The use of hybrid cells in immunotherapy.

HARRIS et. al. isolated somatic hybrid cells (A9/SEWA) between polyoma-induced tumor and mouse fibroblast cell lines. Although these hybrid cells were no longer tumorigenic, we found that their immunogenicity was conserved. It therefore seemed to us that these antigenic, non transplantable, living cells would be an ideal tool for immunotherapy experiments. In our first experiments we assessed the immunoprotection afforded by A9/SEWA cells in both the SEWA tumor/A.SW mouse and C3HPy/C3H mouse systems. However, the efficiency of immunization with hybrid cells is dependent on the stability of the cells, especially in respect to the expression of the TATA. So we also tried to evaluate the immunogenicity as a function of the number of subcultures undergone by the hybrid line. In both systems, the immunogenicity was very good in the early subcultures but, in the C3HPy tumor/C3H mouse system, a loss of immunogenicity was observed as the number of subcultures increased. Thus any clinical application or immunization by hybrid cells would necessitate the verification of the presence of the tumor-associated antigens at each subculture. We are at present experimenting with various in vitro techniques for detection of the expression of these antigens.     

Comparison between different techniques of immuno-protection in the A-SW mouse-SEWA tumour system

Various procedures for immunoprotection in the A-SW mouse SEWA tumour system were compared. Four types of protector agents were used: polyoma virus, allogeneic S724 cells, A9/SEWA hybrid cells and xenogeneic ST_py cells. Several injection schedules were also employed. On the whole, reasonable immunoprotection was obtained. The results, when allogeneic hybrid or xenogeneic cells were used, were comparable with those obtained for polyoma virus. This indicates that the T.A.T.A. of the SEWA tumour had been well conserved. When polyoma virus alone was used as the protector agent, injection 7 days before the tumour challenge, or iterative injection, afforded better protection than a single injection on the day of challenge.     

The influence of the thymus on the development of transplanted mammary tumour in mice

The influence of thymectomy and the grafting of additional thymuses on the development of syngeneic mammary tumour has been studied in high cancer C3H/He strains of mice. The effect on the development of the grafted tumour of replacement of the thymus of F1 hybrid mice (C3H/HexC57BL) by thymus derived from donors of the parental streins has also been investigated. In C3H/He mice thymectomized on the 3rd day after birth the development of mammary tumour grafted at the age of 2 months was delayed. One or three additional syngeneic thymuses from 1-month-old donors grafted subcutaneously to C3H/He mice stimulated the development of grafted tumours, whereas inhibition of tumour development was observed in mice grafted with five additional thymuses. The replacement of the thymus in F₁ hybrid mice (C3H/HexC57BL) at the 5th day after birth by thymus of 5-day-old donors of low cancer C57BL strain led to the inhibition of mammary tumour grafted to mice at 2-3 months of age, while the replacement of the hybrid thymus by that obtained from donors of the high cancer C3H/He strain had no inhibitory effect. The data presented have been interpreted as evidence for genetically determined differences in the functional specificity of the thymus concerning its influence on malignant growth in high and low cancer strain mice. It is suggested that hereditary predisposition to malignant growth is, in particular, related to the functional properties of the thymus in high cancer strains of mice.     

Selective suppression of metastasis but not tumorigenicity of a mouse lung carcinoma by cell hybridization

Somatic cell hybrids were produced by polyethylene glycol-induced cell fusion between metastatic CMT 167 (HGPRT-/OUAR) C57BL/Icrfat mouse lung carcinoma cells and 2 non-metastatic cell lines: C3H/He mouse L-M(TK-) cells of mesenchymal origin and EJ (OUAS) human bladder carcinoma cells. Fusion of 2 different CMT167 (HGPRT-) clones with L-M(TK-) cells followed by selection in HMT medium gave rise to 14 intraspecific hybrids, which were shown to express H-2 antigens specific for both the C57 and C3H mouse strains. Three interspecific hybrids arising from fusion of EJ(OUAS) and CMT167(HGPRT-/OUAR) cells were selected in HMT/ouabain medium and characterized by human isozyme analysis. All the hybrids produced large tumours after subcutaneous inoculation of 5 X 10(5) cells into adult athymic nu/nu mice. The intraspecific hybrid tumours were predominantly sarcomatous (mesenchymal) in structure but a few contained epithelial acini. Metastatic ability (as assessed by production of lung metastases) was completely suppressed in 13 of the 14 mouse/mouse hybrid cell clones. These results suggest that tumorigenicity, tumour structure and the ability to metastasize are expressed independently. The interspecific hybrids, which had not retained a full human chromosome complement, produced metastatic tumours that remained epithelial in structure.     

