Effective Treatment of Transmissible Venereal Tumors in Dogs with Vincristine and IL2.

AIM: To improve treatment of inoperable transmissible venereal tumors (TVTs) in dogs. Recently, we showed that TVT is sensitive to intratumoral treatment with interleukin-2 (IL2). In addition it is known that TVT is sensitive to intravenous treatment with vincristine. In the present study we tried to establish the therapeutic effect of intratumoral treatment with vincristine and IL2. PATIENTS AND METHODS: We treated 12 dogs with TVT with 1-4 intratumoral treatments with vincristine and IL-2. Per treatment we used vincristine (0.5-0.7 mg/m(2)) and IL2 (2×10(6) units). The injections were given at weekly intervals. RESULTS: Early therapeutic effects were: three complete regressions, four partial regressions, three stable disease, and two progressive disease. Late therapeutic effects were established 45-60 months after the first presentation; there were five complete regressions, no partial regressions, nor stable or progressive diseases. Interestingly, all five dogs with late therapeutic effects were in good health. No tumor recurrence was noted. CONCLUSION: Intratumoral treatment of TVT with vincristine and IL2 appears to have impressive therapeutic effects.

Treatment of transmissible venereal tumors in dogs with intratumoral interleukin-2 (IL-2). A pilot study.

AIM: To improve the treatment of transmissible venereal tumors (TVTs) in dogs with intratumoral injections of interleukin-2 (IL-2). PATIENTS AND METHODS: We treated 13 dogs with 18 natural TVTs with IL-2. The tumors were treated with intratumoral application of 2×10(6) units IL-2. RESULTS: Three months after injection of IL-2, the tumors in 2/13 dogs had regressed completely, those in 1/13 had regressed partially, and 4/13 dogs had stable disease. CONCLUSIONS: Local IL-2 treatment of TVT is therapeutically effective, as indicated by complete regression (CR), partial regression (PR) and stable disease (SD) of the tumors of 7 out of 13 dogs. In addition, we observed that the intratumoral treatment with IL-2 did not cause any toxic side-effects.

Inefficacy of therapeutic cancer vaccines and proposed improvements. Casus of prostate cancer.

Prophylactic vaccination is arguably the most effective medical preventative method. After local inoculation, vaccines induce antigen-specific systemic immunity, protecting the whole body. Systemic antitumour immunity can cure advanced cancer, but will therapeutic vaccination suffice? A vaccine for castration-refractory prostate cancer (CRPC) was approved by regulatory authority, but its evidence is disputed. We critically reviewed the clinical efficacy of therapeutic cancer vaccines for prostate cancer, including the results of 31 clinical studies employing vaccines-only, and another 10 studies combining vaccines with immune co-stimulation. Vaccinations yielded immunological responses, but no study showed evidence for clinically relevant therapeutic improvement. Clinical failure of therapeutic vaccination is discussed in the light of immunological dogmas and mechanisms of antitumour therapies. We propose that cancer immunotherapy might be improved by immunological danger, i.e. disturbing tumour homeostasis by destroying the tumour tissue or inducing local inflammation. Such danger might override immunological tolerance, and thereby allow clinically relevant anticancer results.

Therapeutic response of untreatable hepatocellular carcinoma after application of the immune modulators IL-2, BCG and melatonin.

AIM: Application of immunotherapy to a patient with untreatable hepatocellular carcinoma. CASE REPORT: The patient had a tumor of 60 mm in the liver. The pathological anatomic diagnosis was adenoma. However, after surgery of the tumor seven new lesions arose, showing that the original tumor had been a hepatocellular carcinoma. In addition, when hepatocellular adenomas grow to a size of more than 6-8 cm, they are considered cancerous and thus become a risk for hepatocellular carcinoma. The patient was treated with interleukin-2, Bacillus Calmette Guerin, and melatonin. RESULTS: During treatment, the alpha-fetoprotein levels in blood fell from 5,000 IU/ml to zero, at which level it remained during the follow-up period of two years. No tumor was detectable on MRI and CT. Six years after the diagnosis of untreatable hepatocellular carcinoma, the patient remains in a good condition. CONCLUSION: In this case, combined immunomodulating therapy was effective. For patients with metastasized tumors of the liver who are not suitable for conventional therapy, immunomodulation may delay tumor progression, induce tumor regression, or even be curative in some patients. Immunotherapeutic approaches combined with conventional methods for hepatocellular carcinoma treatment may be able to improve therapeutic efficacy.

