Tumour cell variants with increased immunogenicity obtained by mutagen treatment

By treating mouse tumour cells in vitro with mutagens it is possible to obtain at high frequency variants that are rejected by normal syngeneic mice. Such variants have been designated 'tum-' to distinguish them from the original tumorigenic (tum+) cell lines. Most tum- variants appear to express new, individual antigens that can be defined in vivo by cross-protection experiments or in vitro by cytolytic T lymphocytes. The failure of tum- variants to form progressive tumours is apparently the result of an immune rejection response. Mice that have rejected a tum- variant clone are often protected against a subsequent challenge with the original tum+ cells. This was even demonstrated for two spontaneous mouse tumours for which no immunogenicity could be demonstrated otherwise. An analysis of 21 variants derived from mastocytoma P815 with cytolytic T lymphocyte populations showed that the repertoire of tum- antigens probably exceeds 50 specificities. No two variants have been found to express the same new antigen. Variant-specific long-term cytolytic T lymphocyte clones were isolated and used for the immunoselection of secondary antigen-loss variants that could then be analysed for the presence of previously undefined residual variant antigenic determinants. Somatic cell hybrids were prepared between different P815 tum- variants or between tum- variants and the original P815 cells. An analysis of the hybrids with variant-specific cytolytic T lymphocyte clones showed that the expression of tum- antigens was dominant. The intriguing paradox of the high frequency of tum- variants in populations of mutagen-treated tumour cells may require new genetic and immunological approaches before being understood.     

Induction of highly immunogenic variants of Lewis lung carcinoma tumor by ultraviolet irradiation

This study was undertaken to determine whether in vitro treatment of Lewis lung carcinoma (3LL) cells with ultraviolet (UV) radiation could increase their immunogenicity. Tumor cells were irradiated with UV light from a germicidal lamp (254 nm; UV-C) at a dose of 720 J/sq m. After 2 weeks of culture, the surviving cell population was cloned by limiting dilution. Cell suspensions of each clone were injected intrafootpad in C57BL/6 mice at a dose of 2.5 X 10(5) cells per mouse. Eighty independent clones were tested. Fifty-one clones showed decreased tumorigenicity and failed to grow in 20 to 95% of immunocompetent mice, whereas they produced tumors in 100% of irradiated (550 R) and athymic nude mice. These clones were designated "tum-" (nontumorigenic) clones. In contrast, all 25 clones selected from the untreated parental 3LL induced progressively growing tumors in 100% of the mice. After two courses of UV treatment, the uncloned 3LL population was rejected in 45% of inoculated mice. Mice rejecting an inoculum of a tum- clone were completely resistant to subsequent challenge with higher doses of the same or unrelated tum- clones. This resistance was fully expressed even after irradiation of immune mice with 550 R. Mice immune to a tum- clone also were able to prevent the growth of various tum+ clones or untreated 3LL tumor cells. When tum- and tum+ clone cells were simultaneously inoculated intrafootpad in opposite legs, rejection of tum- clone resulted also in the prevention of the growth of tum+ clone. Spleen cells of immune mice caused rapid elimination of radiolabeled 3LL tumor cells from the place of their inoculation (intrafootpad) and prevented tumor growth. In an in vitro cytotoxic assay, spleen cells after in vivo and in vitro immunization with tum- clones demonstrated high cytotoxic activity against various tum+ clones and parental 3LL cells, as well as against tum- clones. In addition, parental 3LL tumor cells and tum- cells were similarly able to inhibit cytotoxic activity in the cold target inhibition assay. However, in contrast to tum- cells, 3LL cells were less efficient in in vitro restimulation of cytotoxic activity of immune spleen cells. Therefore, these data suggest that tum-, tum+, and parental 3LL cells share a common antigenic specificity, which is not immunogenic in 3LL cells. UV treatment presumably converted the antigenic determinants present in the 3LL cells into an immunogenic form.     

