Assessment of an Integrative Anticancer Treatment Using an in Vitro

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Assessment of an integrative anti-cancer treatment using an in vitro perfusion-enabled 3D breast tumor model Vaishnavi Kulkarni, Dhananjay Bodas, and Kishore Paknikar ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00153 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ACS Biomaterials Science & Engineering

Assessment of an integrative anti-cancer treatment using an in vitro perfusion-enabled 3D breast tumor model Vaishnavi Kulkarni, Dhananjay Bodas and Kishore Paknikar Nanobioscience group, Agharkar Research Institute, GG Agarkar road, Pune 411 004 India Tel: +9120-25325000; E-mail: [email protected], [email protected].

KEY WORDS: LSMO nanoparticles, perfused 3D cell culture, tumor-on-chip, magnetic fluid hyperthermia, combination treatment, porous scaffolds, in vitro tumor.

ABSTRACT The study presents observations on anti-cancer therapeutic efficacy of magnetic fluid hyperthermia and a combination of hyperthermia-chemotherapy (i.e. integrative treatment) using an

in

vitro -perfused

and

-non-perfused

3D

breast tumor model.

The

3D in

vitro breast tumor models were simulated using Comsol Multiphysics and fabricated using specially designed chips and treated with doxorubicin loaded chitosan coated LSMO (DCLSMO) nanoparticles for hyperthermia and combination therapy in both, perfused and nonperfused conditions. Computation confirmed uniform heat distribution throughout the scaffold for both the models. The findings indicate that both hyperthermia and combination treatment could trigger apoptotic cell death in the perfused and non-perfused models in varying degrees.

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Specifically, the perfused tumors were more resistant to therapy than the non-perfused ones. The efficacy of anti-cancer treatment decreased with increasing physiological complexity of the tumor model. The combination (hyperthermia and chemotherapy) treatment showed enhanced efficacy over hyperthermia alone. This is a pilot study to investigate the effects of magnetic fluid hyperthermia-chemotherapy treatment using perfused and non-perfused 3D in vitro models of tumor. The feasibility of using 3D cell culture models for contributing to our understanding cancer and its treatment was also determined as a part of this work. INTRODUCTION The use of nanotechnology in cancer treatment offers exciting possibilities such as destroying cancer tumors with minimal damage to healthy tissue and organs e.g. a targeted magnetic fluid hyperthermia (MFH) and chemotherapy treatment. Animal experiments, especially in mice, play a key role continue to be at the forefront of such cancer research. However, comprehensive research can be conducted using the 3D cell culture models before moving to expensive and time-consuming animal models 1. Addition of 3D cancer models for preclinical screening can speed-up discovery and save money by providing more physiologically relevant information; and generating more predictive data for in vivo tests. The 3D cell cultures are complex to develop, and an even challenging-to-model feature is angiogenesis (perfusion) such that it can reflect human-tumor biology as closely as possible and contribute to its long-term survival. For instance, in case of research on an integrated therapy like hyperthermia-chemotherapy, perfusion in tumor can significantly affect the heat dissipation and drug distribution 2. In our previous study we established the pronounced anti-cancer effect of doxorubicin loaded chitosan coated La0.7Sr0.3MnO3 nanoparticles (DC-LSMO NPs) in breast cancer treatment on cell monolayers in our previous studies 3. Now considering the above-discussed factors, it is


