Tumor bed expression in xenografted artificial heterogeneous colon tumors.

Artificial heterogeneous xenograft tumors (AHTs) were produced by injecting admixtures of two clonal cell lines (A and D) originally isolated from the DLD-1 human colon adenocarcinoma. The volumetric growth of unirradiated pure A or D tumors and 2 different admixtures (approximately 90% A + 10% D or 10% A + 90% D) were compared to growth of similar tumors growing in normal tissue stroma that had received 5, 10, or 15 Gy of X rays 1 day prior to cell injections. The X-irradiations produced a dose dependent delay in growth at sizes of 500 mm3 (designated as the tumor bed effect, TBE) for both pure and admixed tumors of about 2 at 15 Gy. There were no significant differences in TBE among the various groups. Also, determination of tumor cell yields, or colony forming efficiencies, showed no differences among tumors growing in undamaged or damaged stroma. However, beginning at about 20 days postirradiation, there was a significant change in the percentage composition of the tumors growing in the irradiated stroma. Both admixtures became significantly enriched in the subpopulation that was originally predominant. In contrast, the percentage compositions of the admixtures growing in unirradiated stroma remained stable over the duration of the experiment period (to about 70 days postirradiation). We interpret these data to indicate that the radiation damage to the normal tissue produced an intratumor environment leading to competitive exclusion dynamics of the minority subpopulation. Clinical implications of these results are discussed.

Changes in X-ray sensitivity and glutathione content of human colon tumor cells after exposure to the differentiation-inducing agent sodium butyrate.

Clone A human colon cancer cells were exposed to concentrations of sodium butyrate (NAB, 0-2 mM) for three passages in vitro, and responses to either graded single doses or split doses of 250 kVp X rays were determined. The survival data were fit to the single-hit, multitarget model of inactivation. For the graded single dose experiments, we found that NAB produced a decrease in the magnitude of the quasi-threshold (Dq) parameter after a concentration of about 0.9 mM was exceeded. Similarly, in split dose experiments, the amount of sublethal damage recovery (SLDR) was reduced in a concentration-dependent manner as shown by a decrease in the Dq parameter. However, the inhibition of SLDR occurred with no apparent threshold NAB concentration. NAB did not affect potentially lethal damage recovery. Paradoxically, increasing concentrations of NAB produced an exponential increase in the intracellular glutathione content, which could be blocked by exposure of the cells to buthionine sulfoximine (BSO). BSO treatment of NAB-adapted cells led to additional cell killing, again most noted by changes in the Dq parameter. We postulate that these responses are associated with NAB-induced changes in chromatin structure, particularly the association between DNA and nucleosomal histones H3 and H4.

X ray responses of a human colon tumor cell line after exposure to the differentiation-inducing agent N-methylformamide: concentration dependence and reversibility characteristics.

The combination of differentiation-inducing agents with conventional cytotoxic agents has been suggested as a potential cancer therapeutic strategy. In this regard, we have chronically exposed (3 passages) a human colon tumor line (clone A) to varying concentrations (0-170 mM) of N-methylformamide and examined the change in sensitivity to ionizing radiation in vitro. The linear-quadratic formalism of survival was used to characterize the single graded dose survival curves. This equation yields two constants (alpha and beta) relating to cellular inactivation produced by either single events (alpha) or by the combination of two events (beta). As the N-methylformamide concentration increased, the alpha parameter increased while the beta parameter concomitantly decreased, yielding a concentration dependent radiosensitization which was most marked in the low dose region of the survival curve. Upon removal of NMF, the original radiation resistance was regained within 2-3 cell culture doubling times.

Compositional stability of artificial heterogeneous tumors in vivo: use of mitomycin C as a cytotoxic probe.

A major part of the overall response of solid cancers to cytotoxic treatments will be due to the differential sensitivities of the neoplastic cell subpopulations present. To quantitatively investigate this, artificial heterogeneous human colon xenograft tumors were constructed using two clonally related cell lines (A and D) which were mixed to create compositions of approximately 9:1 or 1:9 A:D cells. Then, the pure A, D, or admixed tumors were challenged with mitomycin C which kills A cells more efficiently than D cells by a factor of about 2.3 as determined by in vitro survival curve inactivation slopes. This difference was also exhibited in vivo by the post-mitomycin C cell survival responses determined by excision assay and by the shapes of the regrowth curves of the pure A and D or admixed tumors. By approximately 30 days after treatment, artificial heterogeneous tumors had reached a new stable cellular composition which could be quantitatively predicted based on the individual survival of A and D cells from pure clonal tumors measured 24 h after treatment. Thus, in this model system, cytotoxic treatment of heterogeneous neoplasms produced predictable and stable, albeit altered, percentage admixtures of subpopulations. This result is consistent with the observed decreased clinical responsivity of primary tumors to sequential courses of therapy due to selection of preexisting resistant subpopulations.

