Radiosensitivity of the murine NKLy solid tumor and recovery of clonogenic cells from potentially lethal radiation damage

It was shown by the cloning method that radiosensitivity of cells of central zones was lower than that of peripheral zones (D0=6.70 and 2.91 Gy, respectively). Under conditions of recovery from potentially lethal damages radiosensitivity of clonogenic cells of peripheral zones decreased (D0=6.22 Gy) whereas that of cells from central zones changed insignificantly (D0=7.98 Gy).     

Clonogenic capacity, radiosensitivity and recovery from potentially lethal damage in individual subpopulations of the murine NKLy ascitic tumor

Effectiveness of the diffusion chamber cloning of subpopulations of cells isolated by the sedimentation method from intact and exposed (5 Gy) mouse tumors NKLy was determined. It was shown that cells at the G2-M stages were highly radiosensitive. The recovery from potentially lethal damages was maximum at the G0-G1 stages of the cell cycle.     

The density of clonogenic cells in human solid tumors

Considering that tumors are maintained by clonogenic cells, and that the primary target in the therapy of cancer is the clonogenic cell, the density of clonogens in a tumor could become an important parameter in quantitating the response to therapy. Indirect methods for determining the density of clonogenic cells in human tumors based on the response of tumors to radiation suggest there are circa 1 X 10(5) clonogens per gram with a large range. Direct methods, based on the measurement of cloning efficiency of enzymatically disaggregated biopsies of human tumors in soft agar, suggest a clonogen density of approximately 1,500 clonogens per gram. As this value is inconsistent with the prior data, we chose to determine the density of clonogenic cells in human tumors by assaying the enzyme digest of biopsies of human tumors for clonogenic cells using an enriched monolayer clonogenic assay. We determined the average clonogen density to be 1.12 x 10(5) clonogens per gram with a large range. The agreement with the indirect method suggests that the enriched monolayer clonogenic assay supports the proliferation of the cell population responsible for maintaining the tumor.     

Availability, not respiratory capacity governs oxygen consumption of solid tumors

Contrary to conventional belief, the mitochondria of most cancer cells usually function normally, i.e., their respiratory capacity is not fundamentally impaired as compared to normal cells. Strong evidence against the misconception of mitochondrial dysfunction is provided by in vivo data clearly showing that O(2) availability is the major determinant of the O(2) consumption rate of cancer cells, independent of the means for increasing availability (e.g., by increasing blood flow or by elevating arterial O(2) content, the latter being accomplished either by an increase in the hemoglobin level and/or arterial hyperoxia). Additional support against the Warburg effect in its original concept comes from normal temperature coefficients (Q(10)) for O(2) consumption rates of malignant cells. Thus, the Warburg hypothesis postulating that mitochondrial dysfunction in cancer cells forces them to generate energy with a poor ATP yield through glycolysis appears to be elusive. Instead, due to a "reprogrammed" cancer cell metabolism, glycolysis is used to produce intermediates as building blocks for various biosynthetic pathways of cancer cells.     

Effect of x-rays on the sensitivity of cells of the murine NKLy tumor to the membrane toxic effect of natural killers

A study of membrane toxicity of allogenic splenocytes with regard to irradiated mouse NKLy lymphosarcoma cells (tested by the release of 3H-uridine label from target cells in the presence of exogenous RNAses) has displayed an insignificant increase, as compared with the control, in the membrane toxicity with radiation doses of 1-2 Gy and its decrease at higher doses.     

Changes in the clonogenic ability of solid tumor cells during combined exposure to hyperthermia and irradiation

A study was made of the clonogenic capacity of cells from central and peripheral zones of NKLy solid tumors of mice after heating up to 42 degrees C and after the combined effect of hyperthermia and local irradiation of the tumor. Hyperthermia markedly increased the rate of radiation death of cell populations from central tumor zones, having low oxygen tension, and had no effect on radiosensitivity of cells from peripheral well oxygenated zones. In heated tumors, the repair of potentially lethal radiation damages to cells from the peripheral zones was inhibited while in conventional irradiation conditions these damages could be restored.     

Effect of hypoxic cell sensitizer metronidazole on the radiation reaction of clonogenic cells from solid tumors

The clonogenic capacity of cells from peripheral and central zones of solid NKLy tumors of mice treated with metronidazole, a sensitizer of hypoxic cells, and with a mixture of metronidazole and radiation was studied by cloning in diffusion chambers. The cytotoxic effect of metronidazole was only noted during the prolonged interaction with cells under acute hypoxia that was observed in central tumor zones. Metronidazole increased by more than two times the radiosensitivity of cells from the central zones of the tumor and did not influence the radiation response of cells from the peripheral zones. Metronidazole was shown to inhibit the repair of potentially lethal radiation damages.     

