Julia Kahn

Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy

UNLABELLED It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1β. PSCs further secrete IL1β, and inactivation of PSCs reduces IL1β expression and TAN recruitment. Furthermore, depletion of TANs, IL1β inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. SIGNIFICANCE Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852-69. ©2016 AACR.See related commentary by Bronte and Tortora, p. 821This article is highlighted in the In This Issue feature, p. 803.

Studies on fractionated hyperthermia in experimental animal systems I. The foot reaction after equal doses: Heat resistance and repopulation

Abstract The response of the mouse foot to fractionated hyperthermia was studied in vivo . Hyperthermia was given by immersing the mouse foot into a constant temperature water bath kept at 43.5 ± 0.1°C. Foot reaction was scored after treatment according to a numerical score system; at 43.5°C the time to induce loss of one toe or a greater reaction in half the treated animals (RD 50 ) was assayed. The equal dose fractions were used throughout the present experiments. The RD 50 for various time intervals (Ti) between two doses demonstrated rapid development of heat resistance which reached a maximum in 48 hours after first treatment. The resistance subsided thereafter and the complete decay of resistance required almost 2 weeks. If 2 doses were given with a Ti of more than 17 days, further increase of the RD 50 was observed, which might be attributed to repopulation of surviving cells. Isoeffect curves for treatment schedules with various Ti were obtained. These studies indicated that heat resistance develops repeatedly after each treatment and is the most important factor in fractionated hyperthermia. The kinetics of the resistance appeared to depend on the Ti and number of fractions as well as fraction size. In general the resistance developed fully in 48 hours after first hyperthermia, whereas it appeared to develop in 24 hours after the second treatment with a Ti of 2 days or after the third treatment with a Ti of 1 day. The resistance was less extensive for a large number of fractions with Ti of 2 days.

Relationship between energy status, hypoxic cell fraction, and hyperthermic sensitivity in a murine fibrosarcoma.

The energy status, radiobiological hypoxic cell fraction, and hyperthermic sensitivity of a spontaneous murine fibrosarcoma, FSa-II, have been evaluated as a function of tumor size. Tumors were evaluated over the size range of 70 to 800 mm3. The concentration of the high-energy phosphate reservoir creatine phosphate progressively decreased by a factor of 5 with increasing tumor volume, and was matched by an increase in creatine. The concentration of ATP also decreased with increasing tumor size, although this decrease was substantially less pronounced. The sum of ATP, ADP, and AMP did not vary with tumor size, suggesting that the necrotic fraction remained constant. The decrease in energy status occurred in parallel with an increase in the size of the hypoxic cell fraction and with increasing thermal sensitivity. The results suggest that energy status may be an important modifier of hyperthermic sensitivity in vivo and reflect tissue oxygen concentration.

The effect of hyperthermia on the early and late appearing mouse foot reactions and on the radiation carcinogenesis: effect on the early and late appearing reactions.

The effect of hyperthermia on radiation-induced early- and late-appearing foot reactions was studied in C3Hf/Sed mice derived from our defined flora mouse colony. The animal foot was irradiated with 137Cs gamma-rays under hypoxic, air, or hyperbaric oxygen (O2 30 psi) conditions. Hyperthermia of 43.5 degrees C for 45 min was given locally in a water bath where a constant temperature +/- 0.1 degrees C was maintained. Treatment intervals between the 2 treatments were 20 min and 2 days. For the early-appearing reactions scores taken between the 14th and 35th post-irradiation days were averaged. Late-appearing reactions became apparent after approximately the 200th post-treatment day and increased with time. The foot reaction was enhanced by hyperthermia given 20 min before or after irradiation. Dose response curves for radiation given 20 min after hyperthermia for acute-appearing reactions lacked shoulders, whereas those following the same treatment schedule for late-appearing reactions showed significant shoulders. The thermal enhancement ratios (TER) for score 2.0 (complete epilation) early- and late-appearing reactions depended on the treatment interval and sequence. The TER values were greater for a short treatment interval (20 min.) than for a long treatment interval (2 days). Thermal enhancement was greater for hyperthermia given before irradiation compared to the reverse sequence. The TER values were always smaller for the late-appearing reactions than for the acute-appearing reactions. The relationships between early reaction scores and late reaction scores showed that the late reactions following combined heat and radiation are less extensive than those following radiation alone if they were compared at radiation doses which induced an equal level of early reactions. This difference was most significant at low early reaction scores and decreased with increasing score level.

The effect of cis-diamminedichloroplatinum(II) treatment at elevated temperatures on murine fibrosarcoma, FSa-II.