Role of marker lesion when applying intravesical instillations of IL-2 for non-muscle-invasive bladder cancer comparison of the therapeutic effects in two pilot studies.

AIM: Comparison of the therapeutic effect of treatment of non-muscle invasive bladder carcinoma (NMIBC) after intravesical Interleukin-2 (IL-2) instillations in the presence and absence of a marker tumour. MATERIALS AND METHODS: Two pilot studies were performed in patients with NMIBC. The first study (10 patients) was performed in Krakow (Poland), the second (26 patients) in Vilnius (Lithuania). In Krakow the tumours were treated with incomplete transurethral resection (TUR) leaving a marker tumour of 0.5-1.0-cm followed by IL-2 instillations (3 × 10(6) IU IL-2) on five consecutive days. In Vilnius the tumours were treated with complete TUR, followed by IL-2 instillations (9 × 10(6) IU IL-2) on five consecutive days. RESULTS: During 30 months follow-up, the recurrence-free survival was 5/10 (50%) and 6/26 (23%) after incomplete and complete TUR, respectively. So, the ratio of the recurrence-free survival after incomplete/complete TUR of 50/23=2.2. The median of the recurrence-free survival is >20.5 months and 7 months after incomplete and complete TUR, respectively. So, this ratio was >20.5/7= >2.9. The hazard ratio which combines both the chance of the disease recurrence and its timing for both censored and uncensored cases was 0.53, again confirming the better outcome after incomplete TUR. CONCLUSION: A possible explanation for the better therapeutic effects after incomplete TUR compared with complete TUR is that the marker tumour has tumour-associated antigens (TAA) that could lead to an immune reaction that is stimulated by local application of IL-2. After complete TUR, no TAA are available to initiate and to stimulate an immune reaction; consequently, local IL-2 therapy is less effective after complete TUR. The results of these two pilot studies have led to the recent start of a randomised prospective clinical trial in which therapeutic effects of local IL-2 therapy after complete and incomplete TUR are compared.

Prediction of response in cancer immunotherapy.

This review focuses on the relationship between pre-treatment immune parameter values and outcome of immunotherapy of cancer patients. The evidence presented in this review suggests that there is a relationship between pre-treatment immune parameter values and survival of cancer patients treated with immunotherapy. Tumour-infiltrating immune cells may have a predictive value for immunotherapy, but predictive power might be obtained from peripheral blood leukocytes. Use of peripheral blood may be preferable due to the convenience of collection and analysis compared to using tumour-infiltrating cells. In vivo numbers of cells of the immune system correlate better with clinical outcome than their functional activity ex vivo. This suggests that immunological antitumour mechanisms in vivo are not always related to generally accepted functional parameters of lymphocytes, such as cytotoxicity or cytokine production, ex vivo. The proliferative status of CD8(+) T lymphocytes seems promising for prediction of response in cancer immunotherapy.

Prognostic significance of peripheral blood CD8highCD57+ lymphocytes in bladder carcinoma patients after intravesical IL-2.

BACKGROUND: The objective of this study was to evaluate the recurrence-preventing effect of intravesical instillations of interleukin-2 (IL-2) in patients with non-muscle-invasive bladder carcinoma. In addition, this study aimed to determine the significance of immune parameters for recurrence-free interval. PATIENTS AND METHODS: Twenty-six patients with non-muscle-invasive bladder carcinoma were treated with intravesical instillations of IL-2 (Proleukin®, Novartis, formerly Chiron) in doses of 9 × 10(6) IU on 5 consecutive days, beginning on the second day after transurethral resection (TUR) of tumours. CD8(high)CD57(+) lymphocytes in peripheral blood were determined before TUR and compared with the recurrence-free interval after treatment. RESULTS: The multivariate analysis showed that CD8(high)CD57(+) lymphocytes had a prognostic significance in combination with number of bladder tumours, prior recurrence rate and age of patients. CONCLUSION: Peripheral blood CD8(high)CD57(+) lymphocytes have prognostic significance for recurrence-free survival in patients with non-muscle-invasive bladder carcinoma after TUR and intravesical IL-2.

