Hyperthermia (HT) and molecular targeting agents can be used to enhance the effect of radiotherapy (RT). The purpose of this paper is to evaluate radiation sensitization by HT and different molecular ...targeting agents (Poly ADP-ribose polymerase 1 inhibitor, PARP1
; DNA-dependent protein kinase catalytic subunit inhibitor, DNA-PKcs-
and Heat Shock Protein 90 inhibitor, HSP90
) in cervical cancer cell lines. Survival curves of SiHa and HeLa cells, concerning the combined effects of radiation with hyperthermia and PARP1
, DNA-PKcs
or HSP90
, were analyzed using the linear-quadratic model: S(D)/S(0) = exp - (αD + βD²). The values of the linear-quadratic (LQ) parameters α and β, determine the effectiveness at low and high doses, respectively. The effects of these sensitizing agents on the LQ parameters are compared to evaluate dose-dependent differences in radio enhancement. Combination of radiation with hyperthermia, PARP1
and DNA-PKcs
significantly increased the value of the linear parameter α. Both α and β were significantly increased for HSP90
combined with hyperthermia in HeLa cells, though not in SiHa cells. The Homologous Recombination pathway is inhibited by hyperthermia. When hyperthermia is combined with DNA-PKcs
and PARP1
, the Non-Homologous End Joining or Alternative Non-Homologous End Joining pathway is also inhibited, leading to a more potent radio enhancement. The observed increments of the α value imply that significant radio enhancement is obtained at clinically-used radiotherapy doses. Furthermore, the sensitizing effects of hyperthermia can be even further enhanced when combined with other molecular targeting agents.
In patients with limited peritoneal metastasis (PM) originating from colorectal cancer, cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a potentially ...curative treatment option. This combined treatment modality using HIPEC with mitomycin C (MMC) for 90 minutes proved to be superior to systemic chemotherapy alone, but no benefit of adding HIPEC to CRS alone was shown using oxaliplatin-based HIPEC during 30 minutes. We investigated the impact of treatment temperature and duration as relevant HIPEC parameters for these two chemotherapeutic agents in representative preclinical models. The temperature- and duration- dependent efficacy for both oxaliplatin and MMC was evaluated in an
setting and in a representative animal model.
In 130 WAG/Rij rats, PM were established through i.p. injections of rat CC-531 colon carcinoma cells with a signature similar to the dominant treatment-resistant CMS4 type human colorectal PM. Tumor growth was monitored twice per week using ultrasound, and HIPEC was applied when most tumors were 4-6 mm. A semi-open four-inflow HIPEC setup was used to circulate oxaliplatin or MMC through the peritoneum for 30, 60 or 90 minutes with inflow temperatures of 38°C or 42°C to achieve temperatures in the peritoneum of 37°C or 41°C. Tumors, healthy tissue and blood were collected directly or 48 hours after treatment to assess the platinum uptake, level of apoptosis and proliferation and to determine the healthy tissue toxicity.
results show a temperature- and duration- dependent efficacy for both oxaliplatin and MMC in both CC-531 cells and organoids. Temperature distribution throughout the peritoneum of the rats was stable with normothermic and hyperthermic average temperatures in the peritoneum ranging from 36.95-37.63°C and 40.51-41.37°C, respectively. Treatments resulted in minimal body weight decrease (<10%) and only 7/130 rats did not reach the endpoint of 48 hours after treatment.
Both elevated temperatures and longer treatment duration resulted in a higher platinum uptake, significantly increased apoptosis and lower proliferation in PM tumor lesions, without enhanced normal tissue toxicity. Our results demonstrated that oxaliplatin- and MMC-based HIPEC procedures are both temperature- and duration-dependent in an
tumor model.
Cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment with curative intent for peritoneal metastasis of colorectal cancer (CRC). Currently, there is ...no standardized HIPEC protocol: choice of drug, perfusate temperature, and duration of treatment vary per institute. We investigated the temperature-dependent effectiveness of drugs often used in HIPEC.
The effect of temperature on drug uptake, DNA damage, apoptosis, cell cycle distribution, and cell growth were assessed using the temperature-dependent IC50 and Thermal Enhancement Ratio (TER) values of the chemotherapeutic drugs cisplatin, oxaliplatin, carboplatin, mitomycin-C (MMC), and 5-fluorouracil (5-FU) on 2D and 3D CRC cell cultures at clinically relevant hyperthermic conditions (38-43 °C/60 min).