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imperative that perfusion-induced variation in treatment efficacy should be investigated at the actual tumor level. But it is difficult to control the tumor generation and vasculature in vivo, and it is equally difficult to monitor the post-treatment changes in real time in animals. 3D cell culture models can be more realistic and controllable than human cancer animal xenografts because the tumor cells are generally transplanted into sites that are convenient for tumor observation but do not reflect the microenvironment of the original tumour. Thus, in continuation to our previous research, we have conducted a pilot study on effects of DC-LSMO NPs using in vitro models of 3D tumor. Structures bearing similar properties like tumors can be reproducibly generated in vitro in 3D for studying and understanding phenomenon like nanoparticle induced MFH and chemotherapy. For instance, during MFH, the therapeutic temperature achieved in the tumor would be a function of the heat generated by magnetic nanoparticles and the dissipation of that heat across the tumor mass 4. Another parameter is the surface to volume ratio, which increases with decreasing size, resulting in superior heat dissipation 5. Even within the tumor structure, the heatinduced damage may vary across the distinct heat zones viz. the cells in the region farthest from the nanoparticles being the least affected. Another decisive factor affecting the temperature distribution is the presence of blood vessels 6. A well-perfused tumor would result in increased heat losses owing to enhanced heat dissipation. Tumor blood vessels contribute to uneven blood flow distribution and hence, perfusion heterogeneity

7

. These variations are also more

pronounced from tumor to tumor. Such physiological and microenvironment based factors can be different for different cells at different locations in the same tumor tissue 8. And all such forms of biological and physical heterogeneity within the tumor microenvironment can lead to failure of treatment. Hence, a requirement of appropriate computational model to derive accurate


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parameters required for treatment. Computation will enable us understand the heat distribution the amount of nanoparticles required for uniform distribution of temperature. The addition of perfusion to the model allows us to understand anomalies in the temperature due to associated flows. Here we have investigated the effects of a combined MFH-chemotherapy treatment using a perfused and non-perfused in vitro 3D breast tumor model. This system consists of perfusionenabled PDMS cell culture chips housed in a specially designed device with controlled conditions of temperature and perfusion. These 3D breast tumor models (referred to as spheroidal tissue structures or STS) were subject to MFH alone, and in combination with chemotherapy. Simulation studies on flow patterns and heating effects were also included in this work as a part of hyperthermia treatment planning. Such MFH treatment planning was considered important since it is rapidly moving towards clinical application. Furthermore, molecular effects of treatment were studies by investigating the generation of heat shock proteins (HSP), reactive oxygen species (ROS), mitochondrial dysfunction, and generation of caspases in treated tumors. MATERIALS AND METHODS Generation of 3D cell culture chips and STSperfused cell culture device The 3D cell culture chip was fabricated by a one-step fabrication of all the components viz. scaffold, media reservoir and connecting microchannels. PDMS base and curing agent (Sylgard 184, Dow Corning, USA) were mixed in the ratio 10:1, poured into the mold and allowed to cure at 60°C for 3 h to produce the chips. Sucrose crystals (~ 400 µm) were used as porogens to introduce pores in the scaffolds (Figure 1 a,b). The cell culture chip was functionalized using fibronectin (Sigma, USA) to aid cell adherence.


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Mold is fabricated in aluminium of accurate dimensions. Figure 1c shows a well with a notch to hold the porogen and copper wire. After the curing process, copper wire is removed to create channel and the porogen is dissolved to generate the scaffold (see Figure 1c).

Figure 1: a) Schematic depicting generation of porosity in PDMS polymer to develop the porous scaffolds; the b) model of the cell culture chip (circular, green and red) along with dimensions and housing (cuboidal, blue), and c) photograph of the mold used for fabrication of the cell culture chip, fabricated chip after removal of porogen (showing a channel on the sides and central porous scaffold) and the functional device; d) Entire set-up of the STSperfused with device, cell culture chips, external pump and temperature controller. The STSperfused cell culture device was fabricated using polycarbonate. It was divided into two separate chambers, a medium reservoir and a culture chip-housing chamber. The scaffold chamber held three cell culture chips at a time, facilitating experimentation in triplicates. The system was designed to run without any supervision and monitoring. The temperature was maintained by using two external heaters set at 37°C using a controller; the heaters were placed both, above and below the STS cell culture device to maintain thermal uniformity (Figure 1 c,d).


