Hypoxic Jumbo Spheroids On-A-Chip (HOnAChip): Insights into Treatment Efficacy.

Hypoxia is a key characteristic of the tumor microenvironment, too rarely considered during drug development due to the lack of a user-friendly method to culture naturally hypoxic 3D tumor models. In this study, we used soft lithography to engineer a microfluidic platform allowing the culture of up to 240 naturally hypoxic tumor spheroids within an 80 mm by 82.5 mm chip. These jumbo spheroids on a chip are the largest to date (>750 µm), and express gold-standard hypoxic protein CAIX at their core only, a feature absent from smaller spheroids of the same cell lines. Using histopathology, we investigated response to combined radiotherapy (RT) and hypoxic prodrug Tirapazamine (TPZ) on our jumbo spheroids produced using two sarcoma cell lines (STS117 and SK-LMS-1). Our results demonstrate that TPZ preferentially targets the hypoxic core (STS117: p = 0.0009; SK-LMS-1: p = 0.0038), but the spheroids' hypoxic core harbored as much DNA damage 24 h after irradiation as normoxic spheroid cells. These results validate our microfluidic device and jumbo spheroids as potent fundamental and pre-clinical tools for the study of hypoxia and its effects on treatment response.

Synergy between Non-Thermal Plasma with Radiation Therapy and Olaparib in a Panel of Breast Cancer Cell Lines.

Cancer therapy has evolved to a more targeted approach and often involves drug combinations to achieve better response rates. Non-thermal plasma (NTP), a technology rapidly expanding its application in the medical field, is a near room temperature ionized gas capable of producing reactive species, and can induce cancer cell death both in vitro and in vivo. Here, we used proliferation assay to characterize the plasma sensitivity of fourteen breast cancer cell lines. These assays showed that all tested cell lines were sensitive to NTP. In addition, a good correlation was found comparing cell sensitivity to NTP and radiation therapy (RT), where cells that were sensitive to RT were also sensitive to plasma. Moreover, in some breast cancer cell lines, NTP and RT have a synergistic effect. Adding a dose of PARP-inhibitor olaparib to NTP treatment always increases the efficacy of the treatment. Olaparib also exhibits a synergistic effect with NTP, especially in triple negative breast cancer cells. Results presented here help elucidate the position of plasma use as a potential breast cancer treatment.

Heat shock induces the cellular antioxidant defenses peroxiredoxin, glutathione and glucose 6-phosphate dehydrogenase through Nrf2.

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and/or chemotherapy. Heat (42-45 °C) can kill cancer cells. Low doses of heat at milder temperatures (39-41 °C) induce thermotolerance, an adaptive survival response that upregulates defense molecules to protect cells against subsequent exposure to toxic stress. Although hyperthermia has proven effective in clinical trials, there is still much to learn about its cellular mechanisms. This study aims to understand the role of reactive oxygen species (ROS), antioxidants and the antioxidant transcription factor Nrf2 in cellular stress responses to mild and lethal heat shock. Mild thermotolerance (40 °C) and hyperthermia (42-43 °C) caused increased expression of the antioxidants peroxiredoxin-3 (Prx3) and Prx2, and its hyperoxidized form Prx-SO3. Cellular levels of superoxide and peroxides increased at 40 °C and 42 °C. Heat shock (42 °C)-induced increases in Prx3 and Prx-SO3 were inhibited by antioxidants (PEG-catalase, MnTBAP) and a Nrf2 shRNA. Glucose metabolism by the pentose phosphate pathway produces NADPH, which maintains the antioxidant glutathione in its reduced form, GSH. Heat shock (40°C-42 °C) increased GSH levels, expression of glucose transporter GLUT1, and enzymatic activity and expression of glucose 6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose cycle. Heat-induced increases in GSH levels and G6PD expression were inhibited by antioxidants and Nrf2 knockdown. These results suggest that heat shock-generated ROS were involved in induction of cellular defense molecules Prxs, GSH and G6PD through Nrf2 activation. Our study sheds new light on the role of Nrf2 and antioxidants in cellular responses to heat shock at mild and lethal temperatures.

On-chip combined radiotherapy and chemotherapy testing on soft-tissue sarcoma spheroids to study cell death using flow cytometry and clonogenic assay.

