David P. Houchens

Human brain tumor xenografts in nude mice as a chemotherapy model

Two human brain tumors which were previously established in nude mice were used to determine antitumor efficacy of various therapeutic agents. These tumors were a medulloblastoma (TE-671) and a glioma (U-251) with mass doubling times of 3.5 and 5.5 days respectively as subcutaneous implants in nude mice. Intracranial (i.c.) tumor challenge was accomplished by inoculating tissue culture-grown cells of either tumor into the right cerebral hemisphere to a depth of 3 mm. Median survival time (MST) in untreated mice with 10(5) i.c. injected TE-671 cells was approximately 30 days and 53 days in the U-251 tumor. With 2 X 10(5) U-251 tumor cells the MST was 27-31 days. Groups of mice which had been inoculated with tumor were treated with various doses and schedules of antineoplastic compounds by the i.p. route. The TE-671 tumor responded to AZQ treatment with an increase in life span (ILS) of 37% compared to untreated controls and an ILS of 30% with CCNU treatment. BCNU and PCNU were ineffective. With the U-251 tumor BCNU produced an ILS of greater than 60%, with 75% cures, greater than 112% ILS with PCNU and 49% ILS with CCNU. Neither tumor responded to procarbazine, PALA, dianhydrogalactitol, D-O-norleucine or dibromodulcitol. The U-251 tumor was treated on various schedules and doses with BCNU and found to respond well on late as well as early treatment. A new drug (rapamycin) being investigated by the NCI was found to be very effective against the U-251 tumor. This model system should prove valuable in assessing the effects of various chemotherapeutic modalities against brain tumors.

Chemotherapy of human colon cancer xenografts in athymic nude mice

The lack of effective chemotherapy in advanced colon cancer and difficulties in evaluating response in clinical trials indicate a need for experimental models to screen new agents for activity against this specific type of cancer. Xenografts of human colon cancer in nude mice reproduce many features of the original tumor specimen and could be expected to predict drug response for colon cancer with more specificity than previously used screens. NIH‐Swiss nude mice bearing subcutaneous (s.c.) trocar implants of tumor tissue were used in these studies. Three serially transplantable lines of human colon cancer (designated “HT”, “CA”, and “BE”), which range in their degree of differentiation from well‐differentiated adenocarcinoma to undifferentiated carcinoma, were used for drug testing. Treatment was delayed until tumors had reached 60‐600 mg in mass to correspond to advanced tumors in patients. Drugs being developed for clinical trial by NCI were selected for testing against the xenografts based on promising activity against transplantable murine tumor lines and difference in mechanism of action. Two antimetabolites, PALA and Bakers antifol, a mitotic spindle poison, maytansine, and a DNA intercalating agent, AMSA, produced no significant regression in any of the three colon xenograft lines. From the group of the nitrosoureas, methylCCNU and chlorozotocin were tested. MethylCCNU caused no regression of line HT, a transient response of line CA and complete regressions of line BE. Chlorozotocin also caused regression of line BE but had no effect on the other tumor lines. The results of chemotherapy studies with human colon cancer xenografts in nude mice reflect the clinical situation where few objective responses are achieved with presently available chemotherapy. We would conclude from these studies that information about activity against colon cancer of a new drug may be gained by testing the drug against a panel of human colon cancer xenografts. Careful clinical followup studies and parallel studies with xenograft systems should establish the correlation between individual clinical response and response in the xenograft system.

Intraoperative radioimmunodetection of colorectal tumor with a hand-held radiation detector

A hand-held gamma detection probe was used intraoperatively to localize primary and recurrent colorectal tumors in 28 patients 48 to 72 hours after they received an intravenous injection of 2.2 mCi of iodine-131 labeled anticarcinoembryonic antigen polyclonal baboon antibody. Preoperative evaluation included determination of serum carcinoembryonic antigen, barium enema, colonoscopy, chest film, computerized axial tomography, liver, spleen, and bone scans, and endoscopy when indicated. Preoperative whole-body imaging correctly localized primary tumors in only 33 percent of the patients, whereas it correctly demonstrated tumor in 64 percent of those with recurrent disease. Intraoperative tumor-to-background ratios derived from the detector probe were elevated in all patients, averaging 3.97:1 in primary lesions and 4.18:1 in recurrent tumors. Postoperatively, carcinoembryonic antigen was localized in tissues with the avidin-biotin peroxidase staining technique to confirm intraoperative readings. Variations in stain uptake in a patient could be correlated with variations in radiation detector readings in the same patient. Results support our previous work in nude mice, demonstrating the improved sensitivity and specificity of the hand-held gamma detection device over whole-body imaging for intraoperative localization of immunoradiolabeled tumors.

Impaired growth of a radiation-induced lymphoma in intact or lethally irradiated allogeneic athymic (nude) mice.

Radioinduced lymphomas were maintained in mice by serial transplantation of neoplastic cells and mortality of mice was recorded for 60 days. Mice were x- irradiated at a dose rate of 137 R/min. Lethally irradiated mice were resistant to an incompatible lymphoma as determined by 125I-IUdR uptake values of the spleen. The lack of influence of an immunodepressive factor and the observation that lethal irradiation did not abrogate the relative resistance to tumor challenge appeared to exclude the possibility that the reduced growth of the lymphoma in nude mice is the result of a conventional allograft reaction.