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Cell culture Breast cancer cell lines, MCF-7, and MDA-MB-231 were obtained from National Centre for Cell Science, Pune, India. MCF-7 was maintained in supplemented Dulbecco’s modified Eagle’s medium and MDA-MB-231 in Leibovitz’s medium (Invitrogen, USA), respectively. Cells were maintained at 37°C in 5% CO2 atmosphere. 24 h cell culture was used for all the experiments. Synthesis of DC-LSMO nanoparticles LSMO nanoparticles were synthesized by the thermal decomposition method. Chitosan was coated on LSMO nanoparticles in a 1:1 (w/w) ratio using electrostatic interactions. Doxorubicin hydrochloride (Sigma, 15 µg) was added to C-LSMO NPs, 1 mg/mL in phosphate buffered saline, pH 7.4 at 4°C for 2 h with gentle shaking. Doxorubicin loaded C-LSMO nanoparticles (DC-LSMO NPs) were collected by magnetic separation. Qualitative assessment of LSMO, CLSMO and DC-LSMO NPs was performed by Fourier transform infrared spectroscopy (Model Spectrum One, Perkin Elmer Incorporation, USA). DC-LSMO NPs suspensions were subjected to RF at 365 kHz for 3, 5 and 10 min. After exposure, nanoparticles were separated using a magnet, and the drug release in the supernatant was estimated by fluorescence spectrophotometry. Unexposed nanoparticles suspension was used as a control. Percentage release of drug was calculated using the formula given below: % =

( ) ∗ 100 − − − (1) ( )

Anti-cancer treatment using DC-LSMO nanoparticles Uptake of DC-LSMO nanoparticles


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MCF-7 STSnon-perfused cultures (14-days) were treated with DC-LSMO NPs (i.e. exposed to the radio frequency of 365 kHz for 5 min) and incubated at 37°C for 2-4 h. Cells were then washed with phosphate-buffered saline, fixed with 3.7% paraformaldehyde. DAPI (4',6-diamidino-2phenylindole, Invitrogen, USA) and Alexa flour 488 Phalloidin (Invitrogen, USA) were used for nucleus and cytoskeletal actin staining respectively. Lysotracker Blue (Invitrogen, USA), a specific stain for lysosomes was used. Cells were incubated for 2 h for uptake of nanoparticles. Images were acquired using confocal- microscope (Model TCS SP8, Leica, Germany). Studies on cellular responses of breast cancer STSnon-perfused and STSperfused cultures to hyperthermia and combination treatment Hyperthermia studies on STS (perfused and non-perfused) cultures MCF-7 and MDA-MB-231 cells (1 x 106) were seeded on PDMS scaffolds and allowed to grow for 21 days. C-LSMO nanoparticles (1 mg in 50 µL medium) were then introduced in STS and exposed to a radiofrequency of 365 kHz. The STS cultures were treated for 10 min. After 24 h, the cell viability, HSP and caspase generation was evaluated. The error bars represent the SD value (n=3). Combination treatment studies on STS cultures: MCF-7 and MDA-MB-231 cells (1 x 106) were seeded on PDMS scaffolds and allowed to grow for 21 days. DC-LSMO nanoparticles (1 mg in 50 µL medium) were then introduced in STS and exposed to a radiofrequency of 365 kHz. The STS cultures were treated for 10 min. After 24 h, the cells were assessed for viability, HSP and caspase generation. The error bars represent the SD value (n=3). Viability of STS cultures after treatment


