Clonal variation in tumorigenicity of L1210 lymphoma cells: nontumorigenic variants with an enhanced expression of tumor-associated antigen and Ia antigens

Clonal variations in the tumorigenicity and in the expressions of tumor-associated antigens (TAA) as well as normal cell surface antigens were studied using clones of a highly tumorigenic DBA/2 lymphoma, L1210, which were isolated by limiting dilution in vitro. The majority of the clones were highly tumorigenic (tum+) in normal syngeneic mice, as was the parent L1210. The rest were nontumorigenic (tum-) in such mice; these clones, however, were tumorigenic in host mice that had been immunosuppressed by irradiation with 450 rads. Moreover, these tum- variants were shown to have an ability to elicit, in syngeneic mice, strong host resistance specifically directed against challenge with the parent L1210 and tum+ cloned cells and an ability to generate an in vitro primary syngeneic cytotoxic T-cell response against L1210 clones, indicating an enhanced immunogenicity in tum- variants. The expression of TAA by tumor clones was defined by determining the reactivity of monoclonal antibody, raised in syngeneic mice against an immunogenic L1210 subline, L1210/GZL. Marked clonal variation in the expression of monoclonal antibody-defined TAA was demonstrated, while no significant variation was seen in the H-2Dd expression. There was an inverse relationship between the TAA expression and the tumorigenicity. Furthermore, the enhanced expression of the TAA and the increased immunogenicity was associated with the I-Ad expression on the tum- variants. The unique characteristics of the tumor variants were very stable and heritable although occasional revertant phenotypes were detected on some clones. The results suggest that the tumor variants bearing distinct immunological properties exist in the parent L1210 line and carry a potential to modulate host immune responses directed against tumor cells.     

Human tumor spontaneous metastasis in immunosuppressed newborn rats. II. Multiple selections of human melanoma metastatic clones and variants

Using a recently developed model of human tumor spontaneous metastasis in immunosuppressed newborn rats, we selected variants with different metastatic abilities from the human melanoma cell line M4Be. We used 4 in vivo selection approaches, by direct serial tumor transplantations or by techniques involving in vitro reculture of the cells recovered from s.c. tumors or lung and lymph-node metastases. In addition, 3 series of clones were derived in vitro from the M4Be cell line, either by limiting-dilution or by cloning in semi-solid agar and harvesting small and large colonies. A considerable amount of heterogeneity in tumorigenicity and metastatic ability was demonstrated among variants and clones following in vivo selections and in vitro cloning. Four main malignant phenotypes were identified among those expressed by the selected cells: poorly tumorigenic and poorly metastatic; poorly tumorigenic and highly metastatic; highly tumorigenic and poorly metastatic; and highly tumorigenic and highly metastatic. However, while malignant phenotype (i.e., tumorigenicity and metastatic ability) did not appear to be grossly influenced by in vitro cloning procedure, it appeared greatly influenced both by the in vivo selection procedure (direct transplantations or use of in vitro culture between the in vivo passages) and by the origin of the cells under selection (s.c. tumor or metastases). Our study provided us with a large panel of variants and clones with varying metastatic abilities, which represent a model of human melanoma spontaneous metastasis allowing the study of critical determinants in human tumor metastasis.     

Cytoplasmic suppression of tumorigenicity in reconstructed mouse cells

Previous cybrid studies aimed at demonstrating cytoplasmic suppression of tumorigenicity have been generally inconclusive because of (a) the use of mutagens or carcinogens to introduce nuclear-coded and cytoplasmic-coded genetic markers and (b) dilution of putative cytoplasmic suppressors with tumorigenic cytoplasm of whole cells used in the cybrid construction. We have circumvented these potential problems by examining tumorigenicity in reconstructed cells made from tumorigenic karyoplasts and nontumorigenic cytoplasts and by using a ricin-antiricin selection to obtain the reconstructed cells. Karyoplasts from tumorigenic NIH/3T3 cells that were derived from a clone that had survived incubation with benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxy (anti) and been passaged 17 times were fused to NIH/3T3 cytoplasts derived from nontumorigenic cells. The cytoplasts were loaded with antiricin antibody prior to fusion. Ten clones which survived ricin selection were not tumorigenic in nude mice. These findings offer support for the presence of cytoplasmic factors in nontumorigenic mouse cells that suppress benzo(a)pyrene epoxide-induced tumorigenicity.     

