By the use of a variety of cell culture and separation methods, four cell lines were isolated from a single autochthonous BALB/cfC3H mammary tumor. These lines differ markedly from each other in culture morphology, various in vitro growth properties, expression of murine mammary tumor virus antigen, and karyotype, yet all four lines are tumorigenic in normal, syngeneic hosts, yielding tumors of generally similar histology, although distinct from the original neoplasm. Three of the four lines have been cloned from soft agar. The clones exhibit the same growth properties as the lines from which they were derived. Karyotypic analysis of the parent tumor revealed the presence of cells with heterogeneous numbers of chromosomes similar to those seen in the isolated lines, suggesting both the presence of these distinct cell types in the original neoplasm and a genetic origin of the diversity.

https://cancerres.aacrjournals.org/content/canres/38/10/3174.full.pdf

Heterogeneity of tumor cells from a single mouse mammary tumor.

It is now appreciated that cancers can be composed of multiple clonal subpopulations of cancer cells which differ among themselves in many properties, including karyotype, growth rate, ability to metastasize, immunological characteristics, production and expression of markers, and sensitivity to therapeutic modalities. Such tumor heterogeneity has been demonstrated in a wide variety of animal tumors of differing etiology, tissue and cellular origin, and species. It has been shown in autochthonous, as well as transplanted, tumors. Similar results have been reported for human cancers, although much of the evidence that heterogeneity of human cancers, also reflects, at least in part, the existence of clonal subpopulations, is still indirect. Heterogeneity is not a unique property of malignancy. Preneoplastic tumors, as well as normal tissues, are also composed of cellular subpopulations. Proposed mechanisms for the origin of tumor heterogeneity include coalescence of multiple foci of cancer clones and the generation of diverse subpopulations from a single clone. This latter process could be due to genetic errors arising from classical genetic mechanisms or to the production of cellular variants as in normal tissue differentiation. Indeed, certain tumor subpopulations have been shown to produce variants at high frequency. In some cases this frequency can be modified by environmental circumstances. Nontumor cells may also contribute to production of cancer cell variants, perhaps, in the case of infiltrating phagocytic cells, by producing mutagens or by somatic hybridization with cancer cells. Production of tumor cell variants is a dynamic process which can occur at any time. Although tumors are mixed populations of cells, knowledge of the characteristics of individual components is not sufficient to predict the behavior of the whole. Individual cancer subpopulations can interact to affect each other's growth, immunogenicity, ability to metastasize, sensitivity to drugs, and clonal stability. The existence of multiple, interactive subpopulations provides a basis for the well-known phenomenon of ‘tumor progression’ in which tumors undergo qualitative changes in characteristics over the course of time. Selection of subpopulations better able to survive changing environmental circumstances allows for such changes as autonomy in regard to endogenous growth regulation, more ‘malignant’ behavior, and loss of response to therapy. Tumor subpopulation interactions may play a regulatory role in this process. Tumor heterogeneity has obvious consequences to the design of effective therapy. It provides one rationale for combination therapies and suggests that initial treatment should be early and comprehensive. The continuing emergence of new clones suggests that treatment which is unsuccessful at one point might be effective later. Assays to predict effective therapy for individual patients need to address the multiplicity of tumor subpopulations and the ability of these subpopulations to influence each other. Subpopulation interactions may also be useful in therapy design, as may be efforts to control the extent of tumor heterogeneity by agents which effect cellular differentiation. Thus, tumor heterogeneity presents both problems and, perhaps, new solutions for control of cancer.