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The viability of MCF-7 and MDA-MB-231 as monolayer and STS cultures was estimated after hyperthermia and combination treatment using Prestoblue reagent (Invitrogen, USA). Appropriate controls with untreated cells were also run in parallel. The error bars represent the SD value (n=3). Generation of heat shock proteins After 24 h, the cells were washed with ice-cold PBS and treated with RIPA lysis buffer (Sigma, USA) for 30 min on ice. Cells were centrifuged, and protein content of the supernatant was estimated by Bradford assay. Heat shock proteins were studied by ELISA method. For ELISA assay, 20 µg/mL of cell lysate was coated in microtiter plate wells and incubated at 4°C overnight. After washing and blocking, 100 µL of primary antibody (Sigma, USA) against HSP70 and 90 was added, incubated for 2 h at RT and washed 3 times with PBS. Then, 100 µL of horseradish peroxidase-conjugated secondary antibody (Sigma, USA) in blocking buffer was added, and the plate was incubated for 2 h at RT and washed 3 times with PBS. TMB solution was added to each well and incubated for 15 min. Stop solution (2M H2SO4) was added and the optical density was read at 450 nm (Details are provided in supplementary information). The error bars represent the SD value (n=3). Generation of caspases MCF-7 and MDA-MB-231 STS models were subjected to 10 min of hyperthermia. After 24 h, cells were collected, lysed, and caspase levels were estimated using ApoTarget Caspase colorimetric protease assay kit (Invitrogen, USA). The error bars represent the SD value (n=3). Scanning Electron Microscopy Studies


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STS cultures were fixed with 4% paraformaldehyde and visualized with a desktop SEM (Hitachi TM-1000, USA), after 10 min of MFH treatement. Reactive oxygen species generation MCF-7 STSnon-perfused were subjected to 5 min hyperthermia. After 24 h ROS generation was detected using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) based kit (Invitrogen, USA). The samples were visualized using confocal fluorescence microscope (Model TCS SP8, Leica, Germany). The error bars represent the SD value (n=3). Mitochondrial membrane permeability transition MCF-7 monolayers and STSnon-perfused were subjected to 5 min hyperthermia. After 24 h, the effect of treatment on mitochondrial permeability was determined using a kit-based assay (Image-IT® LIVE Mitochondrial Transition Pore Assay Kit, Invitrogen, USA). The samples were visualized using confocal fluorescence microscope (Model TCS SP8, Leica, Germany). Transmission Electron Microscopy Studies The cells were treated with hyperthermia using 300 µg/mL LSMO for 10 min. After 24 h, the cells were harvested and fixed using 2.5% glutaraldehyde solution. The fixed cells were embedded in an epoxy resin, cut into ultra-thin sections with an ultramicrotome (Leica EM UC 6, Austria), mounted on 150 mesh copper grids and stained (with uranyl acetate and lead citrate). The stained sections were then observed under TEM (JEOL 2100F, USA). Statistical analysis All statistical calculations were performed using Graph Pad Prism software. Analysis of variance (ANOVA) was used to test the statistical significance of difference between control and treated


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groups. Bonferroni’s multiple comparisons test results are expressed as mean values with 95% confidence intervals. P-values less than 0.05 were considered statistically significant. RESULTS AND DISCUSSION The STSnon-perfused and STSperfused: static and dynamic culture In an attempt to bring the studies closer to real in vivo situation, a 3D cell culture model was used for the hyperthermia and hyperthermia-chemotherapy combination study. PDMS was poured on to in-house designed molds (Figure 1c) and allowed to cure at 60°C to obtain the scaffolds. These PDMS scaffolds were spherical; 15 mm in diameter, 3 mm in height, with three distinct zones. The core was 5 mm wide and porous for holding the cells. A negative etching principle was used to create pores in the scaffolds. Sugar crystals were used as a porogen that could be easily dissolved in water. Around the core was a reservoir, 2 mm wide and 3 mm deep, that could hold a volume of ~120 µL. Around the reservoir was a 3 mm wide elevated ridge. An advantage of this design was that all the seeded cells were retained onto the scaffold, as it had restricting PDMS walls. A channel of 500 µm was created across the scaffold to enable perfusion of media at the interior of the porous network. The scaffold was characterized for the porosity, tortuosity, and mechanical strength. Porosity and tortuosity were calculated using the following equations 9–11. () =

− − − − (2)

( ) = !