Characterization of "H-2K,D-like structures" on MM2 x mouse L cell hybrids. II. An "H-2Kd-like" structure on C3H-derived tumor

Anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, immunoprecipitated "H-2K,D-like" molecules on DBA/2 and C3H.H-2 degrees lymph node cells as well as on somatic cell hybrids between MM2 tumor and mouse L cells. Preclearing of lysates from C3H.H-2 degrees lymph node cells with anti-H-2.31 serum removed all "H-2K,D-like" molecules reactive with anti-MM2 serum, indicating that the molecules detected by anti-MM2 serum are H-2Kd antigens. The anti-H-2.31 serum detected an "H-2K,D-like structure" on the hybrid cells and absorption of the anti-H-2.31 serum with the hybrid cells deprived the serum of anti-H-2Kd reactivity. The hybrid cells could induce antibodies against the H-2Kd antigen in C3H/He mice. These results indicate that on the hybrid cells, whose parental cells were both derived from C3H mice, there is an "H-2K,D-like structure" that has the H-2Kd private specificity. Absorption of anti-MM2 serum with EL4 cells did not affect the capacity of the serum to detect the H-2Kd antigen on C3H.H-2 degrees lymph node cells, indicating that the "H-2Kd-like structure" is distinct from "H-2K,D-like structure A" which was previously reported. Nine isozymes were examined and MM2 cells, mouse L cells, and the hybrids were found to have the same isozyme markers as those of the C3H/He mouse.     

Influence of milk source on transplantability of histocompatible mammary tumours in mice.

It is confirmed that C3H mammary tumours are much more easily transplantable in histocompatible recipients when these have been reared on C3H milk, than when they have been reared on milk from the inbred Swiss/B strain. By contrast, A.CA mammary tumours transplanted in histocompatible hosts reared on A.CA or Swiss/B milk, grow almost equally well in both sorts of recipient. Thus, rearing on Swiss/B milk has different effects on the transplantability of mammary tumours of C3H and A.CA. On the other hand, recipients which were reared on C3H or A.CA milks accept grafts of C3H mammary tumours about equally, suggesting that milks from A.CA and C3H have the same effect on the transplantability of C3H mammary tumours. The different action of Swiss/B milk on tumours of C3H and A.CA seems best attributed to differences between C3H and A.CA tumours or between mouse strain genotypes. By contrast, the transplantability of C3H mammary tumours is significantly changed when the recipients were reared on milk from the RIII strain instead of C3H. These facts suggest that the milk from RIII has an action which differs from that of both C3H and A.CA in this respect. The data are discussed on the basis of a differential tollerance-inducing action of mammary tumour viruses (MTVs) which infect C3H, A.CA and RIII, and have an important role in tumour induction.     

Strain and tumour specific variations in the effect of hypoxia on osteopontin levels in experimental models.

PURPOSE: To investigate the relationship between tumour hypoxia and serum and tumour osteopontin (OPN) levels. MATERIALS AND METHODS: Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma (CDF1) or SCCVII squamous cell carcinoma (C3H/Km), respectively. Mice were either untreated or gassed with 10% oxygen for 1-72 h. Serum and tumour OPN levels were measured with an ELISA and tumour OPN mRNA levels using RT-PCR. Tumour oxygenation was estimated using the Eppendorf histograph with the percentage of pO(2) values 相似文献   

The semistability of centric chromatin bodies during long-term passage of multidrug resistant mouse-Chinese hamster cell hybrids

In a cell fusion experiment with multidrug resistant (MDR) mouse SEWA tumor cells and sensitive Chinese Hamster CHO cells, the resistant hybrid cells were completely without recognizable mouse chromosomes. Instead, numerous chromatin bodies (CB) were found that contained copies of a high molecular weight P-glycoprotein gene (PGY1) associated with the MDR condition. The present paper reports on the CB under long-term selective and nonselective growth. The CB were of a stability intermediate between that of double minutes (DM) and homogeneously staining regions (HSR). The stability of the CB was different in the two hybrid lines studied.     

BW12C-induced changes in haemoglobin-oxygen affinity in mice and its influence on the radiation response of a C3H mouse mammary carcinoma.