Biologic and biochemical differences between in vitro and in vivo passaged Friend erythroleukemia cells. I. Tumorigenicity and capacity to metastasize

Cloned interferon-sensitive (745) and interferon-resistant (3Cl-8) Friend erythroleukemia cells (FLC) passaged in vitro, are not very tumorigenic when first injected intraperitoneally (i.p.) into syngeneic DBA/2 mice although they do form solid tumors when injected subcutaneously (s.c.). By serially passaging FLC (either 745 or 3Cl-8 cells) i.p. in DBA/2 mice, we obtained two different FLC lines capable of growing i.p. and inducing tumor ascites. The s.c. injection of DBA/2 mice with these in vivo passaged FLC resulted in tumor metastases in the liver and spleen, whereas metastases were not observed in mice inoculated s.c. with in vitro passaged FLC. The capacity of in vivo passaged FLC to metastasize was acquired after several i.p. passages. This highly malignant behavior was a stable characteristic of these cells. All the clones derived from in vivo passaged FLC and passaged more than 14 times in vitro induced hemorrhagic ascites when injected i.p., and metastasized to the liver and spleen when injected s.c. The phenotype of sensitivity or resistance to the inhibitory effect of alpha/beta mouse interferon on virus replication and cell multiplication was conserved during serial i.p. passages and maintained in the clones derived from in vivo passaged cells. These FLC showed a decreased capacity to differentiate in vitro upon treatment with dimethylsulfoxide (DMSO) and a reduced production of Friend leukemia virus with respect to the original clones passaged in vitro.     

Decrease in tumor-producing capacity of mouse cell lines following infection with mouse leukemia viruses

Two neoplastic cell strains derived from the C57BL/6 mice, P4bis originating from normal adult lung tissue “spontaneously” transformed in vitro, and TBLC2 developed from a methylcholanthrene-induced sarcoma, were both infected in vitro with Rauscher leukemia virus. Consecutive to this infection, the tumor-producing capacity of these cells, which continued to multiply normally in vitro, dropped considerably when checked in syngeneic mice. This was evidenced by a dramatic decrease of takes and a significant prolongation of latency period. The high leukemogenic RRL⁺ and the low leukemogenic RCL^? virus variants were equally efficient in infecting cells in vitro and producing a decrease of their tumorigenic potential in vivo. This decrease coincided with the massive appearance in the cells and culture supernatant of C-type particles and of a new surface antigen reacting specifically in the immunofluorescence test with anti-Rauscher virus serum. The specific immunological nature of decreased tumorigenicity was confirmed by the fact that the rejection of the virus-infected cells could be prevented by total irradiation of intact recipient animals, whereas animals preimmunized and later irradiated continued to reject the infected cells. On the other hand, the rare tumors which appeared in non-irradiated adults after a long latency period were composed of virus-free cells whereas cells from tumors obtained by inoculation of new-born animals regularly contained the virus and were rejected when grafted on normal adults. In another series of experiments, in P4bis cell cultures, subjected in vitro to the action of methylcholanthrene for several months, the appearance of C-type particles and of a new cell-surface antigen reacting specifically in the immunofluorescence test with anti-Gross leukemia serum, was observed. The untreated control cultures remained negative for C particles and antigen. The appearance of this chronic C-type virus infection coincided, here again, with a considerable decrease in tumor-producing capacity of the P4bis cells as checked in syngeneic mice. The reported results are discussed in the context of the possible role played by chronic infections, especially by widely spread non-pathogenic variants of leukemia viruses, in the “heterogenization” of tumor cells contributing to their rejection triggered by an immune reaction of the host.     

Tumorigenicity of T lymphoma/T lymphoma hybrids and T lymphoma/normal cell hybrids