2

3[1 − $(1 − )

%' &]

+

1 − − − (3) 3


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As the present scaffold has a non-regular arrangement in 3-D space, B is calculated as a free parameter to be 1.09

9,11

. The scaffold possesses a porosity of 0.99 and tortuosity of 0.54 which

indicate highly interconnected network structure. The porous scaffold can hold liquid up to 203.5% of its own weight. The scaffold possesses a Young’s modulus of 2.2 MPa as it was prepared using 10:1 base to curing agent ratio. The gas permeability of PDMS aided cell growth, and its optical transparency enabled direct visualization of cells

12

. The hydrophilic surface of scaffold material readily mimics the in vivo

microenvironment and facilitates cell adhesion, thus, PDMS was made hydrophilic by oxygen plasma 13, which allowed surface patterning with biological molecules to enhance cell adherence. Surface of PDMS scaffold was successfully modified with fibronectin after 18 min of oxygen plasma exposure (Figure S1). The device was divided into two separate chambers, a medium reservoir and a cell culturescaffold housing chamber (Figure 1). The dimensions of these chambers were 6 x 6 x 1.5 cm and 4 x 6 x 1.5 cm, respectively. The cell culture scaffold housing chamber holds three scaffolds at a time, facilitating experimentation in triplicates. Standard polytetrafluoroethylene (PTFE) tubing of diameter 500 µm connects the outlet of the reservoir chamber to scaffolds (inlet) placed the scaffold chamber. The other end of the scaffold (outlet) was connected by PTFE tube of same dimensions to a peristaltic pump. This tube served as an outlet for spent medium in case of perfused model. The entire set-up was devised to run without any supervision and monitoring. Temperature was maintained by using two external heaters set at 37°C using a temperature controller with feedback system; the heaters were placed both, above and below the STS cell culture device. For the perfused model, perfusion rate was maintained at 0.28 mL/min/g, which is the reported rates of blood flow in breast tumors

14,15

.


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Temperature of the core was measured using a Pt100 probe (ø5 mm) and that at the outlet was recorded using a digital thermometer. For a non-perfused model, outlet from the scaffold was not connected to the external peristaltic pump. The medium flow from the reservoir will be initiated by capillary when the nutrient medium is consumed by the growing cells. In this model, cellular material doesn’t leave the scaffold and no external flow is used ensuring static condition. The MCF-7 and MDA-MB-231 STSnon-perfused could be successfully maintained till 35 days (Figure S1). The fluorescence studies showed that cells migrated deeper inside the scaffold to form STS masses within seven days (Figure 2a, b). Such 3D organization of cells emulated an in vivo tumor-like environment in vitro and could function as a more realistic cancer-testing model 16

. We have also conducted studies on cell cycle (using Premo™ FUCCI Cell Cycle Sensor) at

different time points in the 35-day study. The showed that cells were proliferating during the 35day experiment, and were present in different stages of cell cycle (Supplementary Information, Figure S3). The presence of mostly green (S, G2, and M phases), with intermittent red (G1) and yellow (G1 to S transition) fluorescence at 7, 14, and 35 day time point indicated progression through the cell cycle and division. Further, it has been reported that the cellular organization in 3D tumor models show characteristic homotypic cell-cell connections and imitate conditions of hypoxia

17

. The

expression of VEGF from MCF-7 spheroidal tissue structures increased from 19 pg/mL up to ~400 pg/mL in 35 days. It could be likely that their responses to treatment could be extrapolated to the in vivo cancers to a certain degree. Heat generation by C-LSMO nanoparticles in STSperfused culture Conventional anti-cancer treatments have limited therapeutic efficacy owing to the complex nature of cancer pathogenesis, metastasis, and drug resistance

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. Therefore, alternatives like


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hyperthermia are the need of the day. In our previous studies, we have shown that LSMO nanoparticles are promising hyperthermia mediators

20,21

. It was also established that chitosan

polymer enhanced the colloidal stability of LSMO nanoparticles (C-LSMO). In a radiofrequency field of 365 kHz, LSMO nanoparticles (5 mg) showed a specific absorption rate (SAR) value of 57.4 W/g, indicating optimal heating capacity. In our previous work, we have already shown that doxorubicin could be loaded on C-LSMO nanoparticles with a 76.4% percent drug loading 3.