Radiotherapy (RT) and chemotherapy (CT) are the major therapeutics to treat cancer patients. Conventional in vitro 2D models are insufficient to study the combined effects of RT and CT towards optimized dose selection or drug screening. Soft-tissue sarcomas (STS) are rare cancers with profound social impacts as they affect patients of all ages. We developed a microfluidic device to form and culture STS spheroids to study the combined cytotoxicities of RT and CT. Uniformly-sized spheroids of two different cell lines, STS 93 and STS 117, were formed in the device. RT doses of 0.5 Gy, 2 Gy, and 8 Gy were used in combination with CT, doxorubicin at 2 µM and 20 µM. The spheroids culture chambers within the device were arranged in a 3 × 5 matrix form. The device was made "peelable", which enabled us to collect spheroids from each treatment condition separately. Collected spheroids were dissociated into single cells and evaluated using flow cytometry and clonogenic assays. Through this workflow, we observed that STS 93 spheroids treated with doxorubicin die through apoptosis, whereas RT induced death through other pathways. Spheroids from the p53 mutant STS 117 cell line were more resistant to RT and doxorubicin. The developed device could be used for the discovery of new drugs and RT synergies.

The antioxidant transcription factor Nrf2 contributes to the protective effect of mild thermotolerance (40°C) against heat shock-induced apoptosis.

The exposure of cells to low doses of stress induces adaptive survival responses that protect cells against subsequent exposure to toxic stress. The ability of cells to resist subsequent toxic stress following exposure to low dose heat stress at 40°C is known as mild thermotolerance. Mild thermotolerance involves increased expression of heat shock proteins and antioxidants, but the initiating factors in this response are not understood. This study aims to understand the role of the Nrf2 antioxidant pathway in acquisition of mild thermotolerance at 40°C, and secondly, whether the Nrf2 pathway could be involved in the protective effect of thermotolerance against heat-shock (42°C)-induced apoptosis. During cell preconditioning at 40°C, protein expression of the Nrf2 transcription factor increased after 15-60min. In addition, levels of the Nrf2 targets MnSOD, catalase, heme oxygenase-1, glutamate cysteine ligase and Hsp70 increased at 40°C. Levels of these Nrf2 targets were enhanced by Nrf2 activator oltipraz and decreased by shRNA targeting Nrf2. Levels of pro-oxidants increased after 30-60min at 40°C. Pro-oxidant levels were decreased by oltipraz and increased by knockdown of Nrf2. Increased Nrf2 expression and catalase activity at 40°C were inhibited by the antioxidant PEG-catalase and by p53 inhibitor pifithrin-α. These results suggest that mild thermotolerance (40°C) increases cellular pro-oxidant levels, which in turn activate Nrf2 and its target genes. Moreover, Nrf2 contributes to the protective effect of thermotolerance against heat-shock (42°C)-induced apoptosis, because Nrf2 activation by oltipraz enhanced thermotolerance, whereas Nrf2 knockdown partly reversed thermotolerance. Improved knowledge about the different protective mechanisms that mild thermotolerance can activate is crucial for the potential use of this adaptive survival response to treat stress-related diseases.

Mild thermotolerance induced at 40 °C protects cells against hyperthermia-induced pro-apoptotic changes in Bcl-2 family proteins.

PURPOSE: Despite clinical progress, mechanisms involved in cellular responses to low and high doses of hyperthermia are not entirely clear. This study investigates the role of Bcl-2 family proteins in control of the mitochondrial pathway of apoptosis during hyperthermia at 42-43 °C and the protective effect of a low dose adaptive survival response, mild thermotolerance induced at 40 °C. MATERIALS AND METHODS: Levels of Bcl-2 family proteins were detected in HeLa cells by western blotting, caspase activation by spectrofluorimetry and apoptosis by chromatin condensation. RESULTS: Hyperthermia (42-43 °C) decreased total and mitochondrial expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, while expression of pro-apoptotic proteins Bax, Bak, Puma and Noxa increased. Hyperthermia perturbed the equilibrium between these anti- and pro-apoptotic Bcl-2 family proteins in favour of pro-apoptotic conditions. Hyperthermia also caused activation of caspases-9 and -3, and chromatin condensation. Disruption of the balance between Bcl-2 family proteins was reversed in thermotolerant (40 °C) cells, thus favouring cell survival. Bcl-2/Bcl-xL inhibitor ABT-737 sensitised cells to apoptosis, which indicates that Bcl-2 family proteins play a role in hyperthermia-induced apoptosis. The adaptive response of mild thermotolerance (40 °C) was still able to protect cells against hyperthermia (42-43 °C) when Bcl-2/Bcl-xL were inhibited. CONCLUSIONS: These results improve knowledge about the role of Bcl-2 family proteins in cellular apoptotic responses to hyperthermia (42-43 °C), as well as the adaptive survival response induced by exposure to mild stresses, such as a fever temperature (40 °C). This study could provide rationale to explore the manipulation of Bcl-2 family proteins for increasing tumour sensitivity to hyperthermia.