Leo E. Gerweck

Cellular Responses to Combinations of Hyperthermia and Radiation

The two principal rationales for applying hyperthermia in cancer therapy are that: (a) the S phase, which is relatively radioresistant, is the most sensitive phase to hyperthermia, and can be selectively radiosensitized by combining hyperthermia with x-irradiation; the cycling tumor cells in S phase which would normally survive an x-ray dose could thus be killed by subjecting these cells to hyperthermia; and (b) the relatively radioresistant hypoxic cells in the tumor may be selectively destroyed by combinations of hyperthermia and x-irradiation. Both of these rationales have been mentioned as reasons for using high LET irradiation in cancer therapy; therefore where such irradiation may be of use, hyperthermia may also be advantageous.

Tumor pH controls the in vivo efficacy of weak acid and base chemotherapeutics

The extracellular pH of tumor tissue is significantly lower than the extracellular pH of normal tissue, whereas the intracellular pH of both tissues is similar. In principle, extracellular acidity may be expected to enhance the intracellular uptake and cytotoxicity of weak acid chemotherapeutics that are membrane permeable in their uncharged state and inhibit the efficacy of weak bases. However, procedures for assessing the role of the gradient as a determinant of drug efficacy in vivo by altering the pH gradient may also alter drug availability and thus mask or exaggerate the effect of the gradient change. In the present study, we have altered the extracellular pH of tumors and compared the effect of the resultant pH gradient change on the efficacy of a weak acid versus a weak base. This experimental design gives rise to a change in the ratio of chlorambucil- to doxorubicin-induced tumor growth delay, independent of possible changes in drug availability. The extracellular pH of the 54A human tumor in NCr/Sed/nu/nu mice was altered by administration of 5 mg/g i.v. glucose. The resultant 0.2 pH unit increase in the tumor cell pH gradient gives rise to a predicted 2.3-fold increase in the ratio of chlorambucil to doxorubicin growth delay. The experimentally measured change in the growth delay ratio was 2.1. The results provide compelling evidence that the pH gradient in a determinant of the efficacy of weak electrolytes in the complex in vivo environment and may be exploited for the treatment of cancer. [Mol Cancer Ther 2006;5(5):1275–9]

Relative biological effectiveness of proton beams in clinical therapy

PURPOSE In clinical proton beam radiation therapy, an RBE of 1.1 relative to megavoltage X-rays is currently being employed at most treatment centers. This RBE pertains to radiation in the spread out Bragg-peak (SOBP) for all tissue systems, all dose levels per fraction and all proton beam energies. As the number of centers and treatment sites for which proton beam therapy continues to increase and additional experimental data is accrued, a re-assessment of the justification for a generic RBE is warranted. In this paper we address: (1) the constancy of the RBE along the central axis from the plateau entrance to the distal SOBP (upstream of the distal edge); (2) RBE as a function of dose (or cell survival level); and (3) the target cell or tissue (alpha/beta) dependency of the RBE. This analysis pertains to modulated proton beams of initial energies of approximately 70-200 MeV and SOBPs of approximately 2-10 cm, respectively. RESULTS AND CONCLUSIONS With exceptions, the available experimental data indicate that the RBE of SOBP protons increases with decreasing dose or dose per fraction and increasing depth in the SOBP, with the magnitude of both effects likely being dependent on the alpha/beta ratios of the target cells or tissues. The use of a generic RBE of 1. for all tissues, especially those exhibiting low alpha/beta values such as CNS, may be too low, especially at dose levels of < or = 2 Gy/fraction. Systematic determination of the RBE values dependent upon the three interdependent variables identified in this manuscript (beam depth, dose size and target tissue) will provide an enhanced data base for detailed treatment planning and institutional trial comparisons, thereby maximizing the therapeutic benefit of proton beams.

Effect of heat and radiation on synchronous Chinese hamster cells: killing and repair.