Effects of the differentiating agents sodium butyrate and N-methylformamide on the oxygen enhancement ratio of human colon tumor cells.

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio.

Cell cycle responses of heterogeneous human colon adenocarcinoma subpopulations to X-irradiation.

The cell cycle responses of two exponentially growing subpopulations of cells (clones A and D), originally obtained from a human colon adenocarcinoma to X-irradiation, were studied using centrifugal elutriation. Cell suspensions were separated by changing counter-current flow rate while keeping the rotor speed constant (1600 rpm) and the composition of eluted fractions was determined using flow cytometry. The X-ray sensitivity of unseparated clone D cells was somewhat greater than that of clone A cells (e.g. 10% greater at the 10% level of survival). This difference appeared to be due to a greater value of the alpha parameter (one-hit cell killing), using the linear-quadratic equation in which the relative survival S/S0 = exp - (alpha D + beta D2) with dose (D) in Gy. This finding was confirmed in the cell cycle studies where the alpha parameter was always greater for the clone D cells than for the clone A cells. The beta parameter was essentially the same for both cell lines through the cell cycle.

Growth properties of artificial heterogeneous human colon tumors.

Two clonal cell lines (designated as clones A and D), originally isolated from the heterogeneous DLD-1 human colon adenocarcinoma, were used to produce xenograft tumors in nude mice. Neoplasms produced from either A or D cells alone were compared to those produced from a range of percentage admixtures of the two subpopulations. Then, Gompertzian growth parameters (initial growth rates, retardation rates) were determined, along with estimation of the final asymptotic volumes. It was found that the growth kinetics of the various artificial heterogeneous tumors could not be predicted from knowledge of the growth parameters of the pure clonal xenograft tumors. Additionally, both pure clonal and artificial heterogeneous tumors were enzymatically disaggregated as a function of time postinjection, and it was found that the admixed tumors became more zonal in composition as time progressed. Further, admixtures of extreme composition (i.e., 9% A plus 91% D or 88% A plus 12% D) remained stable with time, while those of intermediate initial composition (i.e., 50% A plus 50% D) did not. All of these data (growth kinetics, zonality, compositional stability) indicate that the growth properties of heterogeneous tumors are very complex.

Induction of doxorubicin resistance in heterogeneous human colon tumor cells by N-methylformamide.

Two clonal subpopulations of cells (termed A and D) obtained originally from a heterogeneous human colon adenocarcinoma were studied in vitro with regard to possible modification of responsivity to graded doses of doxorubicin (ADM) after long-term growth in medium containing the differentiating agent N-methylformamide (NMF). Non-NMF-adapted clone A and D cells exhibited biphasic response curves after exposure to graded doses of ADM (0-1.4 micrograms/ml, 1 hour, 37 degrees C). The inactivation slopes for the initial regions of the survival curves were 0.13 and 0.39 g/ml for clone A and D cells, respectively. NMF-adapted cells, however, exhibited decreased sensitivity to ADM killing as the inactivation slopes for the initial region of the survival curves increased to 0.26 and 0.59 g/ml for clone A and D cells, respectively. The final slopes of the biphasic response curves (doses above about 0.8 micrograms/ml) were not significantly different between clone A and clone D tumor cells in either the non-NMF- or NMF-treated conditions. These data on ADM responses after differentiation induction by NMF indicate that the combination of differentiation-inducing agents with certain chemotherapeutic agents may produce therapeutically undesirable effects and that preclinical data are necessary prior to implementation of possible combined-modality protocols.

Similarity of thermotolerance characteristics in heterogeneous human colon tumor subpopulations after exposure to fractionated heat doses (44 degrees C).

We have studied the thermotolerance (TTR) responses of three tumor cell populations obtained from a heterogeneous human colon cancer after exposure to 44 degrees C water bath hyperthermia given in a split dose regimen. The cell populations studied consist of the original tumor line (DLD-1) obtained from surgical biopsy, and two subcloned populations termed clones A and D. All tumor lines were treated with initial heat doses at 44 degrees C which reduced the initial survival level to about 30, 10, or 1%, after which cultures were returned to 37 degrees C. Complete survival curves were then determined for all three lines as a function of time after the initial treatment (0, 2, 4, 8, 12, 14, 20, and 24 h), and the magnitude and kinetics of thermotolerance development and its decay were determined. It was found that the DLD-1 line was more heat sensitive and exhibited a lesser degree of induced thermotolerance than did the A and D subpopulations, indicating intraclonal heterogeneity. Also, the rate of thermotolerance induction, the maximal thermotolerance reached (TTRmax), and the rate of thermotolerance decay were strongly dependent upon the survival level produced by the initial heat exposure. The importance of these findings to the treatment of heterogeneous cancers by heat is discussed.