Tumor heterogeneity: biological implications and therapeutic consequences

The histological pleomorphism of human malignant gliomas as well as their variable response to therapy suggests that such tumors are not homogeneous but are composed of a heterogeneous population of cells. To search for heterogeneity in these tumors, we developed a protocol to identify by karyotype, isolate, and clone the subpopulations of the tumor. Freshly resected tumors were mechanically dissociated into single cells that were grown in suspension and short-term monolayer cultures. Chromosomal preparations were collected over the first 6 to 72 hr postresection. The karyotypes prepared comprised the “reference set” of chromosomes found in each tumor. The dissociated single cells were also dilution plated for monolayer culture. Cells that attached were marked and isolated as clones whose karyotypes were compared to those in the reference set in order to identify those clones that were cellular representatives of the tumor. Explant cultures established from the same tumor were grown in monolayer culture and cloned; their karyotypes were likewise compared to those in the reference set. Eight gliomas were analyzed. Each had metaphases that ranged in chromosome number from hypodiploid to hyperploid, but the frequency distribution of the subpopulations differed among the tumors. Defining a cellular subpopulation in the tumor as one yielding at least five karyotypically identical cells in the reference set, we found that each tumor contained from three to 21 subpopulations. All but one tumor produced 100 or more clones, and in seven tumors, more than 50% of the clones could be expanded for karyotyping; 7.6 to 25% of these clones had karyotypes identifiable in the reference set of their tumor. There was little relationship between the morphology of the clones and their karyotypes or growth kinetics. While explant cultures grew well, no clone derived from such cultures could be identified in the reference set. The protocol permits studies of clonal lines identifiable as cellular representatives of a patient9s tumor and not variants generated in tissue culture. These studies confirmed that human malignant gliomas are comprised of karyotypically heterogeneous cellular subpopulations. Preliminary work suggests that their phenotypic behavior may be similarly heterogeneous.

https://cancerres.aacrjournals.org/content/canres/41/6/2349.full.pdf

Isolation, Karyotype, and Clonal Growth of Heterogeneous Subpopulations of Human Malignant Gliomas

Three distinct subpopulations of tumor cells derived from a single parent strain BALB/cfC3H mammary adenocarcinoma were tested in vivo for sensitivity to cyclophosphamide, methotrexate, and 5-fluorouracil. Treatment was begun either 2 days after s.c. tumor cell injection or at the time when the tumors became palpable. It was given on a weekly basis for 4 weeks. The mice were observed for growth of the primary implant and for development of spontaneous metastases. The three subpopulations differed markedly in their sensitivity to the drugs. The effects of the drugs ranged from induction of regression of the "primary" to enhancement of metastases. The effect on primary growth was independent of that on metastasis. The effect of the time of administration of the drugs also varied among the subpopulations. The sublines were also tested in vitro with methotrexate and 5-fluorouracil. Again there were marked differences in sensitivity to inhibition of cell division by the drugs. The relative sensitivities in vitro did not correlate with observations in vivo. The existence of subpopulations of tumor cells, differing in sensitivity to therapeutic agents, within a single neoplasm, presents a challenge to development of assays capable of predicting drug response and to the selection of combination therapies.

https://cancerres.aacrjournals.org/content/canres/38/11_Part_1/3758.full.pdf

Heterogeneity in Drug Sensitivity among Tumor Cell Subpopulations of a Single Mammary Tumor

Ten permanent clones derived from a single biopsy specimen of an untreated human adenocarcinoma of the stomach were established and characterized in vitro. Tissue culture growth properties, doubling times, plating efficiencies, growth fractions, cell cycle phase distributions, DNA indices, modal chromosome numbers, and ploidies were determined. Growth fractions were nearly 100%, and doubling times ranged from 23 to 37 hr. The plating efficiencies were generally high for tumor cells in culture, ranging up to 70%. Modal chromosome numbers varied from 45 to 48, with a wider range of variability in about 25% of the cells studied in each clone. In addition, the parent cell line (from which the clones were isolated) was shown to grow in athymic mice and to have the same histochemical and cytological characteristics as the specimen taken from the patient. It is important to characterize human tumor cells in vitro in this detailed manner, since they serve as excellent model systems for other studies involving the heterogeneous responses to drugs and radiation. The identification of mechanisms of drug sensitivity and resistance and the testing of drug and radiation combination treatment schedules in such in vitro systems can provide valuable insight into the design of clinical protocols for treatment of stomach cancer in humans.

https://cancerres.aacrjournals.org/content/canres/43/4/1703.full.pdf

S.C. Barranco

Papers

Differential response by human melanoma cells to 1,3-bis-(2-chloroethyl)-1-nitrosourea and bleomycin