The effect of the substituted benzaldehyde BW12C on haemoglobin-oxygen binding affinity, tumour radiation response and blood perfusion were investigated in a C3H mouse mammary carcinoma grown in the feet of CDF1 mice. Mouse P50 (partial pressure of oxygen at half saturation) was estimated using an ABL blood gas analyzer; radiation response determined from tumour regrowth and local tumour control assays; and tumour blood perfusion measured with a 86RbCl extraction procedure. A single intravenous injection of BW12C substantially decreased mouse P50. This effect was dependent on the time after injection with the nadir observed within 15 min and only returning to normal after several hours. It was also dependent on drug dose, the decrease becoming larger with increasing concentration, reaching a maximum 50% reduction at 70 mg/kg. The decrease in P50 could be maintained for at least 6 h following injection of 70 mg/kg, if mice were also given 25 mg/kg at hourly intervals. However, no changes in radiation response or tumour blood perfusion were observed with either single or multiple administrations of BW12C. These results suggest that BW12C induced changes in tumour hypoxia reported by several groups of workers, may not be entirely the result of a change in haemoglobin-oxygen affinity.     

Lung tumour incidence in mice treated with hydrazine sulphate

Lung tumour incidence in mice fed with hydrazine sulphate (1.1 mg/day/mouse) was studied in male and female mice of Swiss, Strong “A” and F₁ cross of ICRC (female) × C3H (Jax) (male), as well as in C17 males. Swiss strain mice showed 100% lung adenocarcinomas. None of the treated mice of different strains had liver tumours. Hydrazine sulphate also induced adenocarcinomas of lung in Strong “A” and F₁ cross of ICRC females × C3H (Jax) males but it produced lymphomas of lung in C17 strain. Female mice of Swiss strain and F₁ hybrids showed greater susceptibility to hydrazine sulphate than the males. It was interesting to observe that protein and vitamin B deficiency in the diet shortened the tumour induction period in Swiss strain mice.     

The effect of methotrexate on spontaneous mammary adenocarcinomata in female C3H mice.

The effect of methotrexate on solid rodent tumours has been investigated using the spontaneous mammary adenocarcinoma of the female C3H/Bts mouse. As these tumours exhibit a wide range of volume doubling times, calculations of tumour response to methotrexate must be related to doubling time. When methotrexate is injected into the tumour a dose-dependent tumour response is obtained. Systemic citrovorum "rescue" prevents methotrexate lethalities but reduces tumour response by a factor of 2. Methotrexate-treated tumours have an increased volume doubling time post treatment.     

Protection against MUC1 expressing mouse tumours by intra-muscular injection of MUC1 cDNA requires functional CD8+ and CD4+ T cells but does not require the MUC1 tandem repeat domain

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background.     

Persistence of the immunoprotective effects of leukemia X fibroblast hybrid cells toward leukemia in histocompatible mice

Hybrids of ASL-1 murine leukemia cells and LM(TK-) cells, a cultured line of mouse fibroblast origin, stimulate partial immunity toward ASL-1 cells in (A/J X C3H/HeJ)F1 mice (F1 mice). Such mice ordinarily exhibit no resistance to the malignant proliferation of ASL-1 cells. Unprotected animals invariably die within 14-18 days after receiving as few as 200 ASL-1 cells. The hybrid cells, the mice used in the experimental studies and the leukemia cells used for challenge all share the same histocompatibility antigens. ASL-1 cells are H-2a; LM(TK-) cells are H-2k, both ASL-1 X LM(TK-) hybrid cells and A/J X C3H/HeJ)F1 mice are H-2a/k. The long-term persistence of the immunoprotective properties of the hybrid cells toward murine leukemia was investigated by using cells that had been in continuous culture for approx. 36 months. (A/J X C3H/HeJ)F1 mice injected previously with hybrid cells in continuous culture and then challenged with up to 10(7) ASL-1 cells survived longer (p less than 0.001) than mice who had not received hybrid cells previously. Some mice challenged with lesser number of ASL-1 cells survived indefinitely (greater than 200 days). The median survival time of F1 mice injected simultaneously with 10(7) hybrid cells and 200 or 2000 ASL-1 cells was significantly (p less than 0.001) prolonged as well, although the differences between experimental and control groups are less pronounced than if the hybrid cells were injected before challenge with ASL-1 cells. The hybrid cells like those freshly prepared continue to be rejected by histocompatible precipients. In no instance has there been evidence of a progressively growing tumor of hybrid cells in immunocompetent F1 mice. Hybrid cells like those investigated previously do form rapidly growing metastasizing tumors in immunodeficient nu/nu (BALB/c) or X-irradiated (550 R) F1 mice. The cells possess approx. 70 chromosomes (reduced from 85) including chromosomes identified as having originated in each parental source. Like (A/J X C3H/HeJ)F1 animals, they continue to express both H-2a and H-2k antigenic determinants.     

