7, + 14, and +21. Treat- ment with poly A-poly U alone resulted in 39 +- 7 lung co- lonies, which was significantly less than the control group. Syngeneic and allogeneic blood transfusion combined with poly A-poly U resulted in 20+4 and 33+_10 lung co- lonies, respectively. These numbers were significantly low- er than the control group. Surgery alone or in combination with blood transfusion and adjuvant poly A-poly U treat- ment also resulted in a significantly lower mean number of lung colonies. Surgery alone resulted in 26_+ 6 mean num- ber of lung colonies, whilst in combination with syngeneic and allogeneic transfusion 40+9 and 28+_4 number of lung colonies were found, respectively. These results indi- cated a profound suppressive effect on the growth of esta- blished lung tumor metastases following adjuvant treat- ment with poly A-poly U, even when the animals under- went a surgical procedure and received allogeneic blood transfusion.

Discussion

Lundy et al. [15] have shown that the use of thiobendazole, an immunorestorative drug, as immunotherapy can be an effective adjuvant treatment to surgery in preventing the growth of micrometastatic loci. Lacour et al. [13] have demonstrated significant benefit of adjuvant treatment with poly A-poly U in operable breast cancer patients, dem- onstrating an increase in both overall and relapse-free sur- vival, particularly in the group with positive axillary nodes. Bonadonna and Valagussa [20] have reported simi- lar success in treating breast cancer patients with cyclo- phosphamide, methotrexate, and flourouracil (CMF) as adjuvant therapy. However, in contrast to poly A-poly U, several side effects following CMF use were reported. The results of this study confirm the beneficial use of poly A- poly U, an immunostimulatory drug, as an adjuvant in cancer therapy. Previously, in the same tumor-host model employed in this study, we have demonstrated significant- ly enhanced tumor growth following allogeneic blood transfusion alone or in combination with abdominal sur- gery [16, 19, 20]. These animals further showed a signifi- cantly diminished mitogen blastogenic response to con- canavalin A and phytohemagglutinin stimulation. Poly A- poly U is shown in the present study to be effective in abrogating the adverse effect of blood transfusion and surgery on tumor growth. In the same tumor-host model, poly A-poly U treatment was found to result in significant stimulation of mitogen blastogenic response and gamma- interferon production capacity [10]. Youn et al. [25] have shown significant enhancement of NK cell activity follow- ing poly A-poly U treatment in operable stomach cancer patients. The antitumor and stimulating effect on cellular immune response, NK cell activity, and interferon produc- tion capacity of poly A-poly U treatment could be the pos- sible mechanisms responsible for offsetting the detrimental effects of surgery and blood transfusions, resulting in sig- nificantly diminished tumor metastases growth [3, 10, 11, 12].

Surgical resection is still the most frequent therapy em- ployed in cancer treatment. Inspite of improved surgical techniques, perioperative blood loss often necessitates ad- ministration of blood transfusions. Recent retrospective studies indicate an adverse effect of blood transfusion on disease-free survival [5, 18]. This blood transfusion effect could lead to restrictions in surgical procedures used in

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cancer patients. Thus the use of immunorestorat ive adju- vant therapy in surgical treatment, especially when blood transfusions are also administered, should be considered. The absence of any reported toxic, pyrogenic, or other sec- ondary effect of poly A-poly U and its demonstrated effec- tive employment as adjuvant chemotherapy in clinical studies makes poly A-poly U a strong candidate for this [4]. The results of this study and other experimental studies further substantiate the effectiveness of its use.

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Received May 18, 1987/Accepted July 14, 1987