Fluorescent stains for quantification of DNA by confocal laser scanning microscopy in 3-D.

Confocal microscopy requires the use of fluorophores to visualize structures of interest within a specimen. To perform reliable measurements of the intensity of fluorescence, the stain should be specific, penetrate well into tissue sections, and bind stoichiometrically. Furthermore, emission must be linear with respect to DNA content and brightness, and fluorescence should be stable. Confocal microscopy is used to determine DNA ploidy and to analyze texture of nuclei, which is accomplished in three dimensions, because nuclei can be measured within the original tissue context. For this purpose the sample must be stained with a DNA binding fluorophore with the properties described above. Stains with different properties have been developed for different applications. We review here the advantages and disadvantages of these different stains for analyzing DNA ploidy and nuclear texture using three-dimensional microscopy. We conclude that SYBR green I and TO-PRO-3 are the most suitable stains for this purpose at present.

Intra-arterial embolization of head-and-neck cancer with radioactive holmium-166 poly(L-lactic acid) microspheres: an experimental study in rabbits.

A total of 22 NZW rabbits with VX2 squamous cell carcinomas transplanted into the auricles were intra-arterially (i.a.) embolized with radioactive or inactive holmium-labelled poly(L-lactic acid) (HoPLA) microspheres with a mean diameter of 38-80 microm. The effects on tumour growth, the efficiency of i.a. infusion, the efficacy of retention of microspheres in the primary tumour and the excretion of free holmium-166 were analyzed. Complete tumour remissions were obtained in 79% and 86% following embolization with radioactive and inactive microspheres, respectively. Over 95% of the microspheres were retained in the tumour and the leaching of holmium-166 in urine and faeces was less than 0.1% in 2 days. The injection efficiency was not optimal, as 40% of the microspheres were retained in the cannulation system. Arterio-arteriolar connections should be detected and closed prior to embolization to prevent stray emboli from entering the brain. It is concluded that 166HoPLA microspheres are promising candidates for further studies on radio-embolization of unresectable head-and-neck cancer.

Tumour embolization of the Vx2 rabbit head and neck cancer model with Dextran hydrogel and Holmium-poly(L-lactic acid) microspheres: a radionuclide and histological pilot study.

INTRODUCTION: Intra-arterial embolization of unresectable malignant tumours with biodegradable microspheres is an effective way of selective anti-tumour therapy. Promising candidates are Dextran hydrogel (Dex) microspheres for chemo-embolization and Holmium-166 poly(L-lactic acid) (166HoPLA) microspheres for radio-embolization. This study was performed to investigate the distribution of intra-arterially injected microspheres both in vivo and histologically in order to establish an optimal size of particles for embolization of head and neck tumours. MATERIAL: Twenty rabbits with Vx2 auricular tumours were embolized via the caudal auricular artery with 4 different batches of microspheres: Radioactive (166)HoPLA microspheres sieved between 20 and 50 microm and Dextran hydrogel microspheres sieved between 20 and 100 microm (Dex20), 30 and 100 microm (Dex30) or 50 and 100 microm (Dex50). Dex20 and Dex50 microspheres were labelled with 99mTechnetium in six cases. METHODS: The average particle size of the microspheres was determined. The proportion of microspheres entrapped in the tumour was measured with a gamma camera. The distribution of microspheres around the primary tumour and spill of particles over into lungs or other organs was analysed from histological sections. RESULTS: The mean particle diameter varied from 19 to 66 microm: (166)HoPLA 19+/-11 microm, Dex20 40+/-19 microm, Dex30 50+/-19 microm, Dex50 66+/-21 microm. The 19 microm(166)HoPLA particles proved inadequate for embolization as 51% spilled over into the lungs, whereas over 95% of the 40-66 microm Dex microspheres were retained within the primary tumour area. Particle density in lung tissues proved significantly lower for the Dex50 group. Stray emboli to the brain occurred in two rabbits. CONCLUSION: The results of this investigation show that both Dextran hydrogel and holmium-166 poly(L-lactic acid) microspheres are potential candidates for embolization of head and neck cancer. In future studies, arterio-arteriolar anastomoses which might confound treatment should be identified and occluded. Particles with a number weighted mean diameter of at least 40 microm and a volume weighted mean size up to 70 microm should be used.

