Luka Milas

The proportion of stem cells in murine tumors

Considerable evidence suggests that tumors contain only a minority of cells which are capable of regrowing the tumor (ie. tumor stem cells). Since all tumor stem cells must be killed if treatment is to be successful, the number of stem cells in a tumor can be expected to be an important determinant of curability. We have attempted to examine the proportion of stem cells in a variety of murine tumors by making measurements of three different parameters which might be expected to be related to stem cell content: (a) the radiation dose required to control the tumor (TCD50); (b) the number of cells required to transplant the tumor (TD50) and (c) the in vitro plating efficiency. An inverse correlation has been demonstrated between measured TCD50 and TD50 values for two independent groups of murine tumors of varying histopathological type. An inverse correlation was also obtained between the TD50 value and in vitro plating efficiency for a group of spontaneous murine mammary tumors. These correlations most likely reflect underlying differences in the stem cell content of the tumors, and indicate that there is a wide range (2-3 orders of magnitude) of stem cell proportions in different murine tumors, even those which have been transplanted a number of times.

Potential methods for predicting tumor radiocurability

Several predictors of tumor radiocurability are already integrated into clinical practice, for example, tumor size, gross morphology (that is, infiltrative or exophytic), histologic type and grade. These are nonspecific and relatively imprecise. The aim of research into predictive assays is not only to refine the discrimination of existing predictors, but also to suggest specific experimental approaches for overcoming tumor radioresistance in individual patients. Two broad categories of predictive assays can be defined: direct and indirect measurement of tumor cell survival and/or repair capability following irradiation, and measurement of cellular and extracellular parameters affecting radiosensitivity. The ongoing research at The University of Texas M. D. Anderson Hospital is overviewed to illustrate potential methods for predicting radiocurability.

Protection of acute and late radiation damage of the gastrointestinal tract by WR-2721

WR-2721 was investigated for its protective effect against acute and late damage produced by irradiation of the esophagus, small intestine, and colon of mice. The microcolony assay was used to measure the acute response of the small intestine and the colon, and an LD50 assay (within the 28- to 42-day time range) was used to measure acute esophageal damage. A dose of WR-2721 at 400 mg/kg, injected 30 min prior to irradiation, resulted in a protection factor (PF) of 1.6 against radiation damage in these three regions of the gastrointestinal tract. Lethality and histology scores were applied to determine late radiation damage to the rectum, at times ranging from 3 to 15 months after irradiation. Deaths occurred after doses of 20 Gy and above throughout the postirradiation period. WR-2721 increased the survival of mice; the PF calculated from the LD50 values was 1.5. PFs of animal survival did not vary during the observation period. Histological studies showed evidence of ulceration, fibrosis, and vascular changes as late radiation damage. WR-2721 protected against radiation-induced histological damage with a PF of 1.3. There was no qualitative difference between the types of histological damage observed in the group undergoing only irradiation and the group treated with WR-2721. Biochemical measurements of fibrosis by hydroxyproline determination of collagen 16 months after irradiation showed an increase in collagen per milligram wet weight of rectal tissue in all irradiated groups, but no increase in the amount of collagen per 5 mm segment of the rectum. Thus it appears that the apparent fibrosis is a result of atrophy rather than collagen accumulation. We conclude that WR-2721 is indeed effective at protection against late damage from large single doses of radiation to the rectum as measured histologically and also improves the long-term survival of the mice, although the target cells for this damage are not known.

Relationship between lymph nodal status and primary tumor control probability in tumors of the supraglottic larynx.

A retrospective review of 248 patients with squamous cell carcinoma of the supraglottic larynx was undertaken to determine the relationship between the probability of control of the primary lesion, the extent of neck nodal disease at initial presentation, and its ultimate control. All patients were treated at the U.T. M. D. Anderson Hospital between 1960 and 1980, and had a minimum of 3 years follow-up. The primary lesion was staged T1 in 38 patients, T2 in 132, T3 in 50 and T4 in 28. The initial volume of neck nodal disease was scored on a scale of 0 (no palpable nodes) to 9 (bilateral neck nodes greater than 6 cm in diameter). All primary lesions were treated definitively with megavoltage radiation therapy. Treatment to the neck varied according to the extent of lymph node involvement. There was no significant difference in the range of total radiation doses delivered to the primary lesion, stage for stage, in patients who presented with clinically negative or positive nodes, or in those with controlled versus uncontrolled neck disease. Analysis of the probability of primary tumor control was made by life table methods because of the poorer survival expectation in node positive patients. For T1 and T2 primary lesions, any positive node decreased the probability of primary tumor control (p = 0.06). For T3 and T4 lesions, a single node less than 3 cm in diameter did not worsen the chance of primary tumor control, but any greater degree of lymph node involvement did (p = 0.03). For both T stage groupings, the probability of primary tumor control at 5 years decreased progressively with increasing neck nodal disease. Primary tumor control probability was also significantly associated with control of the neck disease, independent of the modality of neck treatment. No correlation could be demonstrated between the histological grade of the primary tumor and initial lymph node status or tumor control probability. Possible interpretations of this manifestation of biological heterogeneity are discussed.