Stable hybrids formed between clones of established murine T-cell lymphoma lines, and between lymphoma clones and normal spleen or thymus cells were examined for their tumorigenic properties by intravenous (i.v.) and intradermal (i.d.) inoculation into syngeneic AKR mice. Fusion parents consisted of T lymphoma clones of high and low tumorigenicity derived from the SL 12 cell line. In addition, normal spleen cells and thymocytes were fused with poorly tumorigenic T-lymphoma clones. Hybrids tested by i.v. inoculation of 10(6) cells to syngeneic hosts showed that fusion between the lymphoma cells resulted in hybrids which displayed the phenotype of the highly tumorigenic parent. Also, it was shown that fusion of poorly tumorigenic lymphoma cells with normal spleen cells resulted in hybrids with enhanced tumorigenicity. Fusion of poorly tumorigenic lymphoma cells with normal thymocytes resulted in hybrids with the highest tumorigenic potential. The pattern of spread for the tumor/tumor hybrid was that of the highly tumorigenic parent. Tumor spread patterns for the spleen/tumor hybrids were different from those of the thymocyte/tumor hybrids. Intradermal inoculation of 10(5) cells from tumor/spleen or tumor/thymocyte hybrids revealed differences in latent periods between parental and hybrid cells, the tumor/thymocyte hybrids having the shortest latent period. Surface marker studies and T-cell antigen receptor mRNA determinations in the tumor cell/normal cell hybrids indicated that the normal parent was a cell of immature phenotype. Therefore, high tumorigenicity is a dominant characteristic, and poorly tumorigenic but "immortal" T lymphoma cells can derive characteristics which increase their in vivo growth capacity from the putative immature normal cells with which they selectively fuse.     

Cellular heterogeneity in a tissue culture cell line derived from a human bladder carcinoma

To study heterogeneity in a cell line derived from a human bladder carcinoma (EJ), 7 clones were isolated at low passage and examined for differences in culture behaviour, ability to grow in agar and tumorigenicity in nude mice. The parent EJ line had several distinct chromosome populations (both diploid and tetraploid), grew in agar and produced tumours in nude mice. Three of the clones had pseudodiploid modes and 4 had either hypo- or hypertetraploid modes. The 7 clones had 5 marker chromosomes in common but the combination of other marker chromosomes made each clone unique. No significant difference was found between the clones in the in vitro growth rate although analysis of in vitro culture behaviour showed heterogeneity in the pattern of cell movement on plastic substratum. Three clones were composed of static cells, one clone had very mobile cells; the other clones had rates of movement intermediate between the two. Differences were also found in the packing density of the cloned cells and in the cell size. All 7 clones grew in agar but heterogeneity was seen between the clones as shown by widely varying colony-forming efficiencies (0.5-13%). One clone had a high colony-forming ability in agar but failed to produce tumours in nude mice. The other clones were tumorigenic regardless of colony-forming efficiency in agar. Specific chromosome abnormalities were found to be associated with growth in agar and tumorigenicity but not with the growth pattern or the rate of movement of the cloned cells in culture.     

Increased tumorigenicity of polyploid Ehrlich variants during growth in vivo is associated with karyotypic changes

In previous studies, treatment of Ehrlich ascites tumors (EAT) with either Sendai virus, Herpes virus or neuraminidase produced variants that differed significantly from EAT in both tumorigenicity and karyotype.In this study, less tumorigenic variants of EAT were isolated by culturing in media containing low concentrations of serum or without serum. In contrast to the relative stability of tumorigenicity in such variants cultured in vivo, the tumorigenincity of these variant clones passaged in vivo was variable, depending on the variant clone. During growth in vivo, 3 variant closes independently isolated from serum-free medium, low serum-containing medium and after neuraminidase treatment produced markedly heterogeneous tumors, and after 4 passages the tumors were about 1000 times more tumorigenic than the initial variants. In generating new tumors, the tumors always segregated chromosomes. The banded chromosome analysis of a variant clone isolated from serum-free medium and its malignant tumor showed that most of the chromosomes segregated from the clone were normal types. On the other hand, the tumorigenicity of Herpes virus-induced variants was unchanged during growth in vivo.     

SL12 murine T-lymphoma: a new model for tumor cell heterogeneity

It has been observed that subclones from the spontaneous murine AKR/J T-lymphoma cell line SL12 with similar in vitro growth characteristics exhibit stable differences in tumorigenicity. The cell line is composed of at least three distinct cloned cell types that are highly, moderately, or poorly tumorigenic in syngeneic host animals. When healthy, young, syngeneic host animals were given iv injections with the same number of viable growth phase cells, each cloned cell type had a different tumor incidence, latent period, and pattern of tumor spread. The unusual stability of the cloned cell lines is shown by a similar incidence, latency, and spread of the tumors when studied after more than 1 year of continuous in vitro culture. The SL12 clones also differ in several phenotypic characteristics commonly used to classify thymocyte maturation, e.g., a) the expression of three of seven surface antigens examined, b) the cellular response to glucocorticoid hormone, and c) the expression of terminal deoxynucleotidyl transferase.     

Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell-mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3–1.8 × 10^?6. Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100–123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.     