Influence of p53 and bcl-2 on proliferative activity and treatment outcome in head and neck cancer patients.

Knowledge about the influence of biomarkers on cell proliferative activity might explain differences in radiosensitivity between head and neck tumors and might improve patient selection for the most optimal treatment strategy. p53 and bcl-2 protein expression were determined immunohistochemically in 56 head and neck cancer patients, treated by surgery only in five cases and by radiotherapy, with or without surgery, in 51 cases. Relationships with various cell proliferation markers, determined by flow-cytometry (G1-phase fraction, S-phase fraction, BrdUrd-labeling index, duration of S-phase and potential doubling time) were investigated. Associations between these cell proliferation parameters, on the one hand, and both p53 and bcl-2, on the other, were not found. Furthermore, p53 and bcl-2 expression were both not related to clinicopathological parameters (T- and N-stage, site, grade) and did not affect loco-regional recurrence-free survival and/or disease-free survival. We could not find a prognostic value for both p53 and bcl-2 protein expression to differentiate radiosensitive from radioresistant head and neck tumors.

Evaluation of the VX2 rabbit auricle carcinoma as a model for head and neck cancer in humans.

INTRODUCTION: This study investigates whether the VX2 carcinoma cell line, transplanted into the rabbit auricle, can be used as a head and neck cancer model. The biologic behaviour of this model is evaluated, comparing tumour transplantation with either tissue pieces or cell suspensions. MATERIAL: Thirty-six adult NZW rabbits received s.c. injections of VX2-suspensions (Group S) and 11 rabbits received solid VX2-pieces (Group P) into both auricles. METHODS: In Group S, 16 rabbits were sacrificed at various days before (S1) and 15 after (S2) the 28th day following transplantation. In the other five rabbits transplantation failed. Animals from Group P were sacrificed every 2 weeks after the 28th day. At autopsy the size of the primary tumours and of lymph node, lung and other metastases were assessed. If transplantation failed, the maximal tumour size and the time at which regression took place were recorded. Exponential trend lines were used to create growth curves of metastases. Differences between groups were evaluated with the chi2 test, correlations between parameters with Kendall's tau. RESULTS: The tumour take-rate in Groups S and P was 78% and 59% respectively. The maximal size and time at which regression occurred was significantly different, amounting to 83 +/- 7 mm2 at 10.4 +/- 1.6 days (Group S) and 243 +/- 30 mm2 at 20.9 +/- 2.0 days (Group P), respectively. Development of lymph node metastases was not different. In Groups P and S2, over 90% of the necks contained lymph node metastases. There was a higher incidence of lung metastases in Group S2 when compared to Group P (47% vs. 14%) but it was not statistically significant. A significant correlation (p<0.05) between weight loss and the size of lung metastases was found. CONCLUSION: Transplantation of the VX2-tumour with cell suspensions produces a useful head and neck cancer model for locoregional disease in which anti-tumour regimens against both the primary and lymph node metastases can be tested. Transplantation with tumour pieces is not advised as the take-rate is low and spontaneous remissions occur at a late stage.

Prognostic relevance of MIB-1 immunoreactivity, S-phase fraction, 5-bromo-2'-deoxyuridine labeling indices, and mitotic figures in gliomas.