The effect of cis-diamminedichloroplatinum(II) (cis-DDP) on the murine fibrosarcoma cells was investigated in vitro and in vivo. For in vitro experiments tumour cell suspensions containing a given amount of cis-DDP were treated in water bath maintained at a desired temperature, and cell survival was determined by the lung colony assay. The D0 or the time to reduce survival from 1.0 to 0.37 on the exponential portion of the survival curve was determined and 1/D0 was plotted as a function of 1/T, where T stands for the absolute temperature. The slope of this Arrhenius plot indicated that the activation energy for chemical reaction of cis-DDP was 44 kcal/M between the temperature range from 37 to 41 degrees C. For in vivo experiments tumours were transplanted into the foot and treated by immersing the animal foot into a water bath when each tumour reached an average diameter of 4 mm (35 mm3). The drug was injected i.p. immediately before hyperthermia. The tumour growth (TG) time or the time required for a tumour to reach 1000 mm3 from the treatment day was determined, and the median TG time was obtained by logit analysis. Dose-response curves between the TG time and drug dose indicated that the cytotoxic effect of cis-DDP was enhanced at elevated temperatures. This enhancement increased with increasing temperature from room temperature to 43.5 degrees C. Because of short plasma half-time of cis-DDP, continuous infusion and pulse injections were attempted.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytotoxic effect of cis-diamminedichloroplatinum(II) on cultured Chinese hamster ovary cells at elevated temperatures : Arrhenius plot analysis

The effect of cis-diamminedichroloplatinum (II) (cis-DDP) and hyperthermia on cultured Chinese hamster ovary (CHO) cells were investigated. Cells were treated with 6 microM cis-DDP for various durations of time at temperatures ranging from 37 to 43 degrees C, and cell survival curves were determined as a function of treatment time. The cytotoxic effect of cis-DDP increased with increasing temperatures, indicating that hyperthermia enhanced cytotoxicity of cis-DDP. Arrhenius analysis of surviving fraction data (6 microM cis-DDP) yielded activation energies of 61 kcal/M between 37 and 41 degrees C and 213 kcal/M between 41 and 43 degrees C. Further experiments using two different drug concentrations (3 and 12 microM) confirmed these activation energies in these two different temperature ranges. However, the activation energy for 12 microM cis-DDP given in the temperature range 33-37 degrees C was 22 kcal/M, which is smaller than those found above 37 degrees C. This activation energy appeared to be identical to that reported for the degradation or depurination of DNA. The activation energy between 37 and 41 degrees C, i.e. approximately 61 kcal/M, was twice as great as that found for alkylation of thio-TEPA, an alkylating agent. This may indicate that the mechanism of action of cis-DDP differs from that of thio-TEPA. A greater activation energy observed in the range of 41-43 degrees C is most likely attributable to the additive effect of hyperthermia and thermal enhancement for cis-DDP. Although only one data point is available at the temperature above 43 degrees C, it suggests that the activation energy is identical to that for hyperthermia alone.

The accelerated repopulation of a murine fibrosarcoma, FSa-ii, during the fractionated irradiation and the linear-quadratic model

Radiation response of a spontaneous mouse fibrosarcoma, FSa-II, to various fractionated doses was studied in vivo together with single dose cell survival curves. Early generation isotransplants were used. Animals were C3Hf/Sed mice derived from our defined flora mouse colony. Lung colony and TD50 assays were used to determine cell survival. Surviving fractions were determined following fractionated irradiations of 1.0 to 5.0 Gy each per fraction with interfractional time intervals of 4 hr. The alpha/beta ratio based on fractionated irradiations was 8.8 Gy for aerobic FSa-II tumor cells and flexure dose was less than 1.3 Gy. Multiple fractions of 5.0 Gy each given with 4, 12, and 24 hr intervals showed an increase in survival with increasing interfractional time interval, suggesting a rapid repopulation of tumor cells between fractions; namely, cell doubling time was shortened between fractions after the first 5.0 Gy doses. These results indicated that tumor cell repopulation is a critical factor in the fractionated radiotherapy. Linear-quadratic model was fitted to single dose survival data. Single dose survival curve of aerobic FSa-II tumor cells following lung colony assays which allowed determination of minimal survival of approximately 3.0 x 10(-3) showed that alpha, beta, and alpha/beta ratios were 0.25 Gy-1, 0.048 Gy-2, and 8.47 Gy, respectively. Single dose survival curve of the same aerobic cells determined by both lung colony and TD50 assays to a survival level of approximately 3.0 x 10(-6) demonstrated that alpha, beta, and alpha/beta ratios were 0.375, 0.0127, and 29.5, respectively. Similar determination for hypoxic FSa-II tumor cells showed that alpha, beta values were smaller whereas the alpha/beta ratio was much larger than for aerobic cells. The oxygen enhancement ratio calculated by the alpha/beta ratios was greater than 3.0.

