Demonstration of treatment planning software for hyperthermic intraperitoneal chemotherapy in a rat model.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is administered to treat residual microscopic disease after cytoreductive surgery (CRS). During HIPEC, fluid (41-43 °C) is administered and drained through a limited number of catheters, risking thermal and drug heterogeneities within the abdominal cavity that might reduce effectiveness. Treatment planning software provides a unique tool for optimizing treatment delivery. This study aimed to investigate the influence of treatment-specific parameters on the thermal and drug homogeneity in the peritoneal cavity in a computed tomography based rat model. METHOD: We developed computational fluid dynamics (CFD) software simulating the dynamic flow, temperature and drug distribution during oxaliplatin based HIPEC. The influence of location and number of catheters, flow alternations and flow rates on peritoneal temperature and drug distribution were determined. The software was validated using data from experimental rat HIPEC studies. RESULTS: The predicted core temperature and systemic oxaliplatin concentration were comparable to the values found in literature. Adequate placement of catheters, additional inflow catheters and higher flow rates reduced intraperitoneal temperature spatial variation by -1.4 °C, -2.3 °C and -1.2 °C, respectively. Flow alternations resulted in higher temperatures (up to +1.5 °C) over the peritoneal surface. Higher flow rates also reduced the spatial variation of chemotherapy concentration over the peritoneal surface resulting in a more homogeneous effective treatment dose. CONCLUSION: The presented treatment planning software provides unique insights in the dynamics during HIPEC, which enables optimization of treatment-specific parameters and provides an excellent basis for HIPEC treatment planning in human applications.

HyCHEED System for Maintaining Stable Temperature Control during Preclinical Irreversible Electroporation Experiments at Clinically Relevant Temperature and Pulse Settings.

Electric permeabilization of cell membranes is the main mechanism of irreversible electroporation (IRE), an ablation technique for treatment of unresectable cancers, but the pulses also induce a significant temperature increase in the treated volume. To investigate the therapeutically thermal contribution, a preclinical setup is required to apply IRE at desired temperatures while maintaining stable temperatures. This study's aim was to develop and test an electroporation device capable of maintaining a pre-specified stable and spatially homogeneous temperatures and electric field in a tumor cell suspension for several clinical-IRE-settings. A hydraulically controllable heat exchange electroporation device (HyCHEED) was developed and validated at 37 °C and 46 °C. Through plate electrodes, HyCHEED achieved both a homogeneous electric field and homogenous-stable temperatures; IRE heat was removed through hydraulic cooling. IRE was applied to 300 µL of pancreatic carcinoma cell suspension (Mia PaCa-2), after which cell viability and specific conductivity were determined. HyCHEED maintained stable temperatures within ±1.5 °C with respect to the target temperature for multiple IRE-settings at the selected temperature levels. An increase of cell death and specific conductivity, including post-treatment, was found to depend on electric-field strength and temperature. HyCHEED is capable of maintaining stable temperatures during IRE-experiments. This provides an excellent basis to assess the contribution of thermal effects to IRE and other bio-electromagnetic techniques.

Capturing colorectal cancer inter-tumor heterogeneity in patient-derived xenograft (PDX) models.

Patient-derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.

Enhancement of Radiation Effectiveness in Cervical Cancer Cells by Combining Ionizing Radiation with Hyperthermia and Molecular Targeting Agents.

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-i; DNA-dependent protein kinase catalytic subunit inhibitor, DNA-PKcs-i and Heat Shock Protein 90 inhibitor, HSP90-i) in cervical cancer cell lines. Survival curves of SiHa and HeLa cells, concerning the combined effects of radiation with hyperthermia and PARP1-i, DNA-PKcs-i or HSP90-i, were analyzed using the linear-quadratic model: S(D)/S(0) = exp - (αD + ßD²). The values of the linear-quadratic (LQ) parameters α and ß, 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-i and DNA-PKcs-i significantly increased the value of the linear parameter α. Both α and ß were significantly increased for HSP90-i 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-i and PARP1-i, 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.

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells.

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+ßD(2)). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a (137)Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition.

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

Radiosensitization by Hyperthermia Critically Depends on the Time Interval.