The significance of intraoperative periportal lymph node metastasis identification in patients with colorectal carcinoma

Background. Nine patients who underwent Radioimmunoguided Surgery (RIGS) (Neoprobe Corporation, Dublin, OH) procedures for colorectal cancer were found to have disease recurrence in the periportal area. This led to a retrospective study to determine whether periportal lymph node involvement could have been predicted intraoperatively for these patients.

Collagenase Inhibitors Retarding Invasion of a Human Tumor in Nude Mice

Tumor invasion has been correlated with the ability of tumor cells to produce collagenolytic enzymes which are capable of degrading normal host tissues. However, the human small cell carcinoma implanted subcutanouesly and growing progressively in athymic (nude) mice produced large quantities of collagenase but did not appear to significantly infultrate adjacent host tissue. In comparison, subcutaneously implanted murine Lewis lung tumors produced similar quantities of collagenase and were locally invasive. The human tumors were surrounded by a compact layer of fibroblast cells in a fibrous matrix. This fibrous sheath exhibited anticollagenase activity and indicated a mechanism of host tissue resistance to invasion via the formation of inhibitors to degradative enzymes produced by tumor cells.

Portable gamma probe for radioimmune localization of experimental colon tumor xenografts

Tumor radioimmune detection as presently practiced utilizes a gamma scintillation camera to image tumors. A major clinical limitation is the inability to detect tumors smaller than 2 cm. This limitation is due in part to the inverse square law which states: the number of detected radioactive counts is inversely proportional to the square of the distance separating a radioactive source from the detecting device. A hand-held gamma-detecting probe (GDP) suitable for intraoperative use has been developed. The GDP can be placed near radioactive tumors and take advantage of the inverse square law in a way not possible with external scanning cameras. The use of radiolabeled baboon carcinoembryonic antigen (CEA)-specific antisera produced increased tumor isotope localization in CEA-producing tumors compared to the injection of nonspecific antisera. Tumor isotope-antisera localization was not influenced by tumor volume or time since tumor implantation. The GDP probe counts demonstrated a high degree of correlation with gamma well tissue counts. The probe was able to detect preferential tumor localization in doses lower than could be detected with external scintillation cameras.

Radioiodination, quality assessment, in vitro and in vivo stability of iodine‐125 nofetumomab

Iodine-125 radiolabeled monoclonal antibody (MAb) preparations used for radioimmunoguided surgery should be prepared in a manner to insure a stable, efficacious and safe product is used in clinical studies. A number of MAbs have been radioiodinated and used for radioimmunoguided surgery procedures including B72.3 and CC49. Individual MAbs and their fragments differ in their physico-chemical response to conditions of radiolabeling, effects of radiolysis, storage and general handling conditions. A radiolabeling and stability study was completed to evaluate the radiochemical purity and immunoreactivity of NR-LU-10 Fab (nofetumomab) radiolabeled with I-125 at three different specific activities using the IODO-GEN® method. Ratios of 2:1, 1:1, 0.4:1 and 0.2:1(mCi/mg) were used to determine the effects of increasing specific activity on the in vitro stability in a range of potential patient dosages. The radiochemical purity and immunoreactivity of each lot of radiolabeled nofetumomab were determined on days 0, 8, 15, 22, 29 and 36 after radiolabeling using standard tests of instant thin layer chromatography (ITLC), high performance liquid chromatography (HPLC) and whole cell binding assay of immunoreactivity. These standard tests indicated the radiochemical purity on the day of radioiodination was high for all specific activity levels (98–99% for ITLC and >98% for HPLC). Preparations at all specific activities showed a slight decrease in radiochemical purity over time with the amount of unbound I-125 increasing or remaining unchanged. The immunoreactivity net bound showed excellent binding with values ranging from 65–75%. The data indicate I-125 nofetumomab undergoes in vitro physico-chemical changes over time. However, the changes in radio-chemical purity and immunoreactivity were minimal and should prove to be clinically insignificant.

Chemotherapy modalities and immune condition of the host.

It is clear that it is possible to improve the immune condition of the host and to increase its immunological reactivity by employing a range of modalities. These may involve nonspecific immunostimulation of the host, active or passive immunization, adoptive chemotherapy, alteration of tumor cell antigenicity or other types of approaches. New agents may be sought that will serve as immunostimulants or that may prevent immunosuppression by tumor growth or by chemotherapeutic agents. New analogs of the known antitumor agents may be examined to determine whether they are less immunosuppressant than the parent drug. Any means leading to the net improvement of the immune status of the host may result in diminished drug toxicity for the host, increased antitumor response, and augmentation of immunochemotherapy.

Effect of Dimethylsulfoxide and Hexamethylene Bisacetamide on the JOK-1 Hairy Cell Leukemia Derived Cell Line Propagated in the Nude Mouse

The JOK-1 hairy cell leukemia derived cell line has been propagated as a subcutaneous tumor in nude mice. After the tumor had been serially transplanted for at least two successive generations, mice were treated with either dimethylsulfoxide or hexamethylene bisacetamide (HMBA). These agents have been shown to induce terminal leukemic cell differentiation in vitro. Our results indicated that these agents had an in vivo growth inhibitory effect, with HMBA exerting a dose-dependent response. Histopathological examination revealed massive areas of necrosis with no overt signs of cellular differentiation. These data suggest that in vitro inducers of differentiation may act via another mechanism in vivo.