Induction in a murine tumor of immunogenic tumor variants by transfection with a foreign gene

Transfection of the undifferentiated murine colon carcinoma line CT-26 with the gene coding for the hemagglutination antigen (HA) of influenza virus resulted in the generation of highly immunogenic tumor cells. CT-26 cells transfected with HA not only failed to grow in syngeneic mice but also protected normal animals against a challenge with otherwise lethal doses of parental nontransfected cells. The immunogenicity of HA-transfected cells appeared to correlate with surface HA expression in that tumorigenic clones of HA-transfected CT-26 cells expressed little HA, while immunogenic clones were high expressers of HA. Irradiation of immunogenic HA clones did not abrogate their immunogenicity. These observations demonstrate that immune recognition of a poorly immunogenic tumor can be produced by immunization with tumor cells expressing a defined, foreign cell surface antigen.     

Effectiveness of low-dose melphalan treatment against a nonimmunogenic primary induced mouse plasmacytoma

The fourth i.p. passage of the plasmacytoma "PR" induced by repeated i.p. injections was used for testing chemotherapy with melphalan. The development of "PR" ascitic tumours was slower and the survival time of inoculated mice was longer than that of MOPC-315 inoculated mice. Moreover, the myeloma protein secreted by the "PR" tumour cells, differed from MOPC-315 secreted myeloma protein in its electrophoretic mobility (fast gamma-2) and its characteristics as IgG immunoglobulin. Chemotherapy by a single injection of melphalan 7.5 mg/kg was effective in preventing the development of both MOPC-315 ascitic tumours and "PR" ascitic tumours. Mice cured from MOPC-315 tumours, however, developed antitumour response as shown by resistance to challenge with a high tumourigenic dose and by development of cytotoxic response in vitro against MOPC-315 tumour cells by spleen cells taken from the cured mice. On the other hand, mice cured from "PR" tumour by melphalan were highly susceptible to challenge and their spleen cells were not able to develop a cytotoxic response in vitro against target "PR" tumour cells.     

In vivo induction of antitumor immunity and protection against tumor growth by injection of CD154-expressing tumor cells

As they should enhance tumor-specific antigen presentation by dendritic cells, tumor cell lines genetically modified to express CD154 molecules have been used in an attempt to induce protective antitumor immunity. Two murine models were used: the major histocompatibility complex (MHC) class I negative melanoma B16F10 and the MHC class I positive mammary adenocarcinoma TS/A. CD154 or mock-transfected B16F10 or TS/A cells were injected subcutaneously into H-2-compatible B6D2 mice. CD154 expression by tumor cells induced a complete rejection (in the TS/A model) or a striking reduction (in the B16F10 model) of modified tumors growth, but also a significant protection against the growth of mock tumor cells injected simultaneously, either mixed with the CD154-expressing tumor cells, or in the other flank of mice. Thirty days after CD154-expressing tumor rejection, splenic lymphocytes from surviving tumor-free mice were able to inhibit tumor proliferation in vitro and significant amounts of IFN-gamma were detected in the sera of these mice. Growth kinetics of mock and CD154-expressing tumors in immunocompetent versus nude mice suggest that T lymphocytes and natural killer cells responses are implicated in this antitumor immunity. The injection of CD154-expressing tumor cell induced an antitumor protective response, both locally and distant from the injection site. The effect was most pronounced in MHC class I expressing TS/A tumor model.     

Immunostimulation of highly immunogenic target tumor cells by lymphoid cells in vitro.

A highly immunogenic mouse tumor was studied with the use of microcytotoxicity test, performed at lymphoid cell:target tumor cell ratios ranging from 1:1 to 1,000:1 at various times during tumour growth and after tumor excision. Although the tumor was highly immunogenic, in vitro lymphoid cell-mediated cytotoxicity was shown only with lymphoid cells from hyperimmunized mice, i.e., mice that had "seen" the tumor more than once. Instead, the lymphoid cells harvested 2 and 3 weeks after tumor inoculation, as well as after excision of the tumor, stimulated the target cells. At 4 weeks after inoculation of the tumor, there appeared to be neither stimulation nor inhibition of the target tumor cells, though at this point the growth of this highly immunogenic tumor had been arrested in vivo.     

Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of antitumor immunity and for tumor immunotherapy

Dendritic cells (DCs) are potent stimulators of immunity, and DCs pulsed with tumor antigen ex vivo have applications in tumor immunotherapy. However, DCs are a small population of cells, and their isolation and pulsing with antigen can be impractical. Here we show that a crude preparation of plasma membrane vesicles (PMV) from the highly metastatic murine melanoma (B16-OVA) and a surrogate tumor antigen (OVA) can be targeted directly to DCs in vivo to elicit functional effects. A novel metal-chelating lipid, 3(nitrilotriacetic acid)-ditetradecylamine, was incorporated into B16-OVA-derived PMV, allowing recombinant hexahistidine-tagged forms of single chain antibody fragments to the DC surface molecules CD11c and DEC-205, to be conveniently "engrafted" onto the vesicle surface by metal-chelating linkage. The modified PMV, or similarly engrafted synthetic stealth liposomes containing OVA or OVA peptide antigen, were found to target DCs in vitro and in vivo, in experiments using flow cytometry and fluorescence confocal microscopy. When used as vaccines in syngeneic mice, the preparations stimulated strong B16-OVA-specific CTL responses in splenic T cells and a marked protection against tumor growth. Protection was dependent on the simultaneous delivery of both antigen and a DC maturation or "danger signal" signal (IFN-gamma or lipopolysaccharide). Administration of the DC-targeting vaccine to mice challenged with B16-OVA cells induced a dramatic immunotherapeutic effect and prolonged disease-free survival. The results show that the targeting of antigen to DCs in this way is highly effective at inducing immunity and protection against the tumor, with protection being at least partially dependent on the eosinophil chemokine eotaxin.     

The antitumor effect of intraperitoneal treatment with rhTNF-alpha muteins on Ehrlich ascites tumor growth

The intraperitoneal (i.p.) treatment with recombinant human tumor necrosis factor-alpha (rhTNF-alpha) is one of the possible therapies for tumors that are confined to the abdominal cavity. Clinical trials aiming at the exploitation of the antitumor effects of rhTNF-alpha have been largely disappointing. In this model the activity of some rhTNF-alpha derivatives was studied. Ehrlich's ascites tumor (EAT) bearing Swiss albino male mice were treated i.p. three times a week with 10 micrograms/mice of rhTNF-alpha, mutein V or mutein VI for two weeks, starting on the 4th day after tumor inoculation. Control mice received PBS. The effect of the rhTNF-alpha derivatives on the course of EAT was evaluated basing on: total ascites volume (TAV); packed cell volume (PCV); total packed cell volume (TPCV); inhibitory growth rate (IGR); cellular population of EAT fluid; morphological EAT cell changes and mean survival time (MST). In the study mutein VI had only a slight effect on MST but significant on TAV- and TPCV-IGR (p < 0.001). In mice treated with rhTNF-alpha and mutein V the enhancement of MST (p < 0.01) was accompanied by TAV- and TPCV-IGR (p < 0.001). The number of EAT cells in ascites decreased after rhTNF-alpha and mutein V administration (p < 0.001). We conclude that treatment with high-dose of this modified molecule lacking the possibility of binding with p75R and not producing so intensified side effects is likely to find wider application in therapy and prevent the ascites growth just as rhTNF-alpha dosage.     

In vivo induction of tumor variants by phorbol 12-myristate 13-acetate

Malignant cells within solid tumors commonly exhibit phenotypic changes such as alterations in karyotype and acquisition of the ability to invade and to metastasize. We have used a fibrosarcoma, grown subcutaneously in C57BL/6 mice, to study the mechanisms underlying this phenotypic instability. Tumor-bearing animals were injected with phorbol myristate acetate (PMA) and then the tumors were transplanted to animals without further PMA treatment. These tumor cells were tum+, that is, unlike the parental tumors, they were able to grow in animals immunized against the parental tumors. This property was maintained for at least 6 tumor passages after the initial PMA injections. Thus, PMA appears to be able to induce an unstable tum+ phenotype in these cells at relatively high frequency.     