Local therapy of cancer with free IL-2.

This is a position paper about the therapeutic effects of locally applied free IL-2 in the treatment of cancer. Local therapy: IL-2 therapy of cancer was originally introduced as a systemic therapy. This therapy led to about 20% objective responses. Systemic therapy however was very toxic due to the vascular leakage syndrome. Nevertheless, this treatment was a break-through in cancer immunotherapy and stimulated some interesting questions: Supposing that the mechanism of IL-2 treatment is both proliferation and tumoricidal activity of the tumor infiltrating cells, then locally applied IL-2 should result in a much higher local IL-2 concentration than systemic IL-2 application. Consequently a greater beneficial effect could be expected after local IL-2 application (peritumoral = juxtatumoral, intratumoral, intra-arterial, intracavitary, or intratracheal = inhalation). Free IL-2: Many groups have tried to prepare a more effective IL-2 formulation than free IL-2. Examples are slow release systems, insertion of the IL-2 gene into a tumor cell causing prolonged IL-2 release. However, logistically free IL-2 is much easier to apply; hence we concentrated in this review and in most of our experiments on the use of free IL-2. Local therapy with free IL-2 may be effective against transplanted tumors in experimental animals, and against various spontaneous carcinomas, sarcomas, and melanoma in veterinary and human cancer patients. It may induce rejection of very large, metastasized tumor loads, for instance advanced clinical tumors. The effects of even a single IL-2 application may be impressive. Not each tumor or tumor type is sensitive to local IL-2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma were not sensitive in our hands. Also the extent of sensitivity differs: In Bovine Ocular Squamous Cell Carcinoma (BOSCC) often a complete regression is obtained, whereas with the Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) mainly stable disease is attained. Analysis of the results of local IL-2 therapy in 288 cases of cancer in human patients shows that there were 27% Complete Regressions (CR), 23% Partial Regressions (PR), 18% Stable Disease (SD), and 32% Progressive Disease (PD). In all tumors analyzed, local IL-2 therapy was more effective than systemic IL-2 treatment. Intratumoral IL-2 applications are more effective than peritumoral application or application at a distant site. Tumor regression induced by intratumoral IL-2 application may be a fast process (requiring about a week) in the case of a highly vascular tumor since IL-2 induces vascular leakage/edema and consequently massive tumor necrosis. The latter then stimulates an immune response. In less vascular tumors or less vascular tumor sites, regression may require 9-20 months; this regression is mainly caused by a cytotoxic leukocyte reaction. Hence the disadvantageous vascular leakage syndrome complicating systemic treatment is however advantageous in local treatment, since local edema may initiate tumor necrosis. Thus the therapeutic effect of local IL-2 treatment is not primarily based on tumor immunity, but tumor immunity seems to be useful as a secondary component of the IL-2 induced local processes. If local IL-2 is combined with surgery, radiotherapy or local chemotherapy the therapeutic effect is usually greater than with either therapy alone. Hence local free IL-2 application can be recommended as an addition to standard treatment protocols. Local treatment with free IL-2 is straightforward and can readily be applied even during surgical interventions. Local IL-2 treatment is usually without serious side effects and besides minor complaints it is generally well supported. Only small quantities of IL-2 are required. Hence the therapy is relatively cheap. A single IL-2 application of 4.5 million U IL-2 costs about 70 Euros. Thus combined local treatment may offer an alternative in those circumstances when more expensive forms of treatment are not available, for instance in resource poor countries.

Changes in cytokine profile during local IL-2 therapy in cancer patients.