Prognostic relevance of cell proliferation markers was evaluated in 27 glioma patients. By 1) flow cytometry (FCM), i.e., S-phase fraction (SPF), and BrdUrd-labeling index (LIfcm); 2) immunohistochemistry (IHC), i.e., BrdUrd-labeling index (LIihc) and MIB-1 immunoreactivity (MIB-1 LIihc); and 3) histologic examination, i.e., the presence or absence of cells in mitoses, were assessed. A longer local progression free survival (LPFS) was significantly associated with low SPF, low LIfcm, and low MIB-1 LIihc. For LIihc, no significant association was found. LIfcm appeared to be a more promising prognosticator than MIB-1 LIihc. In comparison with this marker, the presence or absence of mitotic figures appeared to be an even stronger prognosticator. Prognostic significance of LIfcm appeared to be of importance in low-grade gliomas. The number of patients in our study is limited. Our findings were: 1) the presence or absence of cells in mitoses (M-phase activity) appeared to be of more prognostic significance than LIfcm (S-phase activity) and MIB-1 LIihc (non-G0-phase activity); 2) of the tested experimental cell proliferation markers, LIfcm appeared to be of more prognostic significance than MIB-1 LIihc, SPF, and LIihc; and 3) LIfcm is likely to be an important prognosticator in low-grade gliomas and is, therefore, not definitive and only of potential interest.

The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial embolization of head and neck squamous cell carcinoma with dextran microspheres.

A head and neck cancer model is developed using the VX2 carcinoma cell line injected s.c. in both ears of New Zealand White (NZW) rabbits. The study is focused on the effects of intraarterial embolization of the carcinomas with a new type of dextran hydrogel microspheres. During the phase of exponential growth the tumour-surface doubling-time was 7.1+/-2.0 days. Standard deviation in growth of the tumours was significantly larger between separate animals than between tumours growing in the left and right auricle of each individual animal (2.0 versus 0.65 days). A fresh cell suspension containing at least 10 x 10(6) vital tumour cells was necessary to yield a tumour-take of 85%. The caudal auricular artery perfuses the caudal half of the external ear and is very suitable for macroscopic cannulation. Histological evaluation shows, that the use of dextran hydrogel microspheres of at least 25 microm in combination with ligation of non-tumour perfusing branches of the central auricular artery yields diffuse embolization of the VX2 carcinoma. This tumour model can be of use in further studies to optimize particle size and dosage for embolization as well as to evaluate the effect of different anti-neoplastic drugs, slowly released by controlled degradation of dextran microspheres.

Interleukin-2: hope in cases of cisplatin-resistant tumours.

To establish whether or not local low-dose recombinant interleukin-2 (rIL-2) therapy might result in therapeutic benefit for ovarian cancer patients treated with cisplatin, the antitumour effects of rIL-2 and of combined treatment with cisplatin and rIL-2 in a mouse ovarian tumour (MOT) model were studied. In addition, some possible mechanistic aspects underlying the observed antitumour responses were analysed. MOT cells were injected i.p. into syngeneic, immunocompetent, female C3HeB mice. Tumour-bearing mice received i.p. treatment with cisplatin, rIL-2 or both. The MOT tumour appeared to be hardly responsive to treatment with cisplatin only or rIL-2 only. In contrast, combined local treatment with low doses of cisplatin (1 and 5 mg/kg body weight) and rIL-2 (60000 U/day) resulted in an effective antitumour response in MOT-bearing mice. Complete rejection of the i.p. (local) tumour occurred in up to 60% of the cases. In vitro studies showed that cisplatin and rIL-2 do not have cumulative direct toxic effects on MOT cells. Mice cured after combined treatment with cisplatin and rIL-2 were not able to reject a rechallenge with tumour cells, indicating that these mice had not developed immunity to the tumour. Analysis of tumour-associated leucocytes, however, showed that combined treatment with cisplatin and rIL-2 did result in enhanced non-specific cytolytic activity of peritoneal leucocytes. We have thus demonstrated that, in the MOT model, combined local treatment with low doses of cisplatin and of rIL-2 is far more effective than therapy with cisplatin alone. Non-specific cytotoxicity of leucocytes appears to be involved in antitumour responses induced by combined treatment with cisplatin and rIL-2. These results suggest that, in human ovarian carcinoma, much better results may be obtained with the combined treatment of cisplatin and low (non-toxic) doses of rIL-2 than with cisplatin only. This may also apply to cisplatin-resistant ovarian carcinoma.

Local low-dose interleukin-2 induces systemic immunity when combined with radiotherapy of cancer. A pre-clinical study.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic.

Priming of the antitumor response promotes efficacy of interleukin-2 therapy.

