Roxan F. C. P. A. Helderman

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.

Hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment modality for patients with peritoneal metastasis (PM) of various origins which aims for cure in combination with cytoreductive surgery (CRS). Efficacy of CRS-HIPEC depends on patient selection, tumor type, delivery technique, and treatment parameters such as temperature, carrier solution, type of drug, dosage, volume, and treatment duration. Preclinical research offers a powerful tool to investigate the impact of these parameters and to assist in designing potentially more effective treatment protocols and clinical trials. The different methodologies for peritoneal disease and HIPEC are variable. This study aims to review the objectives, methods, and clinical relevance of in vivo preclinical HIPEC studies found in the literature. In this review, recommendations are provided and possible pitfalls are discussed on the choice of type of animal and tumor model per stratified parameters and study goal. The guidelines presented in this paper can improve the clinical relevance and impact of future in vivo HIPEC experiments.

Hyperthermic intraperitoneal chemotherapy (HIPEC) is administered to treat residual microscopic disease after debulking cytoreductive surgery. During HIPEC, a limited number of catheters are used to administer and drain fluid containing chemotherapy (41–43 °C), yielding heterogeneities in the peritoneum. Large heterogeneities may lead to undertreated areas, increasing the risk of recurrences. Aiming at intra-abdominal homogeneity is therefore essential to fully exploit the potential of HIPEC. More insight is needed into the extent of the heterogeneities during treatments and assess their effects on the efficacy of HIPEC. To that end we developed a computational model containing embedded tumor nodules in an environment mimicking peritoneal conditions. Tumor- and treatment-specific parameters affecting drug delivery like tumor size, tumor shape, velocity, temperature and dose were assessed using three-dimensional computational fluid dynamics (CFD) to demonstrate their effect on the drug distribution and accumulation in nodules. Clonogenic assays performed on RKO colorectal cell lines yielded the temperature-dependent IC50 values of cisplatin (19.5–6.8 micromolar for 37–43 °C), used to compare drug distributions in our computational models. Our models underlined that large nodules are more difficult to treat and that temperature and velocity are the most important factors to control the drug delivery. Moderate flow velocities, between 0.01 and 1 m/s, are optimal for the delivery of cisplatin. Furthermore, higher temperatures and higher doses increased the effective penetration depth with 69% and 54%, respectively. We plan to extend the software developed for this study toward patient-specific treatment planning software, capable of mapping and assist in reducing heterogeneous flow patterns.

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...

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.

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.

Peritoneal metastasis (PM) originating from gastrointestinal and gynecological malignancies are associated with a poor prognosis and rapid disease progression. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) is an effective treatment option with curative intent. Hyperthermia enhances the cytotoxicity of chemotherapeutic drugs, thereby killing microscopic tumors and reducing the risk of tumor recurrence. Eight parameters potentially have an impact on the efficacy of HIPEC: the type of drug, drug concentrations, carrier solution, volume of the perfusate, temperature of the perfusate, duration of the treatment, the technique of delivery, and patient selection. In this review, a literature search was performed on PubMed, and a total of 564 articles were screened of which 168 articles were included. Although HIPEC is a successful treatment, there is no standardized method for delivering HIPEC: the choice of parameters is presently largely determined by institutional preferences. We discuss the current choice of the parameters and hypothesize about improvements toward uniform standardization. Quantifying the effect of each parameter separately is necessary to determine the optimal way to perform HIPEC procedures. In vivo, in vitro, in silico, and other experimental studies should shed light on the role of each of the eight parameters.

Purpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. Experimental Design: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. Results: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. Conclusions: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations. See related commentary by Becker and LeBleu, p. 6110.