Macrophage-mediated induction of drug-resistant variants in a mouse mammary tumor cell line

The ability of macrophages to induce drug-resistant variants was studied in an in vitro macrophage-tumor cell coculture system utilizing the hypoxanthine-guanine phosphoribosyl transferase locus as measured by resistance to 6-thioguanine. Tumor cells of mouse mammary tumor line 66 were sensitive to macrophage induction of thioguanine resistance as shown by an increase in the frequency of thioguanine-resistant variants which arose following macrophage coculture to levels at least 5- to 10-fold above the spontaneous frequency. Detection of increased numbers of variants depended upon the macrophage:tumor cell ratio, with 50:1 or greater being necessary. The activity of the macrophages was dependent upon their activation stage. The induction of drug-resistant variants could be inhibited by oxygen radical scavengers. The basis for the emergence of thioguanine-resistant cells appeared to be induction of new variants rather than selection of preexisting resistant cells from the parental population, since thioguanine-sensitive and -resistant cells were equally sensitive to macrophage-mediated toxicity. In six of the six macrophage-induced variants tested, resistance was associated with loss of hypoxanthine-guanine phosphoribosyl transferase activity. The reverse variation frequency at the hypoxanthine-guanine phosphoribosyl transferase locus in five macrophage-induced variants was low and similar to that of a stable ethyl methanesulfonate-induced, thioguanine-resistant line. Macrophages isolated directly from growing mammary tumors, as well as activated peritoneal macrophages, were capable of inducing thioguanine resistance in line 66 cells.     

Induction of postsurgical tumor immunity and T-cell memory by a poorly immunogenic tumor

The generation of protective CD8 T-cell memory against tumor-expressed self-antigens is an important but elusive goal of cancer immunotherapy. The possibility that a progressive, poorly immunogenic tumor can induce T-cell memory against self-antigens has not previously been studied. Herein, we report that growth of the poorly immunogenic B16 melanoma in the absence of regulatory T cells (T(reg)) generates CD8 T-cell responses that develop into functional memory after the tumor has been surgically excised. Tumor-primed memory T cells recognized melanocyte differentiation antigens TRP-2/DCT and gp100 and persisted for as long as 5 months following surgical tumor excision. Phenotypic analysis showed that these cells develop into both central and effector memory T-cell subsets, which produce IFN-gamma and interleukin-2 on reencounter with antigen. Most importantly, tumor-primed memory T cells mediated the rejection of intradermal and systemically disseminated challenge tumors given 30 to 60 days following surgery. Tumor-excised mice also developed autoimmune vitiligo, showing that T(reg) cells prevent tissue-specific autoimmunity in tumor-bearing hosts. This study establishes that T(reg) depletion in tumor-bearing hosts drives the natural development of protective T-cell memory. Generating such responses may aid in the clinical management of tumor recurrence and metastasis following surgery.     

Indirect induction of a clastogenic effect in epidermal cells by a tumor promoter

The mechanisms by which tumor promoters exert their effectson target tissues are not clearly understood. Recent studieshave demonstrated that phorbol ester tumor promoters inducean oxidative burst in phagocytes and DNA single-strand breaks(SSB) in leukocytes. The purpose of the research presented herewas to investigate the clastogenic effects of tumor promotersin the target cell population, primary mouse epidermal cellsco-incubated with leukocytes. Using the alkaline elution assayto detect DNA SSB, it was demonstrated that tumor promotersinduce DNA SSB in primary mouse epidermal cells incubated inthe presence of leukocytes. By increasing the ratio of leukocytesto epidermal cells from 1: 2 to 10: 1, in the presence of 1.6x 10^–6 M 12-O-tetradecanoylphorbol-13-acetate (TPA), aratio dependent increase in DNA SSB was observed (from 9 x 10^–2to 121 DNA SSB per 10⁶ nucleotides). A dose response in DNASSB was seen with TPA over a concentration range of 4 x 10^–9–1.6x 10^–6 M. Mezerein, a second stage tumor promoter, inducedsimilar levels of DNA SSB to that of TPA. 4-O-Methyl TPA, afirst stage tumor promoter, induced significantly fewer DNASSB than either TAP or mezerein at similar concentrations. Theinduction of DNA SSB in epidermal cells treated with TPA andco-incubated with leukocytes was inhibited by catalase but notsuperoxide dismutase. These data indicate that tumor promoterscan act indirectly on target epidermal cells by stimulatingthe release of a clastogenic factor from leukocytes througha mechanism involving H₂O₂.     

Changes of the immunogenic properties of a radiation-induced mouse lymphoma following treatment with antitumor drugs.