BACKGROUND: Exogenous interleukin 2 (IL-2) can influence the complex cytokine network in vivo. This study investigated the cytokine profile of patients with different malignancies before and after local IL-2 administration. PATIENTS AND METHODS: The human TH1 / TH2 cytometric bead array (CBA) kit was used to investigate IL-2, IFNgamma, TNFalpha, IL-4, IL-5 and IL-10 in a control group and in 13 patients. RESULTS: The baseline serum IL-4 levels in patients were lower than in healthy controls, while the baseline ascitic IL-10 levels in patients was higher than in serum. The IL-2 applications induced a strong serum increase in IL-2 and IL-5 and even more in ascites, while IL-10 increased weakly and mainly locally. One month after the start of therapy, the serum IFNgamma had increased in patients, reaching the level of the control group. CONCLUSION: After local injection, IL-2 probably leaks into the blood circulation. The higher increases of IL-2, IL-5 and IL-10 in ascites compared to the serum suggests that the injected cytokines and their effects are mainly local. The minor increase of the immunosuppressive IL-10 could explain the therapeutic difference between local and systemic IL-2 therapy since IL-10 levels markedly increase after systemic IL-2 therapy. IL-5 was always increased after IL-2 therapy and, consequently, may be a downstream mediator of antitumour responses.

Locoregional IL-2 low dose applications for gastrointestinal tumors.

AIM: To explore the feasibility of local interleukin 2 (IL-2) in patients with different forms of abdominal cancer. This required experimentation with the time interval between IL-2 applications and the methods of application. METHODS: Sixteen patients with stages III and IV of gastrointestinal malignancies (primary or metastatic) who were admitted to our Department of Gastroenterology were treated with locoregionally applied IL-2 in low doses. RESULTS: No major problems applying locoregional IL-2 were encountered. In 6 out of 16 patients, a modest but clinically worthwhile improvement was obtained. Adverse effects were minimal. The therapeutic scheme was well tolerated, even in patients in a poor condition. CONCLUSION: This study demonstrates the feasibility of low dose locoregional IL-2 application in advanced abdominal cancer. Local IL-2 therapy gives only negligible adverse effects. The results suggest that it is important to apply intratumorally. Local IL-2 may be given adjunct to standard therapeutic regimes and does not imply complex surgical interventions. These initial results are encouraging.

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

BACKGROUND: Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling. METHODS: The proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture. RESULTS: The suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 +/- 1.1 h and 21.6 +/- 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 +/- 7.7% vs. 79.2 +/- 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively. CONCLUSION: The proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

Local interleukin-2 and interleukin-12 therapy of bovine ocular squamous cell carcinomas.

Interleukin-2 and interleukin-12 have been used independently to successfully treat the induced and the spontaneous tumours in animals. This trial was done to determine if a combination of IL-2 and IL-12 in the treatment of spontaneous bovine ocular squamous cell carcinomas (BOSCC) would be more successful than IL-2 or IL-12 therapy by themselves. For this trial, we selected 25 BOSCC tumours seen on Holstein Fresian cows in Beatrice, Zimbabwe. The cows were randomly assigned to a treatment group of 5 days of IL-2 (200,000 U/day), 5 days of IL-12 (0.5 microg/day) or 5 days of IL-2 (200,000 U/day) and IL-12 (0.5 microg/day). At 20 months after treatment, the IL-2 therapy group had 63% complete regressions; the combination group had 38% complete regressions, which were significantly higher than the IL-12 group, which had 0% complete regressions at 20 months, despite having 29% complete regressions at 6 months. These results show that IL-2 therapy by itself and in combination with IL-12 is more successful than IL-12 by itself. However, combination therapy does not improve the outcome in comparison to IL-2 as a single therapy. It also proves that IL-2 is consistently successful in the therapy of BOSCC with over 60% complete regression, which corresponds to a number of other studies we have done on IL-2 therapy of BOSCC [Rutten, V.P.M.G., Klein, W.R., De Jong, W.A., Misdorp, W., Den Otter, W., Steerenberg, P.A., De Jong, W.H., Ruitenberg, E.J., 1989. Local interleukin-2 therapy in bovine ocular squamous cell carcinoma. A pilot study. Cancer Immunol. Immunother. 30, 165--169; Stewart, R.J.E., Hill, F.W.G., Masztalerz, A., Jacobs, J.J.L., Koten, J.W., Den Otter, W., 2003. Local low dose interleukin-2 therapy of bovine ocular squamous cell carcinomas in cattle in Zimbabwe, submitted for publication; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H.M., Rutten, V.P.M.G., Ruitenberg, E.J., 1993. Low doses of interleukin-2 can cure large bovine ocular squamous cell carcinoma. Anticancer Res. 13, 2453-2455; Den Otter, W., Hill, F.W.G., Klein, W.R., Koten, J.W., Steerenberg, P.A., De Mulder, P.H., Rhode, C., Stewart, R., Faber, J.A., Ruitenberg, E.J., 1995. Therapy of bovine ocular squamous cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up. Cancer Immunol. Immunother. 41, 10-14].