We have studied the effect of active specific immunization (ASI) on the antitumor response induced by locoregional, low-dose interleukin-2 (IL-2) therapy. On day 0, mice were injected i.p. with viable, syngeneic tumor cells and with irradiated tumor cells (ASI). Low-dose IL-2 treatment was given i.p. for 5 consecutive days. ASI led to extended survival in two out of seven models tested. In these two models, enhanced efficacy was observed when both ASI and IL-2 were administered. In the five models in which ASI had no therapeutic value, ASI + IL-2 treatment was no more effective than IL-2 therapy. In the SL2 lymphoma model, use of ASI prior to IL-2 therapy given as early as days 1-5 led to at least 60% cure, whereas IL-2 therapy without ASI was only effective when administered after day 9. In the P815 mastocytoma model, however, ASI, IL-2, and the combination caused negative (suppressive) effects when administered on days 6-10. IL-2 administered on days 6-10 was therapeutically effective in this model when mice were treated with cyclophosphamide on day 6. In both the SL2 and the P815 tumor models, cured mice were specifically immune. The positive and negative effects observed were not due to the increased number of cells injected (non-specific inflammation) nor to possible antigenic alteration of the ASI cells by irradiation, as ASI with fragmented tumor cells was also effective in inducing synergy. Investigations into the underlying mechanism indicated that CD4+ cells play an important role. In total, the results indicate that ASI may be a good supplement to locoregional IL-2 treatment if care is taken to alleviate immunosuppressive activities.

Membrane modifications in human erythroleukemia K562 cells during induction of programmed cell death by transforming growth factor beta 1 or cisplatin.

Transforming growth factor beta 1 (TGF beta 1) and cisplatin induce apoptosis (programmed cell death, PCD) in human erythroleukemia K562 cells in an additive manner. After PCD was induced in K562 cells, analysis of phospholipid composition, fatty acids and cholesterol content in their membranes showed a decrease in phosphatidylethanolamine and an increase in phosphatidylserine, cardiolipin and phosphatidic acid. Moreover, cisplatin but not TGF beta 1 enhanced sphingomyeline levels in apoptotic cells, whereas TGF beta 1 increased the amount of linoleic acid and, more remarkably, of cholesterol. The combination TGF beta 1 + cisplatin produced membrane changes similar to those provoked by each inducer individually. Furthermore, the specific activities of 5-lipoxygenase and cytosolic phospholipase A2, both modulating the physical properties of membranes and membrane-lipid-mediated intracellular signalling, were enhanced by treatment with TGF beta 1 or TGF beta 1 + cisplatin. These findings highlight the profound changes in cell membranes during the biochemical events of the apoptotic pathway.

The success of locoregional, low-dose recombinant interleukin-2 therapy in tumor-bearing mice is dependent on the time of rIL-2 administration.

The extremely different administration schedules that are used in testing recombinant interleukin-2 (rIL-2) therapies for cancer may account for the extreme variation in efficacy reported in various studies in animal models. A major point may be the variation of the time interval between tumor transplantation and rIL-2 therapy. We hypothesized that administration of rIL-2 before the immune system has mounted a specific cellular reaction against the tumor (associated antigens) might result in lesser efficacies than later rIL-2 administration. This hypothesis was tested in DBA/2 mice bearing a syngeneic SL2 lymphoma. When 7000 IV/day rIL-2 was administered to tumor-bearing mice for 5 consecutive days starting on day 1, 3, 4, 5, or 6 after tumor inoculation, the survival curve of the mice did not significantly differ from that of diluent-treated mice. In contrast, a significant difference was observed when treatment was begun on day 7, 8, 9, 10, or 12 (p < or = 0.004). rIL-2 therapies begun on day 9 or 10 were most effective, curing up to 80% of mice treated, despite there being an enormous burden of disseminated tumor present at that time (1-4% of the total body weight). When rIL-2 was administered for fewer than 5 consecutive days, beginning on day 10, the efficacy of the therapy dropped radically (p < or = 0.055). Involvement of a specific anti-tumor reaction was also tested. All mice that were cured of the tumor as a result of rIL-2 therapy proved to be specifically immune to the SL2 tumor. Furthermore, day 10-14 administration of rIL-2 was completely ineffective in CD4(+)-cell depleted mice (p = 0.0116 vs. rIL-2 therapy in non-depleted mice). Together, this implies that this form of rIL-2 therapy is mediated by tumor-specific T-cells. As a whole, these results indicate that T-cell mediated rIL-2 therapy of cancer in animal models is sensitive to the time when the rIL-2 is administered and to the length of time for which the rIL-2 is given. This should be taken into account when planning new therapy protocols and when analyzing published data.