Comparative radiosensitivity of clonogenic tumor cells of the transplantable Ehrlich carcinoma in the ascitic and solid forms of growth

Survival of clonogenic cells of solid Ehrlich ascites tumor (EAT) exposed to 60Co-gamma-radiation in vitro under the oxygenation conditions was investigated and the clonogenic capacity and radiosensitivity of these cells and cells of the previously studied EAT ascitic form and Lewis solid tumor comparatively studied to elucidate how the efficiency of colony formation (ECF) would affect their radiosensitivity. ECF for solid EAT cells was 2.6 +/- 0.3%, which was lower, by about an order of magnitude, than that for ascitic form of this tumor and was nearly the same as that for Lewis tumor cells. A median cell lethal dose (D0) was practically the same for all tumors under study. It is suggested that the differences in ECF do not substantially influence the radiosensitivity of clonogenic cells of the studied tumors.     

Radiation induction of drug resistance in RIF-1 tumors and tumor cells

The RIF-1 tumor cell line contains a small number of cells (1-20 per 10(6) cells) that are resistant to various single antineoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), and adriamycin (ADR). For 5FU the frequency of drug resistance is lower for tumor-derived cells than for cells from cell culture; for MTX the reverse is true, and for ADR there is no difference. In vitro irradiation at 5 Gy significantly increased the frequency of drug-resistant cells for 5FU, MTX, and ADR. In vivo irradiation at 3 Gy significantly increased the frequency of drug-resistant cells for 5FU and MTX, but not for ADR. The absolute risk for in vitro induction of MTX, 5FU, and ADR resistance, and for in vivo induction of 5FU resistance, was 1-3 per 10(6) cells per Gy; but the absolute risk for in vivo induction of MTX resistance was 54 per 10(6) cells per Gy. The frequency of drug-resistant cells among individual untreated tumors was highly variable; among individual irradiated tumors the frequency of drug-resistant cells was significantly less variable. These studies provide supporting data for models of the development of tumor drug resistance, and imply that some of the drug resistance seen when chemotherapy follows radiotherapy may be due to radiation-induced drug resistance.     

Effects of gramicidin and hyperthermia on the clonogenic capacity of cultured mammalian cells

The cytotoxic effect of gramicidin on cultured mammalian cells appeared to be accelerated by 43 °C hyperthermia. With 0.1 μM gramicidin, in medium without serum, cellular inactivation was approximately 16 times faster than at 37 °C and with 0.03 μM gramicidin, 9 times. The pH of the cell medium had no influence on the cytotoxic action of gramicidin at 43 °C.     

A hypo-osmotic medium to disaggregate tumor cell clumps into viable and clonogenic single cells for the human tumor stem cell clonogenic assay

A hypo-osmolar medium and tissue processing technique is described which is useful for disaggregation of residual human tumor cell clumps persisting after mechanical or enzymatic treatment of solid tumors and malignant effusions. The addition of the hypo-osmolar procedure to the standard methods for disaggregation increased the viable single cell yield in solid tumors by 47% and in malignant effusions by 67%. In 5 of the 26 solid tumor specimens tested in the human tumor stem cell assay, clonogenic single cells were obtained with the hypo-osmolar procedure, whereas no growth was observed using standard methods. Overall, the success rate for clonogenicity increased from 46% to 65% for the 26 solid tumors, with the major improvement occurring in ovarian cancer. Clonogenicity was obtained in 80% of malignant effusions both by standard methods and the hypo-osmolar techniques. The increased total yield of clonogenic cells obtained with this procedure enhances the opportunity for experimental versatility and in vitro drug testing.     

Directing CAR T cells towards the tumor vasculature for the treatment of solid tumors

For successful application of chimeric antigen receptor (CAR) T cell therapy in solid tumors, major hurdles have to be overcome. CAR T cells have to cross the vascular barrier, which is hampered by the anergic state of the tumor vasculature, characterized by suppressed levels of leukocyte adhesion molecules on the endothelium. Additional immunosuppressive mechanisms in the solid tumor microenvironment can affect infiltration, activity and persistence of CAR T cells. Redirecting CAR T cells towards the tumor vasculature poses a possible solution, as molecular targets of tumor endothelial cells can be directly engaged from within the blood.In this review, we discuss recent advances in CAR T cell therapy against solid tumors, with a focus on targeting the tumor vasculature. Furthermore, we discuss opportunities to overcome challenges and barriers through engineering of CAR T cells to enhance trafficking, safety and efficacy.     