Development and characterization of the BALB/cNIV mouse strain

The strain BALB/cNIV/Crgl was developed by infecting BALB/c/Crgl mice with mouse mammary tumor virus from C3Hf mice. A BALB/c normal mammary duct was transplanted into the gland-free fat pad of a hormone-stimulated female C3Hf X BALB/c F1 mouse. A hyperplastic alveolar nodule was found in the BALB/c ductal outgrowth and was transplanted into another hybrid gland-free fat pad. The resultant hyperplastic alveolar outgrowth was finally transplanted to female BALB/c mice. The hyperplastic alveolar outgrowth contained an exogenous, infectious mouse mammary tumor virus named the nodule-inducing virus, which was thought to be derived from the endogenous low oncogenic mouse mammary tumor virus found in C3Hf mice. The hyperplastic alveolar outgrowth-bearing BALB/c mice were inbred for four generations, and one family was selected as the strain BALB/cNIV/Crgl. It was found that (a) the mouse mammary tumor virus found in the BALB/cNIV strain was milk transmitted, but not transmitted by infected males; (b) the BALB/cNIV breeding females had a low tumor incidence (40%) and a longer latent period (14 months) than did female BALB/cfC3H mice (92% at 8 months); (c) the BALB/cNIV nodule outgrowths had low tumor-producing capabilities (50%) and longer latent periods (13.4 months) than did nodule outgrowths derived from female BALB/cfC3H mice (100% at 7.7 months).     

Studies on lung tumours. V. Susceptibility of mice to dimethylnitrosamine-induced tumour formation in relation to H-2 haplotype

Male mice of the strain A/Sn, of its congeneic partner strain A.SW, C57BL/10ScSnA (abbreviated: B10), and of two congeneic strains on a B10 background (B10.A and B10.AKM) were investigated for their susceptibility to lung tumour induction by dimethylnitrosamine (DMN) administered either by intraperitoneal injection or in the drinking water. Strain B10 (haplotype H-2b) proved to be very resistant, whereas strains A/Sn (H-2a) and A.SW (H-2s) were highly susceptible. The introduction of the haplotypes H-2a and H-2m in the resistant strain B10 resulted in a significant increase in sensitivity towards DMN-induced lung tumour formation. Lung tumour incidences in male (B10.A x B10)F hybrids, receiving DMN in drinking water, were found to be intermediate between, and significantly different from, the incidences of identically-treated parent strains B10.A and B10. Males of back-cross (B10.A x B10) x B10 BC proved to be of low susceptibility to lung tumour formation by DMN, tumour incidence being very low and not significantly different from that observed in identically-treated B10 males. It is concluded that, at least in the model system of B10-derived congeneic strains, H-2 haplotype is one of the factors important in determining susceptibility towards DMN-induced lung tumours. Comparison of C3Hf (H-2k), C3H/Sn (H-2k) and the latter's congeneic strains C3H.B10 (H-2b) and C3H.NB (H-2p) of male mice showed that these strains were moderately susceptible to both lung tumour and hepatoma formation by DMN. Accordingly, the presence of an H-2 haplotype from a lung-tumour-resistant strain (H-2b, B10) on the background of a strain of intermediate susceptibility (C3H) does not decrease susceptibility to lung tumour formation. The results were considered in the light of the H-2 haplotype dependence of spontaneous lung tumours, and consequently attention has been paid to the histological types of the induced and spontaneous lung tumours.     

Novel antigen expression on syngeneic somatic cell hybrids of murine spontaneous mammary tumor cells

Spontaneous mammary carcinoma cells of C3H/He mice were fused with syngeneic L-cells, and two types of hybrid cell clones were obtained. In group A, hybrid cells showed contact inhibition, and parental H-2k and L-antigens were well expressed. Metacentric chromosomes of L-cell origin and estrogen dependency were also well preserved in this group. However, in group B, hybrid cells proliferated to pile up, and the expression of parental antigens, H-2 and L-cell antigens, was strongly suppressed. But, mouse mammary tumor antigens (MM-antigens), which were expressed on ascites mammary tumor cells with hypotetraploidy of C3H/He origin and which were not expressed on both parent cells, were newly expressed. The number of metacentric chromosomes was decreased, and estrogen dependency was lost in this group. The relationship between the expression of MM-antigens and that of H-2 antigens was reciprocal, and tumorigenicity was independent of cellular behavior in vitro and of MM-antigen expression. MM-antigen-positive hybrid cell clones were frequently obtained when tumor cells were fused with metaphase-rich L-cells.     

