In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.     

Host-clonal interactions in the generation of proviral gene deletion variants

A nonproducer clone (clone A1) (from a retrovirus-infected guinea pig fibrosarcoma) has been described that under conditions of in vivo immunologic selection forms variants that lack the proviral gene. One trivial explanation for the apparent loss of the provirus from clone A1 is that clone A1 did not originate from a single cell. For evaluation of this possibility, subclones were derived from clone A1 and tested for tumor recurrence in nonimmune and virus-immune animals. Each of four subclones contained the A1 provirus and exhibited specific viral interference; tumor recurrences formed from each of these four subclones lacked the clone A1 provirus. Possible, when heterogeneous populations of retrovirus-infected cells are injected into nonimmune animals, some clones will elicit immunologic responses to retroviral antigens and subject other clones in the population to immunologic selective pressures. For testing this concept, clone A1 was injected in admixture with a producer clone (clone A4) into nonimmune Sewall Wright strain 2 guinea pigs. Tumors formed in nonimmune guinea pigs inoculated with clone A1 in admixture with clone A4 were shown to lack a detectable clone A1 provirus. The results supported the concept that a somatic mutational event (deletion of the proviral gene) occurs during growth of clone A1. When heterogeneous populations of retrovirus-infected cells are injected into animals, host-clonal interactions may occur leading to outgrowth of proviral gene deletion variants. These results supported the notion that interactions between tumor clones and the host can change the dominant clonal type of the tumor and provide a genetic basis for this change.     

In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.     

In vivo generation and selection of variants with altered sensitivity to natural resistance (NR): a model of tumor progression

The stability of a cloned murine tumor for sensitivity to NR was examined following growth in vivo in order to test the hypothesis that tumor progression proceeds through the generation and selection of variants. Clonal sensitivity to the [131I]-dUrd elimination assay of NR was assessed for the L5178Y-F9 tumor grown in syngeneic DBA/2 mice or maintained solely in tissue culture. Subclones derived from a tumor obtained from the injection site 3 1/2 weeks after the s.c. inoculation of 25 cells were less sensitive to NR in comparison with subclones derived from cells grown only in vitro. Subclones from the cells grown in vivo exhibited increased heterogeneity in sensitivity to NR in addition to their expanded range of susceptibility to complement-mediated lysis by CBA serum natural antibodies. The extent of the heterogeneity argues against tumor "adaptation" forming the basis for the phenotypic alteration while chromosomal studies eliminate the possibility that a new tumor was induced. These data support the hypothesis that tumor progression proceeds through the random generation of variants and host-mediated selection for the proliferation of clones with an increased ability to survive.     

In vivo imaging of gene expression

Imaging of gene expression is increasingly essential for cancer diagnosis, prediction of tumor response to various available therapies, and for monitoring response to therapy, whether it is gene or nongene therapy. Most of the work on developing techniques and methodologies for imaging gene expression has been performed in mice. Sophisticated small animal imaging technologies have been developed for this purpose. Imaging of gene expression in humans is still very limited. Nuclear medicine, positron-emission tomography, magnetic resonance imaging, and optical methods are the modalities that have shown preliminary utility in imaging gene expression. Two major imaging strategies have been investigated: using marker genes encoding either intracellular enzymes or cell-surface receptors. The first approach exploits the ability of certain enzymes to modify imaging prodrugs, so that tissue accumulation of such drugs reflects the expression. The second approach uses cell-surface expression of ligand-binding receptors that can be detected using imaging tracers. In this review, we summarize some of the imaging techniques that have been developed to detect gene expression.     

In vitro functional effects of XPC gene rare variants from bladder cancer patients

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.     

Immuno-selection in vivo of H-2D phenotypic variants from a metastatic clone of sarcoma cells results in cell lines of altered metastatic competence

To find out whether manipulation of H-2 expression on metastatic cells could alter their metastatic properties, we immunoselected in vivo H-2 antigen variants from a metastatic clone of the T10 sarcoma [originating in a (C57BL/6J X C3H.eB)FI mouse] and tested their metastatic capacity. The unselected metastatic cells (IE7) were previously found to express H-2Db and H-2Dk antigens, but they did not express the H-2K antigens of either parental haplotype. Transplantation of IE7 cells into C57BL/6J irradiated mice resulted in loss of H-2Dk expression and a reduction in H-2Db antigen density. Further transplantation of these cells into non-irradiated C57BL/6J mice led to a total loss of H-2 expression. The cells concomitantly lost their metastatic potency. Immunoselection of IE7 cells in C3H.eB irradiated and non-irradiated mice resulted in cells which were H-2Dk-positive but H-2Db-negative. Cells of these selected variants not only retained their metastatic potential, but in fact were far more metastatic than the unselected IE7 cells. Thus, changes in H-2 expression on tumor cells may alter their metastatic potential. In the case of T10 cells, H-2Dk expression seems to be directly involved in their metastatic capacity.     