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Figure 2: a) Schematic depicting the formation of STS in cell culture chips by migration and colonization of cells; b) Confocal micrograph of cellular migration of MCF-7 within porous scaffold as seen after 3 and 7 days (cells stained using Phalloidin-Alexa Fluor 488); and c) A schematic depicting injection of magnetic C-LSMO nanoparticles in the STS followed by radiofrequency exposure and subsequent heat generation patterns due to induced heating perfused and non-perfused STS models. Studies and optimization of heat transfer during magnetic fluid hyperthermia has been enabled by different software simulations and coding techniques

22,23

. However, this study was aimed at


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creating realistic experimental conditions in vitro by using an actual 3D tumor to examine how the heat generated by nanoparticle transfer/alters with blood flow. To study perfusion, nutrient media was used as a blood substitute in STSperfused. In this study, the normal body and blood temperature were assumed to be 37°C, and hence the STS cultures as well as media were maintained at 37°C. It was hypothesized that the heat produced after hyperthermia increases the local temperature at nanoparticles site, while the flowing media (at 37°C) stays at a constant temperature (Figure 2c). The simulation model had exact dimensions as that of the PDMS scaffold. The properties of breast tumor tissue were applied to the model. LSMO nanoparticles (with reported SAR values) were interspersed within the structure, but for convenience of simulation the number of nanoparticles used for study was lesser than real situation. The simulation studies clearly indicated that the increase of local temperature in STS was a function of time. As the time of exposure of nanoparticles to radiofrequency increases, the loco-regional temperature of cells also increases. In 300 s, the core of the STS was seen to attain a temperature of more than 43°C while the outer rim (farthest from nanoparticles) showed a temperature of 1 – 1.4°C lower than the central region. A further increase in radiofrequency exposure time, up to 600 s did not significantly change the temperature of either the STS core or rim (Figure 3a). The rise in temperature was by 1.5°C confirming our experimental data that the heat generation plateaus after continuous radiofrequency exposure. The actual experiment was performed by introducing flow into the STS system (i.e. STSperfused), and the temperatures were measured at the core and the outlet during the hyperthermia treatment (Figure 3b). Magnetic fluid hyperthermia treatment of STSnon-perfused for 10 min using 1 mg C-LSMO nanoparticles leads to temperature of 47°C at the core. When the


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media (36.5 - 37°C) was perfused during MFH, the temperature of STSperfused core region was restricted to 43 - 44°C. The temperature of core was measured using a Pt100 probe. The temperature of media collected at cell culture chip outlet showed rise up to 3 min of radiofrequency exposure, after which an average constant temperature of ~40°C (measured using digital thermometer) was obtained (Figure 3c). This data confirmed our hypothesis that the blood flow has potential to impact MFH treatment significantly.

Figure 3: Simulation studies on heat generation in STSnon-perfused; a) Model of the RF coil and scaffold with magnetic nanoparticles used for simulation; b) A schematic depicting the


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experimental setup and effect of flow on heat generation in STSperfused cultures; c) Profile of temperature at outlet measured using a digital thermometer after MFH under continuous flow conditions. To understand how the flow patterns affected the performance for STSnon-perfused and STSperfused a COMSOL simulation was carried out. A porous scaffold was designed using the software (see Figure 4) with an inlet and outlet for flow of medium. A laminar flow in a porous structure module was used for the simulation study. Boundary conditions such as laminar flow for nonNewtonian liquid was used. The porous scaffold was assigned parameters of poly(dimethyl siloxane) and the liquid parameters was that of medium. A flow of 3 µL/min was set in case of perfused culture and for non-perfused liquid was allowed to flow by capillary forces.