The glycosylation profile of interleukin-2 activated human lymphocytes correlates to their anti-tumor activity.

BACKGROUND: Natural killer cells display spontaneous, non-MHC-restricted cytotoxicity against tumour cells, which is strongly enhanced after incubation with IL-2. The molecular background of the increased anti-tumour activity of these lymphokine-activated killer cells is still only partly understood. MATERIALS AND METHODS: In this paper, investigation has been made of the correlation between cell surface glycosylation and anti-tumour activity of LAK cells by stimulating peripheral blood lymphocytes with interleukin-2, in the presence of inhibitors of N- and O-glycosylation. RESULTS: Inhibition of N- or O-glycosylation of proteins during IL-2 activation leads to a 70-80% decrease in the cytolytic activity of LAK cells against K562 and Daudi tumour cells, coinciding with drastic alterations in their cell surface carbohydrate profile. CONCLUSION: The conclusion is drawn that there is a clear correlation between the glycosylation of LAK cell glycoproteins and their anti-tumour activity which points to the involvement of cell surface glycoconjugates in the development of LAK activity.

Reevaluation of the superiority of polyethylene glycol-modified interleukin-2 over regular recombinant interleukin-2.

Other groups have reported a superior antitumor efficacy of polyethylene glycol-modified interleukin-2 (PEG-IL-2) compared with regular recombinant interleukin-2 (rIL-2). However, detailed comparison of the antitumor efficacies of locally applied PEG-IL-2 and rIL-2 in the well-established DBA/2-SL2 model shows a higher antitumor efficacy of PEG-IL-2 only at doses < 800 micrograms IL-2 protein/kg body weight. At doses > 800 micrograms IL-2 protein/kg body weight, rIL-2 has better therapeutic efficacy. The superiority of rIL-2 at doses > 800 micrograms IL-2 protein/kg body weight is a result of the toxicity of PEG-IL-2 at these doses. With either IL-2 preparation, cure rates of approximately 90% can be obtained at nontoxic doses. We conclude that PEG-IL-2 does not have superior antitumor efficacy to rIL-2. The main advantage of PEG-IL-2 is that for optimal therapeutic efficacy a daily injection schedule is not required as seems to be the case for rIL-2.

Effect of immunomodulators on specific tumor immunity induced by liposome-encapsulated tumor-associated antigens.

Reconstituted membranes consist of liposomal structures formed by removal of detergent from solubilized membrane constituents. The membrane-like configuration of reconstituted membranes makes them attractive as vehicles for presentation of tumor-associated antigens and induction of immune responses. In this study the potential of immunomodulators was assessed to enhance the specific immune response induced by immunization with reconstituted membranes prepared from SL2 lymphosarcoma cells. Reconstituted membranes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) provided better protection against a challenge with SL2 cells than did reconstituted membranes containing alternative immunomodulators. Local administration of IL-2 at the immunization sites further augmented the protection induced by reconstituted membranes with MTP-PE, but was ineffective when administered with plain reconstituted membranes. Immunity elicited by the triple modality of reconstituted SL2 membranes with MTP-PE and IL-2 was specific for SL2 cells. Systemic immunity was obtained against a challenge with a 100-fold higher number of SL2 cells than was reached after immunization with reconstituted membranes alone (10(5) vs. 10(3) SL2 cells). Macrophages isolated from the peritoneal cavity of immunized mice 5 to 7 days after tumor challenge expressed high in vitro cytotoxicity. However, in contrast to the observed specificity of the systemic immunity, macrophages killed both SL2 cells and non-related P815 cells. Neither major cytotoxic lymphocyte activity nor substantial cytotoxic antibody titers were detectable. These results clearly indicate that the approach using reconstituted membranes combined with particular immunomodulators warrants further exploration for the development of safe, well-characterized cancer vaccines.