PURPOSE: Hyperthermia is a potent sensitizer of radiation therapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiation therapy are still under debate. METHODS AND MATERIALS: We investigated the interval and sequence in vitro in cervical cancer cell lines, patient-derived organoids, and SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. RESULTS: All 3 approaches confirmed that shortening the interval between hyperthermia and radiation therapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis and 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥80 minutes) to 54% (interval <80 minutes). In vitro and in vivo results showed that the sequence of hyperthermia and radiation therapy did not affect the outcome. CONCLUSIONS: Shortening the interval between hyperthermia and radiation therapy significantly improves treatment outcomes. The sequence of hyperthermia and radiation therapy (before or after) does not seem to matter.

Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-cell lymphomas.

APRIL (A proliferation-inducing ligand) is a TNF family member that binds two TNF receptor family members, TACI and BCMA. It shares these receptors with the closely related TNF family member, B-cell activating factor (BAFF). Contrary to BAFF, APRIL binds heparan sulfate proteoglycans (HSPGs), which regulates cross-linking of APRIL and efficient signaling. APRIL was originally identified as a growth promoter of solid tumors, and more recent evidence defines APRIL also as an important survival factor in several human B-cell malignancies, such as chronic lymphocytic leukemia (CLL). To target APRIL therapeutically, we developed two anti-human APRIL antibodies (hAPRIL.01A and hAPRIL.03A) that block APRIL binding to BCMA and TACI. Their antagonistic properties are unique when compared with a series of commercially available monoclonal anti-human APRIL antibodies as they prevent in vitro proliferation and IgA production of APRIL-reactive B cells. In addition, they effectively impair the CLL-like phenotype of aging APRIL transgenic mice and, more importantly, block APRIL binding to human B-cell lymphomas and prevent the survival effect induced by APRIL. We therefore conclude that these antibodies have potential for further development as therapeutics to target APRIL-dependent survival in B-cell malignancies.

Monoclonal antibodies against Lgr5 identify human colorectal cancer stem cells.

In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+ Lgr5+ cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5(high) CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.

Local irradiation of patient-derived tumors in immunodeficient mice.

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.

Elevated temperatures and longer durations improve the efficacy of oxaliplatin- and mitomycin C-based hyperthermic intraperitoneal chemotherapy in a confirmed rat model for peritoneal metastasis of colorectal cancer origin.

Introduction: 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 in vitro setting and in a representative animal model. Methods: 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: In vitro 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. Conclusions: 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 in vivo tumor model.

Targeting colorectal cancer stem cells with inducible caspase-9.

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133(+) CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.

Non-Invasive Imaging and Scoring of Peritoneal Metastases in Small Preclinical Animal Models Using Ultrasound: A Preliminary Trial.

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.

Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.

PARP1-Inhibition Sensitizes Cervical Cancer Cell Lines for Chemoradiation and Thermoradiation.

Radiotherapy plus cisplatin (chemoradiation) is standard treatment for women with locoregionally advanced cervical cancer. Both radiotherapy and cisplatin induce DNA single and double-strand breaks (SSBs and DSBs). These double-strand breaks can be repaired via two major DNA repair pathways: Classical Non-Homologous End-Joining (cNHEJ) and Homologous Recombination. Besides inducing DNA breaks, cisplatin also disrupts the cNHEJ pathway. Patients contra-indicated for cisplatin are treated with radiotherapy plus hyperthermia (thermoradiation). Hyperthermia inhibits the HR pathway. The aim of our study is to enhance chemoradiation or thermoradiation by adding PARP1-inhibition, which disrupts both the SSB repair and the Alternative NHEJ DSB repair pathway. This was studied in cervical cancer cell lines (SiHa, HeLa, C33A and CaSki) treated with hyperthermia (42 °C) ± ionizing radiation (2-6 Gy) ± cisplatin (0.3-0.5 µM) ± PARP1-inhibitor (olaparib, 4.0-5.0 µM). Clonogenic assays were performed to measure cell reproductive death. DSBs were analyzed by γ-H2AX staining and cell death by live cell imaging. Both chemoradiation and thermoradiation resulted in lower survival fractions and increased unrepaired DSBs when combined with a PARP1-inhibitor. A quadruple modality, including ionizing radiation, hyperthermia, cisplatin and PARP1-i, was not more effective than either triple modality. However, both chemoradiation and thermoradiation benefit significantly from additional treatment with PARP1-i.