In vivo selection of a highly metastatic cell line from a human pancreatic carcinoma in the nude mouse.

A cell line with high metastatic capacity to the liver was established by sequential passages of a human pancreatic cancer cell line through the nude mouse liver. A subline, L3.5, established after five passages of the fast-growing variant (FG) of the human pancreatic cancer COLO 357 through the nude mouse liver produced extensive hepatic metastases in 100% of experimental animals when injected into the spleen. The incidence of pulmonary metastases decreased from 43% for FG to 9% for L3.5. The L3.5 cell line showed aggressive growth with almost complete replacement of the hepatic parenchyma in one third of the mean time required for the development of macroscopic metastases of FG in the liver after splenic injections of tumor cells. This study indicates that the nude mouse provides a good model for in vivo selection of metastatic cells from human pancreatic cancer. The L3.5 cell line will be valuable in the study of human pancreatic cancer metastasis, particularly in the area of survival and growth of metastatic cells in the microenvironment of the liver.     

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.     

Protein glycosylation in rat fibroblast cells expressing deletion variants of the human hsp70 gene

Elevated heat resistance is often associated with high cellular levels of hsp70. In the rat cell line, M21, increased heat resistance is associated with both the expression of an intact exogenous human hsp70 gene, and of an exogenous stress glycoprotein, GP62. In this study, we examined heat-stress induced alterations in protein glycosylation in two variant lines of M21 that contain specific deletions in the exogenous human hsp70 gene. The deletion mutant, MVHΔSm, lacks the nucleolar localization sequence for human hsp70 gene, whereas the other mutant, MVHΔBg, is characterized by deletion of the ATP binding domain in the human hsp70 gene. After heat induction, the MVHΔSm mutants exhibited constitutive and heat-induced endogenous hsp70 mRNA levels, protein glycosylation, as well as hsp70 and GP62 protein levels comparable to those in the parent cell line, Rat-1. Cellular heat sensitivity of MVHΔSm mutants was also similar to that of Rat-1 cells, although the mutant cells had a reduced capacity for thermotolerance development. On the other hand, MVHΔBg mutants before thermotolerance induction, exhibited constitutive endogenous and human exogenous hsp70 mRNA levels similar to those in MVHΔSm mutants. However, after thermotolerance induction, MVHΔBg mutants showed lower levels of heat-induced endogenous hsp70 mRNA than MVHΔSm mutants, an overall reduction in protein glycosylation, low hsp70 and GP62 levels, and increased heat sensitivity when compared to the parent Rat-1 cell line. Incorporation of D-[2-³H]mannose into oligosaccharide precursor pools and glycoproteins was consistent with protein glycosylation pattern for each cell line. Acute heat stress resulted in the selective glycosylation of the ‘prompt’ glycoprotein, P-SG64, in the two deletion mutants, similar to that in M21 cells expressing the intact human hsp70 gene. The data indicate that both protein glycosylation and hsp70 expression generally correlate with cellular heat resistance and thermotolerance expression. The presence of full or partially deleted copies of human hsp70 modulates thermotolerance and protein glycosylation in a complex manner that is not yet fully understood.     

In frame deletion of intron-7 in the p53 gene in a human tonsil tumor

We here report a unique intron deletion in the p53 gene. Analysis of a human tonsil tumor that expressed elevated levels of p53 showed a deletion of the entire intron 7 in one of the p53 alleles, leaving the coding sequence intact. The significance of the intron 7 deletion for the development of the tonsil tumor is discussed.     