GERWECK, L. E., GILLETTE, E. L., AND DEWEY, W. C. Effect of Heat and Radiation on Synchronous Chinese Hamster Cells: Killing and Repair. Radiat. Res. 64, 611-623 (1975). Synchronous Chinese hamster cells were heated to 45.500C before, during, or after Xirradiation. The lethal effects of heat and radiation treatment were determined by the capacity of single cells to form colonies. Heat treatment prior to irradiation during G, or S was more effective, in terms of cell killing than heat treatment during or after irradiation. The time interval between irradiation and subsequent heat treatment influenced survival. The half-time for maximal survival when heat followed irradiation was 20-30 min in both G1 and S phase cells. Survival of cells which were heated, and then irradiated after heat treatment, indicated an independent, additive or synergistic interaction between heat and radiation, depending on the time of heat treatment and the phase of the cell cycle treated. Nine minutes of heat treatment during G, or 7 min during S decreased the shoulder region and increased the slope of the radiation survival curves. But most important, 7 min of heat prior to irradiation radiosensitized the relatively radioresistant S phase cells more than the relatively radiosensitive G, cells, and thus virtually eliminated the differences in radiosensitivity normally observed between G1 and S. Repair of heat damage as determined by survival to subsequent irradiation, began approximately 6 hr after 9 min heat treatment during Gx, and approximately 12 hr after 7 min heat treatment during S. In both cases, heat damage was repaired in the absence of cell division; for heating in Gi, repair occurred during Gi, but for heating during S, repair may have been associated with movement into G2. Cells exposed to split heat treatments during S repaired heat damage while they were still in S phase, and maximum survival was attained when 6 hr elapsed between the heat treatments.

Proton vs carbon ion beams in the definitive radiation treatment of cancer patients.

BACKGROUND AND PURPOSE Relative to X-ray beams, proton [(1)H] and carbon ion [(12)C] beams provide superior distributions due primarily to their finite range. The principal differences are LET, low for (1)H and high for (12)C, and a narrower penumbra of (12)C beams. Were (12)C to yield a higher TCP for a defined NTCP than (1)H therapy, would LET, fractionation or penumbra width be the basis? METHODS Critical factors of physics, radiation biology of (1)H and (12)C ion beams, neutron therapy and selected reports of TCP and NTCP from (1)H and (12)C irradiation of nine tumor categories are reviewed. RESULTS Outcome results are based on low dose per fraction (1)H and high dose per fraction (12)C therapy. Assessment of the role of LET and dose distribution vs dose fractionation is not now feasible. Available data indicate that TCP increases with BED with (1)H and (12)C TCPs overlaps. Frequencies of GIII NTCPs were higher after (1)H than (12)C treatment. CONCLUSIONS Assessment of the efficacy of (1)H vs(12)C therapy is not feasible, principally due to the dose fractionation differences. Further, there is no accepted policy for defining the CTV-GTV margin nor definition of TCP. Overlaps of (1)H and (12)C ion TCPs at defined BED ranges indicate that TCPs are determined in large measure by dose, BED. Late GIII NTCP was higher in (1)H than (12)C patients, indicating LET as a significant factor. We recommend trials of (1)H vs(12)C with one variable, i.e. LET. The resultant TCP vs NTCP relationship will indicate which beam yields higher TCP for a specified NTCP at a defined dose fractionation.

Tumor pH: Implications for treatment and novel drug design*

Although limited data exist, electrode-measured pH values of human tumors and adjacent normal tissues, which are concurrently obtained by the same investigator in the same patient, consistently show that the electrode pH (believed to represent tissue extracellular pH primarily) is substantially and consistently lower in tumor than in normal tissue. In contrast, the 31P-magnetic resonance spectroscopy-estimated intracellular pH is essentially identical or slightly more basic in tumor compared with normal tissue. As a consequence, the cellular pH gradient is substantially reduced or reversed in these tissues. This difference provides an exploitable avenue for the treatment of cancer. The extent to which drugs exhibiting weakly acid or basic properties are ionized depends on their ionization potential (pKa) and the pH of their milieu. Weakly acidic drugs that are lipid soluble in their nonionized state diffuse freely across the cell membrane and on entering a relatively basic intracellular compartment become trapped and accumulate within the cell. This may lead to substantial (10-fold or more) differences in the intracellular-to-extracellular drug distribution between tumor and normal tissue for cytotoxics, hypoxic cell sensitizers, or other drugs exhibiting appropriate pKa. Experimental in vitro evaluation of these predictions confirms both the predicted pH gradient-dependent changes in cellular drug accumulation and toxicity.

Perivascular nitric oxide gradients normalize tumor vasculature

Normalization of tumor vasculature is an emerging strategy to improve cytotoxic therapies. Here we show that eliminating nitric oxide (NO) production from tumor cells via neuronal NO synthase silencing or inhibition establishes perivascular gradients of NO in human glioma xenografts in mice and normalizes the tumor vasculature, resulting in improved tumor oxygenation and response to radiation treatment. Creation of perivascular NO gradients may be an effective strategy for normalizing abnormal vasculature.