Disaggregation studies of xenograft solid tumors grown from pure or admixed clonal subpopulations from a heterogeneous human colon adenocarcinoma.

Two clonal tumor subpopulations (designated as A and D) obtained originally from a heterogeneous human colon adenocarcinoma (DLD-1) were used to produce xenograft solid tumors in nude mice. First, disaggregation studies were performed to determine the optimal choice of enzyme and time of dissociation for the pure A and D neoplasms, using cell yield (cells/mg/min) and colony forming efficiency (CFE) assays. The enzymes investigated were: 0.5 or 0.2% trypsin, and two cocktails containing pronase (0.5 or 0.05%), collagenase (0.02%), and DNAse I (0.02%). For the 0.5% trypsin treatments, the cell yield from A and D tumor fragments increased until about 30 min, at which time a plateau in cell yield was reached. A plateau in CFE was also reached at this time. In contrast, the cell yields for the 0.2% trypsin treatment did not reach a plateau within the time of the dissociation (120 min), and the CFEs were lower than with the 0.5% trypsin. Whereas no differences in cell yield or CFE were found between the enzyme cocktail studies (0.5% trypsin vs. 0.05% pronase), the cell yield and the CFE from the clone D carcinomas were significantly less than that found with the 0.5% trypsin (the cell yield and CFE from clone A tumors were identical for 0.5% trypsin or enzyme cocktail). These data indicate that, while these clonal neoplasms have somewhat different responses to enzyme disaggregation, it is possible to select an enzyme treatment and treatment time that is appropriate for use on both A and D tumors (i.e., 0.5% trypsin). After determination of an acceptable enzyme procedure, 'reconstructed' heterogeneous tumors produced from an initial injection bolus of 50% clone A and 50% clone D cells were disaggregated as a function of time (days 12-83 postinjection). Over this period, we found that the cell yield decreased exponentially, with a half-time (T1/2) of 20.5 +/- 7.3 days (95% confidence limits), with a maximum extrapolated cell yield at time zero of about 1.2 X 10(5) cells/mg. The CFE was essentially constant over the duration of the assay period. Moreover, it was found that the percentage of clone A cells appeared to decrease exponentially (T1/2 = 20.5 +/- 11.5 days, 95% confidence limits) until about 40 days postinjection. After this time an equilibrium mixture consisting of about 10% clone A cells and 90% clone D cells was reached.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement by N-methylformamide of the effect of ionizing radiation on a human colon tumor xenografted in nude mice.

Polar solvents, which induce differentiation in murine and human tumor cells, enhance the effect of ionizing radiation on cultured mouse mammary and human colon cancer cells. To determine whether this enhancement occurs in vivo, DLD-2 human colon carcinoma xenografts in nude mice were treated with combinations of 6 MV photon irradiation, the polar solvent N-methylformamide (NMF), or combinations of the two agents. Nude mice bearing 300-mg s.c. implants of DLD-2 tumors were treated i.p. with 150 mg NMF/kg daily for 19 days. Local tumor irradiations were administered as graded single doses or as fractionated doses, daily for 4 days, following the third NMF injection. The growth-inhibiting effect of the radiation treatment for both single dose and fractionation protocols was enhanced by the polar solvent. NMF alone increased the time required for a doubling of initial tumor volume by 1.7 days, compared to control tumors. Initial tumor volume doubling times compared to untreated controls were increased by 3.6 and 7.6 days by photon doses of 10.0 and 13.75 Gy, respectively, whereas NMF plus 10.0 or 13.75 Gy increased the DLD-2 regrowth delay time by 7.5 or 12.9 days. NMF caused essentially equivalent enhancements, whether split-dose schedules of 2.5 Gy daily for 4 days, and 3.44 Gy daily for 4 days, or single doses of 10.0 and 13.75 Gy were used; therefore, radiation enhancement was not due to effects on sublethal damage repair. The results support the use of NMF, currently in Phase 1-Phase 2 clinical trials, with radiation in the therapy of selected human neoplasms.

Intrinsic and extrinsic heterogeneity in the responses of parent and clonal human colon carcinoma xenografts to photon irradiation.