Figure 4: Simulation of fluid flow in a culture chip a) Model of scaffold, b) Capillary based flow in non-perfused STS culture at 0 s, c) Flow (3 µL/min) in perfused culture at 0 s, d) Flow pattern


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in non-perfused STS culture at 1 s, e) Flow pattern observed in the perfused STS culture at 1 s. f) Model to depict the extraction of quantifiable data from Comsol simulation, g) Graph of the flow distribution after 0 and 1 s. and h) Graph depicting flow distribution in perfused culture after 0 and 1 s. Figure 4 (b-e) shows output from the simulation data for non-perfused and perfused models. It can be seen that the velocity in non-perfused culture is significantly lower as compared with the perfused one. Once the medium enters the interconnected porous network due to capillary force, a gradual (diffusion) dispersion of the liquid is evident. Whereas for a perfused setup dominance of convection based flow regime is dominant. In non-perfused cultures, the difference between the initial and final flow throughout the scaffold area could be attributed to the underlying low-pressure capillary forces and a further drop in pressure due to ramifications in porous scaffold. In the perfused cultures, although the flow rate drops once medium enters the porous network; it is still sufficiently high to maintain constant flow within the porous scaffold. It can be inferred that cells can be supplied with medium more efficiently in perfused cultures along with removal of dead cells due to constant flow rate. It can be easily deduced that the non-perfused system resembles the classical in vitro model in 3D, whereas the perfused one is closer to in vivo conditions. It should be noted that, both in nonperfused and perfused setup, medium is distributed evenly throughout the porous matrix which also is the case when nanoparticles are allowed to flow in the system. The flow rate ensures uniformity of medium and temperature throughout the porous scaffold. The flow also takes care of the cellular debris and loosely bound cells leaving healthy cells in the architecture. This ensures accuracy of the hyperthermia and drug action against only healthy cells.


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The effect of MFH on STS tumor models Hyperthermia triggered by magnetic nanoparticles, commonly known as magnetic fluid hyperthermia (MFH) for treatment of cancer has recently garnered attention from the medical fraternity

24

. MFH uses selective, inductive heating of magnetic nanoparticles under externally

applied radiofrequency fields to locally heat and destroy cancerous tissue

25

. The MCF-7 and

MDA-MB-231 STSnon-perfused and STSperfused cultures were subject to a single dose MFH treatment and they showed a reduction in cellular viability after 24 h. However, as expected the viability of both cell lines in perfused STS cultures was higher than the STSnon-perfused cultures. The MCF-7 and MDA-MB-231 STSperfused cultures showed a viability of more than ~50% and 30%, respectively. Whereas their non-perfused counterparts showed lower viability of ~35% and 25%, respectively (Figure 5a). In hyperthermia treatment, the cellular response depends on the degree of applied heat and the thermal sensitivity of individual cells. Such thermal dose– response relationship also varies with different cell types

26

. However, the results obtained here

suggest that such a relationship could also exist not only among different cell culture models, but also on their perfusion status. It should be noted that the initially 1 x 106 cells were seeded in both, non-perfused and perfused models. However, as the flow is initiated in the perfused scaffold, the non-adherent cells are lost due to shear forces. Hence, the effective cell density was lower in perfused scaffold as compared to the non-perfused model. The lower %viability obtained for perfused culture model can therefore, be attributed to variable effective seeding density.


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Figure 5: STS models were treated with

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MFH and their a) Viability, b) Expression for

generated heat shock proteins c) Generation of caspases, and d) Reduction in expression of VEGF assessed after 24 h. The studies were conducted using both, MCF-7 and MDA-MB-231 cells lines. Bonferroni’s multiple comparisons test with p

Assessment of an Integrative Anticancer Treatment Using an in Vitro

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