Pre-Operative Decitabine in Colon Cancer Patients: Analyses on WNT Target Methylation and Expression.

DNA hypermethylation is common in colon cancer. Previously, we have shown that methylation of WNT target genes predicts poor prognosis in stage II colon cancer. The primary objective of this study was to assess whether pre-operative treatment with decitabine can decrease methylation and increase the expression of WNT target genes APCDD1, AXIN2 and DKK1 in colon cancer patients. A clinical study was conducted, investigating these potential effects of decitabine in colon cancer patients (DECO). Patients were treated two times with 25 mg/m2 decitabine before surgery. Methylation and expression of LINE1 and WNT target genes (primary outcome) and expression of endogenous retroviral genes (secondary outcome) were analysed in pre- and post-treatment tumour samples using pyrosequencing and rt-PCR. Ten patients were treated with decitabine and eighteen patients were used as controls. Decitabine treatment only marginally decreased LINE1 methylation. More importantly, no differences in methylation or expression of WNT target or endogenous retroviral genes were observed. Due to the lack of an effect on primary and secondary outcomes, the study was prematurely closed. In conclusion, pre-operative treatment with decitabine is safe, but with the current dosing, the primary objective, increased WNT target gene expression, cannot be achieved.

A Four-Inflow Construction to Ensure Thermal Stability and Uniformity during Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in Rats.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Radiosensitization by Hyperthermia: The Effects of Temperature, Sequence, and Time Interval in Cervical Cell Lines.

Cervical cancers are almost exclusively caused by an infection with the human papillomavirus (HPV). When patients suffering from cervical cancer have contraindications for chemoradiotherapy, radiotherapy combined with hyperthermia is a good treatment option. Radiation-induced DNA breaks can be repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Hyperthermia can temporarily inactivate homologous recombination. Therefore, combining radiotherapy with hyperthermia can result in the persistence of more fatal radiation-induced DNA breaks. However, there is no consensus on the optimal sequence of radiotherapy and hyperthermia and the optimal time interval between these modalities. Moreover, the temperature of hyperthermia and HPV-type may also be important in radiosensitization by hyperthermia. In this study we thoroughly investigated the impact of different temperatures (37-42 °C), and the sequence of and time interval (0 up to 4 h) between ionizing radiation and hyperthermia on HPV16+: SiHa, Caski; HPV18+: HeLa, C4I; and HPV-: C33A, HT3 cervical cancer cell lines. Our results demonstrate that a short time interval between treatments caused more unrepaired DNA damages and more cell kill, especially at higher temperatures. Although hyperthermia before ionizing radiation may result in slightly more DNA damage, the sequence between hyperthermia and ionizing radiation yielded similar effects on cell survival.

The Temperature-Dependent Effectiveness of Platinum-Based Drugs Mitomycin-C and 5-FU during Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in Colorectal Cancer Cell Lines.

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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Cyclopentenyl cytosine has biological and anti-tumour activity, but does not enhance the efficacy of gemcitabine and radiation in two animal tumour models.

Cyclopentenyl cytosine (CPEC), targetting the de novo biosynthesis of cytidine triphosphate (CTP), increases the cytotoxicity of gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) alone and in combination with irradiation in several human tumour cells in vitro. We investigated whether CPEC enhances the therapeutic ratio of gemcitabine and irradiation in human pancreatic BxPC-3 xenografts and in rat syngeneic L44 lung tumours. These models were selected because gemcitabine and radiation are used to treat both pancreatic and lung cancer patients and both models differ in growth capacity and in gemcitabine-induced radiosensitisation. A profound dose-dependent CTP-depletion was observed after a single injection of CPEC in both tumour tissue and in normal jejunum. In both models, CPEC alone induced a slight but significant tumour growth delay. The combination of CPEC with gemcitabine, at time intervals that showed CTP-depletion after CPEC, enhanced neither tumour growth delay nor toxicity as compared to gemcitabine alone. In addition, no beneficial effect of CPEC was observed in combination with gemcitabine and radiation. These results suggest that CPEC and gemcitabine alone as well as in combination with radiation target a similar cell population in both tumour models. In conclusion, future clinical development of CPEC as a modulator of gemcitabine combined with radiation is unlikely.