Eight sublines of the radiation-induced lymphoma S-1033 of C57BL/10 (hereafter called B10) origin were established by exposing the cells in vivo to eight antineoplastic agents for a number of transplant generations. The parental and drug-treated sublines were tested for immunogenic properties, i.e., the ability to elicit allograft reactions in the host of origin and in congenic-resistant mice differing for the S-D or K-I-S regions of the H-2 complex. Lymphoma S-1033 and all drug-treated sublines except one were found to be essentially nonimmunogenic for B10 mice. The S-DIC subline, when exposed for 8 to 12 transplant generations to dimethyltriazenoimidazolecarboxamide, became immunogenic for syngeneic B10 mice, as judged from prolongation of survival time. Large i.v. inocula (10(7) cells) of S-1033 and of the drug-treated sublines, with the possible exception of the cyclophosphamide-treated and dimethyltriazenoimideazolecarboxamide-treated lymphomas, were more effectively rejected by K-I-S- than by S-D-incompatible mice. Dilution escape (i.e., tumor rejection after challenge with large inocula, and lethal tumor growth after injection of small inocula of lymphoma cells in allogeneic recipients) occurred in K-I-S-incompatible mice that were inoculated with S-1033 and three drug-treated (5-fluorouracil, cyclophosphamide, and pyrazocarboxamideamino) sublines. No dilution escape occurred with dimethyltriazenoimidazolecarboxamide or bischloroethylnitrosourea sublines. These data favor the hypothesis that various types of immunogenic changes of neoplastic cells may occur in tumor-bearing hosts following treatment with antineoplastic agents in vivo.     

Potent antitumor effect of tumor microenvironment‐targeted oncolytic adenovirus against desmoplastic pancreatic cancer

Pancreatic cancer is a leading cause of cancer‐related death. Desmoplastic pancreatic tumors exhibit excessive extracellular matrix (ECM) and are thus highly resistant to anticancer therapeutics, since the ECM restricts drug penetration and dispersion. Here, we designed and generated two hypoxia‐responsive and cancer‐specific hybrid promoters, H(mT)E and H(E)mT. Transgene expression driven by each hybrid promoter was markedly higher under hypoxic conditions than normoxic conditions. Moreover, H(E)mT‐driven transgene expression was highly cancer‐specific and was superior to that of H(mT)E‐driven expression. A decorin‐expressing oncolytic adenovirus (Ad; oH(E)mT‐DCN) replicating under the control of the H(E)mT promoter induced more potent and highly cancer‐specific cell death compared with its cognate control oncolytic Ad, which harbored the endogenous Ad E1A promoter. Moreover, oH(E)mT‐DCN exhibited enhanced antitumor efficacy compared with both the clinically approved oncolytic Ad ONYX‐015 and its cognate control oncolytic Ad lacking DCN. oH(E)mT‐DCN treatment also attenuated the expression of major ECM components, such as collagen I/III, elastin and fibronectin and induced tumor cell apoptosis, leading to extensive viral dispersion within orthotopic pancreatic tumors and pancreatic cancer patient‐derived tumor spheroids. Collectively, these findings demonstrate that oH(E)mT‐DCN exhibits potent antitumor efficacy by degrading the ECM and inducing apoptosis in a multifunctional process. This process facilitates the dispersion and replication of oncolytic Ad, making it an attractive candidate for the treatment of aggressive and desmoplastic pancreatic cancer.     

Regression of tumor growth and induction of long-term antitumor memory by interleukin 12 electro-gene therapy