Responses to photon irradiation of xenografted human colon tumors derived from the heterogeneous DLD-1 line or its derivative A and D subpopulations were determined using excision assay and tumor regrowth delay assays. Differential responses among the three xenografted carcinomas were demonstrated. Clone A tumors treated with up to 17.5 Gy showed no actual regression below pretreatment volume. In contrast, clone D tumors were sensitive to doses as low as 3.5 Gy, and tumor volumes were reduced by 65% with a dose of 17.5 Gy. The responses of DLD-1 tumors were intermediate between the clone A and clone D tumor responses. The survival parameters obtained in the excision assay studies for the DLD-1, clone A, and clone D tumors were, respectively: n = 3.3, 1.4, and 1.0; D0 (Gy) = 2.1, 2.2, and 2.7; and DQ (Gy) = 2.6, 0.6, and 0.0. These data indicate that the DLD-1 tumors were the most resistant, with clone A of intermediate sensitivity, clone D being the most sensitive tumor. In addition to the interclonal diversity among xenograft lines, intraclonal variation was also observed with clone A (but not clone D or DLD-1) tumors. A biphasic survival curve of cells from clone A xenografts irradiated in air-breathing hosts clearly indicated a minority (approximately 3%) subpopulation of hypoxic cells. Similar results indicating a small percentage of hypoxic cells in clone A solid tumors were obtained from the tumor regrowth delay studies. Also, excision assay data from experiments in which the heterografted carcinomas were irradiated under anoxic conditions support the interpretation that clone A tumors contain a small fraction of hypoxic cells. This study indicates that: (a) heterogeneity in vivo to ionizing radiation exists in the DLD-1 system; and (b) intraclonal variation occurs in vivo due to extrinsic (e.g., environmental hypoxia) factors, such that the intrinsic radioresistance of a subpopulation (clone A) of a heterogeneous human tumor can be further increased.

Response of a human colon adenocarcinoma (DLD-1) to X irradiation and mitomycin C in vivo.

Mice hosting a heterogeneous human colon xenograft tumor produced by subcutaneous injection of the DLD-1 tumor cell line were treated either with x irradiation alone, with mitomycin C alone (4 mg/kg), or with x irradiation given two hours after intraperitoneal injection of mitomycin C (4 mg/kg). Radiation alone produced a dose dependent delay in the time needed for tumors to regrow to twice their size at the time of irradiation, and in the mice receiving mitomycin C plus x irradiation, an additional growth delay equivalent to that produced by 3-3.5 Gy of X rays was seen at all X ray dose levels. As the DLD-1 tumor xenografts do not appear to possess a significant hypoxic fraction, we conclude that the two agents are acting in a simple additive cytotoxic manner by the killing of oxic tumor cells.

Modification of radiation damage in rat spinal cord by mitotane.

Modification of the paralytic response in rats after 6-MV photon irradiation of the spinal cord with either single or split exposures (two equal fractions given in a 24-hour period) by mitotane was investigated. Mitotane was administered as a suspension in physiologic saline (300 mg/kg/day) for either 5 days prior to or 5 days after irradiation. For rats receiving split doses of 6-MV photons, either the last two doses of mitotane were given 2 hours prior to each radiation fraction or mitotane was begun 2 hours after the second fraction and continued for 5 days. The data to 6 months after irradiation indicate that, in rats given mitotane for 5 days prior to single-dose photon irradiation, the paralytic response (as defined by the dose needed to produce paralysis in 50% of the irradiated groups of rats) was enhanced by a dose-enhancement factor (DEF) of 1.40. The DEF in the group of rats given mitotane after single doses of 6-MV photons was 1.15. In the split-dose irradiation experiments, the DEF for the groups of rats given mitotane prior to each radiation fraction was 1.36; while the DEF for the group of rats receiving mitotane beginning after the second fraction was 1.18. These data indicate that mitotane can potentiate the effects of 6-MV photon irradiation to the central nervous system, with mitotane administered prior to irradiation being the most effective sequence.

Modification of the response of mouse skin to x-irradiation by a polar solvent, N,N-dimethylformamide.

The modification of the response of mouse skin to either single or split (24 hrs) graded doses of X rays by topically applied N,N-dimethylformamide (DMF) was investigated. DMF was applied daily for 5 days prior to irradiation. At a radiation dose level producing dry desquamation, DMF enhanced the X ray response by a factor of 1.3. Also, at the same level of response, the fraction of X ray dose repaired in 24 hours was 0.57, whereas for the DMF-treated and irradiated skin, this factor was 0.41, indicating a reduction of about 28% in subeffective damage repair. The times of maximal involvement of the skin reactions were not different in the X ray plus DMF treated mice versus mice receiving x-irradiation only. The data indicate that DMF is able to modify intrinsic radiation sensitivity of mouse skin epithelial cells, possibly through a reduction in the magnitude of the shoulder region of the survival curve.