Local interleukin 2 therapy is most effective against cancer when injected intratumourally.

Local interleukin 2 (IL-2) therapy is more effective against systemic tumours than systemic IL-2 therapy, but it remains unclear whether IL-2 should be injected intratumourally or peritumourally. To investigate this question, we treated DBA/2 mice bearing a large subcutaneous syngeneic SL2 lymphoma with either intra or peritumoural IL-2 therapy. Both applications enhanced survival, but intratumourally injected IL-2 was more effective than peritumourally injected IL-2. Tumours started to regress 4 days after IL-2 injection. Tumour cells died at the IL-2 injection site, although IL-2 is not directly cytotoxic for SL2 cells in vitro. Tumour cell death correlated well with oedema and extravascular erythrocytes, but less with leukocyte infiltrates. In mice bearing two s.c. tumours, intratumoural application therapy of IL-2 in one tumour caused decrease in size of both tumours in 4-9 days after therapy. However, the IL-2 treated tumours regressed more strongly than the untreated tumours. We conclude that vascular leakage and/or tissue destruction inside the tumour may contribute to the enhanced effect of intratumoural IL-2 therapy compared to peritumoural IL-2 therapy. Hence, we recommend applying of intratumoural rather than peritumoural IL-2 therapy.

Treatment of stage III-IV nasopharyngeal carcinomas by external beam irradiation and local low doses of IL-2.

The therapeutic effect of intratumoural application of Interleukin-2 (IL-2) was studied in patients with stage III-IV nasopharyngeal carcinoma (NPC) that received radiotherapy. Patients with stage III-IV NPC receiving a standard treatment of 7,000 cGy external beam irradiation have a mean disease-free survival of about 1.5 years. In this paper, we describe ten of these patients who were treated with additional peritumoural and intratumoural injections with 3 x 10(4) U IL-2 on 5 days in weeks 2, 4, and 6 of the 7-weeks' irradiation period. This combined treatment group was compared with a historical group of patients treated with standard irradiation alone. Local IL-2 therapy showed a marked clinical and statistical significant improvement of disease-free survival. After 5 years, 63% of the IL-2 treated patients were disease-free versus 8% of the control patients. These results suggest that the therapeutic results of radiotherapy can be significantly improved by combining it with local IL-2 treatment. To our knowledge, this is the first clinical report showing that local IL-2 therapy is effective against an infiltrative and locally metastasizing tumour in human patients.

In situ crosslinked biodegradable hydrogels loaded with IL-2 are effective tools for local IL-2 therapy.

We investigated the therapeutic efficacy of recombinant human interleukin-2 (rhIL-2)-loaded, in situ gelling, physically crosslinked dextran hydrogels, locally applied to SL2 lymphoma in mice. The physical crosslinking was established by stereocomplex formation between d-lactic acid oligomers and l-lactic acid oligomers grafted separately to dextrans. The stereocomplex hydrogel as described in our manuscript has several favourable characteristics, which enables its use as system for the controlled release of pharmaceutically active proteins. Firstly, the hydrogel system is a physically crosslinked system. In physically crosslinked gels, the use of chemical crosslinking agents is avoided. Such agents can potentially inactivate the protein and can covalently link the protein to the hydrogel network. Secondly, the hydrogel formation takes place at room temperature and physiological pH, and, importantly, in an all-aqueous environment. All factors are important to preserve the three-dimensional structure, and thus the biological activity, of the protein to be entrapped and released from the gels. Thirdly, the gel formation does not occur instantaneously. This means that a liquid formulation can be injected which solidifies after injection (in situ gel formation is possible). Fourthly, no pH drop during degradation is expected during degradation. As a control, free rhIL-2 was administered locally in either a single injection or at five consecutive days. All mice received the same total dose of rhIL-2. The rhIL-2-loaded hydrogels released most IL-2 over a period of about 5 days. The biocompatibility and biodegradability of the gels were excellent, as there were no acute or chronic inflammatory reaction and as the gels were replaced completely by fibroblasts after 15 days. The therapeutic efficacy of rhIL-2-loaded in situ gelled hydrogels is very good, as was demonstrated in DBA/2 mice bearing SL2. The therapeutic effect of a single application of gels loaded with 1 x 10(6) IU rhIL-2 is at least comparable to the therapeutic effect of injection of an equal dose of free rhIL-2. All mice cured with rhIL-2-loaded hydrogels survived a subsequent challenge, rejecting 10(6) intraperitoneal (i.p.) injected SL2 cells. In conclusion, this study demonstrates that in situ gelling, physically crosslinked dextran hydrogels slowly release encapsulated rhIL-2 in such a way that it is intact and biologically and therapeutically active. These hydrogels may greatly enhance the clinical applicability of rhIL-2 immunotherapy as only a single treatment is required and as these hydrogels are completely biodegradable.