Metastatic properties of murine sarcomas and carcinomas. I. Positive correlation with lung colonization and lack of correlation with s.c. tumor take.

The spontaneous metastatic properties of six sarcomas and seven carcinomas syngeneic to C3Hf/Kam mice were investigated and the correlation between spontaneous metastasis, the lung colony forming efficiency (LCFE) of i.v. injected tumor cells, and s.c. tumor take was determined. The incidence and number of spontaneous metastases in the lung were determined in mice that had primary tumors in the leg removed 17 to 120 days earlier, depending on tumor type. There was a significant positive correlation between spontaneous metastasis and LCFE when all 13 tumors were compared, but the significance was lost when carcinomas and sarcomas were considered separately. No significant correlation between spontaneous metastasis and the s.c. tumor take was observed. Also, no correlation was found between LCFE and the s.c. tumor take of carcinomas, but there was a strong inverse relationship between these two properties of sarcomas. The number of cells shed from primary tumors was estimated and found to be more extensive in tumors with higher metastatic properties. Thus, in general, highly metastatic tumors were characterized by a high LCFE and a significant cell shedding. Furthermore, LCFE was greatly increased by treatment of animals with cyclophosphamide and by admixing heavily irradiated tumor cells to viable cells, implying that local environmental factors are important in determining the establishment of tumor cell clonogens into metastasis.

Protection of spermatogonial survival and testicular function by WR-2721 against high and low doses of radiation☆

The radioprotection of normal cells with WR-2721 at doses of radiation extending down to less than 1 Gy was investigated using testicular cells. Survival of stem spermatogonia after single doses of radiation was measured by counts of repopulating tubules and by sperm head counts, with consistent results obtained for both endpoints. Protection factors (PF) obtained by injection of 400 mg/kg WR-2721 at 15 min prior to irradiation decreased from about 1.4 at radiation doses above 10 Gy to 1.0 at 2 Gy. Similarly, the radioprotection by 300 mg/kg WR-2721 was reduced from a PF of about 1.35 when the drug was given prior to a single high dose of radiation to 1.0-1.1 when the drug was given prior to each of 5 daily fractions of 2 Gy. Thus, less protection of testicular stem cells by WR-2721 was observed at lower doses of radiation. This lowered protection may be explained, at least in part, by a direct cytotoxic effect of WR-2721 on testicular stem cells. Protection of differentiated spermatogonia was observed with 400 mg/kg WR-2721; the PF was 1.4 at 1 Gy and decreased at lower doses. The protection of testicular function by WR-2721, as assayed by the return of fertility and the maximum recovered level of sperm production, was compared to the protection of stem cell survival. At about 8 Gy the PF with 400 mg/kg WR-2721 for both functional endpoints was about 1.5, which was not significantly different from the value of 1.3 obtained using the stem cell assays.