Hyperthermia alone decreased cell viability and clonogenicity of all cell lines. Treatment with platinum-based drugs and MMC resulted in G2-arrest. Platinum-based drugs display a temperature-dependent synergy with heat, with increased drug uptake, DNA damage, and apoptosis at elevated temperatures. Apoptotic levels increased after treatment with MMC or 5-FU, without a synergy with heat.
Our in vitro results demonstrate that a 60-min exposure of platinum-based drugs and MMC are effective in treating 2D and 3D CRC cell cultures, where platinum-based drugs require hyperthermia (>41 °C) to augment effectivity, suggesting that they are, in principle, suitable for HIPEC.
Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore ...undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi 1 μM to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.
Hyperthermic intraperitoneal chemotherapy (HIPEC) after cytoreductive surgery (CRS) is used for treating peritoneal metastases of various origins. Present HIPEC protocols have rarely been validated ...for relevant parameters such as optimal agent, duration and perfusate temperature. In vitro experiments are not completely representative of clinical circumstances. Therefore, a good preclinical in vivo HIPEC model is needed in which temperature distributions can be well-controlled and are stable throughout treatments.
We designed a setup able to generate and maintain a homogeneous flow during a 90-min HIPEC procedure using our in-house developed treatment planning tools and computer aided design (CAD) techniques. Twelve rats were treated with heated phosphate-buffered saline (PBS) using two catheter setups (one vs. four- inflows) and extensive thermometry. Simulated and measured thermal distribution and core temperatures were evaluated for the different setups.
Overall, the four-inflow resulted in more stable and more homogeneous thermal distributions than the one-inflow, with lower standard deviations (0.79 °C vs. 1.41 °C at the outflow, respectively) and less thermal losses. The average thermal loss was 0.4 °C lower for rats treated with the four-inflow setup. Rat core temperatures were kept stable using occasional tail cooling, and rarely exceeded 39 °C.
Increasing the number of inflow catheters from one to four resulted in increased flow and temperature homogeneity and stability. Tail cooling is an adequate technique to prevent rats from overheating during 90-min treatments. This validated design can improve accuracy in future in vivo experiments investigating the impact of relevant parameters on the efficacy of different HIPEC protocols.
Severe combined immunodeficient mice are typically used for xenografting experiments and show reliable tumor engraftment; however, their Prkdscid mutation renders them highly sensitive to ...irradiation. Here, we describe a protocol that allows safe local irradiation of tumor xenografts in immunodeficient mice. We detail the steps for the establishment and handling of patient-derived cancer cultures, subcutaneous injection of cancer cells on the mouse hind limb, localized irradiation in mice, tumor monitoring, and tumor characterization via histological and immunohistochemical assessment.
For complete details on the use and execution of this protocol, please refer to Dings et al. (2022).1
Display omitted
•Protocol for local irradiation of tumor xenografts in SCID mice•Steps for dissociation of patient-derived tissues and establishment of cancer cultures•Subcutaneous injection of patient-derived cells on mouse hind limb and tumor monitoring•Can be widely used for safe irradiation of tumors in immunodeficient mice
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Severe combined immunodeficient mice are typically used for xenografting experiments and show reliable tumor engraftment; however, their Prkdscid mutation renders them highly sensitive to irradiation. Here, we describe a protocol that allows safe local irradiation of tumor xenografts in immunodeficient mice. We detail the steps for the establishment and handling of patient-derived cancer cultures, subcutaneous injection of cancer cells on the mouse hind limb, localized irradiation in mice, tumor monitoring, and tumor characterization via histological and immunohistochemical assessment.
► We examined hat mild hyperthermia inhibited homologous recombination repair. ► We examined translocation formation after hyperthermia. ► Inhibition of HR increased chromosomal translocation ...formation. ► Induction of chromosomal translocation is decreased by inhibition of NHEJ.
In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41°C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).
It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.
Poly-(ADP-ribose)-polymerase1 (PARP1) is involved in repair of DNA single strand breaks. PARP1-inhibitors (PARP1-i) cause an accumulation of DNA double strand breaks, which are generally repaired by ...homologous recombination (HR). Therefore, cancer cells harboring HR deficiencies are exceptionally sensitive to PARP1-i. For patients with HR-proficient tumors, HR can be temporarily inhibited by hyperthermia, thereby inducing synthetic lethal conditions in every tumor type. Since cisplatin is successfully used combined with hyperthermia (thermochemotherapy), we investigated the effectiveness of combining PARP1-i with thermochemotherapy.