Further development of local IL-2 therapy of cancer: multiple versus single IL-2 treatment of transplanted murine colon carcinoma.

We have compared the effect of one and up to four local IL-2 treatments of transplanted MC38 colon carcinoma. A single IL-2 treatment prolonged the survival time ( p=0.015), but no cure was obtained. One local IL-2 treatment inhibited tumor growth for about 1 week. After the start of tumor regrowth, a further IL-2 injection was given. After four IL-2 injections 6 out of 13 mice were cured. Histological studies show that IL-2 induced a local vascular leakage syndrome leading to massive peritumoral edema and subsequent necrosis of tumor tissue. IL-2 also attracted infiltrating cells, mainly macrophages. Subsequent IL-2 injections led to complete tumor regression. We believe that the combination of necrotic tumor debris and the IL-2-induced macrophage reaction enhanced a tumor-specific immune response. This local IL-2 application was not toxic.

Therapeutic effect of a single peritumoural dose of IL-2 on transplanted murine breast cancer.

Interleukin-2 therapy is not clearly effective against breast cancer both in mouse models and in human patients. However, the study of IL-2 therapy of breast cancer remains important, as 3,700 women died from this malignancy in the Netherlands in 2000. Previously we have shown the therapeutical efficacy of a single peritumoural IL-2 application in different experimental models and in veterinary patients. Here we apply this mode of IL-2 therapy to advanced mouse mammary carcinoma models, i.e., severe metastasised tumours in A/Sn mice and non-metastasised carcinomas in BALB/c mice. Mice with advanced transplanted mammary carcinomas were given a single peritumoural treatment with 2.5 x 10(6) IU IL-2 at days 10-14 after i.p. or s.c. inoculation of 10(6) carcinoma cells. Within each experiment it was always possible to distinguish relatively slowly and fast growing tumours which allows the therapeutical effect of IL-2 in tumours with different growth rates to be studied. A new approach to analyse results enabled us to show that survival of mice with transplanted, advanced metastasised breast cancer can be significantly improved after a single local treatment with IL-2. Advanced relatively fast i.p and s.c. growing mammary carcinomas seem to be more sensitive to a single IL-2 treatment than relatively slowly growing tumours. IL-2 was most effective against non-metastasised mouse breast cancer.

Mechanisms of macrophage cytotoxicity in IL-2 and IL-12 mediated tumour regression.

IL-2 and IL-12 are promising anti-tumour agents. However, little attention has been paid to the role of macrophages during IL-2/IL-12 mediated tumour rejection. We studied the role of macrophages during IL-2/IL-12 mediated tumour rejection in DBA/2 mice bearing syngeneic SL2 lymphoma. Local treatment with IL-2 and IL-12 cured 85% of mice with severe metastasised tumour load. In vivo depletion studies showed that macrophages were required for the anti-tumour effect of IL-2 and IL-12. Macrophages could kill tumour cells both non-specifically and by antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2, IL-12 or IL-2/IL-12 enhanced production of specific IgG1 immunoglobulins, while treatment with IL-12 and IL-2/IL-12 additionally induced IgG2a production. FcgammaRII and/or III were essential for ADCC expression after treatment with IL-2 and IL-12. These data show for the first time the essential role of macrophages during IL-2/IL-12 mediated tumour rejection and also suggest that IL-2 and IL-12 act via different mechanisms.