Interleukin-2-containing liposomes: interaction of interleukin-2 with liposomal bilayers and preliminary studies on application in cancer vaccines.

Interleukin-2 (IL-2) incorporation in liposomes was studied under different conditions. Information was obtained on the mechanism of interaction of glycosylated recombinant IL-2 with liposomal bilayers. This information was utilized to formulate liposomes with high levels of incorporated IL-2. Multilamellar vesicles were prepared by hydration of a lipid film with an IL-2 solution. The incorporation efficiency, measured with a bioassay after forced release of IL-2 from the vesicles, was strongly dependent on the charge of the liposomes and the pH and ionic strength of the hydration medium. Negatively charged liposomes composed of phosphatidylcholine/phosphatidylglycerol (9:1) and prepared with IL-2 dissolved in 10 mM NaAc/270 mM glycerol, 0.1% BSA, pH 5, showed the highest incorporation efficiency (81%) among the investigated preparations. This type of liposome was selected for further study. Electrostatics play a crucial role in the process of IL-2 association with this type of liposome. Initial studies concerning induction of protective tumor immunity by immunization with reconstituted membranes with muramyl tripeptide phosphatidylethanolamine indicate that coinjection of IL-2-containing liposomes provided a significant enhancement of the immune response.

Critical factors for liposome-incorporated tumour-associated antigens to induce protective tumour immunity to SL2 lymphoma cells in mice.

Physical and immunogenic properties of reconstituted membranes designed for the presentation of tumour-associated antigens (TAA) to the immune system are described. Proteins and lipids of crude membranes of SL2 murine lymphosarcoma cells were partially solubilized with octylglucoside. Reconstituted membranes, consisting mainly of unilamellar vesicles with a diameter of 0.03-0.15 microns, were formed by detergent removal and were purified by floatation in a discontinuous sucrose gradient to remove non-lipid-bound protein. Subcutaneous immunization of syngeneic mice with reconstituted membranes or with purified reconstituted membranes induced protection against an intraperitoneal challenge with 10(3) viable SL2 cells. Reconstituted membranes were more immunogenic than crude membranes in immunoprotection experiments when compared on the basis of protein dose. Detergent removal was required to obtain an immunogenic presentation form of SL2 membrane antigens and to avoid toxicity associated with the detergent. Reconstitution of SL2 membranes in the presence of exogenous phospholipid slightly increased the fraction of protein that associated with the reconstituted membranes. However, the immunogenicity of the solubilized membrane TAA was not significantly affected by the presence of exogenous phospholipid. The reconstitution procedure described may be useful in identifying membrane factors required for the induction of immune responses against TAA. The versatility of the system may be employed to develop safe alternatives for whole-cell vaccines.

Human natural killer cells: a convenient purification procedure and the influence of cryopreservation on cytotoxic activity.

The recognition of natural killer cells as a lymphoid subpopulation with a distinct set of surface markers has led to the development of a variety of antibody-based purification methods. In this paper we describe a rapid, three-step negative selection protocol for the purification of human natural killer (NK) cells from the mononuclear cell fraction, which is obtained by the centrifugation of peripheral blood on Ficoll-Paque. Subsequently, monocytes and B lymphocytes are removed by adherence to nylon wool and T lymphocytes by panning with anti-CD3. With this procedure, CD3-, CD16/56+ NK cells are purified about five-fold, from 12 +/- 3% in the starting population to a final purity of 61 +/- 11%. A further increase to > or = 70% is obtained, if an extra Ficoll centrifugation step is included. The recovery of NK cells (50%) is significantly higher than is usually achieved by previously described procedures. Furthermore, we show that activation of cytotoxicity, with concomitant changes in target specificity, occurs when frozen/thawed NK effector cells are kept in culture in order to regain their pre-freezing cytotoxicity levels.