Reduced tumorigenicity of threshold syngeneic tumor inocula in XID-bearing mice treated with natural antibodies

Several correlative experimental approaches have provided evidence that polyclonal serum natural antibodies (NAb) participate in the defense against small syngeneic tumor foci in vivo. The threshold subcutaneous tumor inoculum model of incipient neoplasia has consistently revealed an inverse relationship between tumorigenicity in vivo. and the NAb binding capacity of the tumor injected or the anti-tumor NAb levels of the recipient animals, including B cell—deficient mice bearing the xid mutation of the CBA/N mouse strain. Now passive i.v. administration of whole normal syngeneic serum NAb in bolus injections, given I each day beginning on day -2, - I or 0 prior to the threshold s.c. challenge of xid-bearing CBA/N or male (CBA/N × CBA/ J)F₁ mice with syngeneic RI-28 lymphoma cells, consistently and significantly reduced their tumorigenicity assayed as tumor appearance and latency. Three similar injections of an ammonium sulfate—precipitated fraction of whole serum NAb also reduced tumor frequency and latency, while a combination of purified IgG and IgM NAb reduced the appearance of tumors slightly and increased the survival of recipients. The reconstitution of the xid-defect in anti-tumor NAb provides more direct evidence to substantiate a role for NAb in the defense against early tumor development in vivo and establishes a model for the dissection of the in vivo. mechanisms of the NAb anti-tumor activity.     

Suppressed tumorigenicity of human endometrial cancer cells by the restored expression of the DCC gene

To obtain functional evidence for DCC as a tumour suppressor associated with endometrial cancer, the human DCC cDNA encoding a complete open reading frame (ORF) was transfected into highly tumorigenic human endometrial carcinoma cells, HHUA and Ishikawa in which DCC expression was completely deleted. Reconstituted expression of DCC in HHUA had little effect on in vitro growth, but suppressed tumour formation in mice completely. The clones from Ishikawa had abundant DCC expression similar to that in normal endometrium. Their growth in vitro was suppressed and showed apoptotic phenotype. Lower levels of DCC expression in the prolonged passaged clones did not induce apoptosis, but still had the potential to suppress tumorigenicity. These observations imply a role of DCC in regulation of normal endometrial cell growth, and categorize DCC as the tumour suppressor gene for endometrial cancer.     

In vivo acquisition of Fc gamma RII expression on polyoma virus-transformed cells derived from tumors of long latency.

BALB/c 3T3 cells transformed in vitro with polyoma virus were cloned and passaged once in syngeneic mice. Resulting tumors from each clone were explanted and recultured. Expression of receptor for Fc of IgG (Fc gamma RII) in the original in vitro maintained clones and in cells derived from tumors elicited by the respective cells was measured at the protein level as well as at the mRNA level. Clones were assayed in pairs. The ancestor in vitro maintained clones [designated cultured cells (C)] were compared with cells derived from the same clones after a single passage in vivo followed by explantation and reculturing [designated cultured-tumor-cultured cells (CTC)]. C cells of any of the tested clones did not express Fc gamma RII. On the other hand, certain CTC cells were positive. The Fc gamma RII-positive cells were derived from tumors appearing after a long precancer latency period (greater than 140 days). CTC cells derived from tumors that appeared after shorter latency periods (less than 80 days) were Fc gamma RII negative. These results were obtained both by using radioimmunoassay and monoclonal antibodies against mouse Fc gamma RII as well as by Northern blot analysis using the Fc gamma RII complementary DNA probe. The involvement of macrophages as the Fc gamma RII-expressing cells in CTC cells was excluded. Fc gamma RII expression was down-regulated in CTC cells as a function of time following their explantation into culture. Fc gamma RII expression could be up-regulated in these cells and induced on C cells by maintaining the cultured cells in the presence of normal mouse serum or recombinant interferon. We also tested the expression of Fc gamma RII on CTC cells following their inoculation into syngeneic mice for a second time (CTCx2 cells). The results showed a positive correlation between Fc gamma RII expression in the inoculated ancestor CTC cells and on the CTCx2 cell progeny.     