The Effect of Step-Down Heating on Murine Normal and Tumor Tissues

Cultured cells can be sensitized to low-temperature hyperthermia (below 43.0 degrees C) by a prior heat shock at a high temperature (above 43.0 degrees C) if the heat above 43.0 degrees C is immediately followed by the heat below 43.0 degrees C. This effect has been termed step-down heating (SDH). We have studied the effect of SDH on the response of murine tumor and normal tissues treated at 45.5 degrees C. Animal tumors were eighth-generation isotransplants of a spontaneous fibrosarcoma in C3Hf/Sed. mice. Tumor response was studied by TG (tumor growth) time assay, i.e., determination of the time required for half the treated tumors to reach 1000 mm3 from the first day of treatment. Normal tissue response was studied in the mouse foot. End point was the time to induction of a score 4.0 (loss of a toe) or greater reaction in half the treated animals, RD50. The SDH at 41.0 degrees C sensitized the response of tumor and normal tissues to 45.5 degrees C. The enhancement ratios were congruent to 1.7 for both tissues, indicating no differential sensitization between tumor and normal tissues. No sensitization was observed if the SDH was given immediately after a second dose of 45.5 degrees C given 6 hr to 5 days following the first dose of 10 min at 45.5 degrees C. The SDH appeared not to inhibit the development of thermal resistance as evidenced by no appreciable changes in the thermal resistance ratio.

Effect of thermochemotherapy on the development of spontaneous lung metastases

Effect of local hyperthermia given alone or in combination with cyclophosphamide and/or hyperglycaemia on the development of lung metastasis was studied using a non-immunogenic mouse fibrosarcoma, FSa-II. Incidence of lung metastasis was dependent upon the tumour size, and was increased when tumour-bearing mice were restrained in animal holders which were used for heat-treatment of animal tumours. The frequency of the metastatic spread was decreased following local hyperthermia at 41.5 degrees and 45.5 degrees C when compared to that following sham treatment. This decrease was independent of the heat dose. Similar reduction in the incidence of metastasis was observed after hyperthermia given following glucose administration. The administration of cyclophosphamide effectively inhibited the development of lung metastasis. However, the magnitude of the inhibition was identical to that following hyperthermia alone. In conclusion, hyperthermia given alone, or in combination with hyperglycaemia or cyclophosphamide, inhibited the development of lung metastasis.

Cytotoxic effect of 1,3 bis (2-chloroethyl)-N-nitrosourea at elevated temperatures: Arrhenius plot analysis and tumour response.

The effect of hyperthermia on the cytotoxicity of 1,3-bis-(2-chloroethyl)-N-nitrosourea (BCNU) was investigated in vitro and in vivo. Tumour cells were early-generation isotransplants of a spontaneous C3Hf/Sed mouse fibrosarcoma, FSa-II. For in vitro studies, single cell suspensions containing 1.0 x 10(6) cells/ml were treated in a water bath where a desired temperature was maintained by a constant-temperature circulator. Cell survival was determined by lung colony assay immediately thereafter. For in vivo studies the tumour cell suspensions were transplanted into the dorsal site of the C3Hf/Sed mouse foot. Tumours were treated by immersing animal feet into a constant-temperature water bath when tumours reached an average diameter of 4 mm (35 mm3). The tumour growth (TG) time or the time for one-half of the treated tumours to reach 1000 mm3 from initial treatment day was used as an endpoint. BCNU dose-cell survival curve at 37 degrees C was exponential with a D0 of 1.1 microgram/ml. Dose-cell survival curves at 37-43 degrees C were determined as a function of treatment time at pH 6.7 and 7.4. BCNU of 1 microgram/ml was added immediately before treatment. The slope of the survival curve became steeper with increasing temperature, indicating that the cytotoxic effect of BCNU was enhanced by hyperthermia. The Arrhenius plot analysis showed that activation energies at pH 6.7 and 7.4 were 53 and 51 kcal/M, respectively (no significant difference). Of interest in this analysis was that the Arrhenius plot did not show a breaking point which has been observed for other agents. Further investigation demonstrated that the decomposition of BCNU, which has been reported to be essential for production of reactive intermediates, occurred in aqueous medium at elevated temperatures. The magnitude of this decomposition depended on treatment temperature. As a result, preheated BCNU became less cytotoxic with an increase in preheating temperatures. The activation energy for this decomposition was about one-half of the activation energy for BCNU cytotoxicity. Studies in vivo indicated that the effect of BCNU was enhanced with increasing temperatures, and the enhancement was greatest when BCNU was administered i.p. immediately before hyperthermia. A glucose dose of 5 g/kg administered i.p. 60 min before hyperthermia further enhanced the antitumour effect of BCNU.