The in vitro data demonstrate a decreased in cell survival after addition of PARP1-i to thermochemotherapy, which can be explained by increased DNA damage induction and less DSB repair. These in vitro findings are in line with in vivo model, in which a decreased tumor growth is observed upon addition of PARP1-i.
Survival of three HR-proficient cell lines after cisplatin, hyperthermia and/or PARP1-i was studied. Cell cycle analyses, quantification of γ-H2AX foci and apoptotic assays were performed to understand these survival data. The effects of treatments were further evaluated by monitoring tumor responses in an in vivo rat model.
Our results in HR-proficient cell lines suggest that PARP1-i combined with thermochemotherapy can be a promising clinical approach for all tumors independent of HR status.
Hyperthermia can transiently degrade BRCA2 and thereby inhibit the homologous recombination pathway. Induced DNA-double strand breaks (DSB) then have to be repaired via the error prone non-homologous ...end-joining pathway. In the present study, to investigate the role of hyperthermia in genotoxicity and radiosensitization, the induction of chromosomal aberrations was examined by premature chromosome condensation and fluorescence in situ hybridisation (PCC-FISH), and cell survival was determined by clonogenic assay shortly (0-1 h) and 24 h following exposure to hyperthermia in combination with ionizing radiation. Prior to exposure to 4 Gy γ-irradiation, confluent cultures of SW‑1573 (human lung carcinoma) and RKO (human colorectal carcinoma) cells were exposed to mild hyperthermia (1 h, 41˚C). At 1 h, the frequency of chromosomal translocations was higher following combined exposure than following exposure to irradiation alone. At 24 h, the number of translocations following combined exposure was lower than following exposure to irradiation only, and was also lower than at 1 h following combined exposure. These dynamics in translocation frequency can be explained by the hyperthermia-induced transient reduction of BRCA2 observed in both cell lines. In both cell lines exposed to radiation only, potentially lethal damage repair (PLDR) correlated with a decreased number of chromosomal fragments at 24 h compared to 1 h. With combined exposure, PLDR did not correlate with a decrease in fragments, as in the RKO cells at 24 h following combined exposure, the frequency of fragments remained at the level found after 1 h of exposure and was also significantly higher than that found following exposure to radiation alone. This was not observed in the SW‑1573 cells. Cell survival experiments demonstrated that exposure to hyperthermia radiosensitized the RKO cells, but not the SW‑1573 cells. This radiosensitization was at least partly due to the induction of apoptosis, which was only observed in the RKO cells and which may have been induced by BRCA2 degradation or different types of chromosomal aberrations. An important observation of this study is that the genotoxic effect of hyperthermia shortly after combined epxosure (to hyperthermia and radiation) is not observed at 24 h after treatment.
Background: The peritoneum is a common site for the formation of metastases originating from several gastrointestinal and gynecological malignancies. A representative preclinical model to thoroughly ...explore the pathophysiological mechanisms and to study new treatment strategies is important. A major challenge for such models is defining and quantifying the (total) tumor burden in the peritoneal cavity prior to treatment, since it is preferable to use non-invasive methods. We evaluated ultrasound as a simple and easy-to-handle imaging method for this purpose. Methods: Peritoneal metastases were established in six WAG/Rij rats through i.p. injections of the colon carcinoma cell line CC-531. Using ultrasound, the location, number and size of intraperitoneal tumor nodules were determined by two independent observers. Tumor outgrowth was followed using ultrasound until the peritoneal cancer index (PCI) was ≥8. Interobserver variability and ex vivo correlation were assessed. Results: Visible peritoneal tumor nodules were formed in six WAG/Rij rats within 2–4 weeks after cell injection. In most animals, tumor nodules reached a size of 4–6 mm within 3–4 weeks, with total PCI scores ranging from 10–20. The predicted PCI scores using ultrasound ranged from 11–19 and from 8–18, for observer 1 and 2, respectively, which was quite similar to the ex vivo scores. Conclusions: Ultrasound is a reliable non-invasive method to detect intraperitoneal tumor nodules and quantify tumor outgrowth in a rat model.
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