Sister chromatid exchanges induced by two radiosensitizing platinum compounds (cis-dichloro-bis isopropylamine trans dihydroxy platinum IV (CHIP) and cis platinum metronidazole2Cl2(FLAP] in CHO cells in vitro

A high proportion of females of the C3H strain of mice develop tumours of the mammary gland which are caused by mouse mammary tumour virus (MMTV) transmitted through the milk. We have examined whether administration of mouse interferon (IFN) to nursing mothers and/or their suckling offspring only during the period of nursing, can affect the incidence of tumours developing in these animals. In two separate experiments, animals receiving IFN by direct injection while suckling, and remaining virgin showed a marked and statistically significant decrease in tumour incidence. Mice receiving the same or a tenfold higher dose of IFN while lactating showed no such reduction in tumour incidence, even if they had also received IFN while suckling. The results suggest that IFN can affect the initial establishment of the MMTV infection in suckling mice sufficiently to delay tumour development provided the animals are not exposed to the hormonal stimulus of pregnancy and lactation.     

Attenuation of exogenous murine mammary tumor virus virulence in the C3H/HeJ mouse substrain bearing the Lps mutation

A major change in mammary tumor incidence (MTI) and latency has occurred in the C3H/HeJ mouse substrain maintained at The Jackson Laboratory. The average time required for 50% of the C3H/HeJ mice to develop a mammary tumor changed from 40 weeks of age to the current 61 weeks of age. This 61-week median MTI in the C3H/HeJ substrain is significantly different from the less than 40-week median MTI seen in other C3H substrains infected with an exogenous milk-transmitted murine mammary tumor virus (MuMTV). The median MTI of over 80 weeks in MuMTV-negative C3H substrains also is significantly longer than that seen in C3H/HeJ mice. Although the median MTI for the C3H/HeJ substrain has changed significantly, the continued presence of an exogenous MuMTV in C3H/HeJ mice was confirmed by the presence of high levels of gp52 antigen in the milk of lactating females. Presence of an exogenous MuMTV in C3H/HeJ female mice also was confirmed by their ability to pass their exogenous MuMTV(HeJ) via their milk to exogenous MuMTV-negative BALB/cByJ, C3H, or C3H hybrid mice. These foster-nursed mice exhibited the reduced tumor frequency and the increased median MTI seen in their C3H/HeJ foster mothers. The change in tumor incidence and latency in the C3H/HeJ substrain is not due to the loss of the exogenous MuMTV but to the occurrence of an attenuated MuMTV. Selection of this attenuated MuMTV may be related to the presence of the Lps mutation that occurred during the same time period that the MTI changed in the C3H/HeJ substrain.     

Characterization of "H-2K,D-like structures" on MM2 X mouse L cell hybrids. I. "H-2K,D-like" alloantigen encoded by the D region and/or to the right of the D region of the H-2 gene complex

It was found that anti-MM2 serum, which had been prepared by immunizing C3H/He mice with syngeneic MM2 mouse mammary ascites tumor, detected molecules of 44-46,000 and 12,000 daltons on EL4 leukemic cells and on C57BL/6 lymph node cells, as well as on somatic cell hybrids between the MM2 and mouse L cells. Experiments with a known rabbit anti-mouse beta2-microglobulin serum showed that the two molecules detected by anti-MM2 serum were hydrophobically associated with each other in membrane extracts; thus, the antigen detected by anti-MM2 serum was structurally similar to H-2K and D antigens. Preclearing of lysates from C57BL6 lymph node cells with a mixture of anti-H-22, anti-H-2.33, and anti-H-2.28 sera did not remove the "H-2-like" antigen, indicating that the antigen was distinct from the H-2Kb and Db molecules. Neither C3H/He nor B10.BR lymph node cells expressed the "H-2-like" antigen, but B10.A(4R) and B10.AM lymph node cells did possess the antigen. Absorption of anti-MM2 serum with EL4 cells abolished the capacity of the absorbed serum to precipitate the "H-2-like" antigen activity on C57BL/6 lymph node cell extracts and reduced the "H-2-like" radioactive peaks of the hybrid cells. These results indicate that there are at least two components being recognized by the anti-MM2 serum in hybrid cells between MM2 tumor and mouse L cells, both of which had originated in the C3H/He mouse (H-2k). One is the same as or cross-reactive with an "H-2-like" alloantigen of normal C57BL/6 lymph node cells (H-2d) and the other is another "H-2-like" antigen. Experiments with recombinant mice show that the "H-2-like" alloantigen on lymph node cells is coded for by the D region and/or to the right of the D region of the major histocompatibility complex (MHC).