Transfer of tumor immunity by both CD4+ and CD8+ tumor infiltrating T lymphocytes activated in vivo by IL-2 therapy of tumor bearing mice.

DBA/2 mice were inoculated i.p. with syngeneic SL2 lymphoma or P815 mastocytoma on day 0 and treated i.p. with 20,000 units IL-2/day on day 10-14. This treatment is curative for 70-90% of the tumor bearing mice. Peritoneal cells and/or spleen cells were isolated from responding mice at the last day of IL-2 therapy. The in vivo antitumor activity of these cells was tested in Winn Assays (i.p.) and by adoptive transfer (i.v.) into mice injected s.c. with tumor previously. Peritoneal exudate cells isolated on day 14 (PEC14) from mice cured of SL2 tumor were highly effective in Winn Assays. Up to 5 x 10(7) SL2 cells could be eliminated in naive mice when injected i.p. together with 2 x 10(7) PEC14. Adoptive transfer (i.v.) of PEC14, without the addition of IL-2, into mice s.c. injected with SL2 tumor cells 1 or 3 days earlier, also prevented outgrowth of the tumor cells. T cells isolated from the spleens were less effective. Only at E:T ratios of 100:1 and 10:1 was tumor outgrowth inhibited. The adoptive transfer of PEC14 resulted in a long lasting immunity of the recipient mice. Furthermore, it was shown that depletion of both the CD8+ and CD4+ T cells from the suspensions used in the transfer studies, resulted in a significant decrease of tumor inhibition. However, the effect was not abrogated completely. PEC14 isolated from IL-2-treated DBA/2 mice cured of P815 tumor, protected naive mice against P815 tumor at E:T ratio 20:1. Adoptive transfer (i.v.) of these PEC14 into mice bearing s.c. P815 did not have an antitumor effect. In conclusion, low dose i.p. IL-2 therapy predominantly induces locally both CD4+ and CD8+ T cells with a strong antitumor activity in vivo. The potency of the IL-2-induced immunity seems related to the type of tumor used.

Dualistic effects of cis-diammine-dichloro-platinum on the anti-tumor efficacy of subsequently applied recombinant interleukin-2 therapy: a tumor-dependent phenomenon.

The efficacy with which disseminated SL2 and P815 tumors, in syngeneic DBA/2 mice, can be eradicated with low-dose recombinant interleukin-2 (rIL-2) therapy is about equal. Treatment (i.p.) of DBA/2 mice, injected i.p. with SL2 or P815 cells on day 0, with rIL-2 (Proleukin) on days 10 to 14 results in a cure rate of 50 to 60% in each case. The in vitro sensitivity of SL2 and P815 cells to cis-diammine-dichloro-platinum [II] (cisplatin) is also comparable, although P815 appears to be slightly more sensitive. In vivo, however, therapy with cisplatin is far less effective against P815 than against SL2. In the DBA/2-SL2 model, at all doses tested, combination therapy with cisplatin (administered on day 2) and rIL-2 (administered on days 10-14) resulted in anti-tumor efficacy greater than that of either drug separately. In contrast, in the DBA/2-P815 model, cisplatin decreased the anti-tumor efficacy of subsequently applied rIL-2 therapy. As the only difference between the 2 tumor models is the tumor itself, the success of combination therapy with cisplatin and rIL-2 was dependent on tumor characteristics. We suggest that in these 2 tumor models, neither the sensitivity of these tumors to cisplatin nor their growth and dissemination patterns were responsible for the contrasting results of combination therapy in these models. Instead, tumor-dependent immune-modulating effects of cisplatin may be the cause of these effects. These immune-modulating effects may comprise (a) effects of cisplatin on the tumor cells, resulting in changes in their susceptibility to immune effector cells, or changes in their immunogenicity; (b) activating or suppressive effects of cisplatin on immune effector cells; or (c) a combination of these effects. These effects could then either synergize or antagonize with the immune activating properties of rIL-2.