Effect of tumor type, size, and endpoint on tumor radioprotection by WR-2721

Experiments are reported showing that the degree of tumor radioprotection afforded by WR-2721 varies with the type of tumor and assay endpoint, and that for a given tumor system, microaggregates are protected better than larger cell masses. The tumors used were a methylcholanthrene-induced fibrosarcoma (FSa), and two tumors of spontaneous origin, another fibrosarcoma (NFSa), and a mammary carcinoma (MCa-4), all syngeneic to C3Hf/Kam mice. WR-2721 was given in a dose of 400 mg/kg 30 minutes before irradiation in all experiments. In TCD50 assays, WR-2721 protected 5 mm diameter and impalpable 3 day-old transplants of 5 X 10(5) FSa cells growing in the leg by factors of 1.11 and 1.13, respectively. Using the tumor latency endpoint, 3 day-old s.c. transplants of 10(3) FSa in the abdominal wall were protected by a factor of 1.27, a degree of protection similar to that reported earlier for sterilization of lung micrometastases of the same tumor. MCa-4 tumors growing in the leg were protected better than FSa in TCD50 assays with protection factors of 1.3 for 4 day-old transplants, 1.24 for 5 mm tumors, and 1.23 for 8 mm tumors. MCa-4 tumors recurrent after irradiation as 4 day-old transplants grew more rapidly in mice that had received WR-2721, and this was shown to be most likely due to protection by the drug against expression of the tumor bed effect. Using the lung micrometastases assay, NFSa was protected by a factor of 1.22. This variability in protection with different tumor types, sizes, and assay endpoints is discussed in terms of drug delivery and uptake, and also in relation to the influence of tumor hypoxia on the radioprotective ability of WR-2721.

Spontaneous metastasis: random or selective?

Spontaneous lung metastases from an infrequently metastasizing fibrosarcoma and a more highly metastatic mammary carcinoma showed no increased capacity for generating primary tumors with greater metastatic potential when transplanted into either normal or whole-body-irradiated syngeneic mice for up to six isotransplant generations. Rather, successive generations varied in their metastatic behavior, tending towards decreasing metastatic efficiency. These data support the view that spontaneous metastases are determined more by random survival of intravasated tumor cells than by the selection of preexisting metastatic cell variants.

The tumor bed effect: Dependence of tumor take, growth rate, and metastasis on the time interval between irradiation and tumor cell transplantation

Experiments were designed to investigate the influence of time interval between leg irradiation and tumor cell transplantation on 3 different aspects of the tumor bed effect (TBE): tumor take, growth rate, and metastasis formation. MCA-4 tumor cells were injected subcutaneously into the legs of syngeneic C3Hf/Kam mice that had been locally irradiated with 30 Gy gamma rays 1, 50, 100, or 200 days previously. Interim TD50 values were higher in the day 1, 50, and 100 preirradiated mice than in controls for about 100 days after tumor cell transplantation. However, the final TD50 values determined 220 days after cell transplantation were marginally lower than controls in the day 1, and 50 preirradiated groups. TD50 values in the day 200 preirradiated group were similar to those of controls at all times after tumor cell injection. Retardation of tumor growth rate was observed in all preirradiated groups, but with a progressive decrease in effect as the time between irradiation and tumor cell injection was increased. In the day 200 preirradiated group it was noted that the degree of tumor growth rate retardation decreased as the number of injected tumor cells was increased. The incidence of lung metastases when the primary tumor reached a size of 20 mm was higher than in controls in mice irradiated 1, 50, and 100 days before tumor cell injection, but not in the 200-day preirradiated group. Thus, in this tumor system TBE influences tumor take, growth rate, and lung metastasis formation differently; but for all parameters, the effect decayed with increasing time between irradiation and tumor cell transplantation.

Radioprotectors in tumor radiotherapy: factors and settings determining therapeutic ratio

WR-2721 and DDC have been used most frequently in our studies on radioprotective agents. WR-2721 was a much more potent radioprotector of murine normal tissues, both against early and late injuries of several organs and tissues, than was DDC. Protection factors for WR-2721 usually ranged between 1.5 and 2.5. Both agents protected solid murine tumors only minimally. While WR-2721 increased therapeutic ratios commonly, DDC did so only rarely. Micrometastatic foci were amenable to radioprotection more than established solitary tumors. Additional factors that influenced the degree of therapeutic benefit included dose of WR-2721, dose of irradiation (single versus fractionated), and time of WR-2721 administration in relation to radiation delivery. The ability of WR-2721 to prevent radiation-induced immunosuppression, metastatic spread, and carcinogenesis are additional benefits in the therapeutic use of this agent. Our current research on the improvement of radioprotectors for therapeutic use is focused on (a) a search for new radioprotective agents that are equal to or better than WR-2721 but less toxic and/or more specific for normal tissue, (b) understanding the basic mechanisms of action of these radioprotective agents at the molecular level, both in cells and tissues, and thus understanding the mechanisms leading to selective or preferential radioprotection of normal tissues, and (c) in vitro testing of primary human tumor cultures for their (non)susceptibility to radioprotection.