Estimates of the number of clonogenic cells in crypts of murine small intestine

There is a proliferative cell hierarchy in the mouse intestinal crypt with ancestral stem cells which can regenerate all components of the lineage after injury (clonogenic cells). The number of these clonogenic or regenerative cells per crypt can be estimated from radiobiological experiments where doses of radiation are used to kill cells and ablate crypts. Various approaches can be adopted which provide different estimates of this number of cells. One of the conventional approaches used in the past provided estimates of about 70-80 clonogenic cells per crypt (i.e. about 50% of the proliferative or 30% of all crypt cells). A technically simpler approach has recently been suggested. This has been used here to provide many independent estimates of the number of crypt clonogenic cells. These suggest about 32 clonogenic cells exist per crypt i.e. about half the previous estimate and about twice the number of putative "functional" stem cells (those which operate as stem cells in the normal steady-state crypt). The reasons for the differences are discussed. The new estimates are compatible with the hypothesis that the crypt contains a ring of about 16 functional stem cells which are expected to be clonogenic, besides which there is a second ring of 16 clonogenic cells which represent early transit cells (the immediate daughters of the stem cells) which can act as clonogenic cells if required after radiation injury.     

Radiation adaptive response of glial cells.

There are various types of radiation in space including high energy particles. It is, therefore, becoming to be important to study the low dose and low dose-rate effects in space radiation biology. Radiation adaptive response (RAR) for cell growth and its mechanism were examined using cultured glial cells. The cells from hippocampus of Wistar rats were irradiated with a low dose (0.1 Gy) of X-rays and 3 h after with a high dose (2 Gy). Decrease in the rate of cell growth with 2 Gy was suppressed by the 0.1 Gy preirradiation, when cells were counted 2 days after irradiation. The inhibitors of protein kinase C (PKC) and DNA-dependent protein kinase (DNAPK) or phosphatidylinositol 3-kinase (PI3K) suppressed RAR. The treatment with the activators of PKC instead of 0.1 Gy-preirradiation also caused adaptive response to 2 Gy-irradiation. Moreover, glial cells cultured from severe combined immunodeficiency (scid) mice, which have lost DNAPK activity, and AT-2KY cells, fibroblasts of an ataxia-telangiectasia (AT) patient, showed no RAR. These results indicated that PKC, ATtM, DNAPK and/or PI3K were involved in RAR for growth of cultured glial cells. Proteomics [correction of preteomics] analysis of these cells exposed to low dose irradiation in now underway.     

Study of factors determining the clonogenic capacity of irradiated cells by computer simulation

The methods of the multifactor disperse analysis of the results of studies of the simulation model of the effect of ionizing radiation on cell populations were used to study the role of some characteristics of the stationary culture in its response to a single radiation effect. The clonogenic capacity of cells was used as a criterion for assessing the biological effect of radiation. "The share of resting cells" was a predominant factor influencing the survival of irradiated cell populations.     

Effects of murine tumor necrosis factor on heterotransplanted human tumors.

A partially purified glycoprotein fraction (the G-200 II fraction) obtained from sera of CD-1 mice sensitized with Corynebacterium parvum and treated with endotoxin was designated as tumor necrosis factor (TNF). Human melanoma cells exposed to this factor in vitro had decreased tumorigenicity when injected into nude mice. Human melanoma, embryonal adenocarcinoma of the testis and colon carcinoma heterotransplanted in nude mice exhibited regressions in size following intraperitoneal injections of TNF. The responses were related to dose and duration of exposure.     

Generation of reactive oxygen species and radiation response in lymphocytes and tumor cells

Several types of lymphoid and myeloid tumor cells are known to be relatively resistant to radiation-induced apoptosis compared to normal lymphocytes. The intracellular generation of reactive oxygen species was measured in irradiated spleen cells from C57BL/6 and BALB/c mice and murine tumor cells (EL-4 and P388) by flow cytometry using dichlorodihydrofluoresceindiacetate and dihydrorhodamine 123 as fluorescent probes. The amount of reactive oxygen species generated per cell was low in the tumor cells compared to spleen cells exposed to 1 to 10 Gy of gamma radiation. This could be due to the higher total antioxidant levels in tumor cells compared to normal cells. Further, the changes in mitochondrial membrane potential and cytoplasmic Ca2+ content were appreciable in lymphocytes even at a dose of 1 Gy. In EL-4 cells, no such changes were observed at any of the doses used. About 65% of spleen cells underwent apoptosis 24 h after 1 Gy irradiation. However, under the same conditions, EL-4 and P388 cells failed to undergo apoptosis, but they accumulated in G2/M phase. Thus the intrinsic radioresistance of tumor cells may be due to a decreased generation of reactive oxygen species after irradiation and down-regulation of the subsequent events leading to apoptosis.