BACKGROUND: Interleukin 12 (IL-12) is a proinflammatory cytokine with antitumor activity. Plasmid-based intratumoral gene therapy for treating malignancy with IL-12 or other genes is safe, inexpensive, and simple to carry out. However, effective delivery methods for injecting DNA plasmid into a tumor to generate therapeutic levels of gene product are lacking. To overcome this obstacle, we used electroporation to deliver the IL-12 gene intratumorally in a murine squamous cell carcinoma (SCC) model, SCCVII. METHODS: Plasmids with or without mouse IL-12 gene were injected into SCCVII tumors of C3H/HeJ mice (n = 5 per group). Electric pulses were then applied to the tumors, a process termed electro-gene therapy. The first treatment was administered when the tumor reached 4-6 mm in diameter, and the second treatment followed a week later. The tumor size, survival, and ability to generate systemic antitumor memory were assessed at various time intervals. Changes in gene expression were measured using northern blot analysis, and vessel density and T-cell infiltration were examined by immunostaining. The results were analyzed by two-sided Student's t tests. RESULTS: Electroporation of 20 microg or 40 microg of IL-12 DNA plasmid eradicated tumors in 40% of mice (P =.031 and.022, respectively). A total of six mice from two separate experiments with regressed tumors were challenged with homologous SCCVII tumor cells multiple times; three of six mice showed no tumor growth for more than 11 months and thus indicated the generation of antitumor memory in these mice. IL-12 electro-gene therapy was associated with increased expression of IL-12, interferon-gamma, monokine induced by interferon-gamma, and interferon-inducible protein 10. IL-12 electro-gene therapy was also associated with decreased vessel density and increased infiltration of CD8(+) T cells after the second administration (P =.02 and.03, respectively). CONCLUSION: IL-12 electro-gene therapy appears to be effective in reducing tumor growth by triggering both antiangiogenic effects and an immune response. The antitumor memory was seen to last more than 11 months. Because IL-12 electro-gene therapy is easy to administer and is effective, it could potentially be applicable in the treatment of electrode-accessible malignancies, such as head and neck SCCs.     

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.     

Modification of immunogenic tumor growth by adrenalectomy in a syngeneic murine system

The immunosuppressive action of adrenal glucocorticosteroids is well-known, and depressed cell-mediated immunity and adrenal cortical hyperplasia have been described in tumor-bearing animals. This study was designed to evaluate the effect of removing the source of lympholytic steroids by adrenalectomy upon tumor growth rate, thymus weight, and thymocyte incorporation of iodine 125 (125I) deoxyuridine into DNA. Newly derived methylcholanthrene-induced immunogenic fibrosarcomas were used in male syngeneic mice. Log dosages of 10(4), 10(5), and 10(6) viable tumor cells as single cell suspension were injected subcutaneously into the popliteal space of adrenalectomized and control mice. Tumor size was followed serially with caliper measurements, and the animals were killed 4 weeks after inoculation. Adrenalectomized mice inoculated with 10(4) cells had smaller tumors (P less than 0.02), heavier thymi (P less than 0.01), and more thymic DNA synthesis (P less than 0.05) than their tumor-bearing controls. No differences were seen between populations receiving 10(5) or 10(6) tumor cell inoculations. A second experiment was carried out in which intact controls, adrenalectomized animals, and sham adrenalectomy animals were inoculated with 10(4) tumor cells and killed 28 days later. Tumor growth rate and volume were significantly decreased for the adrenalectomized mice, which had higher thymus weights and DNA synthesis. These findings suggest that pretreatment adrenalectomy slows the growth of antigenic tumor cells and prevents thymic involution after tumor growth in a syngeneic murine system.     

The effect of hyperthermia and antitumor drugs on brain tumor cell lines

The effects of heat and antitumor drugs on malignant brain tumor cell lines were studied. A human glioblastoma cell line (SKMG1) and rat malignant brain tumor cell lines (T9, EB 679 and TR 481) were used in this experiment. Five different modalities of treatment with heat and drugs were used as follows: (Group 1) exposure to heat alone at 42 degrees C for one hour; (Group 2) exposure to antitumor drug alone for one hour (ACNU 2.5 or 5 micrograms/ml, ACR 0.02 micrograms/ml and CDDP 1 microgram/ml); (Group 3) simultaneous exposure to heat at 42 degrees C and drug for one hour; (Group 4) heat at 42 degrees C given first for one hour, followed by one hour exposure to drug one hour later ("preheating"); (Group 5) drug given first for one hour, followed by one hour exposure to heat at 42 degrees C one hour later ("postheating"). After each treatment, the inhibition rate at 4 days was evaluated and compared for each group. A synergistic effect was observed in Group 3. For example, when T9 cells were exposed to ACNU and to heat at 42 degrees C at the same time for one hour, inhibition rate was 78%, while the rates for Group 1 and Group 2 were 7% and 21%, respectively. The cytotoxicity of simultaneous treatment with antitumor drugs (ACNU, ACR and CDDP) and hyperthermia at 42 degrees C was apparently superior to that of other treatment modalities.