遗传工程化细胞在小鼠体内长期产生和递送抗肿瘤重组蛋白

目的:探索荷载含Icon基因小鼠纤维母细胞的免疫隔离装置在体内长期产生和递送肿瘤基因疗法重组免疫偶合Icon蛋白的可能性。方法:Icon基因质粒稳定转染小鼠纤维母细胞(NIH3T3/Icon)和中国仓鼠卵巢细胞(CHO/Icon),经免疫沉淀法、亲和层析法、Western blot筛选高效稳定表达的NIH3T3简称(NIH3T3/Icon)和CHO简称(CHO/Icon)细胞克隆;FACS测定其生物活性;选用NIH3T3/Icon细胞质入免疫隔离装置里并移植入小鼠皮下,采用荧光-ELISA检测小鼠血浆Icon中的浓度。结果:NIH3T3/Icon细胞表达Icon蛋白的产量为CHO/Icon细胞产量的23倍,但生物活性前者为后者的5%~6%。含NIH3T3/Icon细胞的免疫隔离装置(TheraCyte)在移植入小鼠体内后的第7天至第39天,在小鼠血浆中可检测到高水平Icon蛋白,其浓度维持在120~150ng/ml;此后,其Icon蛋白水平开始下降,在第53天时的血浆浓度为50ng/ml,至第59天时血浆中无可检测的Icon蛋白。而在对照组,在小鼠血浆中无可检测的Icon蛋白。结论:NIH3T3/ICON细胞的免疫隔离装置在移植入小鼠体内后可较长时间产生和递送具抗肿瘤作用的重组Icon蛋白。     

In vivo and ex vivo gene therapy strategies to treat tumors using adenovirus gene transfer vectors

The adaptation of gene therapy strategies to treat tumors has broadened the potential armamentarium of anticancer strategies to include approaches for local control of tumor growth as well as to enhance systemic antitumor immunity to treat metastases. A major focus of the author and colleagues has been to use replication-deficient adenovirus vectors, both in vivo and ex vivo, to enhance local control of and systemic immunity against cancer. Several examples will be used to demonstrate these strategies. Using prodrugs, systemically administered drugs converted to toxic metabolites in the local tumor milieu, has proven to be a useful strategy for achieving high local concentrations of the toxic product while avoiding the systemic toxicity that limits the use of chemotherapy agents. Transfer of genes encoding cytosine deaminase (with 5-fluorocytosine) and carboxylesterase (CE) (with irinotecan) are two paradigms that have been used in our laboratory. The data demonstrate that using adenoviruses to deliver these genes to the tumor site leads to production of the active chemotherapeutic agent, which diffuses from the cell in which it was produced to suppress tumor growth and attain regional control in a single organ. Extensive experimental and clinical data now exist to support the concept that tumor growth is critically dependent on angiogenesis and that vascular endothelial growth factor (VEGF) appears to play a central role in the process of tumor neovascularization. Data generated in our laboratory have shown that adenovirus-mediated regional anti-VEGF therapy using a gene encoding a soluble form of flt-1 (one of the VEGF receptors) can be used for regional control of tumor growth. The critical dependence of many tumors on VEGF for neovascularization and dissemination predicts the general applicability of this strategy for treatment of many solid tumors. Another paradigm involves dendritic cells, potent antigen-presenting cells that play a critical role in the initiation of antitumor immune responses. Immunization of mice with dendritic cells genetically modified using an adenovirus vector transferring a gene encoding a tumor antigen confers potent protection against a lethal tumor challenge, as well as suppression of preestablished tumors, resulting in a significant survival advantage. One clinical scenario to which this approach is relevant is treating micrometastases present at the time of primary detection of many malignancies. A possible clinical strategy would be to modify dendritic cells from such patients using an adenovirus vector encoding the relevant tumor antigen, and then administering the genetically modified dendritic cells as adjuvant treatment following primary therapy.     

In vivo and in vitro generation of a new altered HLA phenotype in melanoma-tumour-cell variants expressing a single HLA-class-I allele

A new HLA-class-I altered phenotype is described in melanoma. This phenotype is the result of a combination of HLA-B-locus down-regulation and HLA-haplotype loss. The alteration was found in 2 melanoma cell lines generated from 2 patients; one was derived from an in vivo lesion (FM37) and the other was obtained after in vitro immunoselection (R22.2). The R22.2 cell line was isolated from FM55P, a cell line derived from a primary melanoma, after in vitro treatment with a heterologous HLA-A2-restricted cytotoxic-T-lymphocyte (CTL) clone. Two additional cell lines from patient 55 were obtained from 2 s.c. metastases (FM55M1 and FM55M2). Iso-electric focusing and flow-cytometric studies showed a significant reduction in the expression of both HLA-B alleles in all cell lines studied. The expression of HLA-B-locus products recovered completely after IFN-γ treatment of FM55P, M1 and M2. In contrast, FM37 and R22.2 tumour cells showed an additional HLA defect: the absence of one HLA haplotype. Simple tandem-repeat polymorphism markers spanning chromosome 6 showed that DNA from the 2 samples (FM37 and R22.2) showed loss of heterozygosity (LOH). In both cases, homozygosity was observed on 6p, which maps the HLA region, the final consequence being a tumour cell that expressed a single HLA-class-I allele (HLA-A3 and HLA-A1 respectively). FM37 cells may thus reflect the in vivo counterpart of resistance to lysis by HLA-A2-restricted tumour-infiltrating lymphocytes. Int. J. Cancer 75:317–323, 1998. © 1998 Wiley-Liss, Inc.     