Tumor size dependent changes in a murine fibrosarcoma: Use of in vivo31P NMR for non-invasive evaluation of tumor metabolic status☆

Tumor tissue contains viable hypoxic regions that are radioresistant and often chemoresistant and may therefore be responsible for some treatment failures. A subject of general interest has been the development of non-invasive means of monitoring tissue oxygen. Pulse Fourier transform 31P NMR spectroscopy can be used to estimate intracellular nucleotide triphosphates (NTP), phosphocreatinine (PCr), inorganic phosphate (Pi) and pH. We have obtained 31P NMR spectra as an indirect estimate of tissue oxygen and metabolic status in a C3H mouse fibrosarcoma FSaII. Sequential spectra were studied during tumor growth in a cohort of animals and peak area ratios for several metabolites were computed digitally by computer. During growth, tumors showed a progressive loss of PCr with increasing Pi, and most tumors greater than 250 mm3 in volume had little or no measurable PCr. The smallest tumors (38 mm3 average volume) had PCr/Pi ratios of 1.03 +/- .24, whereas tumors 250 mm3 or more had an average PCr/Pi ratio of 0.15 +/- .04. Similarly derived NTP/Pi ratios decreased with tumor size, but this change was not significant (p = .17). Radiobiologic hypoxic cell fractions were estimated using the radiation dose required to control tumor in 50% of animals (TCD50) or by the lung colony technique. Tumors less than 100 mm3 had a hypoxic cell fraction of 4% (TCD50) while tumors 250 mm3 had a 40% hypoxic cell fraction (lung colony assay). These hypoxic fraction determinations correlated well with the depletion of PCr and decline in NTP/Pi ratios seen at 250 mm3 tumor volumes. Tumor spectral changes with acute ischemia were studied after ligation of the tumor bearing limb and were similar to changes seen with tumor growth. PCr was lost within 7 minutes, with concurrent increase in Pi and loss of NTP. Complete loss of all high energy phosphates occurred by 40 minutes of occlusion. In vivo tumor 31P NMR spectroscopy can be used to estimate tissue metabolic status and may be useful in non-invasive prediction of hypoxic cell fraction, reoxygenation, and radiation treatment response.

Enhancement of mammalian cell sensitivity to hyperthermia by pH alteration.

The effect of pH on the growth rate and reproductive capacity of Chinese hamster cells was examined at 37° and 42°C. At 37°C, the population doubling time did not vary between pH 7.4 and 7.0, but w...

IN VITRO INTRINSIC RADIATION SENSITIVITY OF GLIOBLASTOMA MULTIFORME

Glioblastoma multiforme is one of the most resistant of human tumors to radiation whether used alone or in combination with surgery and/or chemotherapy. This resistance may be caused by one or more of several different factors. These include inherent cellular radiation sensitivity, an efficient repair of radiation damage, an increased number of clonogens per unit of volume, a high hypoxic fraction, high [GSH] concentration, and rapid proliferation between fractions. In the present study, we evaluate the intrinsic radiation sensitivity (surviving fraction at 2 Gy or mean inactivation dose) of malignant human glioma cells in vitro. The in vitro radiation sensitivity of 21 malignant glioma cell lines (early and long term passages) has been measured using colony formation as the end-point of cell viability. The survival curve parameters (SF2 measured and calculated, alpha, beta, D0, n and MID) have been determined for single dose irradiations of exponential phase cells (18-24 hr after plating) under aerobic conditions and growing on plastic. The mean SF2 of the 21 cell lines is 0.51 +/- 0.14 (with a range of 0.19 to 0.76). This value may be compared to the mean SF2 of 0.43-0.47 for SCC, 0.43 for melanoma, and 0.52 for glioblastoma as reported from other authors when using colony formation of cells in exponential phase on plastic. Although glioblastoma is almost invariably fatal, our data demonstrate a very wide range of intrinsic radiosensitivities. These broadly overlap the radiation sensitivities of cell lines from tumors that are often treated successfully. We conclude that standard in vitro measurements of cellular radiation sensitivity (SF2) do not yield values that track in a simple manner with local control probability at the clinical level and that, for at least some of the tumors, other parameters and/or physiological factors are more important.