Evidence for the role of 34-kDa galactoside-binding lectin in transformation and metastasis

The endogenous Mr 34,000 galactoside-binding lectin (L-34) is found at elevated levels in a wide variety of neoplastic cells and correlative evidence suggests that it is involved in tumor metastasis in vivo and in transformation in vitro. We demonstrate here that introduction of recombinant L-34 into tumorigenic, weakly metastatic UV-2237-cl-15 fibrosarcoma cells results in an increased incidence of experimental lung metastases in syngeneic and nude mice. Transfection of normal BALB/c-A31 cloned fibroblasts with functional L-34 results in acquisition of anchorage-independent growth and in morphological transformation in vitro but not in tumorigenicity in vivo. These results provide direct evidence that the cellular expression of L-34 is associated with some aspects of transformation and with metastasis, but not with tumorigenicity per se.     

5-Azacytidine-induced uncoupling of differentiation and tumorigenicity in a murine cell line

Highly tumorigenic, myogenically defective T984-15 murine cells were treated with the hypomethylating agent 5-azacytidine (5-azaC). In response to drug treatment, T984-15 cells formed colonies that were myosin-positive and contained fused myotubes. When cloned, these differentiated colonies gave rise to cells that maintained their myogenic potential even after prolonged growth in tissue culture. The myogenic differentiation observed in response to 5-azaC treatment was not the result of selection of a preexisting myogenic subpopulation, inasmuch as treatment of a subcloned population of nondifferentiating cells with 5-azaC also resulted in the induction of myogenesis. In addition to inducing myogenic potential, 5-azaC generally suppressed the tumorigenic potential of the treated cells. Whereas 19 of 19 untreated T984-15 clones when injected into BALB/c nude mice produced tumors, 8 of 9 of the 5-azaC-treated clones injected displayed suppressed tumorigenicity under identical conditions. Tumorigenic suppression, however, was independent of the induction of differentiation: 1 myogenic clone remained tumorigenic and 5 clones whose tumorigenic potential was suppressed were nonmyogenic. Thus treatment with the hypomethylating agent 5-azaC not only affected the expression of differentiated and tumorigenic phenotypes but also dissociated their usually coordinate regulation.     

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.     

Role of anchorage in the expression of tumorigenicity of untransformed mouse cell lines

Cultured mouse cells were tested for tumorigenicity in nude mice with both a conventional assay (injection of cell suspensions) and a new test involving implantation of cells grown on gelatin sponges. Sublines of Balb/3T3 cells, obtained from different sources, varied in their tumorigenic potential with either assay. One subline (A) formed distinctive precancerous nodules only in the sponge assay; these nodules often became progressive after a latent period of 3-4 months. However, suspensions of cells of this subline also caused tumors after a similar latent period, but no nodular phase preceded tumor formation. Another subline of Balb/3T3 (M) has failed to form tumors in either assay. The Balb/3T3 sublines did not differ in vitro properties, such as low saturation density, failure to grow in methylcellulose, and monolayer morphology. A second experimental approach involved tests on nude BALB/c mouse-embryo fibroblasts at various passage levels. The cells were passaged from primary culture, through crisis, to heteroploid, established cell lines. Tumorigenicity was demonstrable earlier in the sponge assay, at which time in vitro parameters putatively associated with malignant behavior were unchanged. Possible relationships with the in vivo phenomenon of solid-surface sarcomagenesis are discussed.     

Modulation of angiogenesis and tumorigenicity of human melanocytic cells by vascular endothelial growth factor and basic fibroblast growth factor

Human melanoma cells express two prominent angiogenic factors, e.g., vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF/fibroblast growth factor-2). In this study, we report on the relative contribution of these two factors to in vitro and in vivo growth of a tumorigenic melanoma cell line (WM164) and nontumorigenic, immortalized melanocytes (FM516SV). Overexpression of either cytokine significantly boosted tumorigenicity of WM164 cells in immunodeficient SCID mice. Attempting to overexpress bFGF antisense sequences produced no viable clones confirming earlier reports that autocrine bFGF is obligatory to melanoma cell survival and growth. By contrast, down-regulation of endogenous VEGF production did not affect growth of WM164 cells in vitro. In vivo expansion of WM164 cells expressing VEGF antisense was delayed but not abrogated. Forced expression of either bFGF or VEGF in immortalized but nontumorigenic melanocytes did not induce sustained tumor growth in vivo highlighting that neither of the two factors is sufficient for induction of tumorigenicity in this model system. Overexpression of either cytokine in WM164 cells led to the development of atypical large vessels but not to an increase in microvessel density. Taken together our results confirm an essential autocrine role of bFGF in human melanoma and indicate a beneficial but nonessential role of VEGF in the tumorigenic phenotype of human melanoma cells.     

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.