In situ immunologic characterization of follicular lymphomas

Surface markers were studied in a series of follicular lymphomas with immunofluorescence on frozen sections (39 cases) and on cell suspensions (21 cases), and with immunoperoxidase on frozen sections using a panel of 15 monoclonal antibodies (17 cases). With immunofluorescence on frozen sections, 22/39 cases showed monotypic sIg (IgMK: 14 cases, IgML: 7 cases, M: 1 case). In the remaining 17 cases the neoplastic follicles were negative. Nevertheless, even if sIg is not detected, the absence of an extracellular immunoglobulin network is indicative of the neoplastic, and not of the reactive nature of lymphoid follicles. The results obtained with immunofluorescence on frozen sections and on cell suspensions were identical in about half of the cases. In 9/21 cases monotypic sIg were detected by only one of these two methods. All the 17 cases studied with immunoperoxidase on frozen sections using monoclonal antibodies demonstrated monotypic sIg. On low magnification 6/17 sIg+ exhibited a nodular staining pattern while 7/17 cases this staining was diffuse. In 4/17 cases the staining pattern for heavy and light chains was different. A thin mantle zone, with sIgM plus sIgD cells, was observed in only 4 cases. Anti-HLA-DR and Leu-10 were positive in all cases. T cells positive for OKT3 were mainly distributed in the interfollicular areas; OKT4 + cells outnumbered OKT8 + cells. Within the neoplastic follicles, T cells stained mainly for OKT4 and OKT8 + cells were scarce. Leu-7 + cells predominated within the neoplastic nodules in 5 cases. With the anti-dendritic reticulum cell monoclonal antibody, all 17 cases showed a network, usually more loosely arranged than in reactive follicles. In 4 cases, of follicular and diffuse lymphoma, this network was extremely dissociated and in some areas these cells were scanty or lacking. We concluded that immunoperoxidase on frozen sections, using monoclonal antibodies, appears to be the most reliable method for the immunological phenotyping of follicular lymphomas.     

In vivo selection of human tumor cells resistant to monoclonal antibody-Vinca alkaloid immunoconjugates

UCLA-P3 human lung adenocarcinoma cells were grown in nude mice and given repetitive treatments of a monoclonal antibody-Vinca alkaloid immunoconjugate. Although this therapy resulted in a greater than 4-fold reduction in mean tumor mass of the established tumors, some animals experienced a reinitiation of tumor growth after cessation of conjugate treatment. Two such animals were treated again with high doses of monoclonal antibody-Vinca but one of the tumors was no longer regressed by the drug conjugate. The tumor was excised, enzymatically dissociated, and grown in tissue culture. Cultured cells were reimplanted in nude mice and subjected to further therapy with a monoclonal antibody-Vinca conjugate. The resulting tumors were also refractory to the immunoconjugate therapy. This cycle was repeated for a total of three times and resulted in the serial in vivo selection of three conjugate resistant variants. The mechanism responsible for the in vivo resistance of human tumor cells to the monoclonal antibody-Vinca immunoconjugate is unknown but does not appear to involve antigen modulation, altered tumor cell growth rate, or an apparent decrease in tumor targeting in vivo. The resistance was also not accompanied by any detectable elevation in multidrug resistance 1 mRNA or P-glycoprotein expression. Significantly, the resistance pattern was observed only in vivo and was not maintained by cells grown in vitro.     

In vitro and in vivo selection of two Lewis lung carcinoma cell lines

Two tumor cell lines adapted to grow in vitro were originated from an explant of lung metastases of Lewis lung carcinoma. These lines differ in their malignancy when reinoculated into syngeneic animals; nevertheless, they do not show any difference for their in vitro clonogenic ability. From these lines 2 in vivo sublines of 3LL carcinoma were developed. The TD50 of the 2 in vivo sublines are different, and both the values obtained are lower than that of the original line. These results are interpreted as a selection of more malignant tumor cell lines.